iraqi journal of hematology vol.5 issue 1 2016
Transcription
iraqi journal of hematology vol.5 issue 1 2016
Iraqi Journal of Hematology(IJH) IJH is a Peer Reviewed Scientific biannual Medical Journal published by the National Center of Hematology Al-Mustansiriyah University, Baghdad-Iraq. May 2016 Volume 5, Issue 1 Editor director Editor-in-chief Prof. Dr.Ali Muhammed Jawad FRCP Prof.Dr. Alaa Fadhil Alwan FICMS Secretary Dr.Nidhal Kareem Alrahhal MSc, D.CH Executive editorial Board Prof.Dr.Raad Jaber Musa FICMS Advisory board Prof.Dr. Naseer Al-Allawi Ph.D (univ.Dohuk) Prof.Dr.Ban Abass Abdulmajid PhD Prof.Dr. Khalid Nafee CABM (univ.Mosul) Prof.Dr.Salma Abass Alhadad CABM Prof.Dr. Ali Muslim CABM (USA,Ohaio) Assist.Prof.Dr.Aladin M.Zubair FICMS Prof.Dr. Ahmed Ibraheem M.D (lebanon) Assist.Prof.Dr.Alaadin S.Naji FICMS Prof.Dr.Anwar Sheikha FRCP(univ.sulaymani) Prof.Dr.Mead Kadhim CABM(Univ.Basrah) Prof.Dr.Subh S. Al-Modalal FICMS(nahrain) Prof.Dr.Waseem Fadhil CABM.(univ.Nahrain) Dr.Jaafar Alghaban consultant CABM Ass.Prof. Adeeb abbas PhD(Uni.mustansiriya) Ass. Prof. Nabil Salman CABM (Egypt) First issue published in 2011 Ass. Prof. Raheem Mahdi FICMS(univ.Kufa) 1st editor-in-chief Dr.Nabil S. Murad Ass.prof.Alaa Sadik Alawad(Univ.Babylon) 1st editor director Dr. Adeeb Alshami Ass. Prof. Mazin Faisal FICMS(univ.baghdad) Ass. Prof.Haitham AlRubai FICMS(Baghdad) Ass. Prof. Ahmed Kudhair FICMS(univ.Erbil) Dr. Fatin Al-Yassin (Bagdad teach.Hosp) Dr.Bassam Francis FICMS(Bagh.Teach.Hosp.) Dr.Asad A. Eledan FICMS(Basrah Teach. Hos) Dr.Ibrahim K. Ibrahim CABM(Kadhimin Hos) Dr Abdulmajeed Alwan CABM(Yarmouk Hos) Dear doctors and colleagues We would like to congratulate all the colleagues specially those who work in hematology field, clinical and laboratory for publishing volume 5 issue 1 of the Iraqi Journal of hematology. This issue contains 10 original articles deal with different topics concerning hematological diseases. We sincerely hope from the authors to continue their support and cooperation through sending original articles, case reports, scientific comments and criticism to the editors in order to keep the journal going on and to keep raising its standards. Kind regards Editor in chief Instructions to Authors Editor Director Prof.Dr.Ali Muhammed Jawad FRCP,CABM Editor in-chief Prof. Dr.Alaa Fadhil Alwan FICMS jnt med, FICMS clin hem Secretary Dr. Nidhal K. Al-Rahhal M.S.C. (Physiology)-D.CH Executive Editorial Board Prof. Dr.Raad Jaber Mosa M.Sc,FICMS(hempath) Prof.Dr. Ban Abbas Abdulmajid Ph.D Molecular path Prof.Dr.Salma Abass AlHadad CABM ped Ass.Prof.Dr.Alaadin Mudafar FICMS(hempath) Ass.Prof.DrAladdin Sahham Naji FICMS jnt med, FICMS clin hem The Iraqi Journal of Hematology is a periodic peer-reviewed journal published biannually by the National Center of Hematology in collaboration with the Iraqi Society of Hematology. The journal welcomes original articles, case reports and letters to editor in all fields relevant to Hematology. Review articles are also welcomed. However, review articles of high standards will be considered.English language is the standard language of the journal yet Arabic abstracts should be accompanying the original articles. Papers are accepted on the understanding that the subject matter has not and will not be submitted simultaneously to another journal. The following notes are expected to be considered carefully in writing manuscripts. 1- Manuscripts preparation: the format of the Iraqi Journal of Hematology complies with the by-standard of the International Committee of Medical Journal Editors (ICMJE) in Vancouver, British Colombia, in 1979 and its last update in February 2006, available on the website www.icmje.org. 2- Three clear and complete copies (including figures and tables) should be submitted. Manuscripts and figures will not be returned to the authors irrespective of the editorial decision to accept, revise or reject them. 3- Manuscripts must be accompanied by a covering letter signed by all authors that the paper has not been published and will not be submitted to another journal if accepted in the Iraqi Medical Journal. 4- The title page should include: Titles of the paper in Arabic and English. Correct first name, middle name and family name of all authors in Arabic and English as well as a maximum of two highest academic degrees for each author. Name(s) and address (es) of the institution(s) where the work was carried out. The name and address of the author responsible for correspondence together with telephone number, fax number and e-mail address (if any). 5- Abstracts for original articles should contain a structured abstract of no more than 250 words in Arabic and English, Abstract headings include: Background, Objectives, Methods, Results and Conclusions. Abstracts in English of the review articles and case reports should be unstructured and of not more than 150 words. 6- The main text of the original article should be divided into sections; each section should be started on a new page after the title page: A. Introduction: should state clearly the purpose and rationale of the study. B. Methods: should include selection of subjects, identifications of the methods, apparatus and chemicals used and include statistical analysis. C. Results: presented in a logical sequence preferably with tables and illustrations emphasizing in the text only the important observations. D. Discussion: emphasizes new findings of the study, implications and reference to other relevant studies. E. Acknowledgements: only to persons who have made substantive contribution to the study. F. References: should be in the Vancouver style. They should appear in the text by numbers in the order. List all authors when six or less; when seven or more, list only first six and add et al. Journal titles should be abbreviated in accordance with index Medicus. Examples of correct reference forms are given as follows: Journal: Al-Salihi AR, Hasson EH, Al-Azzawi HT. A short review of snakes in Iraq with special reference to venomous snake bite and their treatment. Iraqi Med J 1987; 36:57-60. Book chapter: Pen AS. Immunological features of myasthenia gravis. In:Aguayo AJ, Karapti G, editors. Topics in Nerves and Muscle Research. 31st ed. Amsterdam: Experta Medica; 1975; p.123-32. 7- Illustrations: photographs unmounted on glossy paper should be provided with magnification scale if appropriate. Lettering should be in either letraset or stencil of comparable size. Illustrations should be marked on the back with the figure number, title of the paper and name(s) of the author(s) with soft pencil. All photographs, graphs and diagrams should be referred to as figures and should be numbered consecutively in the text in Arabic numerals. The legends to illustrations should be typed on a separate sheet. Tables should be numbered consecutively in the text in Arabic numerals and each typed on a separate sheet. Vertical lines normally will not be printed. 8- Measurement is preferably expressed in SI units. 9- Use only standard abbreviations in the title and abstract. The full term for which the abbreviations stand should precede its first use in the text. 10- Page proof will be sent to the corresponding author for proof correction. Major alterations from the text cannot be accepted. All submission and correspondence should be sent to: Editor-in-chief Iraqi Journal of Hematology (IJH) National Center of Hematology. Hay AlQadisyia - st 14 E-mail: [email protected] Phone: 07901860817 Or you can submit you work online through web site below: www.nchiraq.org/journals Table of Contents Title Inducing of Interleukins -10 and 8 by Epstein Barr Virus in Chronic Lymphocyte Page 1 Leukemia………………… Luma Amer Yassir ,Amer Alnajjarr, Dawood S. Dawood ,Alaa Fadhil Alwan Immunohistochemical expression of VEGF in relation to VEGFR and CD34 in NHL using digital image analysis system……….. 19 Bassam Mohammad Hameed, Hind Shaker Al-Mamoori, Raad Jaber Musa Evaluation of the changes in iron homeostasis and hepcidin concentration in 32 preeclamptic patients…………Noora Abd Ali Muhsin, Subuh Salim Al-Mudalal The results analysis of complete blood count during the first half of 2015 at the 42 Central Child Teaching Hospital in Baghdad………………. Rasha Tariq Jawad , Ebtehal Ali Hussien Distribution of red cell antigens according to ABO, Rh and other rare blood 55 group systems in Kurdish ethnicity…………….Hisham A. Getta, Shaema S.Amin, Najmaddin Khoshnaw , Belal A. Muhammad Review of Congenital Factor XIII Deficiency in Single Iraqi Teaching Hospital 81 Lubna Foad Hussain, Obeida Amir Abid CD38 and ZAP-70 as prognostic immunological parameters In patients with 90 chronic lymphocytic leukemia…………..… Intisar Sh. Ali ,Ghassan A.Al-Anni ,Salman A. Al-Jubury Evaluation the Role of Bone Marrow Examination in Diagnosis of Hematological 98 Diseases in Hemato-Oncology Centers in Kurdistan Region……….Shorsh Jameel Ridha , Nawsherwan Sadiq Mohammad , Hoger Ismael Muhammed Sarhang Types of Anaemia in Patients with Rheumatoid Arthritis in Sulaymaniyah- 114 Kurdistan Region of Iraq……………. Hisham A. Getta, Najmaddin Khoshnaw , Alaa Fadhil Alwan ,Sundus F.A, Raouf R. Mirza A Clinical-Hematological Study of Pancytopenia Patients Attending Nanakaly Hospital in Erbil City………… Alan Isaac Isho , Nawsherwan Sadiq Mohammad, Saran Abdulqadir Nooruldin 129 Original article Inducing of Interleukins-10 and 8 by Epstein Barr Virus in Chronic Lymphocytic Leukemia Luma A. Yassir 1, Amer Alnajjar2, Dawood S. Dawood 3, Alaa Fadhil Alwan 4 1 MSc microbiology/ The national center of hematology/Almustansiriya University/ Baghdad/Iraq 2 Prof. of medical virology/ head Dept. of microbiology / Almustansiriya medical college/Baghdad/Iraq 3 Assistant prof. of medical virology/ Dean of medical technical college/Baghdad / Iraq 4 Prof. of clinical hematology/ director of national center of hematology/Baghdad/ Iraq Received: 2/12 /2015 Accepted: 14/ 12/ 2015 Abstract: Background: Many newer studies reported that Epstein- Barr virus (EBV) has association with chronic lymphocytic leukemia (CLL). The average age of presentation is of patients with CLL is between 65 and 70 years with male to female ratio is 2:1. Notably, several studies have reported that expression of Epstein Barr encoding RNA (EBERs) is associated with progressive or accelerated clinical courses. This type of RNA increases the level of IL-8 and IL-10 in serum of newly diagnosis of CLL patients. Objectives: the aims of study were to determine the interleukins 10 and 8 level in newly diagnosed CLL patients and determine the incidence of EBV infection in patient of CLL. Materials and Methods: A prospective study conducted at department of clinical hematology in the national center of hematology in Baghdad, Iraq from January 2013 to January 2014. It included thirty samples of formalin-fixed, paraffin-embedded tissue of bone marrow aspirates samples and blood from newly diagnosis B-CLL. They were diagnosed with CLL according to Binet criteria. The detection of EBV encoded RNAs (EBER1, EBER2) and also detection for the level of Interleukin (8 and 10) in the serum of CLL patients were done by in situ hybridization technique. Results: Histopathological study revealed that all the controls were negative for EBERs and 46.7% were found to be EBERs positive. There was a correlation between positive EBERs and tumors stage and also EBERs and IL-10 and with IL-8. Conclusion: The highest incidence of CLL occurs in the age group 40-80 years old and males are more liable than females (male/female: 70/30).In situ hybridization technique is successful method to detect of EBV and positive EBERs. IL-8 is highly significant in CLL patients and correlates with EBERs and LMP1. There is a correlation between IL-10 EBERs and LMP1 in CLL patients. Keywords: Epstein-Barr virus, CLL, EBERs, IL-8, IL-10 Iraqi J. Hematology, May 2016, vol.5, Issue 1 1 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. Introduction Chronic lymphocytic leukemia (CLL) regularly results from neoplastic proliferation of a immortalized after exposure to EBV, mature B although this can be achieved after dividing by the accumulation of nonSmall characterized lymphocytes. by a It is persistent lymphocytosis of more than 10 x 109/ liter and lymphoid intrusion of the bone marrow of at least 40% (1,2) With the use of immunological markers, it is likely to establish the diagnosis of CLL by B cell clonality, even with lymphocyte counts of less than 5 x 109/liter. CLL is documented as a disease entity in the WHO organization and as the leukemic counterpart of small activated or cytokine activation.(4) Epstein-Barr virus is the first virus described to be linked with the human pathogenesis of tumor. The topographical distribution of Burkitt lymphoma were related to areas endemic with falciparum malaria is supposed to cause chronic excitement or suppression of the immune system, making children more susceptible to the oncogenicity of EBV.(5,6) In 1968, EBV was recognized to be lymphocytic lymphoma and CLL the most common type of adult leukemia in become the etiological factor of infectious (3) mononucleosis. At the same time, EBV the was reported to alter infected B cells to immunoglobulin heavy chain variable uncontrolled proliferation.(7) A large study region (Ig VH) genes can differentiate in 2009 determined around one in every ten between these two groups: CLL patients stomach cancers contained EBV. Studies are with unmutated immunoglobulin high ongoing to decode what role the virus is the United States and Western Europe. The mutation status of variable (Ig VH) genes have a negative prognosis with rapid progression of the disease.(3) playing in this type of cancer and how it weaves together with other risk factors like nutrition, genetics, and infection of H. pylori.(8) Epstein- Barr virus (EBV) infection is only occasionally detected in CLL by Initiation of EBV lytic program occurs in conventional diagnostic approaches. This memory B cells recirculating during the is consistent lymphoid with in vitro results suggesting that CLL cells do not Iraqi J. Hematology, May 2016, vol.5, Issue 1 tissue related with the oropharyngeal mucosa. Host 2 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. immunosuppression may also Material and Methods: generate viral reactivation in lately infected B cells, which leads to creative infection. Nevertheless, the original mechanism of viral reactivation in vivo is not clearly understood.(9) EBV alters causing This study was designed as a prospective (case-control) study conducted on the following main groups during the period from January 2013 to January Thirty B-lymphocyte formalin-fixed, 2014. paraffin growth, embedded tissue blocks were obtained growth from Bone marrow biopsies and blood permanent transformation by regulated expression samples of B-CLL patients. of multiple viral genes. These genes The age of the patients ranged between comprise 40-80 years, and the three integral membrane proteins, latent membrane proteins 1, collected 2A, and 2B (LMP), 6 EBV nuclear Baghdad Teaching Hospital, antigens (EBNA1, 2, 3A, 3B, 3C, and National Center of Hematology and EBNA-LP), then two small, non-coding Twenty Bone marrow biopsies and blood nuclear RNAs (EBERs). The gene linked sera products relate with or present homology hematological problem other than CLL to as control. various cytokines, endorsing antiapoptotic and signal EBV molecules, transducers, infection, immortalization, and alteration.(10) EBV-1 and EBV-2, which vary depend the EBV nuclear antigen (EBNA) genes.(11) .EBV-1 is more common in most populations and is more competent in transforming B cells in vitro. EBV-2 is mainly found in parts of Africa and is related endemic Burkitt lymphoma. of 20 from patients patients in from the who have We use In situ hybridization to detected EBERs in Bone marrow samples In situ hybridization detection kit from abcam There are two types of EBV viruses , on directly samples were (12) Iraqi J. Hematology, May 2016, vol.5, Issue 1 lot-S01_M61, REF _ WB. 005.50: Hybridization / detection system for EBV was purchased from ZytoFast /Germany Cat. Numbers (T-1070-40) and ELISA kit for detection Human IL-10 and Il-8 by (abcam46059 lot:GR162207- 4) and (abcam46032 the lot: GR151489-6) . 3 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. Results: significantly The distribution of age in the studied group ranged between 40 - 80 years with a mean of (60.4) year (table 4.1). Males constituted 70% of cases and female 30%. The ages of control groups ranged between 42 - 70 years with a mean of (55.8) year. Males constituted 76.2% of control groups and female constituted 23.8%. (Table 1). As shown in table (2), all controls were negative for EBERs. Although the median score and intensity for EBERs was negative for cases group, the mean rank for EBERs score, intensity and composite score (a score resulting from multiplying the score by intensity) was higher among cases compared to controls, figure (1),(2) ,(3) and Figure( 4 ) show the result of In situ hybridization. As shown in table (3), the median IL8 was significantly higher in CLL cases group (33.1 pg/ml) compared to control group (22.1 pg/ml),also in figure (5). A similar pattern was applicable to IL10. The median IL10 was also significantly higher in CLL cases group (29.1 pg/ml) compared to control group (0 pg/ml), figure (6) . As shown in table (4), the positive test for EBERs had no obvious or statistically significant association with IL8 concentration. IL10 concentration also failed to show any noticeable linear correlation with similarly measured IL8 concentration. Table (1): CLL patients distribution according the age and gender Age (years) Gender <50 50--60--=>70 Mean±SD(Range ) P value Male Female P value CLL Control No % 5 16.7 7 23.3 14 46.7 4 13.3 60.4±9.2 (40-80) No % 4 19.0 9 42.9 7 33.3 1 4.8 55.8±8.1 (42-70) 0.392 21 9 70.0 30.0 16 5 76.2 23.8 0.626 . *Significant difference between proportions using Pearson Chi-square test at 0.05 level Iraqi J. Hematology, May 2016, vol.5, Issue 1 4 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. Table 2 : The case-control difference in median score and intensity and composite score for EBERs viral marker. Study group Controls Cases (CLL) N % N % P 1. EBERs-score Negative 20 100.0 16 53.3 + 0 0.0 11 36.7 ++ 0 0.0 3 10.0 Total 20 100.0 30 100.0 (Negative to Range Negative) (Negative to ++) <0.001 Median Negative Negative (Negative to Inter-quartile range Negative) (Negative to +) Mean rank 18.5 30.2 2. EBERs-intensity Negative Weak Moderate High Total Range Median Inter-quartile range Mean rank <0.001 20 100.0 0 0.0 0 0.0 0 0.0 20 100.0 (Negative to Negative) Negative (Negative to Negative) 18.5 16 8 5 1 30 53.3 26.7 16.7 3.3 100.0 (Negative to High) Negative (Negative to weak) 30.2 EBERs-composite score (score x 3. intensity) Range Median Inter-quartile range Mean rank Iraqi J. Hematology, May 2016, vol.5, Issue 1 <0.001 (Negative to Negative) Negative (Negative to Negative) 18.5 (Negative to 6) Negative (Negative to 1) 30.2 5 Relative frequency (%) Inducing of Interleukins -10 and 8 by Epstein Barr 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 Luma A.Y., Amer A., Dawood S. D., Alaa F.A. 10 36.7 ++ + Negative 100 53.3 Controls Cases (CLL) Figure 1: Component bar chart showing the case-control difference in EBERs-score. 100% 0 90% 3.3 16.7 Relative frequency (%) 80% 70% 26.7 60% 50% High Moderate Weak Negative 100 40% 30% 53.3 20% 10% 0% Controls Cases (CLL) Figure 2: Component bar chart showing the case-control difference in EBERsintensity. Iraqi J. Hematology, May 2016, vol.5, Issue 1 6 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. 6 5 4 3 2 1 0 Controls Cases (CLL) Study group Figure 3 : Dot diagram with error bars showing the median (with its inter-quartile range) EBERSs in cases with CLL compared to controls. Iraqi J. Hematology, May 2016, vol.5, Issue 1 7 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. a b : Representative expression of EBV-encoded small nuclear early region (EBERs) in Bone Marrow of CLL patients d . a : positive result show the Nuclear localization of the EBERs in neoplastic cells by ISH . b : Negative result. Figure 4 Iraqi J. Hematology, May 2016, vol.5, Issue 1 8 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. Table 3 : The case-control difference in median interleukin concentration. Study group Controls Cases (CLL) IL8 (pg/ml) Range Median Inter-quartile range N Mean rank (12.2 - 55.3) 22.1 (17.9 - 35.4) 41 20.6 IL10 (pg/ml) Range Median Inter-quartile range N Mean rank P 0.03 (7.8 - 1215) 33.1 (21.9 - 70.1) 30 29.8 <0.001 (0 - 24.9) 0 (0 - 0) 41 12 (0.9 - 43) 29.1 (7.7 - 33.9) 30 35.8 10000 IL8 1000 100 10 1 Controls Cases (CLL) Study group Figure 5 : Dot diagram with error bars showing the median (with its inter-quartile range) IL8 (pg/ml) in cases with CLL compared to controls. (Logarithmic scale was used) Iraqi J. Hematology, May 2016, vol.5, Issue 1 9 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. 45 40 35 IL10 30 25 20 15 10 5 0 Controls Cases (CLL) Study group Figure 6 : Dot diagram with error bars showing the median (with its inter-quartile range) IL10 (pg/ml) in cases with CLL compared to controls. Table 4 : The median IL8 concentration (pg/ml) by selected explanatory variables among cases with CLL. Range IL8 Interquartile Median range N Mean Rank Age group (years) <60 60+ (10.6 to 79.4) (7.8 to 1215) 31.9 36 (22.2 to 61.3) (21.9 to 130.3) 12 18 14.5 16.2 Gender Female Male (7.8 to 1215) (10.6 to 346.2) 55.6 32.8 (20 to 401.5) (24.3 to 46.9) 9 21 17.2 14.8 Positive EBERs Negative Positive P 0.6[NS] 0.5[NS] 0.1[NS] (10.6 to 1215) (7.8 to 401.5) 43.1 30.4 Iraqi J. Hematology, May 2016, vol.5, Issue 1 (25.3 to 104.9) (17.7 to 44.4) 16 14 18 12.7 10 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. Table 5 : The median IL10 concentration (pg/ml) by selected explanatory variables among cases with CLL. IL10 Interquartile Median range Range Age group (years) <60 (0.9 to 38.3) 60+ (2.5 to 43) Positive EBERs Negative Positive 20.35 32.3 (6.25 to 29.85) (9.3 to 34.8) N Mean Rank 12 18 12.9 17.3 P 0.18[NS] 0.63[NS] (0.9 to 43) (2.2 to 43) 20.75 32.3 (8.5 to 33.9) (4 to 33.9) 16 14 14.8 16.3 Discussion : Epstein- Barr virus has strong association with variety in B-cell tumors including Burkitt's lymphoma, Hodgkin lymphoma, human immunodeficiency virus, post transplantation lymphoma disorder and chronic However EBERs is also found in quiescent EBV latency where no protein is produced and that may be a suboptimal marker for proliferation or transformation capability. lymphocytic leukemia. Many studies reported that In this study all controls showed CLL patient had evidence of EBV negative result EBERs but in patients the infection by In situ hybridization for result show that 14 (46%) out of 30 CLL EBERs and detection of EBV-encoded patients were positive with EBERs. EBER transcripts is considered the gold standard for localizing latent EBV in tissue samples, as EBER transcripts are universally expressed in all EBV associated tumors.(13,14) Results obtained are nearly compatible to previous study who reported that (38%) of CLL patients had evidence of EBV infection proved by EBERs positively in tumor cells (16). The presence of EBERs has been shown or Result demonstrated in this study were in accelerated clinical course including accordance with 16) Tsimberidou et al transformation to Richter's large cell who stated that 12 out of 32 CLL lymphoma.(15,16) patients has appositive result.(16) to correlate with progressive Iraqi J. Hematology, May 2016, vol.5, Issue 1 11 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. On the other hand another study found In our study we found that IL-8 was that 8 of 75 (10.7%) cases showed significantly higher in CLL cases group EBERs expression restricted to 5–10% the compared with control group. This of tumor cells. (17) result is compatible with other study This controversy in the above results may be related to that ISH process depends on the RNA staining and the concentration of RNA in the cell .this method affected by many factors, including the RNA present in the cell and concentration of RNA. This technique is very sensitive. Interleukins8 and it's receptor increased in cancer published (23,24) which found that plasma IL-8 level enhances in CLL patients. The same result was studied by Yoshizaki et al who found that IL-8 increased in CLL patients .(25) On the other hand another study found that serum IL-8 level was not increased in significant level in patient with CLL when she compared with healthy control.(26) cells, infiltrating neutrophils, endothelial The association of EBERs and IL-8 did cells, not and tumor-associated macrophages (18) by normal B cells but many studies showed that the natural cellular source of IL-8 production have been described to be monocyte/macrophages, T cells, large granular lymphocytes, fibroblasts, cells, mesothelial cells, keratinocytes, neutrophils ,hepatocytes and chondrocytes.(19,20,21) al showed that elevated IL-8 levels may be founds in the serum of untreated B cell patient which may be release by B cells and superannuated of purified Bcells contain level This of statistical result is not compatible with study done by William et al who found that the stages were associated with significantly higher plasma IL-8 levels (P < 0.0001) but There were no significant difference between IL-8 production and gender. (27) While another study found compatible result with this study where IL8 level in CLL patients not correlated with CLL stages. (24) A study which has been done by Celle et CLL the significance. There is no evidence of IL-8 production endothelial reach IL8 released chemotactic activity for neutrophils. (22) Iraqi J. Hematology, May 2016, vol.5, Issue 1 Notably, serum IL-10 levels are increased in CLL patients and correlate with adverse disease features and short survival.(28,29) Results obtained in this study revealed that the IL-10 was significantly higher in CLL cases group median rang (29.1 pg. 12 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. /ml) compared to control group (0 pg/ml) new approach for the treatment of B- and the mean rank 35.8 with p<0.001 CLL.(33,34) figure (6) IL-10 was derived from EBV infected This result is compatible with other tumor cells and demonstrated in serum study(30),Which found that IL-10 levels of CLL and Hodgkin lymphoma patients. were higher in CLL patients (median, (30,34) 5.04 pg/mL; range, undetectable to 74 association between EBERs, LMP1and pg/mL) IL10 than normal volunteers undetectable; range, enhanced production of viral IL-10 and undetectable to 13.68 pg/mL) (P < may also contribute to a local immune .00001) ,and another study found that IL- suppression 10 levels increased in CLL patient of hydrophobic peptides derived from the Iraq and significantly than control group first transmembrane domain of LMP-1 P<0.05 same study was studied by [31] (35) who found that Serum levels IL-10 in 20 transcription CLL patient depending (median, in . Several studies have reported an severely dropped in untreated group (27±11.47 and 0.65±0.23 pg/mL respectively) and differed significantly stimulation. EBV by infection production of In contract EBERs induce the of on various cell type, cytokines such as interleukin-10 (IL-10). healthy in 20 Conclusion: The highest incidence of control group (1715.66±1014 pg/mL CLL occurs in the age group 40-80 years respectively) (26) and David at al who old and males are more liable than found that Serum IL-10 levels were also females (male/female: 70/30).In situ significantly elevated in hybridization technique is successful CLL patients. (18) method to detect of EBV and positive Other explanations for the increase in EBERs. IL-8 is highly significant in CLL the level of IL-10 in patient with CLL patients and correlates with EBERs and was demonstrated in other researches LMP1. There is a correlation between which found that IL-10 EBERs and LMP1 in CLL IL-10 is increased in production by culture of CLL and that patients. serum IL-10 levels were elevated in five of the eleven B-CLL patients. These findings suggest that IL-10 acts as an autocrine growth factor for B-CLL cells and cytokine-based therapy might be a Iraqi J. Hematology, May 2016, vol.5, Issue 1 13 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. References: 1. Estella M. Claire D. Chronic 7.Diehl V. , Henle G. , Henle W. , and lymphocytic leukemia, The Medicine Kohn G. Demonstration of a herpes Publishing Company Ltd , (2004) . group virus in cultures of peripheral 125(2):P15-38. leukocytes from patients with infectious 2. Garcia-Muñoz R. & Galiacho V. R . mononucleosis. Journal of virology. Immunological (1968); 2(7): p. 663-9. aspects lymphocytic in chronic leukemia (CLL) 8.Murphy G. , Pfeiffer R. , Camargo development. Ann Hematology .(2012) . M.C. , and Rabkin C.S. Meta-analysis 91:981–996. shows that prevalence of Epstein-Barr 3. Byrd J.C. , Stilgenbauer S. , and Flinn virus-positive I.W. Chronic lymphocytic leukemia. based on sex and anatomic location. Hematology. Am. Soc. Hematology. Gastroenterology. (2004) Educ.V,11(3): p. 163-183. 824-33. 4. Mao Z. , Quintanilla-Martinez L. , 9.Ayumi K., Masatoshi F., Tohru K. et al Raffeld M. , Richter M. , et al. IgVH . Inhibiting Cellular DNA Replication mutational status and clonality analysis Cyclin-Dependent Kinase Activity while of Richter's transformation: diffuse large Virus Occurs with High S-Phase Cells B-cell Latently Infected with Epstein-Barr: lymphoma and Hodgkin lymphoma in association with B-cell chronic lymphocytic leukemia (B-CLL) represent 2 different pathways of disease evolution.(2007). Am J Surg Pathol. 31(10): p. 1605-14. 5.Hjalgrim H , and Engels EA. Infectious aetiology of Hodgkin and non-Hodgkin lymphomas: a review of the epidemiological evidence. Journal of internal medicine. (2008); 264(6): p. 537-48. 6.Burkitt D. Etiology of Burkitt's lymphoma--an alternative hypothesis to a vectored virus. Journal of the National Cancer Institute. (1969) ;42(1): p. 19-28. Iraqi J. Hematology, May 2016, vol.5, Issue 1 gastric cancer differs (2009);137(3): p. Journal of virology .(2003) ;77(2):851. 10.Ayln F. K., Özyar EN. S., Ayfie A. A. University, Epstein-Barr virus genes and nasopharyngeal cancer. Turkish Journal of Cancer. (2006). Volume: 36, No.3. 11. Bortolin M T , Pratesi C , Dolcetti R et al., Clinical value of Epstein–Barr virus DNA levels in peripheral, blood samples of Italian undifferentiated patients carcinoma with of nasopharyngeal cancer type. Letters 233. (2006) ; 247–254. . 12.Young LS , Yao QY , Rooney CM , Sculley TB , et al. New type B isolates of 14 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. Epstein-Barr Youwei Chen, Guglielmo M. Venturi, virus from Burkitt's lymphoma and from normal individuals Giandomenico in endemic areas. The Journal of general Gockerman, Joseph O. Moore, Louis F. virology. (1987); 68(Pt 11): p. 2853-62. Diehl,Alicia D. Volkheimer, Daphne R. 13.Tsimberidou AM , Keating MJ , Friedman, Mark C. Lanasa, Russell P. Bueso-Ramos CE , and Kurzrock R Hall, and Thomas F. Tedder,Chronic Epstein-Barr virus in patients with Lymphocytic Leukemia and Regulatory chronic lymphocytic leukemia: a pilot B Cells ShareIL-10-Competence and study. Leuk Lymphoma. .(2006); 47(5): Immunosuppressive p. 827-36. Leukemia. (2013) ; 27(1): 170–182 14.Neparidze N , and Jill M.D. 19.Smyth Russo, M.J., Jon P. Function Zachariae , C.O.C., Malignancies Associated With Epstein- Norihisa Y., Ortaldo J.R., Hishinu A., BarrVirus: Clinical Matsushima K . IL-8 gene expression Features, and Evolving Treatments , and production in human peripheral Clinical Advances in Hematology & blood Oncology.(2014); 12(6). Immunology.(1991); 146:3815. Pathobiology, lymphocyte subsets. J 15.Ansell S.M., Li C.Y., Lloyd R.V., Phyliky RL. Epstein-Barr virus infection in Richter’s transformation. Am J 16.Tsimberidou AM , Keating MJ , Bueso-Ramos CE , and Kurzrock R. Epstein-Barr virus in patients with chronic lymphocytic leukemia: a pilot study. Leuk Lymphoma. (2006) ; 47(5): L, Mantovani A. Expression of adhesion molecules and chemotactic cytokines in cultured human mesothelial cells. J Exp Med . (1992). 176:1165. 21.Strieter RM, Chensue SW, Standiford TJ, Basha MA, Showell HJ, Kunkel SL p. 827-36. vaccines, Sciacca FL, Colotta F, Pelicci PG, Lanfrancone Hematol. (1999) ;60(2):99-104. 17.Cohen 20.Jonjic N, Peri G, Bernasconi S, J.I. Epstein–barr Clinical & virus Translational .Disparate gene chemotactic cytokines mononuclear expression phagocytes. by of human Biochem Immunology ; doi (2014):10.27. Biophys Res Commun (1992) .1662386. 18.David J. DiLillo, J. Brice Weinberg, 22.Celle P.F. , Carbone A. , Marchis D., Ayumi Yoshizaki, Mayuka Horikawa, Zhou D, Zupo, Massimo Pini et al Jacquelyn .Cytokine Gene Expression in B-Cell M.Bryant, Yohei Iwata, Takashi Matsushita, Karen M. Matta, Iraqi J. Hematology, May 2016, vol.5, Issue 1 Chronic Lymphocytic Leukemia 15 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. Evidence of Constitutive Interleukin-8 27.William G. Wierda,1 Marcella M. (IL-8) mRNA Expression and Secretion Johnson,2 Kim-Anh Do,et al. Plasma of Biologically Active IL-8 Protein, interleukin 8 level predicts for survival Blood.(1994); 84(1) : p 220-228. in chronic lymphocytic leukaemia , British Journal of Haematology. (2003) ; 23.Wierda, G, William Marcella M. Johnson,2 Kim-Anh Do, et al., Plasma interleukin 8 level predicts for survival in chronic lymphocytic leukaemia , British Journal of Haematology.(2003) ; 120, 452–456. 28.Dolcetti R. , and Masucci M.G. Epstein-Barr virus: induction and control of cell transformation. J Cell Physiol. (2003). 196(2): p. 207-218. 120, 452–456. 24.Kara I.O., Sahin B., Gunesacar R 29.Vockerodt M , Morgan SL , Kuo M , and Wei W , et al. The Epstein-Barr virus interleukins-8 and -10 in B-cell chronic oncoprotein, latent membrane protein-1, lymphocytic leukemia. Correlation with reprograms germinal centre B cells disease towards a Hodgkin's Reed-Sternberg-like .Expression of stage soluble and CD27 prognosis.(2007); 24(1) : pp 29-40. phenotype. J Pathol. (2008); 216(1): p. 25.Yoshizaki T, Horikawa T, Qing-chun 83-92 R, Wakisaka N et al. Induction of 30.Fayad L., Keating M. J., Reuben M. J. Interleukin-8 et al. Interleukin-6 and interleukin-10 by Epstein-Barr Virus Latent Membrane Protein-1 and Its levels Correlation leukemia:correlation to Nasopharyngeal Angiogenesis Carcinoma, in Clinical in chronic with lymphocytic phenotypic characteristics and outcome .by The Cancer Research. (2001);Vol. 7, 1946– American Society 1951. Blood. (2001); 97 (1). of Hematology. 31.Al-Dabagh M. A. and Al-Mayah K. 26.Ahmed N. Sh., Saad M. Nada, Hadi Sh. Evaluation of immune response in Y. A. , Ibraheem A. N., Jaafar M.R. patients Expression of Cytokine of Chronic leukemia II: Humoral immune response. Lymphocyte Leukemia from Baghdad / Iraqi journal of science , (2011) ;Vol Iraq Population, Iraqi Journal of Cancer .52,No.3, ,P.388-393. and Medical GeneticsVolume. .(2014);7 32.Kitabayashi A., Kirokawa M., Miura (2). A.B . The role of interleukin-10 (IL-10) with chronic lymphocytic in chronic B-lymphocytic leukemia: ILIraqi J. Hematology, May 2016, vol.5, Issue 1 16 Inducing of Interleukins -10 and 8 by Epstein Barr Luma A.Y., Amer A., Dawood S. D., Alaa F.A. 10 Exprazession prevents leukemic cells from of the Transcription apoptotic cell death. Int J Hematology. Factors ATF-2/c-Jun in B Lymphocytes .(1995 ;62:99-106. but not in B-CLL Cells Virus Genes 33.Tangye S.G., Weston K.M., Raison ,(2005); 30:3, 323–330. R.L .Interleukin- 10 nhibits the in vitro 35.Naji A. S. Outcome of 49 Iraqi adult proliferation patients leukemic of CD51 human activated B-cells. Leuk with Chronic Lymphocytic Leukemia treated with oral alkylating Lymphoma. .(1998) ;31:121-130. agent:, J Fac Med Baghdad. (2012); 54 34.Blay J.Y., Voorzanger N., Favrot M., (2). Burdin N., Rousset F., Banchereau J. Presence of Epstein-Barr virus viral interleukin 10 in the serum of patients with non-human-immunodeficiency- Correspondance to Luma Amer MSc. virus related diffuse large-cell nonHodgkin’s lymphomas. Blood. (1995). 86:4702-4704. Dept. of molecular genetic The national center of hematology 34.Bandobashi K. , Liu A. , Mi Nagy N. et al .EBV Infection Induces Iraqi J. Hematology, May 2016, vol.5, Issue 1 Almustansiriyah university 17 Luma A.Y., Amer A., Dawood S. D., Alaa F.A. Inducing of Interleukins -10 and 8 by Epstein Barr تحفير االنترلوكين 01-و 8بواسطة فيروس ابشتاين بار في سرطان الدم الليمفاوي المزمن لمى عامر ياسر ،0عامر النجار ، 2داود سلمان داود ، 3عالء فاضل علوان 4 1ماجستير علم األحياء المجهربة /المركز الوطني ألمراض الدم /الجامعة المستنصرية /بغداد /العراق 2أستاذ علم الفيروسات الطبية /رئيس قسم األحياء المجهربة /كلية طب المستنصرية /بغداد /العراق 3استاذ مساعد .علم الفيروسات الطبية /عميد كلية التقنية الطبية /بغداد /العراق 4أستاذ أمراض الدم السريرية /مدير المركز الوطني لألمراض الدم /بغداد /العراق الملخص: الخلفية :اثبت الدراسات ان االبيشتاين بار فايرس له عالقة ويعتبر احد المسببات لمرضى سرطان الدم اللمفاوي المزمن في هذه الدراسة كان معدل عمر المرضى يتراوح بين ( )56-07عام وكانت نسبة الذكور الى االناث تتراوح بين % 1/2في مرضى سرطان الدم اللمفاوي المزمن وكما اثبت هذه الدراسة ان EBERsيزيد من افراز IL-10و IL-8في مصول المرضى . المواد والطرق :دراسة مستقبلية أجريت في قسم علم أمراض الدم السريري في المركز الوطني لألمراض الدم في بغداد ،العراق خالل الفترة من يناير 3102إلى يناير .3102وشملت ثالثين عينات من واألنسجة الثابتة بالفورمالين جزءا ال يتجزأ من البارافين من عينات نخاع العظام والدم من حديثي التشخيص ب سرطان الدم اللمفاوي المزمن ..وقد أجريت للكشف عن الرنا EBV المشفرة ( ،)EBER2 ،EBER1وكذلك الكشف عن مستوى انترلوكين ( 8و )01في مصل الموقعي. التهجين بتقنية المزمن اللمفاوي الدم سرطان مرضى النتائج :كشفت الدراسة النسيجية أن جميع الضوابط كانت سلبية EBERsووجد ٪2..4 إيجابية من EBERsفي سرطان الدم اللمفاوي المزمن .كان هناك ارتباط بين EBERsاإليجابية .IL-8 مع و IL-10 و EBERs وأيضا األورام ومرحلة االستنتاج :يحدث أعلى معدل النتشار سرطان الدم اللمفاوي المزمن في الفئة العمرية 81-21سنة والذكور أكثر عرضة من اإلناث (ذكور /إناث . )21/41 :تقنية التهجين الموقي هي طريقة ناجحة للكشف عن EBVو EBERsاإليجابية IL-8 .مهم للغاية في المرضى الذين يعانون من سرطان الدم اللمفاوي المزمن ويرتبط EBERsو .LMP1هناك عالقة بين EBERs IL-10 المزمن. اللمفاوي الدم سرطان مرضى في وLMP1 كلمات البحث :فيروس ابشتاين بار ،سرطان الدم اللمفاوي المزمن IL-10 ،IL-8 ، 18 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Original article Immunohistochemical expression of VEGF in relation to VEGFR and CD34 in NHL using digital image analysis system Bassam Mohammad Hameed1, Hind Shaker Aboob2, Raad Jaber Musa3 1 M.B.Ch.B., Msc. Pathology (haematology), Ph.D. Pathology (haematology) Lecturer at Department of pathology and forensic medicine /collage of medicine /AL-Nahrain university 2 M.B.Ch.B., FIBMS(path/haemaology) Lecturer at Department of pathology and forensic medicine /collage of medicine /AL-Nahrain university 3 M.B.Ch.B., M. Sc. Path. (Haem.); F.I.C.M.S.Path.(Haem.) Professor of Haematology at Department of pathology and forensic medicine /collage of medicine /AL-Nahrain university Received 31/12/2015 accepted 15/2/2016 Abstract Background: Lymphoma growth and progression appear to be promoted by at least two distinct angiogenic mechanisms: autocrine stimulation of tumor cells via expression of Vascular Endothelial Growth Factor(VEGF) and Vascular Endothelial Growth Factor Receptor(VEGFR) by lymphoma cells, and paracrine influences of the proangiogenic tumor microenvironment on local tumor vascularity. Objectives: To assess autocrine effect of VEGF, by studying the correlation of VEGF expression with its receptor VEGFR expression in NHL. And to assess paracrine effect of VEGF, by studying the correlation of VEGF expression with CD34 expressed on endothelial cells in Non-Hodgkin Lymphoma (NHL). Materials and Methods: A cross sectional study was designed. A total of 66 bone marrow tissue samples were included in the study, all diagnosed as having NHL according to working formulation. From each block, 3 sections were taken, and were immunohistochemically stained for CD-34, VEGF and VEGFR. Scoring of Immunohistochemical staining was performed using specialized automated cellular image analysis system, Digimizer software, version 3.7.0. Results: VEGF Immunohistochemical digital parameters named digital labeling index (DLI) was significantly correlated with the followings; VEGFR (DLI) [P =0.042, r =0.324], CD34 stained area (A) [P=0.037, r =0.556]. Also VEGFR (DLI) was significantly correlated with CD34 (A). Conclusion: Autocrine and paracrine effect of VEGF is evident in NHL, as there is positive correlation between VEGF expression and VEGFR expression, and as tumor vascularity increase with the increase in VEGF expression. Keywords: NHL, VEGF, VEGFR Iraqi J. Hematology, May 2016, vol.5, Issue 1 19 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa Introduction quantification while overcoming manual Tumor angiogenesis mediated by many mediators in tumor microenviroment, of these VEGF (vascular endothelial growth scoring method that requires considerable expertise and is interobserver variability. susceptible to (4) Aims of the study factor) is the most important factor for angiogenic switch through its interaction with receptors regulating different aspects of tumor angiogenesis. Lymphoma growth and progression as many other tumor promoted by angiogenesis, this occurs by two main mechanisms; autocrine stimulation of tumor cells by expressing both VEGF and its receptor VEGFR, and second by paracrine influences of the proangiogenic tumor microenvironment. (1) Many researchers stated that there is positive expression correlation with between studying the correlation of VEGF expression with its receptor VEGFR expression, in bone marrow biopsy of nonHodgkin lymphoma patient using automated image analysis system. 2. To assess paracrine effect of VEGF, by studying the correlation of VEGF expression with CD34 expressed on endothelial cells, in bone marrow biopsy of non-Hodgkin lymphoma patient using automated image analysis system. Materials and methods expression A cross sectional study was designed, a in DLBCL lymphoma cells supporting the total of 66 tissue samples (paraffin block concept of autocrine-paracrine role for of bone marrow biopsy) were included in VEGF as growth factor for DLBCL the study. All the samples were taken from promoting the (Medical city/ teaching laboratories), cell VEFGR VEGF 1. To assess autocrine effect of VEGF, by survival and proliferation.(2,3) cases presented during the period 2008- In this study we tried to study angiogenesis via VEGF, VEGFR and microvessel density in non-Hodgkin lymphoma using digital analysis system, Advantages of using digital image system in that scoring immunohistochemical staining in this method offer objectivity, reproducibility, Iraqi J. Hematology, May 2016, vol.5, Issue 1 2010 as all diagnosed having NHL according to working formulation by histopathological examination for primary lymph node biopsy. From each block, 3 sections of 5µm thickness were taken, each section were immunohistochemically stained for CD-34, VEGF and VEGFR. 20 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa The procedure was carried out according immunostaining intensity was done by to manufacturer's instructions. Taking using the Magic Wand tool in the toolbar sections and mounted on Fisher brand menu in digimizer program (see figure 1). positively charged slides. Than slides The tolerance level of the Magic Wand deparaffinized, and placed in DAKO tools was adjusted so that the entire antigen retrieval (PH 6 for VEGF, PH 9 positive for VEGFR, 1700 for CD34) after heating measurements comprised: slides with antigen retrieval solution in microwave for 20 min, slides let to cool down. Later on LSAP (DAKO staining kit) used for staining, after blocking endogenous peroxidase, and incubation of primary antibody for 30 min. Scoring of immunohistochemical staining was performed using specialized automated cellular image analysis system, Digimizer software, version 3.7.0. a were selected. The 1. Color Intensity (I): which measures the average intensity of the brown color for the selected objects depending on the expression of antigens in the cells. 2. Fractional area stained (A) = [(mean area× Number of objects) / area of a single image field] 100% 3. Digital Labeling Index (DLI): first used by Al-Sinjery, G. M. [5], this tool is calculated according to the following Image capture Using cells formula: light microscope, each DLI= [Fractional area(A) × reverse immunohistochemically stained slide was Intensity(I)]. scanned with 10 × objectives for the This positive brown immunostaining, and with representative for the expression because it 40× objective three fields that reflect the combines both the Fractional area and the best of the overall immunostaining of the Intensity entire slide were chosen and captured staining. using a Sony digital camera (cyber-shot DSC-W510). Captured images of digital parameter, of is the best immunohistochemical Statistical analysis: 4000×3000 pixels were saved on PC in an Statistical analysis was performed with uncompressed JPG format. SPSS Image analysis (statistical package for social sciences) version 16 and Excel 2007 programs. Each image was analyzed by Digimizer software (Version 3.7.0). Determination of Iraqi J. Hematology, May 2016, vol.5, Issue 1 21 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa Figure 1. showing snap shot for digimizer softwear window Results Correlation between digital parameters of correlated with the followings; VEGFR digimizer softwear of VEGF and other digital angiogenic markers in NHL: Digital staining area (A), as well as VEGFR (DLI) parameter for VEGF digital labeling index was significantly correlated with the CD34 (DLI) which combines staining intensity staining area (A). figure (II) labelling index (DLI), CD34 and area staining was significantly Iraqi J. Hematology, May 2016, vol.5, Issue 1 22 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa y = 0.553x + 21.406 120 VEGF DLI 100 80 60 40 20 0 0 20 40 60 80 100 120 VEGFR DLI Figure II. Correlation chart shows positive correlation between VEGF (DLI) and VEGFR1(DLI),(P =0.042, r =0.324). 140 y = 0.9586x + 13.552 120 VEGF DLI 100 80 60 40 20 0 0 10 20 30 40 50 60 CD34 area Figure III. Correlation chart shows positive correlation between VEGF (DLI) and CD34 (A),(P=0.037, r =0.556). Iraqi J. Hematology, May 2016, vol.5, Issue 1 23 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa 120 100 VGEFR DLI 80 y = 1.1056x - 1.5145 60 40 20 0 0 10 20 -20 30 40 50 60 CD34 area Figure IV. Correlation chart shows positive correlation between VEGFR-1 (DLI) and CD34(A),(P=0.041, r = 0.303). Figure V. Trephine biopsy section from a patient with Folliculer lymphoma showing paratrabecular infiltration& random focal . H&E, x 4 objective. Iraqi J. Hematology, May 2016, vol.5, Issue 1 24 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa Figure VI. Trephine biopsy section from a patient with B-SLL showing nodular infiltration . with D20 + immunohistochemistry (X 4 objective.). Figure VII. Trephine biopsy section from a patient with B-DLL showing diffuse infiltration. With CD20 + immunohistochemistry (X 10 objective.) Iraqi J. Hematology, May 2016, vol.5, Issue 1 25 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa Figure VIII. Trephine biopsy section from a patient with B-DLL stained with immunohstochemistry showing positive VEGF-A lymphoma cells (arrows). With cytoplasmic brown staining (40X). Iraqi J. Hematology, May 2016, vol.5, Issue 1 26 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa Figure IX. Trephine biopsy section from a patient with B-DLL showing endothelial cells positive for CD34 stained with immunohstochemistry (40X). Discussion In Iraq digimizer software for CD34+ blood many researchers studied angiogenesis in different malignancies like prostate adenocarcinoma, multiple myeloma , renal cell carcinoma, gastric adenocarcinoma.(6-9) Kareem and Jaafer studied angiogenesis in haematolymphoid tumors, Kareem studied MVD in NHL, while Jaafer studied VEGF in CLL.(10,11) But most of these studies used microvessel density as a marker of angiogenesis, with manual count, restricting their result to blood vessel count. Another study done by Qasim B. et al pushed the work step forward by using Iraqi J. Hematology, May 2016, vol.5, Issue 1 vessels , this make assessment more subjective and computer based.(12) This work tried to assess angiogenic markers in NHL, by studying VEGF expression and its autocrine effect via its receptor VEGFR expression and the paracrine effect via CD34+ vessel expression, using Digimizer software to analyze their immunohistochemical expression, instead of manual count for blood vessels and manual score for VEGF/VEGFR expression. Many researcher suggest that VEGF may play dual roles in tumor angiogenesis, first through signaling to endothelial cells 27 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa promoting them to form new blood vessels paracrine role of VEGF elaborated by resulting in angiogenesis and second via lymphoma cells in tumor angiogenesis.(3) signaling to tumor cells as an (2) This autocrine/paracrine growth factor. Conclusion research assessed the interaction among Autocrine and paracrine effect of VEGF is vascularity local evident in NHL, as there is positive expression of VEGF and VEGFR in NHL correlation between VEGF expression and cases. VEGFR expression, furthermore as tumor (CD34+ area) and vascularity increases expressed by CD34 VEGF expression had positive correlation with VEGFR expression in different stained area with the increase in VEGF expression. digimizer softwear parameters (intensity "I", area "A", digital labeling index "DLI"). This may be due to autocrine effect of VEGF. Both VEGF and VEGFR had positive correlation with lymphoma vascularity expressed by CD34 area "A", also reinforcing the concept of paracrine effect of VEGF. These findings are similar to findings of previous work.(10,13-18) The VEGF reflective of immunohistochemistry effective local is VEGF signaling. It is expected that MVD increases with VEGF expression. Average lymphoma vascularity labeled by CD34+ endothelial cells did increase with strength of VEGF staining with statistical significant in the present work, and this was also demonstrated in the study by Berthold et al . (19) Dita et al have shown that higher MVD is present in DLCL specimens expressing higher levels of VEGF.(16) This finding is consistent with a Iraqi J. Hematology, May 2016, vol.5, Issue 1 28 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa References: 1. Ruan, J., et al., Angiogenesis and 6. Nabeel, W.R.,Sahira A.A., and Usama antiangiogenic therapy in non-Hodgkin's T.A. Usama, neuvasculrization in prostatic lymphoma. Ann Oncol, 2009. 20(3): p. adenocarcinoma as determined by CD34: a 413-24. retrospective 2. Gratzinger, D., et al., Microvessel study. Iraqi journal of medical sciences 2011. 9(4): p. 371-375. Density and Expression of Vascular 7. Al-Mudallal, S.S., Assessment of bone Endothelial Its marrow angiogenesis using F VIII-related B-Cell antigen and its relationship to proliferating American cell nuclear antigen (PCNA) in multiple Journal of Pathology, 2007. 170(4): p. myeloma. Journal of the Faculty of 1362-1369. Medicine 2011. 53(2): p. 180-185. Receptors Lymphoma 3. Growth in Factor Diffuse Large Subtypes. Alshenawy, and The H.A., Prognostic 8. Mazin, J.I., NabeelA.Rasheed, and A.H. significance of vascular endothelial growth Manal, Microvessel density in Renal Cell factor, basic fibroblastic growth factor, and Carcinoma. Journal of the Faculty of microvessel density and their relation to Medicine 2010. 52(4): p. 424-425. cell proliferation in B-cell non-Hodgkin's lymphoma. Annals of Diagnostic Pathology, 2010. 14(5): p. 321-327. 9. Al-kaptan A.H,I., Uses of CD31 monoclonal Antibody for the Assessment Of Angiogenesis as a prognostic Factor in 4. Ghaznavi, F., et al., Digital Imaging in Gastric Adenocarcinoma. Journal of the Pathology: Faculty of Medicine 2005. 47(1): p. 42-49. Whole-Slide Imaging and Beyond. Annual Review of Pathology: Mechanisms of Disease, 2012. 8(1): p. 121116135809005. 10. Kareem, G.M., A Study of Angiogenesis Measured by Expression of CD34 antigen in Non-Hodgkin's 5. Al-Sinjery, G.M., Immunohistochemical Lymphoma, in A thesis submitted to the Expression of Epstein Barr virus Antigen council of the College of Medicine at Al- Latent Membrane Protein-1 and Bcl-2 in Nahrain University. 2006, AL-Nahrain: Classical pathology Hodgkin’s and Lymphoma, forensic in medicine department. 2011, AL -Nahrain: Baghdad. baghdad. 11. Ja’afar A.M., In Situ Hybridization Analysis of p-53 and Bcl-2 Oncogenes and Angiogenesis Factors VEGF and MMP-9 Iraqi J. Hematology, May 2016, vol.5, Issue 1 29 Immunohistochemical expression of VEGF Bassam M.Hameed, Hind Sh. Aboob, Raad J. Musa in Chronic Lymphocytic Leukemia, in A cell lymphoma treated with anthracycline- Thesis Submitted to College of Medicine based chemotherapy. Lab Invest, 2007. and Committee of Graduate Studies 88(1): p. 38-47. University of Baghdad. 2008, Baghdad. 12. Qasim, 16. Dita G, et al., Microvessel density and B.J., Assessment of expression of vascular endothelial growth Immunohistochemical Expression of factor and its receptors in diffuse large B- Matrix cell lymphoma subtypes. Am J Pathol, Molecular Markers: Metalloproteinase -7 (MMP-7), CD34, p53, bcl2, Proliferating Cell Nuclear Antigen (PCNA), Estrogen Progesterone Receptors in Colorectal Carcinogenesis and Human Using 2007. 170: p. 1362-9. 17. Tzankov, A., et al., Angiogenesis in nodal B cell lymphomas: a high throughput study. Journal of Clinical Pathology, 2006. 60(5): p. 476-482. Specialized Automated Cellular Image Analysis System. , in A thesis submitted to 18. Zhang, W., et al., Expression of tumor- the Council of college of Medicine/ Al- associated Nahrain University in partial fulfillment of endothelial growth factor correlates with the requirements for the degree of Doctor poor of Philosophy in pathology. 2011, Al- lymphoma, Nahrain: Baghdad. Leukemia & Lymphoma, 2011. 52(1): p. macrophages prognosis of not and vascular peripheral otherwise T-cell specified. 46-52. 13. Wang, E.S., et al., Targeting autocrine and paracrine VEGF receptor pathways 19. Streubel, B., et al., Lymphoma-specific inhibits human lymphoma xenografts in genetic vivo. Blood, 2004. 104(9): p. 2893-2902. endothelial cells in B-cell lymphomas. N 14. Lee, Y.K., et al., VEGF receptor aberrations in microvascular Engl J Med, 2004. 351(3): p. 250-9. phosphorylation status and apoptosis is modulated by a green tea component, Correspondence to epigallocatechin-3-gallate (EGCG), in B- Dr. Bassam Mohammad Hameed cell chronic lymphocytic leukemia. Blood, 2004. 104(3): p. 788-94. Department of pathology and forensic medicine /collage of medicine /ALNahrain university 15. Gratzinger, D., et al., Prognostic Email: significance of VEGF, VEGF receptors, [email protected] and microvessel density in diffuse large B Mobile: 07700664914 Iraqi J. Hematology, May 2016, vol.5, Issue 1 30 Bassam M.Hameed, Hind Sh. Aboob, Raad J. Immunohistochemical expression of VEGF Musa التقين الوناعي النسيجي للوعاهل ( (VEGFوارتباطه ب ( VEGFRو ( CD34في االورام اللوفاوية عذا هوجكن باستخذام نظام رقوي لتحليل الصور م.د.بسام دمحم حويذ ,1م.د.هنذ شاكر عبعوب , 2ا.د.رعذ جابر هوسى 3 1يذسط فً فشع ػهى االيشاع وانطة انؼذنً\كهٍح انطة\ جايؼح انُهشٌٍ 2يذسط فً فشع ػهى االيشاع وانطة انؼذنً\كهٍح انطة\ جايؼح انُهشٌٍ 3اسرار فً فشع ػهى االيشاع وانطة انؼذنً\كهٍح انطة\جايؼح انُهشٌٍ الولخص الخلفية :اٌ ًَى األوساو انهًفاوٌح وانرقذو تركىٌُها ًٌكٍ اٌ ٌرى يٍ قثم اثٍٍُ ػهى األقم يٍ اَنٍاخ انًشضٍح :انرحفٍض autocrineانخالٌا انسشطاٍَح ػٍ طشٌق انرؼثٍش األوػٍح انذيىٌح غشائً ػايم انًُى ( )VEGFواألوػٍح انذيىٌح غشائً ػايم ًَى يسرقثالخ ( )VEGFRيٍ قثم خالٌا سشطاٌ انغذد انهًٍفاوٌح ،وانرأثٍشاخ َظٍش انظًاوي يٍ انًكشوٌح انىسو proangiogenicػهى األوػٍح انًحهً. انسشطاٍَح انذيىٌح األهذاف :ذقٍٍى ذأثٍش autocrineيٍ ،VEGFيٍ خالل دساسح ػالقح انرؼثٍش VEGFيغ يسرقثالخ انرؼثٍش VEGFRفً .NHLونرقٍٍى ذأثٍش َظٍش انظًاوي يٍ ػايم ًَى تطاَح االوػٍح، يٍ خالل دساسح ػالقح انرؼثٍش VEGFيغ CD34أػشب ػٍ انخالٌا انثطاٍَح فً سشطاٌ انغذد (.)NHL هىدجكٍٍ غٍش انهًٍفاوٌح الوواد والطرق :ذى ذظًٍى دساسح يقطؼٍح .أدسجد يا يجًىػه 66يٍ ػٍُاخ األَسجح نُخاع انؼظى فً انذساسحَ ،ى ال ذشخٍض ل NHLوفقا .working formulationيٍ كم ػٍُح أخزخ 3 أقساو ،وطثغد immunohistochemicallyل ،CD-34ػايم ًَى تطاَح االوػٍح و.VEGFR انخهىٌح طىسج وذحهٍم َظاو يرخظض انً تاسرخذاو انرشقٍى ذى ذُفٍز .3.7.3 اإلطذاس ،Digimizer تثشَايج النتائج :اسذثظ VEGFانًُاػى انًؼهًاخ انشقًٍح اسًه يؤشش انؼالياخ انشقًٍح ( )DLIإنى حذ كثٍش يغ يا ٌهً ،VEGFR (DLI) [P = 0.042 :،ص = CD34 ،]3.324يُطقح انًهىٌ ( )A ،[P = 0.037ص = .]3.556أٌضا )VEGFR (DLIاسذثظ تشكم يهحىظ يغ .)CD34 (A الخالصة :ذأثٍش Autocrineوَظٍش انظًاوي يٍ ػايم ًَى تطاَح االوػٍح هى واضح فً ،NHL كًا أٌ هُاك ػالقح إٌجاتٍح تٍٍ انرؼثٍش VEGFوانرؼثٍش ،VEGFRوكًا صٌادج األوػٍح انذيىٌح .VEGF انرؼثٍش فً صٌادج يغ ٌرُاسة نهىسو ألكلوات الوفتاحيةVEGFR ،VEGF ،NHL : 31 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Original article Evaluation of the changes in iron homeostasis and hepcidin concentration in preeclamptic patients Noora Abd Ali Muhsin1, Subuh Salim Al-Mudalal2, Bassam Mohammad Hameed3 1 M.B.CH.B /AL-Imamin AL-Kadhmin medical city 2 Professor of Haematopathology/ M.B.Ch.B /M.Sc /F.I.C.M.S. (Pathology/Haematology)/ Department of pathology and forensic medicine /collage of medicine /AL-Nahrain university 3 Msc. Pathology (haematology), Ph.D. Pathology (haematology) Lecturer Department of pathology and forensic medicine /collage of medicine /AL-Nahrain university Received 31/12/2015 accepted 15/2/2016 Abstract Background: Plasma iron is increased in preeclampsia (PE) in comparison to normal pregnant women. The relation between iron homeostasis and inflammation is hepcidin. Hepcidin is an acute phase reactant protein which has major role in iron hemostasis. Objectives: To evaluate serum iron, total iron binding capacity (TIBC), serum ferritin and serum hepcidin levels in preeclamptic pregnant women in relation to non preeclamptic pregnant women, who were not on any iron supplement regimen. Materials and Methods: This case control study was conducted on twenty pregnant women in the third trimester of pregnancy suffering from preeclampsia that had not received iron supplement or had blood transfusion within last three months, those patients attended the obstetrics and gynecology department at (AL-Imamin AL-Kadhmin medical city) between May to August 2013. Along with those patient twenty non preeclamptic pregnant women who were age and gestational age matched were included as control group. Moreover any subject presented with active infection, chronic diseases, chronic blood loss or twin pregnancy was excluded. A total of 5 ml of venous blood sample was obtained from each patient and control and tested for measurement of Hb, PCV, and MCHC by automated device whereas, serum of iron and TIBC, ferritin and hepcidin were measured by ELISA technique. Results: The mean level of Hb, PCV, MCHC, serum iron, serum ferritin and serum hepcidin in preeclamptic patients were higher than those of control group (P value of < 0.05).There was a nonsignificant correlation between serum iron and hepcidin in preeclamptic patients (r=0.234, P= 0.32) whereas there was a significant strongly positive correlation between serum iron and hepcidin in the control group (r=0.839, P = 0.003). Conclusions: In preeclamptic patients serum iron concentration is increased in spite of high hepcidin concentration which might indicate a resistance to the iron-decreasing action of hepcidin. Keywords: preeclampsia, hepcidin; pregnancy; iron regulation Iraqi J. Hematology, May 2016, vol.5, Issue 1 32 Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H. Introduction Preeclampsia, a systemic syndrome The aims of the study were to evaluate manifested primarily by hypertension and serum iron, total iron binding capacity proteinuria, presents mainly in the second (TIBC), serum ferritin and serum hepcidin half of pregnancy, and affects approximately levels in pregnant women suffering from 3% to 5% of pregnancies worldwide. (1) preeclampsia and to compare them with non Normal women has a decrease in serum iron and ferritin during the third trimester of pregnancy as their stores of iron are depleted because of fetoplacental demand and required expansion of red cell mass. (2,3) preeclamptic pregnant women. Materials and methods This study was conducted on twenty preeclamptic pregnant women in the third trimester of pregnancy who were attending Plasma iron concentrations and ferritin are the obstetrics and gynecology department at increased, whereas total (AL-Imamin AL-Kadhmin medical city) capacity (TIBC) is iron binding in between May to August 2013. Along with preeclampsia. The increase in plasma iron is twenty non preeclamptic pregnant women in contrast to inflammation characteristic for served as control and who were age and preeclampsia. iron gestational age matched and both groups had (4) a parity of three or less and twin pregnancy Hepcidin, a negative regulator of iron were excluded. All of the preeclamptic The link decreased between homeostasis and inflammation is hepcidin. absorption and Recycling (5) , is a small patients were diagnosed previously by peptide produced by the hepatocytes in obstetrical specialist and the mean arterial response to increased body iron and pressure (MAP) was measured for all inflammation.(6) The increase in plasma iron pregnant women. concentrations despite concentrations in high preeclampsia hepcidin might MAP = [(2×diastolic blood pressure)+systolic blood pressure] / 3. indicate a resistance to the iron-decreasing action of hepcidin. (4,7) A written consent for participation in the study was obtained from each subject included in the study. Iraqi J. Hematology, May 2016, vol.5, Issue 1 33 Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H. Moreover pregnant women included in the ARCHITECT, Abbott,USA). The TIBC was study were not receiving iron supplement calculated from serum iron concentration and had not received blood transfusion plus unsaturated iron binding capacity within the last three month. Also both (UIBC).Ferritin was calculated by enzyme groups should not suffer from active immunoassay based on standard method infection, any chronic disease, and chronic using blood loss. (Minividas, automated immunoanalyzer Biomerieux, France) and commercially available kit (VIDAS Ferritin, Blood sampling: 30 411, Biomerieux, France). Hepcidin was A total of 5 ml of venous blood sample was calculated in patient and control serum by obtained from each patient and control by Enzyme venipuncture from the cubital fossa under (ELISA) based on biotin double antibody aseptic technique. The blood sample was sandwich technology using standard enzyme divided into two smaller samples as follows: reader 1-First sample comprised of two milliliters of blood in Ethylene diamine tetra acetic acid (EDTA) tube for measurement of Hb, linked (ELISA immunosorbent Reader, assay Diagnostic automation inc, USA) and a commercially available kit (Human Hepcidin ELISA kit, MBS164980, MyBioSource, USA). PCV, MCHC by automated haematology Computerized analyzer (Sysmex KX-21N, Japan). performed using SPSS (statistical package 2-Second sample comprises of three milliliters of blood in plain tube to obtain serum by centrifugation of clotted blood for measurement of iron and UIBC, ferritin and hepcidin. Serum Iron and UIBC was estimated by the direct colorimetric assay using automated analyzer (ARCHITECT of social statistical sciences), analysis version 16 data were expressed as mean±SD. Student ttest was used to estimate the difference between two means. Correlation test was used to find the association between two numerical variables. available kit using for (IRON, 6K95-30 and 6K95-41, ARCHITECT, Abbott, USA), and UIBC (UIBC LIQUID, 4P79-30, Iraqi J. Hematology, May 2016, vol.5, Issue 1 with Microsoft office excel 2007.The numeric c4000, Abbott, USA) and by commercially for were 34 Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H. Results This study included twenty preeclamptic control group. Only the mean level of TIBC pregnant women in third trimester of was lower in preeclamptic patients than pregnancy with mean age was 28.5±4.9 control group. years. Along with twenty non preeclamptic pregnant women who were age and gestational age matched and who served as control group, whose mean age was 28.3±6.3years. The mean arterial pressure was significantly higher in preeclamptic patient (114.9±8.9 mmHg ) than the mean arterial pressure control group (87.8±6.2 Furthermore , there was a non significant correlation between serum iron and hepcidin in preeclamptic patients (r=0.234, P= 0.32) whereas this correlation was strongly positive in normal pregnant control group (r=0.839, P = 0.003). (Figure 1) mmHg) Haematological and biochemical parameters of preeclamptic patients and control group were presented in Table 1, showing that the mean level of Hb,PCV,MCHC as well as mean level of serum iron, serum ferritin, serum hepcidin of preeclamptic patients were significantly higher than those of Iraqi J. Hematology, May 2016, vol.5, Issue 1 35 Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H. Table 1: Haematological and biochemical parameters of preeclamptic patients and control group *Significant P value(<0.05) Iraqi J. Hematology, May 2016, vol.5, Issue 1 36 Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H. Figure 1. Correlation between serum iron and hepcidin in control group (P = 0.003) Iraqi J. Hematology, May 2016, vol.5, Issue 1 37 Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H. Discussion The present study had revealed that iron profile (includes S.iron, TIBC, S.ferritin was within normal range in preeclamptic patient although they were not receiving any iron supplement. Whereas the iron profile in normal pregnancy women was that of iron deficiency anemia, which was expected since they were supplements. Those not receiving results iron were in agreement with studies done by Tasneem zafar et al (3) , Gergely Toldi et al (4) and Margaret P. Rayman et al(8). These results may be attributed to that, in preeclampsia there is impairment of trophoblast invasion to the maternal spiral arteries resulting in necrotic and hemorrhagic areas in the placental tissue. The injured red blood cells in those area will be a source of free iron radical. (8,9) Additionally, Margaret P. Rayman et al in their study had found that, iron released from red-cell destruction of damaged placenta are clearly capable of initiating and propagating lipid peroxidation, results in endothelial-cell damage in preeclamptic patients.(8) So we may propose that disturbance of iron status is a cause and result for preeclamptic changes. Consequently, preeclamptic patients had normal Hb, PCV and MCHC levels, whereas Iraqi J. Hematology, May 2016, vol.5, Issue 1 control group had low Hb, PCV and MCHC levels since those patients had not received iron supplement. Those results were in agreement with studies done by Tasneem zafar et al.(3,10) This study showed that, the mean level of serum hepcidin in preeclamptic patients was within normal range and significantly higher than that control group and there was no correlation of its level with S. iron. This result was in agreement with study done by Gergely Toldi et al, who found that increased in the pro-inflammatory cytokine; interleukin-6 (IL-6) in preeclamptic patients as a result of inflammatory reaction, results in high hepcidin concentration in those patients.(4) despite of high S. iron level which might indicate a resistance to the iron-decreasing action of hepcidin. This resistance to hepcidin may be due to functional or structural abnormalities of ferroportin. It is well known that normally the chronic inflammation decreases iron availability through decreasing the release of iron from the macrophages to the plasma as a result of raised serum hepcidin (11), but this drive is absent in preeclamptic patients.(4,7) Thus we may propose that in preeclamptic patients the ongoing inflammation had no 38 Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H. effect on iron status through hepcidin. (7,12) Where as in normal pregnant women hepcidin level was significantly decrease in relation to low S. iron level . Conclusion In preeclamptic patients serum iron concentration is increased despite of high hepcidin concentration thus we may suggest that pregnant women not receiving iron therapy and even more had normal Hb , high S.iron ,and high hepcidin may suggest an impeding preeclampsia , close follow up is recommended , thus for these pregnant women iron status should be assessed before giving iron supplement. Iraqi J. Hematology, May 2016, vol.5, Issue 1 39 Evaluation of the changes in iron homeostasis Noora A.A.M, Subuh S. Al-Mudalal, Bassam M.H. Reference: 1. Maynard, S.E. and S.A. Karumanchi, 8. Rayman, M.P., et al., Abnormal iron Angiogenic factors and preeclampsia. Semin parameters in the pregnancy syndrome Nephrol, 2011. 31(1): p. 33-46. preeclampsia. Am J Obstet Gynecol, 2002. 2. Malek-mellouli, M., et al., [Iron status in 187(2): p. 412-8. pregnant women and its changes during 9. Valenzuela, F.J., et al., Pathogenesis of preeclampsia]. Tunis Med, 2013. 91(10): p. preeclampsia: the genetic component. J 577-82. Pregnancy, 2012. 2012: p. 632732. 3. Zafar, T. and Z. Iqbal., Iron status in 10. Koenig, M.D., et al., Hepcidin and iron preeclampsia. Professional Medical Journal, homeostasis during pregnancy. Nutrients, 2008. 15(1): p. 74-80. 2014. 6(8): p. 3062-83. 4. Toldi, G., et al., Hepcidin concentrations 11. Choudhry, V.P., Hepicidin and its role in and iron homeostasis in preeclampsia. Clin iron metabolism. Indian J Pediatr, 2010. Chem Lab Med, 2010. 48(10): p. 1423-6. 77(7): p. 787-8. 5. Collins, J.F., M. Wessling-Resnick, and 12. M.D. Knutson, Hepcidin regulation of iron iron status in pre eclampsia. Mymensingh transport. J Nutr, 2008. 138(11): p. 2284-8. Med J, 2006. 15(1): p. 22-4. 6. Camaschella, C. and L. Silvestri, Molecular mechanisms regulating hepcidin revealed by hepcidin Basher, K. and K. Deb, Alteration in disorders. ScientificWorldJournal, 2011. 11: p. 135766. 7. Kandi, S., et al., Pre Eclampsia and Iron Status: A Review. American Journal of Correspondence to Dr. Bassam Mohammad Hameed Department of pathology and forensic medicine /collage of medicine /AL-Nahrain university Email: [email protected] Medical and Biological Research, 2014. 2(6): p. 121-123. Iraqi J. Hematology, May 2016, vol.5, Issue 1 Mobile: 07700664914 40 Noora A.A.M, Subuh S. Al-Mudalal, Evaluation of the changes in iron homeostasis Bassam M.H. تقييم التغيرات في تركيز الهبسيدين وتوازن الحديد عند الحوامل المصابات بتسمم الحمل د.نورا عبد علي محسن M.B.Ch.B ا.د.صبح سالم عبد اللطيف المدلل م.د.بسام محمد حميد )M.B.Ch.B/M.Sc/F.I.C.M.S.(Haematology )Msc. Pathology (haematology), Ph.D. Pathology (haematology الملخص الخلفية :.يتم زيادة بالزما الحديد في تسمم الحمل بالمقارنة مع النساء الحوامل الطبيعيين و العالقة بين توازن الحديد وااللتهاب هي هيبسيدين .هيبسيدين هو بروتين تفاعلي حاد المرحلة الذي له دور كبير في توازن الحديد. األهداف :تقييم مستويات حديد المصل ،ومجموع الحديد ملزم قدرة ،مصل الفيريتين وهيبسيدين المصل في النساء الحوامل المصابات بنسمم الحمل فيما يتعلق بالنساء الحوامل االصحاء ،الذين لم يكونوا على أي نظام عالجي للحديد. المواد والطرق :أجريت هذه الدراسة على عشرين من النساء الحوامل في الثلث الثالث من الحمل اللواتي يعانين من تسمم الحمل و لم يعطين الحديد و لم يتم نقل الدم لهم في األشهر الثالثة األخيرة ،هوالء الحوامل حضرن الى قسم أمراض النساء والتوليد في مستشفى االمامين الكاظمين بين مايو-أغسطس عام .3102وإلى جانب هؤالء المرضى عشرين امرأة حامل غيرمصابات بتسمم الحمل الذين كانوا في نفس العمر والعمر الحملي وأدرجت كمجموعة مراقبة مطابقة .وعالوة على ذلك تم استبعاد أي حامل مع عدوى نشطة ،او لديها أمراض مزمنة ،اوفقدان الدم المزمن أو الحمل التوأم .تم الحصول على ما مجموعه 5مل من عينة من الدم الوريدي من كل مريض ومراقبة واختبار لقياس الهيموغلوبين ،PCV ،و MCHCبواسطة جهاز آلي في حين تم قياس مصل الحديد ،TIBCالفيريتين وهيبسيدين .ELISA تقنية بواسطة النتائج :كان متوسط مستوى الهيموجلوبين ،MCHC ،PCV ،حديد المصل ،الفيريتين في المصل وهيبسيدين المصل في المرضى الذين يعانون تسمم الحمل أعلى من تلك التي المجموعة الضابطة (قيمة Pمن < )1.15كان. هناك عالقة غير ذات داللة إحصائية بين الحديد في الدم وهيبسيدين في المرضى الذين يعانون تسمم الحمل (= r ) P = 0.32 ،1.320في حين كان هناك ارتباط إيجابي قوي كبير بين الحديد في الدم وهيبسيدين في السيطرة على .)P = 0.003 ،1.820 (=r المجموعة االستنتاجات :كان الحوامل مع تسمم الحمل لديهم زيادة تركيز الحديد في الدم على الرغم من تركيز هيبسيدين العالية هيبسيدين. من الحديد خفض على للعمل مقاومة وجود إلى تشير قد التي ألكلمات المفتاحية :تسمم الحمل ،هيبسيدين .حمل؛ تنظيم الحديد 41 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Original article The results analysis of complete blood count during the first half of 2015 at the Central Child Teaching Hospital in Baghdad. Rasha Tariq Jawad1 , Ebtehal Ali Hussien2 1 MD. M.B.Ch.B. F.I.C.M.S/Hematopathology ,Senior Specialized Hematopathologist, Manager of the Hematology Unit, Laboratory Department / Central Child Teaching Hospital ,Baghdad ,Iraq. 2 BMLT, Laboratory technician / Hematology Unit, Laboratory Department / Central Child Teaching Hospital ,Baghdad ,Iraq. Received 5/1/2016 accepted 15/2/2016 Abstract: Background: Complete blood count (CBC) is the most commonly requested test by physicians. The central child teaching hospital is the second largest pediatric hospital in IRAQ. We review the CBC for the first half of 2015 at that hospital. Objectives: this study was arranged to explore the prevalence of anemia, leucocytosis, leucopenia, thrombocytosis, thrombocytopenia, using automated CBC counter Emerald & Ruby Abbott for outpatient and inpatients respectively. Patients and methods: A retrospective study included a total of 19341 patient attended central child teaching hospital (from the 1st of January -1st of July 2015). All of them did complete blood picture test using ruby Abbott and emerald Abbott hematology autoanalyzer, 14418 of them were inpatients and 4723 patients attended to the outpatient clinic. Results: the results were analyzed as anemia, leucocytosis, leucopenia, thrombocytosis, thrombocytopenia for the whole patients and for each month to the inpatients so that to analyze the result according to season. Anemia was seen more commonly in the inpatients (30.79%) than the outpatient (20.8%). Leucopenia was seen more in outpatients (17.15%) than in inpatients (8.1%), while leukocytosis shown to be (10.8%) of inpatients and 2% of outpatients. Thrombocytopenia was (8.3%) of inpatient and (3.1%) of outpatients. Regarding the monthly incidence we found that the highest percentage of anemia in outpatients seen at May (42%), while the lowest was seen at January (19.76%).Leucocytosis and thrombocytosis both showed the highest percentage at January (19.76%), (15.29%) respectively. Conclusion: anemia, leucocytosis, and thrombocytosis seen in inpatients more than outpatients ,leucopenia seen in outpatients more than inpatients ,thrombocytosis showed near results for in and out patients but a little higher for inpatients. Keywords: analysis, CBC, children Iraqi J. Hematology, May 2016, vol.5, Issue 1 42 The results analysis of complete blood count .Rasha Tariq Jawad , Ebtehal Ali Hussien Introduction: Complete blood picture is the most commonly requested test by physician as it is easy and highly informative for patient’s health. It requires minimum amount of anti-coagulated whole blood and done on automated blood count machine lasting few minutes to get the results. more global failure of hematopoiesis, caused by conditions such as aplastic anemia, whether the patient have anemia, anemia, myelofibrosis, or leukemia, or may suggest a rapid destruction or trapping of all blood elements, such as hypersplenism, localized coagulopathy in The primary field of interest is Fanconi a large hemophagocytic (HLH) or hemangioma or lymphohistiocytosis macrophage (2) activation whether the WBC count have evidence syndrome (MAS) . The WHO show of infection and whether the platelets that the prevalence of anemia is hemostasis.(1)Pediatric (47.4%) anemia refers to a hemoglobin or children hematocrit level lower than the age- Iraq in children under 5 from 1995- adjusted reference range for healthy 2011 is 36% children. Physiologically, anemia is a of anemia seen more than general condition in which reduced hematocrit population ,a study in India show that or anemia in inpatient in one tertiary count affect hemoglobin levels lead to among (3) preschool aged prevalence of anemia in (4) . Inpatients prevalence hospital was( 72.79%)(5) , diminished oxygen-carrying capacity pediatric that does not optimally meet the another study in Brazil showed the metabolic demands of the body. prevalence of anemia in pediatric Anemia is not a specific disease entity hospital to be (56.6% ) 10(6).while it is but is a condition caused by various 59%,and 24% respectively in the underlying pathologic processes. (2) In general population of these countries(4). addition, conditions are emphasized in Leukocytosis is a common laboratory which anemia is the only hematologic finding abnormality. of practice, in the emergency department anemia with leucopenia, neutropenia, or a medical or surgical setting. or thrombocytopenia may suggest a Generally, the vast majority of cases of The combination Iraqi J. Hematology, May 2016, vol.5, Issue 1 encountered in hospital 43 .Rasha Tariq Jawad , Ebtehal Ali Hussien The results analysis of complete blood count leukocytosis are reactive and benign— is resulting from liberation of various problem in routine practice than is cytokines the leukocytosis. n general, leukopenia development of leukocyte precursors may result from decreased marrow and release of mature cells from the production of leukocytes precursors, marrow, endothelium, by that stimulate and spleen. a less commonly encountered peripheral destruction or Most reactive episodes of leukocytosis sequestration of circulating leukocytes, are or by autoimmune cellular damage or neutrophilic in nature (“neutrophilia”) and result from a myriad of stressors. (7) destruction. The most common Occasionally, etiology of leucopenia is decreased the total leukocyte count will exceed marrow production due to a variety of 25,000 cells/mm3, with the majority of disorders that damage the developing cells leukocyte mass in the bone marrow (7). being polymorphonuclear leukocytes. This scenario is referred to as a leukemoid reaction, and may be difficult to differentiate from a A low platelet (thrombocytopenia) may count also be leukemic picture, hence the name detected in the complete blood count. “leukemoid.” As such, in patients with Thrombocytopenia which is defined as leukemoid platelet less than 150× 103 per µL. It is reactions, the main differential diagnostic concerns include often chronic myelogenous leukemia, acute complete blood picture test myelogenous other with platelet count greater than 50×103 myeloproliferative disorders. A typical per µL rarely have symptoms. Platelet leukemoid reaction, however, occurs in count from 30 to 50 × 103 per µL the presence of an acutely stressful or rarely manifest as purpura. A count inflammatory/infectious event, such as from 10 to 30× 103 per µL may cause hemorrhage, febrile bleeding with minimal trauma .a episodes, sepsis, trauma, pancreatitis, platelets count less than 5×103 per µL or difficile may cause spontaneous bleeding This infection. Clostridium difficile has the may be due to bone marrow problems, unique some medications or , immunologic or leukemia, or hemolysis, Clostridium propensity of causing discovered on patient leukemoid reactions in cases of severe genetic colitis, from disease, or cancers such as leukemia(8) . Leukopenia thrombocytosis is often associated with and likely liberation of cytotoxins results (7) Iraqi J. Hematology, May 2016, vol.5, Issue 1 problems, incidentally advanced liver 44 .Rasha Tariq Jawad , Ebtehal Ali Hussien The results analysis of complete blood count What Affects the Test (9) infection or an inflammation such as osteomyelitis or rheumatoid arthritis ,hematological causes include chronic blood loss red cell occurs as consequence of WBC destruction agglutination ,splenectomy and rebound following recovery from suppression. bone marrow Moderately increased platelet count does not usually have Factitiously low WBC occasionally storage ,prolonged sample ,abnormally fragile cells(leukemia) A very high WBC count is more common and usually result from pathological implication (9). failure of RBC lysis .this occurs The complete blood count values are with RBC of neonates or uremia or usually reported based on the number the of cells in a specific volume of blood. hemoglobin such as S or C Hb. Fully automated multichannel presence of abnormal Low platelets count may be due to instrument usually measure from 8 to giant platelets. Identified as red cells 20 component .automated instruments or have high level of precision ,if Clumping instrument are carefully calibrated and of EDTA-induced platelets. High platelet count may be due to their correct operation is ensured by marked microcytosis or fragmented quality control procedure they produce red cells or fragmented WBC or due test that are generally accurate (9) .A complete blood count may be done as to bacteria or fungi. The automated MCV& Hct are part of a regular physical examination. prone Normal values for the complete blood resulting from microclots or partial count (CBC) tests depend on age, sex, clotting ,extreme microcytosis and how high above sea level you live, and the presence of cryoglobulin or cold the type of blood sample .each ethnic agglutinins the latter cause factitious group has its normal range and many elevation literatures establish their own normal agglutinated cells counted as single range for specific hospitals in these cell. towns (10,11,) to certain of errors. MCV as those the . Table (1) shows the normal value of infant and children Patients and Methods: which clarify the difference with age. A total of 19141 patients attended and did complete blood count (CBC) at the Iraqi J. Hematology, May 2016, vol.5, Issue 1 45 The results analysis of complete blood count .Rasha Tariq Jawad , Ebtehal Ali Hussien Central Child Teaching Hospital in light or change the potential between Baghdad Iraq for the period from the tow electrodes (depending on the 1st of January to the 1st of July 2015. technology used) which last as long as We retrospectively analyzed CBC the cell take to pass ,the number of results of those patients. 14418 was pulses indicate the count ,the height of hospitalized (inpatients), 4723 was the pulse indicate the volume of the outpatients. 2 ml of venous blood was cell passing through (MCV) .WBC is collected in k3 EDTA tubes from the determined in whole blood in which patients, and the CBC for inpatients red cells have been lysed .Platelets are made on Rupy Abbott autoanalyzer counted in whole blood using the same working on principle of electrical technique with an upper threshold is impedance. the outpatient CBC done needed to separate platelets from red on Emerald Abbott Autoanalyzer. The cells and a lower threshold to separate test done at the hematological units of plate lets from debris and electronic the outpatients laboratory and the noise. Three part differential count central laboratory. the result was assigns cells to categories usually compared designated: granulocytes (large cells), with hematological the normal schedule of values for lymphocytes (small cells), and normal infants and normal children monocytes (mononuclear or middle that is published on Dacie and Lewis cells .three part differential counts practical hematology eleventh edition from a single channel instrument (1,9,12) (9) The results of the first half of 2015 is analyzed according to the incidence of anemia ,leucocytosis , leucopenia, thrombocytosis , thrombocytopenia. The automated counters measure the hemoglobin concentration by modification of the manual HiCN method with cyanide reagent ,RBC and other blood cells counted by aperture impedance , blood is highly diluted in a buffered electrolyte solution under controlled condition the diluted blood The normal values may differ slightly based on the reference range and the machine used in the laboratory and, therefore, the results may be slightly different from one laboratory to the next (10,11) . The normal reference range is typically provided and printed with the results of the complete blood count for accurate interpretation. Different laboratories may report slightly different reference ranges.(13) passed through the aperture scattering Iraqi J. Hematology, May 2016, vol.5, Issue 1 46 .Rasha Tariq Jawad , Ebtehal Ali Hussien The results analysis of complete blood count Table (1); hematological values for normal infants and normal children (Dacie and Lewis 11th edition) (9) Parameter RBC×10 12 /L birth 5-7 Day Day Day 1 2 3-6 1 year 3 7 14 month month months 4-6.6 4.9-6.3 3.6-6.2 3-5.4 3.1- 4.1-5.3 4.3 14-22 Hb g/dl 45-75 Pcv(range) 100-120 MCV 15-21 13.5−21.5 45-67 92-118 42-66 88-126 12.5− 11.5- 20.5 16.5 31-71 86-124 31-37 MCH 11.1-14.1 28-42 92- 87- 116 103 6-12 years years 4-5.2 5.1 9.4-14 33-53 3.9- 2-6 30-36 30-40 30-38 11- 11.5- 14 15.5 34-40 35-45 68-84 72-84 75-78 75-95 24-30 25-29 24-30 25-33 9 120-400 50-350 50-100 20-60 30-50 40-100 30-100 9 WBC×10 /L 10-26 7-23 6-22 5-21 5-15 6-18 6-16 5-15 5-13 N×109 4-14 3-5 3-6 3-9 1-5 1-6 1-7 1.5-8 2-8 L×109/L 3-8 2-8 3-9 3-16 4-10 4-12 3.5−11 6-9 1-5 M×109/L 0.5-2 0.5-1 0.1-1.7 0.3-1 0.4− 0.2-1.2 retics×10 /L 3-7 0.2 -1.0 1.2 E×109/L 0.1-1 0.1-2 0.1−0.8 0.1−0.9 0.2-1 Platelets 100−450 210−500 160−500 170−500 200- 210- 500 650 The reported values are either directly 0.2 - 1.0 200-550 200- 170- 490 410 The calculated values are: measured or calculated by the machine. The measured values are: WBC number RBC number Mean Corpuscular Hematocrit (HCT) Mean Corpuscular Hemoglobin (MCV) Volume (MCV) Hemoglobin (Hg) Platelet count Mean platelet volume(MPV) Iraqi J. Hematology, May 2016, vol.5, Issue 1 Mean Corpuscular Hemoglobin Concentration (MCHC) Mean Corpuscular Hemoglobin (MCH) Red Cell Distribution Width (RDW) 47 The results analysis of complete blood count .Rasha Tariq Jawad , Ebtehal Ali Hussien Results A total of 19141 patients included in Table (3) showed that the highest this study with age range from (1day- percentage of anemia was at May 14 years old). About third of the (42,9%) and the lowest was seen at inpatients (30.79 %) and only 20.8% of January the low percentage of leucocytosis was seen at (table January (19.76%) for inpatients, the outpatients were with hemoglobin concentration 2).leucopenia seen in 8.1% of (19.76%). The highest lowest percentage seen at June (4.1%). inpatient and 17.15% of the outpatients Leucopenia was seen comparable while leucocytosis seen in 10.8%. of the whole five months and a lower the percentage in June (5.1%) (Table 4). inpatients and 2% of the for outpatients . Thrombocytopenia seen in Patients with thrombocytosis were 8.3% of the inpatients and 3.11% of higher in January (15.9%) and the the outpatients while thrombocytosis lowest percentage was seen in June seen in 6.8% of the inpatients and (2.35%), 5.14% of the out patients (Table 2). while thrombocytopenia showed the highest percentage in April (12.72%) and the lowest in January (2.68%) (Table 5). Table (2): percentage of high Hb, WBC, platelet, and low Hb, WBC, platelet for inpatient and outpatient. Test IP(N=14419) % OP(N=4723) % Low Hb. 4438 30.79% 984 20.8 Low WBC 1166 8.1% 810 17.15% High WBC 1562 10.8% 96 2% Low plt. 1194 8.3% 147 3.11% High plt. 981 6.8% 243 5.14% Iraqi J. Hematology, May 2016, vol.5, Issue 1 48 .Rasha Tariq Jawad , Ebtehal Ali Hussien The results analysis of complete blood count Table (3): the percentage of anemic patients in the inpatients for each month and the total of the 1st half of 2015. Month No. of patient Patients with low Percentage Hb. January 2419 478 19.76% February 2560 873 34.1% March 2227 767 34.49% April 2367 569 24.03% May 2383 1023 42,93% June 2462 728 29.56% Total 14418 4438 30,79% Table (4) the percentage of leucopenia& leucocytosis for each month and the total of the 1st half of 2015. Month No. of Patients patients Patients with with low Percentage high WBC WBC Percentage January 2419 212 8.76% 478 19.76% February 2560 203 7.92% 218 8.51% March 2227 183 8.21% 306 13,74% April 2367 231 9.75% 269 11.36% May 2383 212 8.9% 191 8.01% June 2462 125 5.1% 100 4.1% Total 14418 1166 8.1% 1562 10.8% Iraqi J. Hematology, May 2016, vol.5, Issue 1 49 .Rasha Tariq Jawad , Ebtehal Ali Hussien The results analysis of complete blood count Table (5): the percentage of thrombocytopenia and thrombocytosis for each month and the total of the 1st half of 2015. Month Pat. No. Low plt. Percentage High plt. Percentage January 2419 65 2.68% 370 15.29% February 2560 137 5.35% 87 3.39% March 2227 192 8.62% 261 11.71% April 2367 301 12.72% 78 3.29% May 2383 249 10.45% 127 5.32% June 2462 250 10.15% 58 2.35% Total 14418 1194 8.3% 981 6.8% Discussion The result of our study showed that become anemic at some point during anemia seen in 30% of the hospitalized their childhood and adolescence(5) . patient and 20% of the outpatient was anemic. prevalence of anemia seen differ from one country to another 10% of hospitalized adult patients seen to be anemic in one study(14) while another study in Brazil show that 56% of hospitalized children under the age of 5 are anemic.(6).in a study in India which chose the same age as ours they show that anemia seen in 72% of the hospitalized patients (5) . The studies show that anemia worldwide seen in 43%undre 5 years(14) , even in the developed world 21-26% Iraqi J. Hematology, May 2016, vol.5, Issue 1 Leucopenia in this study seen in (8.1% ) of the inpatient and (17.15%) of the outpatient . as we depend on the table of Dacie and Lewis for CBC count ,a WBC below 5×109 /L considered leucopenia that why we as middle eastern people should establish our own normal range (10,12) ,even in the west black people have a lower normal leukocytes than white and the books ask the physician not to do unneeded test for those patients and to put this in mind (7) 50 The results analysis of complete blood count Leucocytosis hospitalized seen patient in .Rasha Tariq Jawad , Ebtehal Ali Hussien 10.8% of cause in children the incidence 100 2% of cases per million and 50% usually hospitalized patients are more Psuedothrombocytopenia from EDTA ill than outpatients. dependent agglutinin should be put in inpatients and 5.14% of the outpatients .thrombocytosis in children is common but usually without symptoms the cause mostly infection ,trauma, surgery ,renal disease and chronic inflammation .in a study in Taiwan thrombocytosis hospitalized hospital result seen in patient in 7.6% of (KMCH) in annually outpatients. That is seems logical as Thrombocytosis seen in 6.8 % of the occur person children . mined (15). Conclusion : anemia, thrombocytosis leucocytosis, and seen in inpatients more than outpatients ,leucopenia seen in outpatients more than inpatients ,thrombocytosis show near results for in and out patients but a little higher for inpatients. which is very near to our (8) . Thrombocytopenia which is defined as platelet less than 150× 103 per µL . It is often discovered incidentally on complete blood picture test patient with platelet count greater than 50×103 per µL rarely have symptoms. Platelets count from 30 to 50 × 103 per µL rarely manifest as purpura. A count from 10 to 30 may cause bleeding with minimal trauma .A platelets count less than 5 may cause spontaneous bleeding in the current study 8% of hospitalized patients seen to be thrombocytopenic .this include all thrombocytopenia below 150× 103 which have so many reasons. all the literatures consider ITP as the main Iraqi J. Hematology, May 2016, vol.5, Issue 1 51 The results analysis of complete blood count .Rasha Tariq Jawad , Ebtehal Ali Hussien References 1. Fischbach FT, Dunning MB. hospital in Recife, Brazil Rosemary Manual of Laboratory and Diagnostic Ferreira Tests, 8th ed. Philadelphia: Lippincott Hematol.2011; 33(2): 100–104. Williams and Wilkins.2009 dos Santos,. Rev Bras 7. Mark A, Marinella MD. Infectious 2. Susumu I , Robert J Arceci. disease. Leukocytosis and leucopenia. Pediatric http://www. Acute Anemia. http://emedicine.medscape.com/article/954 antimicrobe.org /new /e19.asp 506-overview 8. Chen HL, Chioou SS, Sheen JM. 3. WHO. Worldwide Prevalence of Anemia 1993-2005: Global Database ON Anemia. Geneva :WHO;2008. Regil LM, et al. Global, regional, and trends in hemoglobin concentration and prevalence of total and severe anemia in children and pregnant and non-pregnant women for 1995-2011: a systematic analysis of population-representative data. medical center of southern Taiwan. Acta 4. Stevens GA, Finucane MM, De- national Thrombocytosis in children at one The Lancet Global Health 2013; 1(1): e16-e25. Paediatrca Tiawinaca1999 , 40(5):309-313. 9. Carol Briggs ,Barbara J.Bain. Daicie and Lewis .Basic .Practical Hematology Hematological Techniques Eleventh Edition .2012 10. Buseri FI, Siaminabo IJ, Jeremiah ZA, Reference values of hematological indices of infants, children, and adolescents in Port Harcourt, Nigeria, 5. Firdose S, Siddaraju P. Anemia July 2010 Volume 2010:2 among hospitalized children at a 65—70. multispecialty 11. Alexander H, Judith K, Benjamin hospital, Bangalore. India. Journal of family medicine and M, primary care .2014 Jan-Mar;3(1):48-53 Haematological 6. Eliane S. , Campos G, Emídio C. de Albuquerque, Ilma K, Grande de Arruda, Prevalence of anemia in under five-year-old children in a children's Iraqi J. Hematology, May 2016, vol.5, Issue 1 Peter GK, and Pages Bertrand L, biochemical reference intervals for infants and children in Gabon. Tropical Medicine and International Health, 2011. March ;volume 16 (3) pp 343–348 52 The results analysis of complete blood count 12. Chris Higgins. Understanding laboratory investigations: a guide for nurses, midwives and healthcare professionals’ .third edition. Part three Hematology .Rasha Tariq Jawad , Ebtehal Ali Hussien tests.2013.Wiley- Blackwell publication Correspondence to: Dr.Rasha Tariq Jawad Manager of the Hematology Unit, Laboratory Department / Central Child Teaching Hospital, Baghdad, Iraq. 13. Rachoin JS, Cerceo E, Milcarek B, Hunter K, Gerber DR Prevalence and impact of anemia in hospitalized patients. South Med J. 2013 Mar;106(3):202-6. 14. Rehemah. Simbauranga, Erasmus kamugisha. Prvalence and factors associated with severe anemia amongst under 5 children at bugando medical center ,mwanza, tanzania. BMC Omack WA.. Hematology.2015. 15. Ropert guaer. Thrompocytopenia. American Family Physicion. Medical Center Family Medicine Residency ,Fort Bragg, north carolina. 2012 march15; 85(6): 612622 Iraqi J. Hematology, May 2016, vol.5, Issue 1 53 .Rasha Tariq Jawad , Ebtehal Ali Hussien The results analysis of complete blood count تحليل نتائج تعداد الدم الكامل خالل النصف األول من عام 5102في مستشفى الطفل المركزي التعليمي في بغداد رشا طارق جواد ، 1ابتهال علي حسين 2 / F.I.C.M.S /M.B.Ch.B / MD0اختصاص امراض الدم المختبري ،مدير وحدة أمراض الدم ،قسم المختبرات /مستشفى الطفل المركزي التعليمي في بغداد ،العراق. ،BMLT 5فني مختبري /وحدة أمراض الدم ،قسم المختبر /المستشفى الطفل المركزي التعليمي في بغداد، العراق. الملخص: الخلفية :تعداد الدم الكامل ( ) CBCهو االكثر طلبا عادة كتحليل دم من قبل األطباء .المستشفى التعليمي الطفل المركزي هو ثاني أكبر مستشفى لألطفال في العراق .نستعرض نتائج CBCللنصف األول من عام 5102في المستشفى. هذه األهداف :تم ترتيب هذه الدراسة الستكشاف مدى انتشار فقر الدم ،فرط الكريات البيض ،نقص الكريات البيض، كثرة الصفيحات ،نقص الصفيحات الدموية ،وذلك باستخدام المحلل اآللي CBCلمرضى العيادة االستشارية التوالي. على للمستشفى الداخلين والمرضى المرضى والطرق :دراسة استعادية ،كان ما مجموعه 04390مريض حضورا المستشفى التعليمي المركزي (من 0يناير من 0يوليو) لكل واحد منهم تم اجراء اختبار صورة الدم باستخدام روبي ابوت المحلل التلقائي. وحضر 9753المرضى إلى العيادة الخارجية. 09901من المرضى كانوا داخلون للردهات النتائج :تم تحليل النتائج على النحو التالي :فقر الدم ،فرط الكريات البيض ،نقص الكريات البيض ،كثرة الصفيحات ،نقص الصفيحات الدموية للمرضى كلهم وعن كل شهر للمرضى الراقدين بحيث تكون تحليل النتيجة وفقا للموسم .فقر الدم كان أكثر شيوعا في المرضى المنومين ( )٪74 ،31من العيادات الخارجية (.)٪1 ،51 واعتبر نقص الكريات البيض أكثر في العيادات الخارجية ( )٪02 ،07من المرضى المنومين في ( ،)٪1،0في حين أن زيادة عدد الكريات البيضاء أظهرت أن ( )٪01.1من المرضى المنومين و ٪5من العيادات الخارجية. كان نقص الصفيحات ( )٪1.3من المرضى الداخليين و ( )٪3.0من مرضى العيادات الخارجية .وفيما يتعلق اإلصابة الشهرية وجدنا أن أعلى نسبة فقر الدم لدى مرضى العيادات الخارجية شهدت في مايو ( ،)٪95في حين كان ينظر إلى أدنى مستوى في كانون الثاني ( .)٪04.71فرط الكريات البيض والصفيحات سواء أظهر أعلى التوالي. على )٪02.54 ( (،)٪04.71 الثاني كانون في نسبة الخالصة :فقر الدم ،فرط الكريات البيض ،وكثرة الصفيحات شوهدت اكثر في المرضى المنومين من العيادات الخارجية ،نقص الكريات البيض شوهدت اكثر في العيادات الخارجية من المرضى المنومين ،كثرة الصفيحات . للداخلين طفيفة زبادة مع للمجموعتين النتائج متقاربة ;كانت كلمات البحث :تحليل ،CBC ،األطفال 54 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Original article Distribution of red cell antigens according to ABO, Rh and other rare blood group systems in Kurdish ethnicity Hisham A. Getta¹, Shaema S.Amin², Najmaddin Khoshnaw ³ * & 4, Belal A. Muhammad5 & 6 ¹Department of Hematopathology, faculty of sciences, school of medicine, university of Sulaimani, Kurdistan region, Iraq ²Department of Hematopatholgy, Internal Teaching Hospital, Sulaymaniyah, Kurdistan Region, Iraq ³*Department of Hematology, Hiwa Hospital, Sulaymaniyah, Kurdistan Region, Iraq 4 Kurdistan Board of Medical Specialties, Clinical Hematology/Trainee, Ministry of higher education and scientific research, Erbil, Kurdistan Region, Iraq 5 Department of Medical Laboratory Techniques, Technical Institute of Halabja, Sulaimani Polytechnic University, Sulaymaniyah, Kurdistan region, Iraq 6 Department of Medical Laboratory Sciences, College of Science, Komar University of Science and Technology, Sulaymaniyah, Kurdistan region, Iraq Received 23/11/2015 accepted 17/1/2016 Iraqi J. Hematology, May 2016, vol.5, Issue 1 55 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M Abstract: Background: Among more than 30 blood group systems, nine of them namely ABO, Rh, Kell, Kidd, Duffy, MNS, P, Lewis and Lutheran are considered to be clinically significant. The distributions of these blood groups are different between populations across the world. Studies about the frequency of blood groups in Kurdish ethnicity are very limited in the literature. Objectives: to explore the distribution of red cell antigens and phenotypes of various blood groups among Kurdish population using different systems. Materials and Methods: five thousand blood donors attending the central blood bank of Sulaymaniyah province were randomly selected and tested for ABO and Rh antigens (D, C, c, E, & e) by using tube method. 500 donors were randomly selected and further analyzed using other blood group systems. Results: In the ABO system, the most common phenotype was O (37%), followed by A (32.6%), B (22.8%) and AB (7.6%). Among the Rh blood group antigens, e was the most common (95.2%) followed by D (91.3%), C (74.8%), c (69.4%), and E (30.6%) with DCe/DCe(R1R1) and dce/dce(rr) being the most common phenotypes among Rh-D+ve and Rh-D-ve groups, respectively. The most common phenotypes for other blood systems were as follow; Kell(K-k+,94%), Kidd(jk a+b+,44.5%), Duffy(fy a+b+,45%), Lutheran(Lu a-b+,92%), Lewis(Le a-b+,54.5%), P(P1,76%), MNS(M+N+S-s+,40%) Conclusion: the various red cell antigens recorded by different blood grouping systems in this study was intermediate between the European and Asian countries with some specificity to the Kurds population reflecting the distinct geographical area and preserved ethnic background of the Kurds in the region. Keywords: ABO, Rhesus (Rh), red cell antigen, Kurdish ethnicity. Iraqi J. Hematology, May 2016, vol.5, Issue 1 56 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M Introduction: The ABO and Rhesus (Rh) blood group are more immunogenic and active at body systems are among the most clinically temperature (8) important discoveries of the last century in The ABO blood group antigens are encoded the field of hematology. The ABO blood by one genetic locus, the ABO locus, which group system was first discovered by has three alternative (allelic) forms A, B, Landsteiner in 1901(1). Later a joint work of and O, located on the long arm of the same author with Wiener resulted in the chromosome 9 discovery of Rhesus (Rh) blood grouping pattern is the four well defined blood groups system in 1940 (2) (9) . The result of this genetic . In these systems, the (A, B, AB, and O). The Rh system is the determination of blood groups is based on most polymorphic and the most clinically certain inherited antigenic substances on the significant blood group system beside the surface of red blood cells (RBCs) (3) . These ABO system. Currently it is composed of 50 antigens have found to play crucial roles in antigens associated to genes located on several clinical areas such as in transfusion chromosome 1 including RhD, RhC, RhE, medicine, Rhc and Rhe which represents the most organ transplantation, (10,11) autoimmune hemolytic anemia (AIHA), important Rh antigens fetomaternal blood group incompatibility, studies have shown that the frequency of paternity allelic distribution of the ABO/Rh blood identification, and forensic . However, medicine (4-6) group varies among different geographical According to the International Society of areas and also between populations with Blood Transfusion (ISBT), there are now different ethnic background across the more than 270 antigens distributed over 30 world. This is probably due to the genetic distinct blood group systems [1, 7]. Nine of polymorphism nature of the ABO/Rh genes. these systems namely ABO, Rh, Kell, Kidd, Clinically speaking, blood transfusion is a Duffy, MNS, P, Lewis and Lutheran are lifesaving process for some patients, yet it is considered important. not free from transfusion-related risks such Among them, the ABO and Rh systems are as transfusion transmissible diseases (TTD) the most important systems during blood and alloimmunization transfusion due to the fact that their antigens determine to be clinically Iraqi J. Hematology, May 2016, vol.5, Issue 1 the (12) .It is crucial to phenotype of clinically 57 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M significant blood group antigens on the information is very limited regarding the donor RBCs especially in situations when extended population blood groups especially alloimmunization is particularly undesirable. using systems other than the ABO/Rh blood Examples of this include young females, groups. In the present study we, for the first pregnant women, and patients who are time, determined the extended red cell expected blood antigens and phenotype frequencies of transfusion in their life such as in patients various clinically significant blood groups that have thalassemia, sickle cell anemia, amongst regular healthy Kurdish voluntary cancer, dialysis, etc. blood donors in Sulaymaniyah province. Racial differences in blood group antigen This study provides valuable information distribution are common between different about the normal distribution of different populations across the world. Available data blood groups among the Kurdish population in the literature have relatively clarified the and it can be used to establish a foundation genotype/phenotype of donor database for different RBC to require repeated variation of blood group systems in the European, American antigens. and some Asian countries. However, such information in developing countries is still limited. Kurdish population are normal inhabitants of the northern part of Iraq (called Kurdistan or Kurdistan region of Iraq) where only ABO and Rh (D) status of blood donors and recipients are taken into account for compatibility testing. A part of two recent regional studies about the ABO/Rh blood group systems (13,14) Iraqi J. Hematology, May 2016, vol.5, Issue 1 58 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M Materials and methods: Study Design: Sample collection and methods: This work is a cross-sectional study carried Blood samples were collected in ethylene out from 1st of June to 30th of September diamine tetra acetic acid (EDTA) tubes and 2014 in the Central Blood Bank of analyzed Sulaymaniyah province/Iraq. The study was detection using acryl amide gel technique conducted after taking approval from blood following the manufacturer’s instruction. bank and informed consent from blood This system uses ID card “DiaClon ABD- donors for their participation in the present Confirmation for Patients” which contains study. monoclonal anti-A, B and D within the gel Donor Selection: matrix freshly for (Bio-Rad ABO/Rh antigen Laboratories, DiaMed Switzerland). A small portion (3-5%) of the A total of 5000 healthy regular voluntary blood donors (aged between 18-60 years) were included in this study. All the donors were subjected to red cell antigen typing using ABO/Rh (D) blood group antigen systems. Out of them, 500 donors were randomly selected for further antigen typing using other Rh blood group antigens including C, c, E, and e. In addition, 400 donors were also randomly selected for extended antigen typing using other blood group systems including Kell (k-cellano), Kidd (Jka, Jkb), Duffy (Fya, Fyb), MNSs (M, N, S, s), Lewis (Lea, Leb), Lutheran (Lua, Lub) and P (P1). RBCs suspended in isotonic saline solution was further analyzed by Direct Antiglobulin Test (DAT) according to standard protocols. The DAT negative samples were further typed for extended antigen profiling through antigen-antibody agglutination method using kits supplied by Rapid Labs Limited, England following the manufacturer’s guidelines. The ABO and Rh (C, c, E & e) antigen detection were performed indirect Antiglobulin Technique by (IAT), using monoclonal IgG antisera [15]. The tests are interpreted depending on the finding of the agglutination which are graded as 1+ to 4+ positive, or negative which indicating the absence of the of corresponding antigen. Iraqi J. Hematology, May 2016, vol.5, Issue 1 59 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M Results: The results of the ABO system showed that and Rh (D) negative groups, respectively the most common blood group was O (Table 3). (37.0%), followed by A (32.6%), B (22.8%) The frequency of red cell antigens of Kell, and Kidd, Duffy, MNS, Lutheran, Lewis and P AB (7.6%). The results also demonstrated that the majority of donors blood were positive for Rh (D) antigen (91.26%) (supplementary Table 2) and the phenotype while minority (8.74%) were negative based frequencies of these systems are presented in on the Rh blood group system (Table 1). (supplementary Table 3). According to the The frequency of the ABO phenotypes Kell blood group system K-k+ was the most associated to the Rh (D) phenotype was as common phenotype (94.2%) in our donors follow: for the Rh (D) positive individuals; and no K+k- & K-k- phenotypes were group systems are shown in +ve the most common blood group was O +ve (33.2%), followed by A (21.1%), and AB +ve observed in any donors. In the Kidd system, +ve (29.8%), B Jk (a+b+) was predominant (44.5%) and no (7.1%). While for the Jk (a-b-) was observed. Jka and Jkb antigens Rh (D) negative individuals the ratio was were determined in (77%) and (67.5%) of -ve -ve 3.8%, 2.7%, 1.7%, and 0.5% for O , A , -ve B , and AB 1). -ve groups respectively (Table Furthermore, our showed Fy (a+b) as the most common also phenotype (45%). The Duffy null or Fy (a-b- demonstrated that the e antigen had the ) phenotype was observed in (4%) of donors, highest frequency (95.2%), followed by D, while Fya and Fyb antigens were observed C, c & E antigens (91.26%, 74.8%, 69.4% & in 30.6%, respectively) respectively. In the Lutheran blood group as results donors, respectively. The Duffy system shown in (Table 1 & 3). (72%) and (51.5%) of donors, system, the most common phenotype was Our phenotypic studies suggest that eight Lu (a-b+) (92%). Lua and Lub antigens probable phenotypes are possibly found in were observed in (4.5%) and (95.5%) of our population. Among them, the DCe/DCe donors, respectively, while, null phenotype (R1R1), and dce/dce (rr) were the most Lu (a-b-) was determined in (3.5%). The common phenotypes among Rh (D) positive predominant phenotype of the Lewis system was Le (a-b+) (54.5%). Lea and Leb Iraqi J. Hematology, May 2016, vol.5, Issue 1 60 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M antigens were observed in (43.5%) and Some variations may even occur in different (64.5%) areas within one small country of donors, respectively. (16) . Despite Approximately, (76%) of donors were the fact that the antigens involved are stable shown to be positive for P1 antigen. Finally, throughout life, the resultant polymorphism in the MNSs blood group system, (33.5%) remains important in population genetic of donors were homozygous for M antigen studies, (M+N-), were compatible blood, evaluating the probability homozygous for N antigen (M-N+). Blood of hemolytic disease in the newborn, groups (M+N+) and (S-s+) were the most resolving disputes in paternity/maternity and common for forensic purposes (17). while only phenotype 20.5% with the same estimating the availability of percentage (46%) and no S-s- phenotype In this study, we examined the RBC was found. Out of nine possible phenotypes, antigens and phenotype frequencies of the (M+N+S-s+) common ABO and Rh blood groups in local donor phenotype (40%), whereas (M-N+S+s-) was population of Sulaymaniyah province (The the (0.5%) northern part of Iraq) using different blood observed in the MNS blood group system of group systems. All the donors were males least was common the most phenotype the current study (supplementary Table 3). and belong to well-known Kurdish tribes living in the city and surrounding area. Our Discussion: results indicated that the blood group O was The knowledge of prevalence of various blood group antigens and phenotype frequencies in the local donor population is important in transfusion services especially in areas of antenatal serology, paternity testing, and selecting compatible blood in problem transfusions (24). The ABO/Rh blood group system: It is well established that ABO and Rh genotypes and phenotypes vary widely across races and geographical boundaries. Iraqi J. Hematology, May 2016, vol.5, Issue 1 the most frequently encountered phenotype (37.0%) followed closely by the blood group A (32.6%), then B (22.8%), and the lowest one was AB (7.6%). The results also demonstrated that the majority of donors were positive for Rh (D) antigen (91.26%) while minorities (8.74%) were negative. These results are in agreement with the general trend of the ABO blood groups (O > A > B > AB) and Rh (Rh+ve > Rh-ve) recorded for the Kurdish population in 61 Distribution of red cell antigens according to ABO (13,14) northern Iraq (Erbil and Duhok city) Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M , and E (25.1%)] and a part from D, the Arabian population lives in Iraq and some frequency of other Rh antigens was in other neighboring countries (18-21) . However, agreement with observation of Mashaali in variation between the frequencies of the Baghdad in which the frequency of e, C, c blood groups is observed (supplementary and E were (94%, 77%, 67% and 32 %, Table 4). This variation may due to respectively) differences in the geographical area and are the only two studies available for racial background. Similar results were also comparison in Iraq. observed in populations of other ethnicity Comparing to the neighboring countries of such as Caucasian (34) Europeans (33) , Blacks (14-19) . To our knowledge, these and Iraq, the results of the current study are . On the other hand in many comparable with studies performed in Asian populations, there is an increase in the northeast of Iran prevalence of group B, e.g. India and (2012). However, apart from e antigen, the Malaysia (22,23) comparable . Our results were not to that neighboring Turkey (24) reported in , and Syria (25) (26) and Bahrain (Jenan YT results were markedly different from that the reported in other countries such as north , in Indian (27) (see Table 4). which higher prevalence of group A was reported (supplementary Table 3). The lowest frequent phenotype of the ABO system linked to the Rh (D) phenotype was AB -ve (0.5%) which is similar to Jaff’s observation in Erbil, a neighboring city within the Kurdistan Region of Iraq (13). A significant difference was observed in the frequency of C, E c, and e antigens when donors were categorized as D+ve and D-ve. In D+ve donors the distribution of C and E antigens was (81.7% and 34.2%, respectively) while in D-ve donors was (17% and 0%) (P < 0.05), suggesting that C and E Regarding the frequency of Rh antigens (D, antigens are more prevalent on D+ve red C, c, E and e), we found that the e antigen cells. In contrast, the c and e antigens were has the highest frequency (95.2%), followed detected in almost all D -ve donors (100%) as by D (91.3%), C (74.8%), c (69.4%) and the compared to the less frequency occurrence lowest frequent antigen was E (30.6%). This in D+ve donors (65.8% and 94.6% for c and e observation is very close to the results of a respectively) (P < 0.05). These results are in recent study performed in Duhok city [e accordance with a study in north Indian (95.6%), D (91.1%), C (75.9%), c (68.1%) Iraqi J. Hematology, May 2016, vol.5, Issue 1 62 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M (26) population in which the frequency of C and Iran E antigens in D+ve donors were massively respectively), but higher than Blacks (2%) higher than their presence among D-ve (29) donors, while the prevalence of c and e was antigen was detected in almost (100%) in in contrast (27) and Whites (8%, and 9%, . In contrast, frequency of k (Cellano) . Moreover, these results are our donor population, which is similar to the further supported by our observation that results reported in north Indian 100% DCe/DCe (R1R1) was the most common (Thakral et al. 2010), and black populations phenotype (34%), followed by DCe/dce 100% (R1r) (29.6%) and DCe/DcE (R1R2), and obtained from Whites and northeast-Iran +ve donors, while the who showed negative results for k (Cellano) dce/dce (rr) found to be the most common antigen by 0.2%, 2.3% respectively. This phenotype (83%) in D-ve donors (see Table implies that while Whites and northeast 4). This genotype pattern shows strong Iranian similarities to that reported in northeast Iran develop anti-k (Cellano), the likelihood of and some similarities to north India and finding this alloantibody in our population is Caucasians population. However, the pattern negligible (Table 5). (18.3%) in our D was far different from what reported in Blacks as shown in (Table 4). Other blood group systems: The Kell Blood Group System: (29) , but differ from the results population Regarding the might distribution occasionally of Kell phenotypes, the most common phenotype was found to be K-k+ (94.2%), followed by K+k+ (5.8%). None of the donor was found to be K homozygous (K+k-) or (K-k-). The K antigen is very immunogenic (second to the D antigen) in stimulating antibody production. Anti-K is an important antibody as it is nearly always immune, IgG, and complement-binding. It causes severe HTRs and HDFN (28) . Its frequency in this study was low (5.8%), which is similar to that of These results are similar to that reported in north Indian population recorded by two independent studies (27,30) . Again our results were found to be intermediate between Whites/northeast of Iran from one side and the Black population from another side (29) (Table 5). Thakral et al. study in north India (5.56%) (27) , and it occurs between the frequencies reported by Keramati et al. in northeast of Iraqi J. Hematology, May 2016, vol.5, Issue 1 (26) 63 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M The Kidd Blood Group System: 86.75%, 66%, respectively. While the The frequencies of Kidd blood group system frequency of Fyb antigen is much closer to antigens (Jka = 77%, Jkb = 67.5%) observed that reported by Thakral et al. (56.15%), and in this study was similar to those in it is in between Keramati et al. study and northeast of Iran (26) , and comparable to that of Whites (Beadling & Cooling, 2007), and north-Indian population (27) , while there was Whites (49.2%, respectively). However, again our results showed much higher percentage of Duffy antigen than the (Fya = 10%, Fyb = 23%) a remarkable difference with the Black Blacks population (Jka = 92%, Jkb = 49%) (Table 6). (29) 83%, (29) (Table 5). In this system, Fy (a+b-) was the most The most common Kidd phenotype was Jk common phenotype in our study was (a+b+) (44.5%), which is similar to those in (38.5%), which is comparable to the results (26) northeast of Iran north India (27) , and comparable to and Whites (29) (49.21 % and reported in north-India Iran (26) , which are (27) and northeast of (43.9%, 47.4%, 49%, respectively), while much higher than respectively). However, it is much higher what recorded for Blacks (34%). The Jk than Whites (17%) and Blacks (9%) (a+b-) is found to be the most common population phenotype among the Blacks (57%) (29) . No (29) . The most common reported phenotype in Whites is Fy (a+b+) 49%, and Jk (a-b-) phenotype was detected in any in Blacks is Fy (a-b-) (68%) [29]. donor, which is also very rare in White and Duffy antigen is postulated to be the Black people, except for Polynesians (< 1%) receptor for entry of the plasmodium vivax (29) on the red cells (31,32). This probably explains (Table 5). high prevalence of Duffy null phenotype Fy The Duffy Blood Group System: The frequencies of the Duffy blood group system antigens were (Fya =70%, Fyb=57.5%). The Fya antigen frequency is very close to that reported in northeast of Iran (73.8%) (26) , and it is intermediate between results reported in north Indian population (27) , and Whites (29) , which are Iraqi J. Hematology, May 2016, vol.5, Issue 1 (a-b-) in the endemic area of malaria such as among the black people (68%) (29,33,34) . The frequency of Duffy null phenotype Fy (a-b-) in our study was (4%), which is very close to that reported by Keramati et al. in northeast of Iran (3.4%) (26) . While it is very rare in Whites (Beadling & Cooling, 2007) 64 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M (27) while in Thakral et al. study the most (Table 6). This higher rate of null phenotype common phenotype was M+N- and S+s+ frequency in our donors might be related to (Table 6). the existence of endemic areas of malaria in Out of nine possible phenotypes found in the Kurdistan Region of Iraq in the past. our study, M+N+S-s+ (40%) was the most The MNSs Blood Group System: common phenotype; whereas, M-N+S+s- The frequencies of the MNSs blood group (0.5%) was the less common phenotype system antigens M, N, S and s in our study observed in the MNS blood group system. were 79.5%, 66.5%, 54%, and 88.5%, The frequency of M+N+S-s+ in this study, respectively. These results are similar to the is comparable to the results reported by results reported in north Indian population Agarwal et al. in India (28.8%) (Agarwal et (75.4%, 61.5%, 56.5%, and 87.4% for M, N, al. 2013), as well as in Europeans (22.6%) S and s respectively) (27). The results are also and African-Americans (33.4%) (35,36), while comparable in Thakral study in north Indian population and (0%) in north Indian population with the observation of Keramati et al. in northeast of Iran (87%, 56.7%, 56.7%, and 84.5%, respectively) (26) , and Whites (78%, 72%, 55%, and 89%, respectively), while they are different with the most common phenotype was M+N+S+s+ (19.6%) (Table E). Conclusions: what recorded for Blacks, particularly for S The knowledge of these antigen and and s antigen frequencies (29) (Table 6). phenotype frequencies is crucial in the Regarding the phenotype frequency, M+N+ and S-s+ were the most common phenotypes observed in the MNS blood group system in our study which were nearly equal (46%) to each other. These results are comparable to that of Keramati et al. study who reported that M+N+ and S-s+ are the most common phenotypes (43.7% and 43.3%, respectively) (26) . The results are also comparable to that reported in White and Black populations(29), Iraqi J. Hematology, May 2016, vol.5, Issue 1 clinical field. Based on this information, one can predict the common alloantibodies that could be happened in pregnant women and patients receiving blood transfusions. For instance, E antigen frequency in our donor population was the lowest (30.6%), followed by c antigen (69.4%) in ascending order of frequency. Thus, it can be assumed that the most common alloantibodies in Rh blood group system among pregnant women and in patients receiving blood transfusion would 65 Distribution of red cell antigens according to ABO Hisham A.G, Shaema S.A, Najmaddin Kh.,Belal A.M be anti-E and then anti-c. Another advantage Sulaymaniyah for their valuable help and of coordination in completing this research. knowing antigen and phenotype frequency is that it helps in selection of antigen negative blood units for patients Authorship contributions: For HAG have designed the research and shared example, if a patient in our population has in writing the article, SSA did all laboratory alloantibody against C and needs two units work and shared in writing the article, NK of blood, a minimum of 8-10 units of ABO reviewed and shared in writing editing, and and Rh (D) matched blood units will need to designing the article. BAM reviewed the be tested for C antigen to find two units of article, redesigned the paper, and did major antigen negative blood (since C antigen editing of all parts of the writing, also negative donors form about 25% of all our submitted the paper. with pre-formed alloantibodies. donor’s population). This notion is also Conflict of interest: applied to the less common blood group All authors declares that there is no conflict systems included in this study. of interest. Over all, the various red cell antigens recorded by different blood grouping systems in this study was intermediate between the European and Asian countries with some population specificity reflecting to the the Kurds distinct geographical area and preserved ethnic background of the Kurds in the region. Acknowledgment: We have special thanks to the ministry of health of Kurdistan region and the health directorate of Sulaymaniyah governorate. We also thank the director and all laboratory staff of the central blood bank of the Iraqi J. Hematology, May 2016, vol.5, Issue 1 66 Tables Table 1: Distribution of ABO and Rhesus (Rh) blood groups both individually (upper panel) and in combination (lower panel). Upper panel, the total number of donors and the percentage of each group is given. The most common ABO blood group was O (37.0%), followed by A (32.6%), B (22.8%) and AB (7.6%). Most of the donors were positive for Rh (D) antigen (91.26%) and minority (8.74%) were negative. Lower panel, the number and percentage of donors for each blood group is given. The total number of donors also provided. In both Rh+ve (left) and Rh-ve (right) groups the most common ABO blood group was O, followed by A, B, and AB as indicated regardless of the presence/absence of Rh antigen. N = 5000. Blood groups Number of donors Percentage (%) O 1850 37 A 1630 32.6 B 1140 22.8 AB 390 7.8 Rh (D) positive (Rh+ve) 4550 91.3 Rh (D) negative (Rh-ve) 435 8.7 ABO/Rh (D) positive (Rh+ve) ABO/Rh (D) negative (Rh-ve) ABO blood groups Rh (D) blood groups Blood groups Number & (%) of donors Blood groups Total number of Number & (%) of donors donors O+ 1662 (33.2) O- 190 (3.8) 1852 (37.0) A+ 1491 (29.8) A- 137 (2.7) 1628 (32.6) B+ 1055 (21.1) B- 84 (1.7) 1139 (22.8) AB+ 355 (7.1) AB- 26 (0.5) 381 (7.6) Total 4563 (91.3) 437 (8.7) 5000 (100%) Iraqi J. Hematology, May 2016, vol.5, Issue 1 67 Tables Table 2: Distribution of other Rh antigens (C, c, E, and e) and phenotypes. A) The number and percentage of donors for each blood-group antigen is given. The most common antigen was e (95.2) followed by C (74.8), c (69.4), and E (30.6). B) The Rh phenotype in Rh (D) positive (Rh+ve) donors. C) The Rh phenotype in Rh (D) negative (Rh-ve) donors. The most common Rh phenotype was R1R1 (DCe/DCe) (34%) in Rh+ve donors, while rr (dce/dce) was the most common phenotype (83%) among Rh-ve donors. N = 500. Number of positive donors Percentage of positive donors C 374 74.8 E 153 30.6 c 347 69.4 e 476 95.2 A) Rh Antigens B) Phenotypes in Rh (D) positive (Rh+ve) donors R1R1 (DCe/DCe) 152 34 R1r (DCe/dce) 132 29.6 R1R2 (DCe/DcE) 82 18.3 R2r (DcE/dce) 47 10.5 R2R2 (DcE/DcE) 24 5.4 R0 r (Dce/dce) 10 2.2 C) Phenotypes in Rh (D) negative (Rh-ve) donors rr (dce/dce) 44 83 rʹr (dCe/dce) 9 17 Iraqi J. Hematology, May 2016, vol.5, Issue 1 68 Tables Table 3: Phenotype frequencies in systems other than ABO/Rh. Results of Kell, Kidd, Duffy, Lutheran, Lewis, P, and MNSs blood group systems are presented in percentage as indicated, N = 400. Blood group system Kell system Kidd System Duffy System Lutheran system Lewis system P system MNS System Phenotype K-k+ K+k+ Jk (a+b+) Jk (a+b-) Jk (a-b+) Fy (a+b-) Fy (a+b+) Fy (a-b+) Fy (a-b-) Lu (a-b+) Lu (a+b+) Lu (a-b-) Lu (a+b-) Le (a-b+) Le (a+b-) Le(a+b+) Le (a-b-) P1 M+N+ M+NM-N+ S-s+ S+s+ S+sM+N-S+sM+N-S+s+ M+N-S-s+ M+N+S+sM+N+S+s+ M+N+S-s+ M-N+S+sM-N+S+s+ M-N+S-s+ Iraqi J. Hematology, May 2016, vol.5, Issue 1 Donors (%) 94.2 5.8 44.5 32.5 23 38.5 31.5 26 4 92 3.5 3.5 1.0 54.5 33.5 10 2 76 46 33.5 20.5 46 42.5 11.5 5.5 19 18 22.5 17.5 40 0.5 6 14.5 69 Tables Table 4: Frequencies of Rh phenotypes. The observed Rh phenotype of the present study compared to the published data of other countries as indicated. Northeast Iran2 Present study (%) North-India1 (%) DCe/DCe (R1R1) 30.4 35.2 25 18.5 2.0 DCe/dce (R1r) 26.4 30.7 31.8 34.9 21.0 DCe/DcE (R1R2) 16.4 8.1 16.5 13.3 4.0 DcE/dce (R2r) 9.4 5.9 9.6 11.8 18.6 DcE/DcE (R2R2) 4.8 0.7 1.7 2.3 0.2 2 2.2 4.2 2.1 45.8 dce/dce (rr) 8.8 0.3 8.3 15.1 6.8 dCe/dce (rʹr) 1.8 2.5 1.3 0.8 Rare Phenotypes Dce/dce (R0r) 1 : Sarkar et al. 2013 2 : Keramati et al. 2011 (%) Caucasian3 (%) 3 : Reid & Lomas-Frances, 2004 Iraqi J. Hematology, May 2016, vol.5, Issue 1 70 Black3 (%) Tables Table 5: Phenotype frequencies of the Kell and Kidd blood group systems. Antigen frequencies (%) of Kell and Kidd blood group systems are compared with other published results as indicated. The phenotype frequencies (%) of the Kidd 0.0 0.0 Present study North-India 5.56 100 94.3 5.7 0.0 0.0 Thakral et al. 2010 97.7 96.0 4.0 0.0 0.0 (%) (k) (%) (%) North-India K+k- 5.8 (%) 94.2 K+k+ 100 (%) 5.8 K-k+ Sulaymaniyah-Iraq Cellano References People groups Kell (K) K-k- (%) blood group system was also compared. 2.3 0.0 Whites 9.0 100 91.0 8.8 0.2 0.0 98.0 2.0 Rare 0.0 Keramati et al. 2011 Beadling & Cooling, 2007 Beadling & Cooling, 2007 References (%) Jk (%) (%) Jka (%) People groups Jkb (%) 2.0 Blacks &Thapliyal, 1997 (a-b-) 5.7 Jk (a-b+) 92.0 Jk (a+b) 99.8 (%) 8.0 Jk (a+b+) Northeast-Iran Nanu Sulaymaniyah-Iraq 77.0 67.5 44.5 32.5 23 0.0 Present study Northeast-Iran 79.1 65.1 44.4 34.7 20.7 0.2 Keramati et al. 2011 North-India 82.6 66.6 49.2 33.4 17.3 0.0 Thakral et al. 2010 77 74 49 28.0 23.0 Very rare Whites Beadling & Cooling, 2007 Blacks 92 49 34.0 57 9.0 Very rare Beadling & Cooling, 2007 Iraqi J. Hematology, May 2016, vol.5, Issue 1 71 Tables Table 6. Antigen and phenotype frequencies of Duffy and MNSs blood group systems. Antigen and phenotype frequencies (%) of Duffy and MNSs blood group systems in this study compared with other published results as indicated. People groups Fya (%) Fyb (%) Fy (a+b-) (%) Fy (a+b+) (%) Fy (a-b+) (%) Fy (a-b-) (%) References SulaymaniyahIraq 70 57.5 38.5 31.5 26 4.0 Present study Northeast-Iran 73.8 49.2 47.4 26.4 22.8 3.4 Keramai 2011 et al. North-India 86.7 56.1 43.8 42.9 13.3 0.0 Thakral 2010 et al. Whites 66 83 17.0 49.0 34.0 Very rare Beadling & Cooling, 2007 Blacks 10 23 9.0 1.0 22.0 68.0 Beadling & Cooling, 2007 People groups M (%) N (%) S (%) M+N+ % M+N -% M-N+ % S-s+ % S+s+ % S+s% s (%) References SulaymaniyahIraq 79.5 66.5 54 46 33.5 20.5 46 42.5 11.5 88.5 Present study North-India 75.4 61.5 56.5 43.7 43.3 13 43.3 41.2 15.5 87.4 Thakral et al. 2010 Northeast-Iran 87 56.7 56.7 36.9 38.5 24.6 43.5 43.8 12.6 84.5 Keramati et al. 2011 Whites 78 72 55 50 28 22 45 44 11 89 Beadling & Cooling, 2007 Blacks 74 75 31 44 26 30 69 28 3 93 Beadling & Cooling, 2007 Iraqi J. Hematology, May 2016, vol.5, Issue 1 72 Supplementary Data Supplementary data: Supplementary Table 2: Antigen frequencies of the Rh (D) blood groups. Results of the Rh system of the present study compared to other parts of Iraq, neighboring countries and some other populations as indicated. Rh (D) +ve Rh (D) – % ve % Sulaymaniyah-Iraq 91.3 8.7 Present study Erbil-Iraq 91.7 8.3 Jaff, 2010 Babylon-Iraq 90.1 9.9 Salih, 2009 Saudi Arabia 91.2 8.9 Al-Himaidi & Umar, 2002 Bahrain 91.1 8.9 Jenan, 2012 Arians (Pakistan) 91.7 8.3 Ali et al. 2005 Kuwait 92.5 7.5 Al-Bustan et al. 2002 Iran 90.2 9.8 Keramati et al. 2011 India 93.4 6.6 Thakral et al. 2010 Baghdad-Iraq 80 20 Mashaali, 2014 Blacks 92 8 Whites 85 15 Asians 99 1 People groups Iraqi J. Hematology, May 2016, vol.5, Issue 1 References Beadling & Cooling, 2007; Cooling, 2007; Barclay, 2001 Beadling & Barclay, 2001 Reid & Lomas-Frances, 1997 73 Supplementary Data Supplementary Table 3: Frequency of red cell antigens in systems other than ABO/Rh. Results of Kell, Kidd, Duffy, MNSs, Lutheran, Lewis and P blood group systems are presented both in absolute numbers and percentage as indicated, N = 400. Antigens Number Percentage (%) K 29/500 5.8 k 400/400 100 308/400 77 270/400 67.5 280/400 70 230/400 57.5 318/400 79.5 266/400 66.5 216/400 54 354/400 88.5 Kell Kidd Jka b Jk Duffy Fya Fyb MNSs M N S s Lutheran Lua Lub 18/400 4.5 382/400 95.5 Lea 174/400 43.5 b Le 258/400 64.5 P1 304/400 76 Lewis P Iraqi J. Hematology, May 2016, vol.5, Issue 1 74 Supplementary Data Supplementary Table 3: Antigen frequencies of the ABO blood groups. Results of the ABO system of the present study compared to other parts of Iraq, neighbour countries, and some other populations as indicated. People groups O% A% B% AB% Sulaymaniyah-Iraq 37 32 23 8 present study Erbil-Iraq 37 32 24 7 Jaff, 2010 Babylon-Iraq 36 28 28 8 Salih, 2009 Baghdad-Iraq 39 26 24 11 Mashaali, 2014 Iran 35 33 23 9 Boskabady et al. 2005 Iran 34 30 28 8 Keramati et al. 2011 Kuwait 44 27 24 5 Al-Bustan et al. 2002 Saudi Arabia 51 26 19 4 Sarhan et al. 2009 Jordan 37 38 18 7 Hanania et al. 2007 Caucasian 47 41 9 3 Guyton & Hal, 2005 African blacks (e.g. Nigeria) 53 24 20 3 Enosolease & Bazuaye, 2008 European 43 40 12 5 Mollison et al. 1997 Asians (e.g. India) 39 23 33 5 Ali et al. 2005 Asian (e.g. North India) 32 22 37 9 Agrawal et al. 2013 Iraqi J. Hematology, May 2016, vol.5, Issue 1 References 75 Supplementary Data Supplementary Table 4: Antigen frequencies of extended Rh blood groups (e, C, c, E). Results of the extended Rh blood group system of this study compared to the published data of other parts of Iraq and some other countries as indicated. People groups e% C% c% E% 95.2 74.8 69.4 30.6 Baghdad-Iraq 94 77 67 32 Northeast-Iran 97.9 75.9 73.9 29.5 Bahrain 97.3 73.2 71 21 North-India 98.1 90.2 49.5 18.9 Sulaymaniyah-Iraq References Present study Mashaali, 2014 Keramati et al. 2011 Jenan, 2012 Thakral et al. 2010 Supplementary Table 5: phenotype frequencies of MNSs blood group system compared to other published data as indicated. Present study (%) •India (%) *European (%) *African American (%) M+N+S-s+ 40 28.7 22.6 33.4 M+N+S+s+ 17.5 20.9 22.4 13 M+N+S+s- 22.5 5.12 3.9 2.2 M+N-S-s+ 18 13.8 10.1 15.5 M+N-S+s+ 19 15 14 7 M+N-S+s- 5.5 7.1 5.7 2.1 M-N+S-s+ 14.5 5.1 15.6 19.2 M-N+S+s+ 6 3.1 5.4 4.5 M-N+S+s- 0.5 1.2 0.3 1.6 Phenotype •(Agarwal et al. 2013) Iraqi J. Hematology, May 2016, vol.5, Issue 1 * Lal et al. 2000; Cleghorn, 1960. 76 References: 1. Daniels, G. and M.E. Reid, Blood groups: 8. Mohamed, S. the past 50 years. Transfusion, 2010. 50(2): Characterisation of rh and other blood group p. 281-9. systems amongst the and I. Muna, maldivian blood donors. Med J Malaysia, 2013. 68(5): p. 2. Avent, N.D. and M.E. Reid, The Rh blood 393-6. group system: a review. Blood, 2000. 95(2): 9. Chester, M.A. and M.L. Olsson, The p. 375-87. ABO blood group gene: a locus of 3. Daniels, G., Functions of red cell surface considerable genetic diversity. Transfus Med proteins. Vox Sang, 2007. 93(4): p. 331-40. Rev, 2001. 15(3): p. 177-200. 4. Lisowska, E. and M. Duk, Red blood cell 10. Sarkar, R.S., et al., Proportion of Rh antigens responsible for inherited types of phenotypes in voluntary blood donors. Med polyagglutination. Adv Exp Med Biol, 2001. J Armed Forces India, 2013. 69(4): p. 330-4. 491: p. 141-53. 11. Wagner, F.F. and W.A. Flegel, RHD 5. Mota, M.A., Red cell and human gene deletion occurred in the Rhesus box. leukocyte antigen Blood, 2000. 95(12): p. 3662-8. candidates for alloimmunization renal transplantation: in a reality. Rev Bras Hematol Hemoter, 2013. 12. Eder, A.F. 35(3): p. 160-1. Noninfectious and L.A. complications Chambers, of blood transfusion. Arch Pathol Lab Med, 2007. 6. Moulds, J.M., Future of molecular testing 131(5): p. 708-18. for red blood cell antigens. Clin Lab Med, 13. Jaff, M.S., ABO and rhesus blood group 2010. 30(2): p. 419-29. distribution in Kurds. J Blood Med, 2010. 1: 7. Logdberg, L., M.E. Reid, and T. Zelinski, Human blood chromosomal group locations genes and p. 143-6. 2010: cloning strategies revisited. Transfus Med Rev, 2011. 25(1): p. 36-46. Iraqi J. Hematology, May 2016, vol.5, Issue 1 14. Eissa, A., A, ABO and Rh blood group polymorphism among the Kurds of Duhok, IRAQ. Duhok Medical Journal, 2014. 8(1). 77 15. Byrne, T., Nolan T, and R. O'Donnell, A 21. Sarhan, M.A., K.A. Saleh, and S.M. Bin- comparison of two column agglutination Dajem, Distribution of ABO blood groups technologies for routine antibody screening and rhesus factor in Southwest Saudi Arabia. using the indirect antiglobulin technique. Br Saudi Med J, 2009. 30(1): p. 116-9. J Biomed Sci, 1996. 53(3): p. 193-5. 22. Agarwal, N., R.M. Thapliyal, and K. 16. Kolmakova, G.N. and L.L. Kononova, Chatterjee, Blood group phenotype [The prevalence of ABO blood groups frequencies in blood donors from a tertiary among persons of native nationality in care hospital in north India. Blood Res, Buryatia]. Sud Med Ekspert, 1999. 42(2): p. 2013. 48(1): p. 51-4. 15-6. 23. Ali, N., et al., Frequency of ABO and Rh 17. Musa, R.H., et al., Red cell phenotyping blood groups in major ethnic groups and of blood from donors at the National blood casts of Pakistan. Pak J Med Sci 2005. center of Malaysia. Asian J Transfus Sci, 21(1): p. 26-29. 2012. 6(1): p. 3-9. 24. Kaya, H., et al., The distribution of 18. Al-Bustan, S., et al., Allele frequencies blood groups in Erzurum. Med J Atatürk and molecular genotyping of the ABO blood Univ, 1999. 31(1): p. 20-22. group system in a Kuwaiti population. Int J Hematol, 2002. 75(2): p. 147-53. 25. Sakharov, R.S. and K. Nofal' Kh, [The frequency of ABO blood groups and the 19. Mashaali, J., K ABO and Subgroups expression of group antigens Phenotype in Iraqi Paternity Cases. Int J isohemagglutinins in Syrian Arabs]. Sud App Res, 2014. 2(8): p. 57-60. Med Ekspert, 1996. 39(2): p. 34-6. 20. Salih, H., A, L, Frequency distribution 26. Keramati, M.R., et al., Blood group of ABO blood groups and Rh phenotypes of antigens frequencies in the northeast of Iran. blood donors in babylon Governorate-Iraq. Transfus Apher Sci, 2011. 45(2): p. 133-6. Med J Babylon, 2009. 6(2): p. 268-275. Iraqi J. Hematology, May 2016, vol.5, Issue 1 78 and 27. Thakral, B., et al., Phenotype 33. Mohandas, N. and A. Narla, Blood frequencies of blood group systems (Rh, group antigens in health and disease. Curr Kell, Kidd, Duffy, MNS, P, Lewis, and Opin Hematol, 2005. 12(2): p. 135-40. Lutheran) in north Indian blood donors. Transfus Apher Sci, 2010. 43(1): p. 17-22. 34. Reid, M.E. and C. Lomas-Francis, The Blood Group Antigen FactsBook, in The 28. Shaz, B., et al., Transfusion medicine Blood Group Antigen FactsBook (Second and hemostasis : clinical and laboratory Edition), M.E. Reid and C. Lomas-Francis, aspects. Second edition. ed. 1 volume. Editors. 2004, Academic Press: London. p. 3-6. 29. McPherson, R.A., M.R. Pincus, and J.B. Henry, Henry's clinical diagnosis and 35. Cleghorn, T.E., MNSs gene frequencies management by laboratory methods. 21st ed. in English blood donors. Nature, 1960. 187: 2007, Philadelphia: Saunders Elsevier. xxi, p. 701. 1450 p. 36. Lal, S., G.S. Sonal, and P.K. Phukan, 30. Nanu, A. and R.M. Thapliyal, Blood Status of malaria in India. J Indian Acad group gene frequency in a selected north Clin Med, 2000. 5: p. 19-23. Indian population. Indian J Med Res, 1997. 106: p. 242-6. Corresponding Author 31. Langhi, D.M., Jr. and J.O. Bordin, Duffy Dr. Najmaddin Khoshnaw blood group and malaria. Hematology, 2006. Clinical Hematologist, head of Hematology Department 11(5): p. 389-98. Hiwa Hospital -Sulaymaniyah -IRAQ 32. Rayner, J., Getting down to malarial nuts Cell phone: +9647701554184, and P.o.Box:54 email: [email protected] bolts: the interaction between Plasmodium vivax merozoites and their host erythrocytes. Mol Microbiol, 2005. 55(5): p. 1297-9. Iraqi J. Hematology, May 2016, vol.5, Issue 1 79 توزيع مستضدات الخاليا الحمراء وفقا لRh ،ABOوغيرها من أنظمة فصائل الدم النادرة لدى قومية الكورد العراقيين هشام جيتا ، 1شيماء امين ،2نجم الدين خوشناو 3و ،4بالل محمد 5و6 1قسم امراض الدم ،كلية العلوم ،كلية الطب ،جامعة السليمانية ،إقليم كردستان ،العراق 2قسم امراض الدم ،مستشفى الداخلية التعليمي ,السليمانية ,إقليم كردستان ،العراق 3قسم أمراض الدم ،مستشفى هيوا ،السليمانية ،كردستان ،العراق 4مجلس كردستان للتخصصات الطبية والسريرية أمراض الدم /متدرب وزارة التعليم العالي والبحث العلمي ،أربيل ،إقليم كردستان ،العراق 5قسم تقنيات المختبرات الطبية ،المعهد الفني حلبجة ،جامعة السليمانية للفنون التطبيقية ،السليمانية ،إقليم كردستان ،العراق 6قسم علوم المختبرات الطبية ،كلية العلوم ،جامعة كومار للعلوم والتكنولوجيا ،السليمانية ،إقليم كردستان ،العراق الملخص: الخلفية :من بين أكثر من 03نظام لفصائل الدم ،تسعة منهم وهما Lewis،P ،MNS ،Duffy ،Kidd ،Kell ،Rh ،ABO و Lutheranتعتبر هامة سريريا .توزيعات هذه فصائل الدم تختلف بين السكان في جميع أنحاء العالم .دراسات حول وتيرة الطبية. المراجع في للغاية محدودة الكردي العرق في الدم فصائل األهداف :الستكشاف توزيع مستضدات الخاليا الحمراء والظواهر من فصائل الدم المختلفة بين السكان األكراد باستخدام أنظمة مختلفة. المواد والطرق :تم اختيار خمسة آالف من المتبرعين بالدم الذين حضروا بنك الدم المركزي في محافظة السليمانية عشوائيا واختبارها ل ABOو Rhومولدات المضادات ( ،E ،C ،C ،Dوه) باستخدام طريقة األنبوب .وقد تم اختيار 533عينة من األخرى. الدم فصيلة أنظمة باستخدام تحليلها وكذلك عشوائي بشكل المتبرعين النتائج :في نظام ،ABOكان النمط الظاهري األكثر شيوعاهو ،٪(37( Oتليها ِ ، )٪02.6( Aو ))B ٪22.8و AB . )٪(7.6بين المستضدات فصيلة الدم ،Rhكان eاألكثر شيوعا ( ،)٪25.2يليه ،)٪62.4(c ،) C ٪ (74.8،)٪(91.3 D و ))E ٪30.6مع )DCe / DCe (R1R1و )rr( dce / dceهي الظواهر األكثر شيوعا بين RhDااليجابي والسلبي على التوالي .وكانت الظواهر األكثر شيوعا ألنظمة الدم األخرى على النحو التالي: Kell (K-k+,94%), Kidd (jk a+b+,44.5%), Duffy (fy a+b+,45%), Lutheran (Lu a-b+,92%), Lewis (Le a-b+,54.5%), P (P1,76%), MNS )(M+N+S-s+,40% الخالصة :مختلف مستضدات الخاليا الحمراء التي سجلتها أنظمة فصيلة الدم مختلفة في هذه الدراسة كان وسطا بين الدول األوروبية واآلسيوية مع بعض الخصوصية للسكان األكراد يعكس منطقة جغرافية متميزة والخلفية العرقية المحفاظ عليها. كلمات البحث :األكراد ، ABO ,مستضد الخاليا الحمراء Rh, 80 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Original article Review of Congenital Factor XIII Deficiency in Single Iraqi Teaching Hospital Lubna Foad Hussain1 , Obeida Amir Abid 2 1 C.A.B.P.(ped) / Specialist Pediatrician,/ Children Welfare Teaching Hospital, Medical City, 2 F.I.B.M.S(Ped) / Specialist Pediatrician, /Children Welfare Teaching Hospital, Medical City, Received 10/1/2016 revised 24/1/2016 accepted 6/2/2016 Abstract Background: Factor XIII deficiency is one of the rarest bleeding disorders with an estimated prevalence of 1in 3 million populations worldwide. The main clinical manifestations of the disease are delayed wound healing, recurrent miscarriage, intracranial bleeding, and prolonged umbilical cord bleeding. Objectives: The aim of this study was to assess the diagnosis and treatment of factor XIII deficiency in Children Welfare teaching hospital in Baghdad. Patients and Methods: This retrospective study was performed on thirty three patients with the severe factor XIII deficiency. The diagnosis of the disease was done by a wide spectrum of characteristics which is part of inclusion criteria and including family history, clinical manifestations, laboratory tests, clot solubility in 5 M urea or monochloroacetic acid environments. Results: the common manifestations of the disease at time of diagnosis were bleeding after trauma (42.4%), umbilical cord bleeding (21.2%)and less frequently gum bleeding and ecchymosis. Conclusions: factor XIII deficiency is a rare disease in Iraq, most patients are diagnosed at the age of 1-10 years, family history was positive in more than half of the patients. Keywords: congenital Factor XIII deficiency, clot solubility test. Iraqi J. Hematology, May 2016, vol.5, Issue 1 81 Review of Congenital Factor XIII Deficiency Lubna Foad Hussain, Obeida Amir Abid Introduction Congenital factor deficiency is a XIII rare (FXIII) autosomal platelets count and bleeding time (BT) (9) .Diagnosis of the disease based on recessive disease usually associated solubility of blood clot in solution of 5 with a severe bleeding diathesis (1). The M urea or 2% acetic acid (or1% mortality and morbidity are primarily monochloroacetic acid). These tests are related intracranial qualitative tests and show positive hemorrhage can be life threatening. result if the activity of FXIII in plasma Because the clinical bleeding is severe of the patients is absent or close to in most patients with hereditary factor zero. If the results of test become XIII deficiency, the diagnosis is made positive at an early age, often during infancy. analysis of FXIII activity is needed (10). to bleeding; Bleeding from the stump of the umbilical cord within the first days to weeks of life is a characteristic sign that occurs individuals in 80% (2,3) of affected . Additional signs of bleeding include the following: CNS hemorrhage is frequent (25-30%) and may occur spontaneously or after minor trauma (4-7) . Soft tissue bleeding and bruising are very common (4,5) . Hemarthroses occur in 20% of cases .Bleeding that is delayed (ie:12-36h) after trauma or surgery is diagnostic of factor XIII deficiency.(8) by observing quantitative Replacement therapy in this deficiency can be administered through fresh frozen plasma inactivated), (preferably cryoprecipitate virusand pasteurized FXIII concentrates. The first FXIII from human source that used in replacement therapy was produced from placenta but later this product replaced by plasma extracted FXIII concentrates (5,11-13) .The aim of the study is to assess the diagnosis and treatment of factor XIII deficiency in Children Welfare Teaching Hospital, Medical City, Baghdad. FXIII deficiency can be initially diagnosed subsequent Patients and methods bleeding episode with normal routine clotting tests including prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen level, Iraqi J. Hematology, May 2016, vol.5, Issue 1 It is a retrospective study. The study participants included all patients age group who were attend the hemophilia center in Children Welfare Teaching 82 Review of Congenital Factor XIII Deficiency Hospital with the diagnosis of FXIII deficiency, from January 2 000 till April 2015. All patients were identified by using coded discharge records with the diagnosis of factor XIII deficiency. Data collected included: Gender, age at presentation, presenting complaint, family history, type of treatment (fresh frozen plasma, cryoprecipitate), hepatitis B ,C in patients. Lubna Foad Hussain, Obeida Amir Abid Results Thirty three patients were diagnosed to have factor XIII deficiency. 20(58%) were males shown in Figure (1). The diagnosis of FXIII deficiency was established at 3 age groups; first group less than 1 year, second group 1-10 years and third group more than 10 years; most patients diagnosed at the age of 1-0 years as shown in Figure (2) The following investigations were Post traumatic bruises were the major reviewed for all patients who presented presenting complaint in 14 patients to the outpatient clinic to establish the (42.4%) , followed by prolonged diagnosis and included complete blood bleeding from umbilical stump in 7 picture and coagulation screen PT patients(21.2%). ,APTT and intracranial hemorrhage diagnosed by bleeding time which reported to be normal. Diagnosis was confirmed by mol/L urea which was done to all our patients, a qualitative test for diagnosis FXIII deficiency; those found positive were labelled as factor XIII deficient.Results were expressed as frequencies and patient had neuro-imagining study .Table1 performing clot solubility tests in 5 of One percentages and presented in tables and figures using Microsoft Office Excel 2013. Family history was positive in 20 patients (58%). The products used in the treatment of these patients only when presented with bleeding included fresh frozen plasma, cryoprecipitate. Testing for transmission of viral infections was also done in all our patients and only 2 from 6 patients received plasma were found with hepatitis C positive after repeated transfusions of fresh frozen plasma. Iraqi J. Hematology, May 2016, vol.5, Issue 1 83 Review of Congenital Factor XIII Deficiency Lubna Foad Hussain, Obeida Amir Abid The other 27 patients were treated by infection .Hepatitis B was found cryoprecipitate with no hepatitis C negative in all patients .Figure3 Gender of the patients 42% Male 58% Female Figure1:The gender of 33 patients Table1:The presenting complaints at the time of the diagnosis of 33 patients Bleeding type Frequency(no.) Percent(%) Post traumic bruises 14 42.4 Umbilical cord bleeding 7 21.2 Gum bleeding 5 15.2 After circumcision 2 10% of males Muscle hematoma 2 6.1 Ecchymosis 1 3.0 Epistaxis 1 3.0 Intracranial hemorrhage 1 3.0 Total 33 Iraqi J. Hematology, May 2016, vol.5, Issue 1 84 Review of Congenital Factor XIII Deficiency Lubna Foad Hussain, Obeida Amir Abid 17 11 5 LESS 1 YEARS 1-10 YEARS MORE TEN YEARS Figure2:The age of diagnosis in 33 patients 6% negative 94% positive Figure3:The results of Hepatitis C screening Iraqi J. Hematology, May 2016, vol.5, Issue 1 85 Review of Congenital Factor XIII Deficiency Lubna Foad Hussain, Obeida Amir Abid Discussion This disorder is so rare that to date of the patients presented early in their only 33 of 778 patients with congenital life, which is supported by this study bleeding which is the same in Iranian, Saudis disorders, have been identified in our hemophilia center with factor XIII deficiency. So the incidence of this disorder is 0.042%. In Saudia Arabia it is also a rare disease (14) .While in Pakistan it was the fifth most common factor deficiency detected in one study conducted there (15) . In Iran, a Middle Eastern country with a high rate of consanguineous and Pakistani study.(14-16) In this study family history is positive in 94% of patients due to higher incidence of interfamilial marriages. In Iranian and Saudi study (59%) had a family history of FXIII deficiency (14,16) .In Pakisatan positive family history was present in 42.4% of patients.(15) marriages, there are approximately 473 FXIII In Iraq FXIII concentrate is not deficiency. An approximately 12-fold available till now, in Pakistan all higher prevalence of FXIII deficiency patients treated with fresh frozen is estimated in Iran in comparison with plasma and cryoprecipitate patients afflicted with overall worldwide frequency. (14) . In this study, bleeding after trauma is the most common, while in Iranian and (15) and the same applied for Iran until 2009 when FXIII concentrate became available for patient management. (14) Pakistani study showing subcutaneous The challenges that we faced in the bleeding (57%), followed by umbilical diagnosis was the unavailability of cord muscle quantitative assay and in the treatment intracranial was the unavailability of plasma bleeding hematoma (56%), (49%) and hemorrhage (34%) to be the major clinical presentation (14,15) derived FXIII product. . In Saudis the presenting symptoms included ecchymosis in 12 patients (71%), bleeding after circumcision in 6 male patients (55%), umbilical stump bleeding in 7 (41%), and intracranial bleeding in 3 patients (18%) (16). Most Iraqi J. Hematology, May 2016, vol.5, Issue 1 86 Review of Congenital Factor XIII Deficiency Conclusion Lubna Foad Hussain, Obeida Amir Abid 3. Siboni S, Zanon E, Sottilotta G, et Factor XIII deficiency is a rare disease in Iraq with low incidence, most patients diagnosed at the age of 1-10 al. Central nervous system bleeding in patients with rare bleeding disorders. Haemophilia. 2012;18(1):34–8. years, family history was positive in 4. Karimi M, Bereczky Z, Cohan N, more than half of the patients.Thus in editors Factor XIIID. Seminars in children thrombosis traumatic umbilical with history of bruising, bleeding post prolonged along with any family history of easy bruising and consanguinity in parents, it is essential to rule out FXIII deficiency especially in those with normal and hemostasis.2009 Jun;35(4):426-38. 5. Eshghi P, Cohan N, Naderi M, et al. Factor XIII deficiency: a review of literature Iranian Journal of Blood and Cancer. 2010;4: 85–91. 6. Sawlani KK, Chaudhary SC, Roy A, Author contributions: et al. Factor XIII deficiency presenting Dr.Lubna Foad Hussain: Acquisition with intracerebral bleed. BMJ Case of data analysis , interpretation of data Rep. ;Epub 2013/01/15. , critical revision. Dr. Obeida Amir 7. Naderi M, Dorgalaleh A, Alizadeh Abid : Study S, et al. Polymorphism conception, study design, drafting of thrombin‐activatable manuscript, and Coagulation studies. inhibitor and risk of fibrinolysis of intracranial haemorrhage in factor XIII deficiency. Haemophilia. 2014 Jan;20(1):e89-92. References 8. Helge Dirk Hartung, MD; Chief 1. Bolton-Maggs PH. The rare Editor: Max J Coppes, MD, PhD, inherited coagulation disorders. Pediatr MBA.Pediatric Factor XIII Deficiency Blood Cancer. 2012: S37. Clinical Presentation. Medscape. 2. Naderi M, Imani M, Eshghi P, et al. 9. Kessel R, Hu C, Shore-Lesserson L, Factor XIII deficiency in Sistan and et al. A child with acquired factor XIII Baluchistan province. Sci J Blood deficiency case report and literature Transfus Organ. 2013;10(3):282–8 review. 2013 Nov;19(6):814-26. Iraqi J. Hematology, May 2016, vol.5, Issue 1 87 Review of Congenital Factor XIII Deficiency Lubna Foad Hussain, Obeida Amir Abid 10. Bay A, Sirikci A, Dogan A, 16. Al-Sharif FZ1, Aljurf MD, Al- Leblebisatan G, et al. Spontaneous Momen Acute Cerebral Hematoma in a Child laboratory features of congenital factor with Factor XIII Deficiency. Pediatr XIII deficiency.Saudi Med J. 2002 Hematol Oncol. 2013. May;23(5):552-4. AM, et al.Clinical and 11. Opat S, Butler J, Malan E, et al. Factor XIII assays. Methods Mol Biol. 2013;992:171–80. 12. Williams M, Will A, Stenmo C, et al. Pharmacokinetics of recombinant Correspondence to: Dr. Obeida Amir Abid Dept. of hemophilia factor XIII in young children with Children welfare hospital congenital Medical city FXIII comparison deficiency with older and patients. Haemophilia. 2013. 13. Caudill e-mail: [email protected] Mobile; 07727825155 JSC, Nichols WL, Plumhoff EA, et al. Comparison of coagulation factor XIII content and concentration in cryoprecipitate and fresh‐frozen plasma Transfusion. 2009:49(4):765_70. 14. Dorgalaleh A, Naderi M, Hosseini MS, et al.Factor XIII deficiency in Iran: a comprehensive review of the literature.Semin Thromb Hemost . 2015 Apr;41(3):323-9. 15. Anwar M, Iqbal M, Ayyub M ,et al. Prevalence of factor XIII deficiency in patients presenting with a bleeding disorder published in Pakistan.Article online: first DOI: 10.1111/j.1538-7836.2003.0543j. Iraqi J. Hematology, May 2016, vol.5, Issue 1 88 Lubna Foad Hussain, Obeida Amir Abid Review of Congenital Factor XIII Deficiency نقض انعامم انوراثي انثانث عشر في احذى مستشفيات انعراق انتعهيميو /دراسو وطفيو د.نبني فؤاد حسين 1د.عبيذه عامر عبذ2 1,2طبيب اختظاص في طب االطفال /مستشفي حماية االطفال انتعهيمي /مذينة انطب انخالطو: خهفية انبحثَ :قص عبيم انحخرش انربند عششانٕساذً ْٕ يٍ االظطشاببت انُضفٍّ انُبدسِ انحً جحذخ فً انبشش, جقذس َسبة حذٔذّ بُسبة 1نكم 3يهٌٍٕ يٍ عبية انُبط.يٍ اْى عاليبت انًشض جأخش انحئبو انجشٔح ,اسقبطبت يحكشسِ عُذ انحٕايمَ ,ضف يطٕل يٍ يكبٌ انحبم انسشي عُذ حذٌرً انٕالدِ. االىذاف :جٓذف ْزِ انذساسّ انى جحذٌذ عٕايم انحشخٍص ٔانعالز نُقص عبيم انحخرش انربند عشش انُضفً انٕساذً فً يسحشفى حًبٌة االطفبل انحعهًًٍ فً بغذاد. انمواد وانطرق :دساسّ يسحشدِ ٔٔصفٍّ ل 33يشٌط يشخصٍٍ بُقص عبيم انحخرشانربند عشش انٕساذً اجشٌث بٍٍ االٔل يٍ جًٕص 0222انى االٔل يٍ ابشٌم .0212جى انحشخٍص ببالعحًبد عهى انحبسٌخ انعبئهً ٔانعاليبت انسشٌشٌّ ٔانفحٕصبت انًخحبشٌّ. اننتائح :اكرش انعاليبت انًشظٍّ شٍٕعب عُذ انًشظى اذُبء انحشخٍص كبَث انُضف بعذ حذٔخ االصبببت ,انُضف يٍ يكبٌ انحبم انسشي عُذ حذبرً انٕالدَِ ,ضف انهرّ ذى ظٕٓس بقع َضفٍّ جحث انجهذ. االستنتاج :جظٓش ْزِ انذساسّ اٌ َقص عبيم انحخرش انربند عشش انٕساذً ْٕ يٍ االيشاض انُبدسِ فً انعشاق .اكرش االعًبس انحً ٌحى فٍٓب جشخٍص انًشض 12- 1سُٕات .جبسٌخ انعبئهّ كبٌ يٕجبب فً اكرش يٍ َصف انحبالت انًشظٍّ . انكهمات انرئيسيةَ :قص عبيم انحخرش انربند عشش انٕساذًَ ,ضف يطٕل يٍ يكبٌ انحبم انسشي ,اخحببس رٔببٌ انجهطّ. 89 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Original article CD38 and ZAP-70 as prognostic immunological parameters in patients with chronic lymphocytic leukemia Intisar Sh. Ali 1 ,Ghassan A. Al-Anni 2 , Salman A. Al-Jubury 3 1 M.Sc. microbiology/laboratory dept./ Alyarmouk teaching hospital 2 consultant Hematopathologist/ FICMS hematopathology/ head of Lab. dept. /Alyarmouk teaching hospital 3 Prof. Of clinical immunology/ microbiology dept./university of kufa college of medicine Received: 15/1/ 2016 Accepted: 17/ 2/ 2016 Abstract Background: Several immunological parameters have been investigated in patients with chronic lymphocytic leukemia (CLL). Some of them are important in detecting the prognosis of the disease. Objectives: the aim of this study was to evaluate the level of ZAP-70 and CD38 in patients with newly diagnosed chronic lymphocytic leukemia. Materials and methods: The study was done on 50 patients with newly diagnosis CLL 36 male and 14 female. CD38 and ZAP-70 were measured in patients by using flow cytometry. These patients were divided according to Ria stage into 29 patients with group II, 21 patients with group I, group I included Rai stage O, I, II in which the RBC and platelet counts are normal while group II included Rai stage III and IV in which RBC and platelet count are low. Results: ZAP-70 and CD38 were significantly increased in patients with group II when compared to group I. ZAP-70 and CD38 may be regarded as bad prognosis factor for CLL patients. Conclusion: From the results of the current study, we conclude that ZAP-70 and CD38 high level in patients with chronic lymphocytic leukemia is associated with bad prognosis Keywords: CD38, ZAP-70, CLL Iraqi J. Hematology, May 2016, vol.5, Issue 1 90 CD38 and ZAP-70 as prognostic immunological Intisar Sh.Ali,,Ghassan A.Al-Ani, Salman A. Introduction Chronic lymphocytic leukemia is the The expression of ZAP-70 (˃ or = 20% most common lymphoid malignancy, it of B cell) has showed to associated with is a disease predominantly seen in increased risk for bad prognosis in elderly with the incidence twice higher patients with CLL and is regarded as men (1) . CLL showed familial tendency with 3-8 times higher than in normal population (2) .Chronic lymphocytic leukemia characterized by accumulation of terminally different clonal of CD5+ B-cell from proliferation and differentiation in the bone marrow and secondary lymphoid organ. It is the most common type of malignancy in the western world but its incidence is low in Far East (3) . Chronic lymphocytic leukemia has different prognosis where the disease may be growing slowly with minimal changes in blood cell count; other may have a faster growing malignancy (4). great risk factor in those patients (6). CD38 is glycoprotein with small cytoplasmic region (21 amino acids), a single chain transmembrane region (23 amino acids), and 256 amino acid extracellular domain that can be loosely divided into two regions. The extracellular amine protein consists of 156 amino acids that make up 5 αhelices. This region is adjacent to the COOH domain (amino acids 200-300) that contains four parallel P-sheets surrounded by two long and two short α(7) helices .CD38 expression on the leukemic cell regarded as an excellent prognostic indicator because it's ZAP-70 is a member of the tyrosine measured from peripheral blood sample kinase group of protein and is normally easily showed in T-cell and natural killer cells CD38 (NK). ZAP-70 have a vital role in the identification of sub group of patients regulation of normal T-cell functions, with such as T-cell receptor(TCR),signal presumably poor prognostic and the initiation, detection of patients with poor prognosis T-cell activation, cell migration and apoptosis (5). (8) among . Furthermore, measurement of expression better what arrows prognosis is the within considered a better prognostic group (9). Iraqi J. Hematology, May 2016, vol.5, Issue 1 91 CD38 and ZAP-70 as prognostic immunological Intisar Sh.Ali,,Ghassan A.Al-Ani, Salman A. Materials and methods: This study was done of 50 patients with evaluate differences between the studied newly diagnosed chronic lymphocytic groups. leukemia from March 2013 till March considered 2015 included 36 male and 14 female, Analysis of data was carried out using the age of the patients rage from 45-75 the available statistical package of SPSS- years, all of the patients were diagnosed 22 (Statistical Packages for Social morphologically as chronic lymphocytic Sciences-version 22). leukemia. These patients were attending Medical City in Baghdad, National For all test, statistically P≤0.05 was significant. Results : Center of Haematology (University of Table (1) and figure (1) represent the Mustansyria), Teaching comparison of the mean value of ZAP- Hospital and Al-Emammain kadhumain 70 in patients of group I and group II Medical City. chronic lymphocytic leukemia. Those patients were divided into two The ZAP-70 level in patients with group groups according to Rai staging group I II chronic lymphocytic leukemia was included Rai stage O, I, II in which the significantly increased (p=0.0001) when RBC and platelet counts are normal compared to group I. Al-Yarmouk while group II included Rai stage III and IV in which RBC and platelet count are low. ZAP-70 and CD38 were determined in patients of the above group by flow Table (2) and Figure (2) represents the comparison of the mean value of CD38 concentration in the sera of group I and group II chronic lymphocytic leukemia. cytometry method using Four-Colour CD38 value were significantly increased Cyflow® (p=0.001) in patients with group II Statistical analysis:Data were expressed; Student's t-test was used to Iraqi J. Hematology, May 2016, vol.5, Issue 1 chronic lymphocytic leukemia when compared to group I. 92 CD38 and ZAP-70 as prognostic immunological Intisar Sh.Ali,,Ghassan A.Al-Ani, Salman A. Table (1): levels of ZAP-70 in group I and II chronic lymphocytic leukemia ZAP-70 Group I (n=21) Group II (n=29) Mean ± SD 8.82±17.10 37.58±22.29 Standard Error of Mean 3.73 4.14 Range 0.19-61.97 1.22-87.04 50th (Median) 0.90 24.49 P value 0.0001* *Significant difference means using Student-t-test for difference between two independent means at 0.05 level. Table (2): level of CD38 in patients with CLL group I and II CD38 Group I (n=21) Group II (n=29) Mean ± SD 6.25±9.30 36.01±24.60 Standard Error of Mean 2.03 0.574 Range 0.53-39.28 0.96-78.92 50th (Median) 2.49 28.64 P value 0.0001* *Significant difference means using Student-t-test for difference between two independent means at 0.05 level. Iraqi J. Hematology, May 2016, vol.5, Issue 1 93 CD38 and ZAP-70 as prognostic immunological Intisar Sh.Ali,,Ghassan A.Al-Ani, Salman A. Figure (1): Distribution of ZAP-70 (mean±SD) in patients with chronic lymphocytic leukemia group I and II Figure (2): Distribution of CD38 (mean ± SD) in patients with chronic lymphocytic leukemia group I and II Iraqi J. Hematology, May 2016, vol.5, Issue 1 94 CD38 and ZAP-70 as prognostic immunological Intisar Sh.Ali,,Ghassan A.Al-Ani, Salman A. Discussion There are immunological Regarding CD38, the data in this study determine the showed that it might be useful for prognosis at the time of diagnosis of prognosis of the disease since the value CLL that would help the decision about of CD38 increase as the disease progress treatment and to determine the outcome and patients with CD38 high value may parameters many used of the disease (10) to .ZAP-70 is a member of be regarded bad prognosis for the the family of intracellular tyrosine patients kinases, is significantly expressed in leukemia normal T cells. It is also may be express with previous results which indicate that at early stage of chronic lymphocytic expression of CD38 patients with CLL is leukemia (11) . The result in the present study determine that ZAP-70 might be useful for detecting the prognosis of the disease because the level of ZAP-70 in (13) chronic lymphocytic .This observation agreed associated with bad prognosis and poor outcome (14). Conclusion increase in patients with group II chronic From the results of the current study, we lymphocytic leukemia when compared conclude that ZAP-70 and CD38 high to group I chronic lymphocytic leukemia level patients. The data in our study regarding lymphocytic leukemia is associated with ZAP-70 value agreed with previous bad prognosis. result which indicate that ZAP-70 in patients with chronic in patients with chronic References lymphocytic leukemia increase as the disease progress 1. Bain B., and Barbara J. Leukaemia (12) diagnosis 4th ed.;2010 . p65-74. . CD38 is a protein that have many function belongs to the number of molecules independently as ecto-enzymes as receptors, CD38 may split the patients with chronic lymphocytic leukemia into two groups and represent a dependable negative prognostic factor (13) Iraqi J. Hematology, May 2016, vol.5, Issue 1 2. Arena G., Keating Carotenuto C. lymphoproliferative integrated differential M.L. point of and Chronic disorders: an view the diagnosis. for Leukemia Lymphoma 2000; 36,225–237. 95 CD38 and ZAP-70 as prognostic immunological 3. Mir M .Chronic Intisar Sh.Ali,,Ghassan A.Al-Ani, Salman A. Lymphocytic 9. Liu Q., Kriksunov I., Graeff, R., and Leukemia. Medscape 2013; 4:1993- et al.Crystal structure of human CD38 1994. extracellular domain. Structure 2005; 13: 4. Elter T., Hallek M., Engert A. Fludarabine in chronic 1331-1339. lymphocytic 10. Digheiro G.,and Hamblia T.Chronic leukemia, Export Opin Pharmacother lymphocytic leukemia. Lancet 2008; 2006; 7(12):1641-1651. 371(9617): 1017-1029. 5. Chen L., Widhopf G., Huynh L., and 11. Nagwa M.,Kathyrn R.,Felisa Al et al. Expression of ZAP 70 is associated concia, and et al. A single tube, four with increased B cell receptor signaling color in chronic lymphocytic leukemia. Blood evaluation 2002; 100: 4609-14. expression CLL. American Journal of 6. Gobossi S., Laurenti L., Longo P.G., flow cytometry of ZAP-70 assay and for CD38 clinical pathology 2010; 133:708-717. and et al. ZAP-70 enhances B cell 12. Maria Ilaria. .Del principle,American receptor signaling despite absent or society of haematology 2006;108(3). inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cell. Blood 2007;109:20322039. al. ZAP-70 expression chronic lymphocytic leukemia and inhibit B cell receptor ZAP-70 expression is a prognostic factor in chronic lymphocytic leukemia. Leukemia 2003;17:2426-34. 7. Deïlschneidr R., Xiao V., Yoon J., and et 13. Dürig J., Nuckel H., Cremer M. et al. signaling. Cell 14. Sylvan S.Targeted therapy and outcome in chronic lymphocytic leukemia. Ph.D. thesis.2014 death and disease 2014; 5:439-442. 8. Malavasi F., Deaglio S., Funaro A., and et al.Evolution and function of the Correspondence to: Intisar Sh. Ali MSc. Dept. of micerbilogy/ Alyarmouk teaching Lab. ADP ribosyl cyclase / CD38gene family in physiology and pathology. Physiol Rev 2008; 88: 841-886. Iraqi J. Hematology, May 2016, vol.5, Issue 1 e-mail:[email protected] mobile: 07735674539 96 Intisar Sh.Ali,,Ghassan A.Al-Ani, Salman A. CD38 and ZAP-70 as prognostic immunological CD38و 07-ZAPمعلمات مناعية للتكهن في المرضى الذين يعانون من سرطان الدم الليمفاوي المزمن انتصار علي ،1غسان احمد خليل العاني ،2سلمان الجبوري 3 1ماجستير علم األحياء الدقيقة /قسم المختبرات /مستشفى اليرموك التعليمي 2استشاري امراض الدم المختبري / FICMS /رئيس قسم المختبرات /مستشفى اليرموك التعليمي 3أستاذ المناعة السريرية /قسم االحياء المجهرية /كلية الطب الكوفة الملخص الخلفية :هناك عدة انواع من المعلمات المناعيه نم دراستها في مرضى ابيضاض الدم اللمفاوي المزمن .بعض من هذه المعلمات مهمه في تعيين حالة المريض . االهداف :اجريت هذه الدراسه لمعرفة فائدة ZAP-70و CD38في مرضى ابيضاض الدم اللمفاوي المشخصين حديثا. المواد والطرق :تم اجراء هذا البحث على 07مريضا 33ذكور و 14اناث ،حيث تم قياس ZAP-70و CD38في مصول هؤالء المرضى بواسطة جهاز الفلوسايتومتري .تم تقسيم هؤالء المرضى الى قسمين المجموعه االولى تضم ( )21مريض والثانيه تضم ( )22مريض. النتائج :لقد كانت هناك زياده محسوسه في مستوى ZAP-70و CD38في مصول مرضى المجموعه الثانيه مقارنه بالمجموعه االولى وبذلك بين اهميه ال ZAP-70و CD38كمعلمات لسؤء حالة المريض المصاب بابيضاض الدم اللمفاوي. االستنتاج :من نتائج الدراسة الحالية ،فإننا نستنتج أن هناك ارتباط بين ZAP-70و CD38على مستوى عال في المرضى الذين يعانون من سرطان الدم الليمفاوي المزمن مع المسيرة السيئة لهذا المرض. الكلمات الرئيسية ZAP-70 :و , CD38سرطان الدم الليمفاوي المزمن 97 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Original article Evaluation the Role of Bone Marrow Examination in Diagnosis of Hematological Diseases in Hemato-Oncology Centers in Kurdistan Region 1 Shorsh Jameel Ridha , Nawsherwan Sadiq Mohammad 2, Hoger Ismael Muhammed Sarhang3 1 MBChB-MSc Hematology /Hiwa Teaching Hospital 2 MBChB- FICMS Hematology/Pathology Department; College of Medicine / Hawler Medical University Email: [email protected] Mob: 00964(0)7504384101 3 MBChB-MSc Hematology/ Erbil Teaching Hospital Received: 10/2/2016 accepted: 13/3/2016 Abstract Background: Bone marrow aspiration and trephine biopsy have an important role in evaluation and diagnosis of most hematological and some non hematological disorders. Objectives: The aims of this study were to assess the value of bone marrow examination in diagnosis of hematological diseases and determination of frequency and causes of dry tap marrow. Patients and Methods: This study was prospective only and it was conducted from January 2013 to June 2013 at Hiwa and Nanakaly hospitals in Sulaymaniyah and Erbil respectively. A total number of 245 cases were underwent bone marrow examination. Touch imprint was prepared in a state of dry tap marrow. Sudan black stain was used in acute leukemia. Results: The patients comprised 138 (56.3%) males and 107 (43.7%) females, with ages ranging from 1 to 81 years and the mean age of cases was 36.7± 24 years. The most frequent clinical finding of patients underwent bone marrow examination were pallor (71.4%), followed by fever (32.2%). The main indications were present of blast cells in peripheral circulation 69 (28.2%), followed by bone marrow assessment for staging of lymphoma 37 (15.1 %). The most common diagnoses encountered were: Acute leukemia 69 (28.2%), Active marrow (negative for lymphoma) 27 (11.1%) and active marrow 24 (9.8%). The frequency of dry tap was (4.08%) and the most common causes were: acute leukemia and faulty technique (normal marrow). Conclusion: Bone marrow examination is an important diagnostic tool in the diagnosis and staging of various hematological disorders. Keywords: Bone Marrow Aspiration, Trephine Biopsy, Hematology, Kurdistan Iraqi J. Hematology, May 2016, vol.5, Issue 1 98 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. Introduction Bone marrow is a quite cellular marrow examination, there are some connective tissue that occupying the contraindications which are related to spaces between bony trabeculae. The the general condition of the patient. main hemopoietic tissue is bone marrow These contraindications are hemophilia, and it accounts for about (5%) of all disseminated intravascular coagulation, body weight. Bone marrow aspiration skin infection and bone disorders such as and trephine biopsy have an important osteomyelitis role in evaluation and diagnosis of most imperfecta (4,5). hematological and osteogenesis non Both procedures are usually safe hematological disorders. Bone marrow but few adverse effects are encountered examination is needed for staging of after bone marrow examination. The lymphoproliferative In popular complication is bleeding which advanced stage, it shows bone marrow occurs specially in patients with severe involvement. Also trephine biopsy is low platelet count, or taking aspirin, or more important than marrow aspiration receiving anticoagulant in and Other morbidity from posterior iliac These crest are identified and included long procedures are valuable in the diagnostic standing pain, infection in immune work up of pyrexia of unknown origin; it compromised patients and needle-related may accidents such as breaking of needle evaluation metastatic shows of solid some or disorders. lymphoma tumors. granuloma, infection, necrosis and hemophagocytic syndrome. as warfarin. within the bone (6,7). They are useful in follow up evaluation In some certain situation, failure of patients who submit chemotherapy, to take out bone marrow on trying bone marrow transplantation and other marrow aspiration, known as dry tap or patterns of medical management(1,2,3). blood tap and these are generally Bone marrow aspiration and described as faulty technique. This biopsy procedures are complementary to difficulty of aspiration may be occurred each other and superiority of one when histology of marrow is normal. procedure over the other relied on the But usually a dry tap indicates important particular disorders (1). In obtaining bone disorders that involve bone marrow. The Iraqi J. Hematology, May 2016, vol.5, Issue 1 99 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. most prominent pathological disorders and routine investigations (complete are metastatic carcinoma, myelofibrosis, blood count, pictures, and erythrocytes acute leukemia (packed cells), hairy cells sedimentation rate leukemia and lymphoma. In Dry tap tests) were carried out prior to bone status, touch imprint must be prepared marrow examination. for early prime diagnosis before the technique (4) result of trephine biopsy was returned the samples from posterior iliac crest by back (8,9) and biochemical The standard was employed in obtaining . In this study we tried to assess using a biopsy set needle (Modern bone the value of bone marrow examination in marrow biopsy needle set /11 gauge/ diagnosis of hematological diseases and Italian). About 0.5 ml of marrow fluid to determine the frequency and causes of was obtained and nearly about ten dry tap marrow. smears prepared. Two slides were stained with Prussian blue technique by well trained staff to demonstrate iron Materials and methods This prospective study has been conducted at Hiwa and granule and ring sideroblasts when Nanakaly indicated. Sudan Black stain was used hospitals which are only centers for routinely for all patients with acute oncology/hematology in Sulaymaniyah leukemia. and Erbil respectively. All patients When biopsy was performed, the underwent bone marrow examination core biopsy was fixed in formalin and between January 2013 and June 2013, sent for processing in histopathology were enrolled in this study. Relapsed and department (Rizgary Teaching Hospital follow-up cases were excluded. A total in number of 245 cases were studied. One Sulaymaniyah) and was examined by 2 hundred pathologists case was received from Erbil and Shorsh hospital independently. in Touch Nanakaly hospital and 145 cases were imprint smears were usually made in collected from Hiwa hospital. cases with dry tap aspiration. Inform consent was taken from For some patients with patient and their relative (In childhood lymphoproliferative disorders and acute cases). In each case a detailed history leukemia, with general and systemic examination collected in Ethylene diamine tetra Iraqi J. Hematology, May 2016, vol.5, Issue 1 marrow materials was 100 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. acetic acid tube and sent for flow 0.05 was cytometry analysis. Also some patients significant. considered statistical with chronic myeloid leukemia and myeloproliferative neoplasm in whom Results the diagnosis were not straight forward, A total of 245 cases underwent specimens were sent for BCR- ABL bone marrow examination from January fusion 2013 to June 2013 were included in this gene and JAK2 mutation respectively. study. Males were 138 (56.3%) and The marrow was examined and females were 107 (43.7%). The ratio of interpreted by two hematologist and was male to female was (1.29:1). The age of reviewed with consideration of the patients in this project were ranged patient’s between 1 to 81 years with mean age of clinical information. and Marrow laboratory smears are 36.71+ 24 years. examined for determination of the The cellularity, the morphological details of examination were based on the clinical hemopoietic cells, estimation of myeloid and or complete blood pictures. In this – erythroid ratio and careful search for study, abnormal cells. A questionnaire was examination of bone marrow were designed that contains a set of questions presence of blast cells in peripheral including (residence, age, sex, clinical circulation which was seen in 69 features, organomegaly, complete blood (28.2%) cases, followed by bone marrow picture, other related investigations and assessment for staging of lymphoma in bone marrow examination report). The 43 (17.5%) patients, and then anemia in questionnaire 20 (8.2%) cases. Leucocytosis was type is structured interviewer administrated. indications the for main bone marrow indications for another indication and found in 14 Data were analyzed by using the (5.6%) cases, 12 of them showed marked statistical package for social science left shift with increased basophilic count (SPSS) version 19. Chi square test of and associated with organomegaly, these association compare were diagnosed as CML. The remaining between proportions. A P-value of ≤ two cases were leucocytosis without was used to Iraqi J. Hematology, May 2016, vol.5, Issue 1 101 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. shift to left, one of them was diagnosed frequency as active marrow and the other one marrow examination was anemia and regarded as was found in 24 (9.8%) patients and the non diagnostic. Other indications were explained in table 1. of indications for bone outcome of bone marrow examination Various hematological disorders were active marrow in (13) cases, were encountered in this study as the multiple myeloma in (7) cases, pure red result of bone marrow examination. The cells aplasia in (2) cases and erythroid most frequent final diagnosis was acute hyperplasia in (2) cases. Table 3 leukemia and accounting for 69 (28.1%) cases of which 40 patients Out of 245 samples 10 (4.08%) were cases were regarded as dry taps and diagnosed as ALL and 29 cases were touch imprints with trephine biopsy categorized as AML. The next in the material were obtained from posterior descending order of frequency was iliac crest for diagnosis. Of these 10 active marrow (negative for lymphoma) cases, only 3 (30%) patients were and found in 27 (11.1%) cases. Other revealed active biopsy, while others final diagnoses are illustrated in table 2. showed significant marrow pathology. A comparison was prepared Acute leukemias were the commonest between the indication for bone marrow pathological causes of dry taps and examination and final diagnosis that found in 3 (30%) cases, two of them made after examination of the bone were ALL and the remaining one was marrow. Acute leukemias were found in AML. Other causes of dry tap are clearly 69 (28.1%) cases and final diagnoses of explained in (Fig 2). this category were 40 cases of ALL and In total 245 cases were underwent bone 29 cases of AML (fig 1). marrow aspiration and trephine biopsy Of the 37 (15.1%) patients were referred for bone no complication were reported. marrow examination for staging of lymphoma, 27 cases didn't showed marrow involvement by lymphoma and 10 cases were demonstrated bone marrow infiltration by lymphoma cells. The next in the descending order of Iraqi J. Hematology, May 2016, vol.5, Issue 1 102 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. Discussion Both bone marrow aspiration and causes was ranked the first frequent trephine biopsy are essential and safe indication due to high frequency of procedures for diagnosis of various nutritional anemia as iron deficiency and hematological and non-hematological megaloblastic anemias13. Leucocytosis disorders. These are important for follow was also another indication and was seen up received in 14 (5.6%) patients, where it was marrow mostly due to CML that was found in 12 transplantation and/or other medical cases, while the rest two cases were treatment. Bone marrow aspiration and regarded as active marrow and non trephine biopsy are complementary to diagnostic marrow. of patients chemotherapy, each other who or and nowadays, both procedures are performed together and usually on same site routinely (10). The result of this study showed that the acute leukemias were the most frequently encountered diagnosis by The most popular indication in this bone marrow examination and these study was presence of blast cells in were found in 69 (28.1%) cases, 40 peripheral circulation (acute leukemia) patients of them were ALL with mean which were found in 69 (28.2%) cases, age 16.8 years and 31 (77.5%) of them followed by staging for lymphoma in were found in 1st and 2nd decades, while descending order of frequency and was 29 cases were AML with mean age 35 found in 37 (15.1%) cases. Similar years and 10 of them were seen in 3rd findings were reported by Bashawri and and 4th decades; however references set Al–Gwaiz as acute leukemia was the that AML is the commonest acute most common indication and was found leukemia in adults and ALL is the in cases commonest childhood malignancy (14). A . In contrast to these similar study was done by Gupta et al findings, acute leukemia was ranked and Egesie et al in India and Niger fourth common indication (1.25%) in a respectively, in which acute leukemias study done by Bedu-Addo et al in were the largest group disorders Ghana, in which anemia of unknown Acute leukemia was ranked third in a (32.9%) respectively and (11,12) (26.2%) Iraqi J. Hematology, May 2016, vol.5, Issue 1 (15,16) . 103 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. study done by Pudasaini et al from even in cases with bicytopenia and mild Nepal, in which frequency of erythroid anemia (2). hyperplasia (mostly due to iron deficiency and megaloblastic anemia) were high and these were due to higher prevalence of nutritional deficiency in their locality (17). indication for bone marrow examination was staging of lymphoma and found in 37 (15.2%) cases. Similarly, Saeed and have also reported that lymphoma were the second common diagnostic report (18) . Bone marrow was not involved in 27 (72.9%) cases and marrow involved by lymphoma was found in 10 in this study. The diagnosis of ITP is made after exclusion of other causes of low platelet count. Other studies were showed 15.7%, 10.5% and 6.21% cases In this study, the second common Jawhar ITP was seen in 21 (8.6%) cases (27.1%) cases. All specimens of involved marrow were non Hodgkin's lymphoma and the majority of these cases were diffuse large B cell of ITP in Pudasaini et al, Kibria et al and Khan et al respectively (17,20,21). Myeloproliferative neoplasms were commonly diagnosed on bone marrow examination and confirmed by estimation of Janus-associated kinase 2 (JAK2) mutations (14) . MPN was found in 21 (8.6%) cases. This is nearly similar to another study which was done by Saeed and Jawhar in Mosul18, while other study was showed low percentage of MPN that found in (2.7%) cases and was published in Yemen (22). lymphoma. Similar results were reported by Durosinmi et al from Nigeria (19). Another Active bone marrow was 3rd common diagnosis in the present study and found in 24 (9.8%) cases. High percentage of active marrow 125 (35.1%) cases was reported in a study from Kenya. These were due to over induction of bone marrow examination Iraqi J. Hematology, May 2016, vol.5, Issue 1 common disorder in present study was chronic lymphocytic leukemia and was found in 13 (5.3 %) cases. Similarly, 2 (5%) cases were seen in another study done in India15. However in a study done by Chandra, 3.1% of cases were CLL23. This might be related to geographical variation. 104 Evaluation the Role of Bone Marrow Examination Chronic myeloid leukemia was found in 12 (4.9%) cases and was the least common leukemia in studied cases constituting (13%) of all leukemia in this study and it is nearly similar to the fact that CML accounts for around 15% of leukemias (14). finding (0.7% of cases with secondary metastasis) (25). In present study, 6 (2.4%) cases were not diagnosed on bone marrow examination as two of them were with inadequate trephine biopsy and the remaining 4 cases were with inadequate Multiple myeloma was another hematological Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. malignancy that of both aspiration and biopsy. Higher frequency (14.4% cases) of non diagnosed by bone marrow examination diagnostic sample was found in another with other diagnostic criteria and it was study (26). found in 12 (4.9%) patients with mean age 61 years and all cases were associated with high ESR. Other studies were reported a frequency of multiple myeloma as 5.6%, 4% and 3.5% respectively (17,2,24). marrow marrow study showed that aspirations was failed bone to obtained in 10 (4.08%) cases in which touch imprint and adequate trephine biopsies were well done, this is known as dry tap or blood tap. The main causes Another important indication for bone This was technique (active marrow biopsy) in 3 detection of secondary metastasis. In (30%) cases and acute leukemia (packed this study, 8 (3.2%) cases of tumors (4 cells in trephine biopsy) in 3 (30%) cases of neuroblastoma, 2 cases of cases. A similar study was done by Ewing of Humphries in 1990, who reported a rate adenocarcinoma of prostate and 1 case of dry tap as (3.9%)9 and higher of were frequency was recorded by Khanum et al underwent bone marrow examination to in Lahore and found in 50 (10%) cases exclude among sarcoma, hepatocellular examination of dry tap in present study were faulty 1 case carcinoma) marrow involvement. The 500 studied patients8. The marrow involvement by secondary remaining cases of dry tap were metastasis was found in 2 (0.8%) cases. myelofibrosis in 2 cases, secondary A study from Lahore reported a similar metastasis and hairy cells leukemia. Iraqi J. Hematology, May 2016, vol.5, Issue 1 105 Evaluation the Role of Bone Marrow Examination In this study, complications after Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. 2. Okinda NA and Riyat MS. Bone both procedures (marrow aspiration and marrow examination finding at Aga trephine biopsy) were not encountered. KhanUniversity hospital, Nairobi. East Complications Afri Med J 2010; 87(1): 4-8. of bone marrow examination are rare as reported by Bain et al (27). S, Chesney A, Clark BD, et al. Bone Conclusion: Marrow We concluded that bone marrow examination is an important diagnostic tool in the diagnosis and staging of various hematological disorders. The main indication for bone marrow examination in this study was presence of blast cells in peripheral circulation (Acute 3. Riley RS, Williams D, Ross M, Zhao leukemia). The commonest causes of dry tap in this study were faulty technique and packed cell in acute Aspirate Pathologist's and Biopsy: Perspective. A II. Interpretation of the Bone Marrow Aspirate and Biopsy. J Clin Lab Anal 2009;23(5):259-307 4. Bain BJ. Bone marrow aspiration. J. Clin. Pathol 2001a; 54: 657-663. 5. Bain BJ. Bone marrow trephine biopsy. J. Clin. Pathol 2001b; 54: 737742. leukemia. Bone marrow examination in 6. Bain BJ. spite of being invasive procedure it is morbidity and mortality. Br J Haematol safe and post operative complications 2003; 121: 949– 951. were not recorded in this study. Bone marrow biopsy 7. Marti J, Anton E, Valenti C. Complications of bone marrow biopsy. Br. J. Hematol.2004; 124(4): 557–558. References 1. Toi PC, Varghese RG, Rai R. Comparative Evaluation of Simultaneous Bone Marrow Aspiration and Bone Marrow Biopsy: 8. Khanum F, Rehman AU, Ahmad S, Anwar J. `Dry tap` of bone marrow and its clinical important. Pak J Med Health Sci 2007; 1- 3. An Institutional Experience. Ind. J. Hematol 9. Humphries JE. Dry tap bone marrow Blood Transfus 2010; 26(2): 41-44. aspiration: clinical significance. Am J Iraqi J. Hematology, May 2016, vol.5, Issue 1 106 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. Hematol 1990; 35 (4): 247 – 250. disorders. J k science 2010; 12(3): 130- (Abstract) 133 10. Islam A. Bone marrow aspiration before bone marrow core biopsy using the same bone marrow biopsy needle: a good or bad practice? J Clin Pathol 2007; 60: 212 – 215. 11. Bashawri LA. Bone marrow examination; indication and diagnostic value. Saudi Med J 2002; 23(2): 191196. Ewuga OJ. Epidemiology of Anaemia Necessitating Bone Marrow Aspiration Cytology in Jos. Niger Med J 2009; 50(3): 61 – 63 (Abstract) 17. Pudasaini S, Prasad KBR, Rauniyar SK, Shrestha R, Gautam K, Pathak R, et al. Interpretation of bone marrow aspiration in hematological disorder. J Path Nepal 2012; 2: 309-312. 12. Al- Gwaiz LA. Analysis of 3494 bone marrow examinations in a referral hospital: indications and interpretations. Saud Med J 1997; 18: 144- 147 Bates I. The role of bone marrow aspirate and hematological trephine samples diagnosis in in patients referred to a teaching hospital in Ghana. Ghan Med J.2013; 47(2): 74- 78. 14. Hoffbrand AV, Moss PAH and Pettit Essential Hematology. 18. Saeed MS, Jawhar NM. Bone marrow trephine is some hematological and non hematological disorders. Ann. Coll. Med. 2010; 36(1& 2): 63 – 71. 13. Bedu-Addo G, Amoako YA and JE. 16. Egesie OJ, Joseph DE, Egesie UG, 5th ed. Oxford, UK: Wiley-Blackwell 2006. PP 238. 15. Gupta N, Kumar R, Khajuria A. Diagnostic assessment of bone marrow 19. Durosinmi MA, Mabayoje VO, Akinola NO. A review of histology of bone marrow trephine in malignant lymphoma. Niger J Med 2003; 12(4): 198- 201. (Abstract) 20. Kibria SG, Islam MDU, Chowdhury ASMJ, Ali MY, Haque MR, Mustanzid SM, et al. Prevalence of hematological disorder: A bone marrow study of 177 cases in a private hospital at Faridpur. Faridpur Med Coll J 2010; 5(10): 11- 13. aspiration smears, touch imprints and 21. Khan A, Aqeel M, Khan TA, Munir trephine A. Pattern of hematological diseases in biopsy in haematological Iraqi J. Hematology, May 2016, vol.5, Issue 1 107 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. hospitalized paediatric patients based on 26. Naznin M, Wahab AJ, Kalavathy R. bone marrow examination. J Postgrad A review of bone marrow examinations Med Inst 2008; 22(3): 196- 200. in Tengku Ampuan Afzan hospital 22. Al-Ghazaly J, Al-Selwi AH, Abdullah M, Al-Jahafi AK, Al-Dubai W, (HYAA), Kuantan. Pahang. Med. J. Malaysia 2006; 68 (4). Al-Hashdi A. Pattern of haematological 27- Bain BJ, Clark DM, Wilkins BS. diseases diagnosed by bone marrow The normal bone marrow. In: Bone examination in Yemen: A developing Marrow Pathology. 4th ed. UK; Oxford: country experience. Clin Lab Haematol Wiley-Blackwell 2010. pp: 40- 51. 2006;28:376-81 23. Chandra S and Chandra H . Comparison of bone marrow aspirates cytology, touch imprint cytology and trephine biopsy for bone marrow evaluation. Hematol Rep 2011; 3(3): 65Correspondence to: 68. 24. Tariq M, Khan N, Basri R, Amin S. Nawsherwan Sadiq Mohammad Aetiology of pancytopenia. Professional Med J 2010; 17(2): 252-256. 25. Khan FS and Hasan RF. Bone Pathology Department; College of Medicine / Hawler Medical University Email: [email protected] marrow examination of pancytopenic children. J Pak Med Assoc 2012; 62(7): Mob: 00964(0)7504384101 660- 663. Iraqi J. Hematology, May 2016, vol.5, Issue 1 108 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. تقييم دورفحص نخاع العظم في تشخيص أمراض الدم في مراكز أمراض الدم و األورام في إقليم كردستان شورش جميل رضا ،1نوشيروان صادق محمد ،2هوكر إسماعيل محمد سرهنك 3 - MBChB 1ماجستير أمراض الدم /مستشفى هيوا التعليمي FICMS - MBChB 2أمراض الدم /قسم علم األمراض/كلية الطب /جامعة هولير الطبية البريد اإللكتروني: [email protected] - MBChB 3ماجستير أمراض الدم /مستشفى أربيل التعليمي الملخص الخلفية :لعينة نخاع العظم والخزعة دورا هاما في تقييم وتشخيص معظم أمراض الدم وبعض االضطرابات غير الدموية .األهداف :هدف هذه الدراسة هو لتقييم قيمة فحص نخاع العظام في تشخيص األمراض الدموية وتحديد تواتر وأسباب نخاع العظم الجاف. المرضى والطرق :هذه دراسة استطالعية فقط وتمت خالل الفترة من يناير 2113إلى يونيو 2113في مستشفى هيوا التعليمي هيوا و مستشفى أربيل التعليمي في السليمانية وأربيل على التوالي .وكان عدد إجمالي من 242حالة خضعت فحص نخاع العظام .وقد استخدم لمس الخزعة في حالة النخاع الجاف .وقد استخدم صبغة السودان في سرطان الدم الحاد. النتائج :كان عدد المرضى )٪2..3( 131من الذكور و )٪43.1( 111من اإلناث ،تتراوحت أعمارهم 11-1 سنوات ،وكان متوسط أعمارهم 24 ± 3..1عاما .اكثر الحاالت شيوعا من المرضى التي خضعت للتقصي السريري وفحص نخاع العظم كانت الشحوب ( ،)٪11.4تليها الحمى ( .)٪32.2وكانت المؤشرات الرئيسية وجود الخاليا السرطانية في الدم ،)٪21.2( .6يليها تقييم نخاع العظام لتحديد مرحلة سرطان الغدد الليمفاوية 31 ( .)٪12.1وكانت التشخيصات الشائعة كاالتي :سرطان الدم الحاد ،)٪21.2( .6ونخاع نشط (السلبي ليمفوما) 21 ( ،)٪11.1ونخاع نشط .)٪6.1( 24وكانت نسبة النخاع الجاف ( )٪4.11وكانت األسباب األكثر شيوعا :سرطان الدم الحاد وتقنية خاطئة (نخاع العادي). الخالصة :فحص نخاع العظم هو أداة تشخيصية مهمة في تشخيص وتحديد مراحل مختلفة من االضطرابات الدموية. كلمات البحث :نخاع العظم ،خزعة ،أمراض الدم ،كردستان 109 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. Table 1 : Indications for bone marrow examination: Indication Frequency Percentage Blast cells in peripheral circulation 69 28.2 Bone marrow for lymphoma staging 37 15.2 Anemia 24 9.8 Pancytopenia 20 8.1 Thrombocytopenia 19 7.7 Bicytopenia 15 6.1 Leucocytosis 14 5.6 Polycythemia 14 5.6 Lymphocytosis 13 5.2 Thrombocytosis 6 2.4 Hepatosplenomegaly with anemia 4 1.6 Leucoerythroblastic anemia 2 0.8 To exclude marrow metastasis 8 3.2 Total 245 100 Iraqi J. Hematology, May 2016, vol.5, Issue 1 110 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. Table 2: Results of Bone marrow examination of the studied cases. Disorders Number of cases % ALL 40 16.3 AML 29 11.8 Active marrow (negative for lymphoma) 27 11.1 Active marrow 24 9.8 Idiopathic thrombocytopenia purpura 21 8.6 Myeloproliferative neoplasm 21 8.6 Chronic lymphocytic leukemia 13 5.3 Chronic myeloid leukemia 12 4.9 Multiple myeloma 12 4.9 Marrow involvement by lymphoma 10 4.1 Aplastic anemia 10 4.1 Non – diagnostic 6 2.4 Active marrow (negative for solid tumors) 6 2.4 Megaloblastic anemia 5 2 Secondary metastasis 2 0.8 Pure red cell aplasia 2 0.8 Erythroid hyperplasia 2 0.8 Myelodysplastic syndrome 2 0.8 Hairy cells leukemia 1 0.4 Total 245 100 Iraqi J. Hematology, May 2016, vol.5, Issue 1 111 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. Table 3: Indication of bone marrow examination versus final diagnosis Indication Acute leukemia Frequency Final diagnosis 69 ALL AML Bone marrow for staging 37 Anemia 24 Pancytopenia 20 Thrombocytopenia Bicytopenia 19 15 Leucocytosis 14 Polycythemia Lymphocytosis 14 13 Thrombocytosis 6 Hepatosplenomegaly and anemia Leucoerythroblastic anemia To exclude marrow metastasis Total 4 2 8 Cases 40 29 Active marrow (negative lymphoma) Marrow involved by lymphoma Active marrow Multiple myeloma Erythroid hyperplasia Pure red cells aplasia Aplastic anemia Megaloblastic anemia Multiple myeloma Non diagnosis marrow Active marrow Hairy cell leukemia ITP Active marrow Multiple myeloma Myelodysplastic syndrome ITP CLL Non diagnostic marrow Megaloblastic anemia CML Non diagnostic marrow Active marrow Myeloproliferative neoplasm CLL Active marrow Myeloproliferative neoplasm Active marrow Active marrow Non diagnostic marrow Myeloproliferative neoplasm for 27 10 Active marrow (negative metastasis) Secondary metastasis for 6 2 245 245 Iraqi J. Hematology, May 2016, vol.5, Issue 1 13 7 2 2 10 4 2 2 1 1 19 5 3 2 2 1 1 1 12 1 1 14 12 1 5 1 2 2 2 112 Evaluation the Role of Bone Marrow Examination Shorsh J. R, Nawsherwan S.M, Hoger I.M.S. Figure 1:(A) Acute lymphoblastic leukemia.(B) Acute Myeloblastic Leukemia. Marrow aspiration (Leishmann stain) Figure 2 : Causes of dry tap in this study. Iraqi J. Hematology, May 2016, vol.5, Issue 1 113 Original article Types of Anaemia and its Correlation with Disease Activity in Patients with Rheumatoid Arthritis among Kurdish Population of Iraq Hisham A. Getta¹, Najmaddin Khoshnaw ²* & 6, Alaa Fadhil Alwan 3 ,Sundus F.A4, Raouf R. Mirza 5 ¹ Department of hematopathology, faculty of sciences, school of medicine, university of sulaimani, Kurdistan region, Iraq ²*Department of Hematology, Hiwa Hospital, Sulaymaniyah, Kurdistan Region, Iraq 3 Department of clinical hematology, the national center of hematology, Almustansiriya University, Baghdad, Iraq 4 Department of rheumatology and medical rehabilitation, general medical teaching hospital, Sulaymaniyah, Kurdistan Region, Iraq 5 Department of rheumatology and medical rehabilitation, school of medicine, university of sulaimani, Sulaymaniyah Kurdistan region, Iraq 6 Kurdistan Board of Medical Specialties, Clinical Hematology/Trainee, Ministry of higher education and scientific research, Erbil, Kurdistan Region, Iraq Received 8/1/2016 revised 3/2/2016 Iraqi J. Hematology, May 2016, vol.5, Issue 1 accepted 23/2/2016 114 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al Abstract: Background: Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease characterized by articular and extra-articular manifestation as weight loss, fatigue, malaise, and anemia. Objectives: The aim of this study was to find the prevalence of different types of anemia and its correlation with the disease activity among patients with RA in Sulaymaniyah province and to determine the associated risk factors. Patients and Methods: A cross sectional study carried out in Sulaymaniyah general medical Hospital for periods from October-2014 to the end of June-2015.A convenient sample of 100 rheumatoid arthritis patients were selected from patients seen in the rheumatology clinic. One hundred healthy voluntary controls of same age groups were selected and same parameters for diagnosis of anemia are used in both groups. Results: The prevalence of all types of anemias among the rheumatoid Arthritis (RA) patients was 40% which more than that found in control group. The common types of anemia was anemia of chronic disease (28%), iron deficiency (10%), thalassemia minor (1%), and megaloblastic anemia 1%. We found a significant correlation of RA with each low hemoglobin, low hematocrit, high leucocyte count and high ESR. Anemia among RA patients in our study was significantly more prevalent among low socioeconomic status patients. The majority of RA patients had moderately or high active disease, which revealed no association between disease activity and anemia (p value=0.3). Conclusion: the prevalence and types of anemias among RA patients in Sulaymaniyah was comparable to that found in other studies and it was two times common than the normal healthy peoples. In our study we concluded that little evidence found between disease activity and anemia. Keywords: Rheumatoid Arthritis, Anemia, Sulaymaniyah, Kurdistan Region, Iraq Introduction: Rheumatoid arthritis (RA) is chronic systemic auto-immune inflammatory RA is associated with a high risk for morbidity and premature death disease characterized by articular and secondary to the earlier development of extra-articular manifestation, such as cardiovascular, weight anemia loss, (1) fatigue, malaise and malignancy (2) lung diseases and . . Iraqi J. Hematology, May 2016, vol.5, Issue 1 115 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al Extra-articular manifestations of RA with the number of active inflamed occur in about 40% of patients, either in joints the beginning or during the course of sometimes observed their disease (3) . (8) . Lymphadenopathy is in active RA, usually presenting on biopsy as benign (9) Patients with RA, who have high titers follicular hyperplasia of rheumatoid factor, are most likely to anemia in RA may include anemia of have extra-articular manifestations of chronic disease, iron-deficiency anemia, their disease present (4) . Patients with RA may with hematological abnormalities either at the time of diagnosis, or during the course of their illness. Hematological manifestations in RA can be broadly categorized into areas of anemia, thrombocytosis, neutropenia, thrombocytopenia, eosinophilia, hematological malignancies (5) and . Anemia is, by far, one of the most common extra-articular manifestations of RA. The cause of multifactorial activity, anemia including drug-induced, in RA is disease nutritional, gastrointestinal bleeding, bone marrow suppression, erythropoiesis and (6) ineffective . Anemia of chronic disease (ACD) usually normochromic normocytic type observed in RA, where it usually correlates with the disease activity. Eosinophilia in RA reflects active disease or hypersensitivity to drugs (7) . Thrombocytosis is a frequent finding in active RA and is correlated Iraqi J. Hematology, May 2016, vol.5, Issue 1 vitamin deficiency . The types of anemia, anemia, or hemolytic anemia aplastic (10) . The first principle of treating RA associated anemia is to reduce inflammation as much as possible using non-steroidal anti-inflammatory drugs (NSAIDS), anti disease modifying rheumatoid drugs (DMARDs). Erythropoietin stimulating agents (ESA) therapy has been shown to be effective in treating RA induced ACD. However, RA patients tend to have a blunted response to ESA therapy, and higher than normal ESA doses are often required. In these patients, ACD shows improvement when inflammation has decreased. When iron deficiency occurs concomitantly with ACD, iron repletion may be needed, either alone or as adjunct therapy with ESAs. ESA therapy in combination with iron supplementation corrects anemia in most patients with RA, and may improve RA outcomes and quality of life (11) .The 116 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al aims of this study were to find the prevalence, types of anemia among patients with RA in Sulaymaniyah and to determine the associated risk factors. met A cross sectional study carried out in General General Hospital Hospital. Inclusion criteria: Materials and methods Sulaymaniyah Rheumatology ward of Sulaymaniyah in the All patients who 2010 ACR-EULAR classification criteria for Rheumatoid Arthritis were included (12) (table1), a Sulaymaniyah governorate for period score of ≥6/10 is needed for diagnosis of from 1st of October, 2014 to the end of patient as having RA. June, 2015. It included 100 patients with RA who attended to Rheumatology Consultation clinic or admitted to Table 1 : criteria for diagnosis of RA A.Joint involvement SCORE 1 large joint 0 2- 10 larg joint 1 1-3 small joints(with or without large joint) 2 4-10 small joints(with or without large joint) 3 >10 joints(at least 1 small joint) 5 A.Serology(at least 1 test result is needed for classification) Negative RF and Anti ccp 0 Low positive RF and Anti ccp 2 High positive RF and Anti ccp 3 C.Acute phase reactants Normal CRP and ESR 0 Abnormal CRP and ESR 1 D.Duration of symptoms Less than 6 weeks 0 6 weeks or more 1 N.B : A score of ≥6/10 is needed for diagnosis of patient as having RA Iraqi J. Hematology, May 2016, vol.5, Issue 1 117 Types of Anaemia and its Correlation with Disease Exclusion criteria: Any cause of Hisham A.Getta, Najmaddin Khoshnaw et al by HPLC (high performance liquid anemia not related to RA which includes chromatography, patients with active GIT bleedings, Health Organization (WHO) criteria bleeding were tendencies, malignancies, patients, menorrhagia, renal failure, chronic diabetic infections excluded in our study. were used to D10) The define World anemia as hemoglobin threshold of <120 g/L for women and <130 g/L for men (13) . The The data were disease activity evaluated according to collected through direct interview and DAS-28 in which values less than 2.6 using corresponds prepared questionnaire. The for clinical remission, known cases of RA were diagnosed by values ranging (2.6-3.2) are regarded as consultant Rheumatologist. Full medical low disease activity, values ranging(3.2- history and clinical examination were 5.1) are regarded as moderate disease done for all patients. A total of 100 activity and more than 5.1 regarded as healthy controls were selected and high disease activity. All patients gave required information was collected from their oral consent before beginning of them. The questionnaire included: socio- the study and agreement was taken from demographic characteristics as Age, official review ethical committee of gender, residence and socioeconomic Sulaymaniyah General Hospital. status. RA characteristics: disease activity assessed by DAS-28, rheumatoid factor (RF) and duration of RA, treatment modalities of RA, family history of RA and anemia, Laboratory tests done as complete blood count, blood smear, reticulocyte count, coombs test and ESR done by autoanalyzer, serum iron and TIBC done by C111Cobas Roche Company, serum ferritin, serum B12, and folate by E411 Cobas Roche Company, Hb- electrophoresis done for selected cases Iraqi J. Hematology, May 2016, vol.5, Issue 1 Statistical analysis: Statistical Package for Social Sciences (SPSS) version 20 was used. Descriptive statistics presented as (mean ± standard deviation) and frequencies as percentages. Chi-square used for categorical variables and Fishers exact test was used when expected variables were less than 20%. Independence t-test was used to compare between two means. In all statistical analysis, level of significance (p value) set at ≤ 0.05. 118 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al Results: A total of 100 patients with RA were More than half of RA patients were included in present study with mean age living in urban areas. The socioeconomic 50±13 years, 52% of them were ≥ 50 status was presented as following; 21 years. Female were more than males patients were good, 44 were fair and 35 with male to female ratio of 1:9.01. RA patients had low socioeconomic status as shown in (table 2). Table 2: Sociodemographic characteristics of RA patients. Variable No. % 20-29 years 8 8.0 30-39 years 12 12.0 40-49 years 28 28.0 ≥ 50 years 52 52.0 Male 9 9.0 Female 91 91.0 Urban 52 52.0 Rural 48 48.0 Good 21 21.0 Fair 44 44.0 Low 35 35.0 Age mean±SD (50±13 years) Gender Residence Socioeconomic status Mean RA disease duration was 11±9 The treatment types used by RA patients years, 65% of RA patients had disease were duration ≤ 10 years. The Rheumatoid prednisolone factor (RF) was positive for 91% of RA (16.8%), Methotrexate (16.5%), Calcium patients and negative for 9% of them. & Vitamin D (14.2%), combination Iraqi J. Hematology, May 2016, vol.5, Issue 1 distributed as (16.8%), followings; Folic acid 119 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al therapy (12.5%), hydroxychloroquine Mean Hb of RA patients was 12.3±1.4 (10.8%), mg/dl, 40% of them had low Hb level. Alendronate (2.6%), Leflunomide (2.3%), Etanercept (3.1%), Mean PCV of RA patients was Rituximab (1.3%), Adalimumab (0.4%), 40.1±30.8 %, 33% of them had low PCV and Azathioprine (0.4%). Mean DAS-28 level. Mean WBC of RA patients was of RA patients was 5±1.2, while 57% of 8.4±2.9 x109, 20% of them had high RA patients had high disease activity. WBC count as shown in table 3. Table 3: hematological parameter of patient with RA Variable No. % Normal 60 60.0 Low 40 40.0 Total 100 100.0 Normal 67 67.0 Low 33 33.0 Total 100 100.0 Normal 80 80.0 High 20 20.0 Total 100 100.0 100 100 100 100 Hb mean±SD (12.3±1.4 mg/dl) PCV mean±SD (40.1±30.8 %) WBC mean±SD (8.4±2.9 x109) Platelets mean±SD (259.9±65.9x109) Normal Reticulocytes level normal Anemia was found in 40 patients deficiency anemia IDA, 2.5% (40%) with RA patient and 70.0% of megaloblastic anemia and 2.5% had anemic patients had anemia of chronic Thalassemia diseases (ACD), 25% had minor (Fig 1). iron Iraqi J. Hematology, May 2016, vol.5, Issue 1 120 No. Types of Anaemia and its Correlation with Disease 30 25 20 15 10 5 0 Hisham A.Getta, Najmaddin Khoshnaw et al 28 10 1 1 Figure 1: Types of anemia among RA patients. Mean serum iron of anemic RA patients Coombs test. Mean ESR of RA patients was 98.4±35.9 µg/dl, with 72.5% had was 46.3±19.3 mm/hr, all of RA patients low iron level. Mean TIBC level of had high ESR. anemic RA patients was 385.9±67.9 No significant differences were µg/dl, with 47.5% had low TIBC level observed between anemic and non- and 20% had high TIBC level. Mean anemic RA patients regarding DAS-28 serum ferritin of anemic RA patients was and rheumatoid factor (RF) (p>0.05). No 73.3±56.1 ng/ml, with 22.5% had low significant differences were observed ferritin level while 25% had high ferritin between anemic and non-anemic RA level. Only one patient had low B12 patients regarding family history of RA level. Hb-electrophoresis was positive and anemia (p>0.05) only for one patient. All of RA patients (table 4) as shown in had normal folate level and negative Iraqi J. Hematology, May 2016, vol.5, Issue 1 121 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al Table 4: Distribution of RA characteristics according to anemia. Variable Anemic Non anemic No. % No. χ² P 3.0* 0.3 3.4* 0.06 2.3 0.1 0.9* 0.3 % DAS28 Remission 0 - 1 100.0 Low activity 3 50.0 3 50.0 Moderate activity 11 30.6 25 69.4 High activity 26 45.6 31 54.4 RF Sero +ve 39 42.8 52 57.2 Sero –ve 1 11.1 8 88.9 Family history of RA Positive 15 51.7 14 48.3 Negative 25 35.2 46 64.8 Family history of anemia Positive 2 66.7 1 33.3 Negative 38 39.2 59 60.8 *Fishers exact test. Discussion: Rheumatoid autoimmune arthritis (RA) disorder of is an normochromic and normocytic type. unknown Anemia is multifactorial, reflected in etiology characterized by symmetric, dimorphic appearance and wide red cell erosive synovitis and, in some cases, extra distribution width. Anemia of chronic articular disease involvement. Extra-articular (ACD) and iron deficiency manifestations can be detected in almost anemia (IDA) are the most important any organ system, causing considerable types of anemia in RA patients (14). disease related morbidity and interference In this study, prevalence of anemia with quality of life. Anemia is a among studied RA patients was (40%). frequently The result was close to that of Wilson A occurring extra-articular manifestation of RA, being mostly of the Iraqi J. Hematology, May 2016, vol.5, Issue 1 et al (15) who reported the anemia to be 122 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al (22) ranged of 33-66% among RA patients, but Swaak A study was higher than that reported by Muia bases GM, et al in which he reported prevalence released during ACD can alter systemic of 33% (16) that , this explained on inflammatory cytokines . On other hand, other studies iron metabolism by inducing excess found higher prevalence which reached synthesis of hepcidin, the iron regulatory up to 70.6% (17). hormone. Since hepcidin inhibits iron Prevalence ratio of developing anemia export from cells by blocking ferroportin among RA patients was 2:1 as compared activity, excess hepcidin is the root cause to healthy controls. This prevalence ratio of the hypoferremia and iron-restricted is close to that reported by Han C, et al erythropoiesis seen in ACD (23), (24). study who reported a prevalence ratio of anemia among RA patients as 2.2.:1(18) In this study we found that mean age of RA patients was 50±13 years with In this study we have found no predominance of female gender. This is significant association between disease consistent with results of Muia et al study activity and anemia (p value=0.3) ,while (16) in other epidemiological study it was rural living and low socioeconomic status reported that lower hemoglobin levels (P≤0.05). This is similar to results of was associated with increased disease Putrick P, et al study (25).High levels ESR activity as measured by the number of and WBC were significantly associated tender and swollen joints, ESR, CRP with RA patients (P≤0.05). This finding level, and assessments of pain and was close to that of Shenair D, et al study fatigue(DAS-28 score)(19). Han C et al (26) reported independently had high DAS28 activity. This finding is physical disability in higher than that reported by Ganna S that contributes to anemia patients with RA (20). More than two thirds of detected . RA was significantly associated with . More than half of studied RA patients study (24) which reported that 30% of RA patients had high DAS-28 activity. anemic RA cases in present study were Anemia among RA patients in our anemia of chronic diseases (ACD) and study was significantly more prevalent 25% of them were iron deficiency anemia among (IDA). This result was close to many patients (P=0.05). This finding was close studies as Ravindran V, et al study (21) and to that of Gordon MM, et al study (27). Iraqi J. Hematology, May 2016, vol.5, Issue 1 low socioeconomic status 123 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al Bengtsson et al reported the association have more severe joint disease and if the between and anemia is successfully treated, the joint educational status and a lower risk for disease will likely respond to treatment as the development of RA in a population well (15). high socioeconomic representative of the population, suggesting Swedish that environmental factors or lifestyle might influence disease evolution (28). Pedersen et al reported that the educational level was inversely associated to the risk of developing RA in the Danish population and the risk was twice as lower for those individuals with a higher number of years of formal schooling (29). Platelets level among RA patients was normal, although, mean platelets among Conclusion: The prevalence of anemia among RA patients in Sulaymaniyah was comparable to that found in other studies and it was two times common than the normal healthy peoples. Anemia of chronic diseases was the most common type of anemia among RA patients. The majority of RA patients had moderately or high active disease, which revealed no association between disease activity and anemia. anemic patients was significantly higher than that of non-anemic (P=0.03). This is consistent with results of Safak S, et al study (30) . ESR level was significantly increased among anemic RA patients in our study (P<0.001). This is similar to results of Ganna S study(24). Recent studies have revealed a key role of cytokines and other mediators of inflammation in the development not only of the articular syndrome, but also a whole range of systemic manifestations of the disease (31) . Wilson A, et al in their Acknowledgment: We have special thanks to all doctors and medical staffs and all patients and peoples who helped us in collecting the data for this study. Authorship contribution: HAG who designed the project of the study, RRM supervisor of the research article, SFA primary investigator, NK and AFA shared in rewriting and editing of the all parts of the manuscript. systematic review, suggested that patients with RA who have anemia are likely to Iraqi J. Hematology, May 2016, vol.5, Issue 1 124 Types of Anaemia and its Correlation with Disease Conflict of interest: All authors declare that there is no any conflict of interest in publishing this article. References: 1. Lundkvist J, Kastang F, Kobelt G. The burden of rheumatoid arthritis and access to treatment: health burden and costs. Eur J Health Econ 2008; 8 (2):S4960. 2. Crostein BN. Interleukin-6 a key mediator of systemic and local symptoms in rheumatoid arthritis. Bull NYU Hosp J Dis. 2007; 65 (1): S11-S15. 3. Cimmino MA, Salvarani C, Macchioni P. Extra-articular manifestations in 587 Italian patients with rheumatoid arthritis. Rheumatol Int. 2000; 19 (6): 213-7. 4. Turesson C, Weyand CM, Matteson EL. Genetics of rheumatoid arthritis: Is there a pattern predicting extraarticular manifestations? Arthritis Rheum. 2004; 51(5): 853-63. 5. Bowman SJ. Haematological manifestations of rheumatoid arthritis. Scand J Rheumatol. 2002; 31: 251-9. 6. Agrawal S, Misra R, Aggarwal A. Anemia in rheumatoid arthritis high prevalence of iron-deficiency anemia in Indian patients. Rheumatol Int. 2006; 26 (12): 1091-5. Iraqi J. Hematology, May 2016, vol.5, Issue 1 Hisham A.Getta, Najmaddin Khoshnaw et al 7. Rosner J, Mosheimer-Feistritzer B, Gruber J, Herold M, Mur E, Weiss G. Prevalence of Anemia in a Cohort of Rheumatoid Arthritis Patients- An Interim Analysis. Ann Rheum Dis 2015; 74:450. 8. Ustun C, Kallab A, Loebl D. Rheumatoid arthritis and immune thrombocytopenia: a report of two cases. Clin Rheumatol. 2002; 21: 543-4. 9. Agarwal V, Sachdev A, Lehl S. Unusual haematological alterations in rheumatoid arthritis. JPGM. 2004; 50: 60-8. 10. Turesson C, McClelland RL, Christianson T, Matteson E. Clustering of extra-articular manifestations in patients with rheumatoid arthritis. J Rheumatol 2008; 35 (1): 179-80. 11. Kaltwasser JP, Kessler U, Gottschalk R, Stucki G, Möller B. Effect of recombinant human erythropoietin and intravenous iron on anemia and disease activity in rheumatoid arthritis. J Rheumatol. 2001; 28(11):2430-36. 12. Daniel A, Tuhina N, Alan JS, Julia F, David TF. 2010 Rheumatoid Arthritis Classification Criteria. ARTHRITIS & RHEUMATISM.Vol. 62, No. 9, September 2010, pp 2569–2581 DOI 10.1002/art.27584 13. World Health Organization. WHO/NHD/01.3: Iron deficiency anemia. Assessment, prevention and control. A guide for program managers. Geneva, 2001. 114p. 125 Types of Anaemia and its Correlation with Disease 14. Borah DJ, Iqbal F. Anemia in Recent Onset Rheumatoid Arthritis. JK Science 2007; 9 (3): 120-122. 15. Wilson A, Yu HT, Goodnough LT, Nissenson AR. Prevalence and outcomes of anemia in rheumatoid arthritis: a systematic review of the literature. Am J Med. 2004; 116 (7A):50S-57S. 16. Muia GM, Oyoo GO, Kitonyi GW, wanzala P. Anemia in patients with rheumatoid arthritis at the Kenyatta National Hospital, Nairobi, Kenya. African Journal of Rheumatology 2015; 3 (1): 42-47. 17. Misra R, Aggarwal A. Anemia in rheumatoid arthritis: high prevalence of iron-deficiency anemia in Indian patients. Rheumatol Int. 2006; 26(12):1096-7. 18. Han C, Zhao N, Rahman MU, Doyle MK, Bala MV. A case-control study of anaemia in patients with rheumatoid arthritis treated with disease-modifying antirheumatic drugs in an adult population in the US: prevalence and impact on healthcare utilisation. J Med Econ 2008; 11(2):255-64. 19. Wolfe F, Michaud K. Anemia and renal function in patients with rheumatoid arthritis. J Rheumatol 2006; 33:1516-22. 20. Han C, Rahman MU, Doyle MK. Association of anemia and physical disability among patients with rheumatoid arthritis. J Rheumatol 2007; 34:2177-82. Iraqi J. Hematology, May 2016, vol.5, Issue 1 Hisham A.Getta, Najmaddin Khoshnaw et al 21. Ravindran V, Jain S, Mathur DS. The differentiation of anemia in rheumatoid arthritis: parameters of irondeficiency in an Indian rheumatoid arthritis population. Rheumatol Int; 2008; 28:507–511. 22. Swaak A. Anemia of chronic disease in patients with rheumatoid arthritis: aspects of prevalence, outcome, diagnosis, and the effect of treatment on disease activity. J Rheumatol 2006; 33; 1467-1468. 23. Kullich W, Niksic F, Burmucic K. Effects of the chemokine MIP-1alpha on anemia and inflammation in rheumatoid arthritis. Z. Rheumatol 2002; 61:568576. 24. Ganna S. The prevalence of anemia in rheumatoid arthritis. Rev Bras Reumatol 2014; 54(4):257–259. 25. Putrik P, Ramiro S, Keszei AP, Hmamouchi I, Dougados M, Uhlig T, et al. Lower education and living in countries with lower wealth are associated with higher disease activity in rheumatoid arthritis: results from the multinational COMORA study. Ann Rheum Dis doi: 10.1136/annrheumdis2014-206737. 26. Sheanair D, Abdul-Mounther M, Khasim MM, Alshaheen H, Omran YG. The relationship between serum potassium levels and some chemical limitations in Rheumatoid arthritis from Qurna district, Basrah city. Scientific Journal of Kerbala University 2011; 9 (2): 197-203. 126 Types of Anaemia and its Correlation with Disease Hisham A.Getta, Najmaddin Khoshnaw et al 27. Gordon MM, Hampson R, Capell HA, Madhok R. Illiteracy in rheumatoid arthritis patients as determined by the rapid estimate of adult literacy in medicine (REALM) score. Rheumatology 2002; 41: 750-754. 28. Bengtsson C, Nordmark B, Klareskog L, Lundberg I, Alfredsson L, EIRA Study Group. Socioeconomic status and the risk of developing rheumatoid arthritis: results from the Swedish EIRA Study. Ann Rheum Dis 2005; 64:1588-94. 29. Pedersen M, Jacobsen S, Klarlund M, Frisch M. Socioeconomic status and risk of rheumatoid arthritis: a Danish case-control study. J Rheumatol 2006; 33:1069-74. 30. Safak S, Uslu AU, Serdal K, Turker T, Soner S, Lutfi A. Association between mean platelet volume levels and inflammation in SLE patients presented with arthritis. African Health Sciences 2014; 14 (4):919-924. 31. Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 2001; 12:907-916. Correspondence to : Dr. Najmaddin Khoshnaw Clinical Hematologist, Department of Hematology Hiwa Hospital -Sulaymaniyah -IRAQ Cell phone: +9647701554184 Po. Box: 54 Email: [email protected] Iraqi J. Hematology, May 2016, vol.5, Issue 1 127 Hisham A.Getta, Najmaddin Khoshnaw et al Types of Anaemia and its Correlation with Disease أنواع فقر الدم وارتباطها مع نشاط المرض في المرضى الذين يعانون من التهاب المفاصل الرثوي بين السكان األكراد في العراق هشام جتا ،1نجم الدين خوشناو ،6 &2عالء فاضل علوان ،3سندس ف , 4رؤوف ميرزا 5 1قسم امراض الدم ،كلية العلوم ،كلية الطب ،جامعة السليمانية ،إقليم كردستان ،العراق 2قسم أمراض الدم ،مستشفى هيوا ،السليمانية ،كردستان ،العراق 3قسم أمراض الدم السريري ،المركز الوطني لألمراض الدم ،الجامعة المستنصرية ،بغداد ،العراق 4قسم الروماتيزم والتأهيل الطبي ،المستشفى العام التعليم الطبي ,السليمانية وإقليم كردستان ،العراق 5قسم الروماتيزم والتأهيل الطبي ،كلية الطب ،جامعة السليمانية ،السليمانية اقليم كردستان العراق 6مجلس كردستان للتخصصات الطبية والسريرية أمراض الدم /متدرب وزارة التعليم العالي والبحث العلمي، أربيل ،إقليم كردستان ،العراق الملخص: الخلفية :التهاب المفاصل الروماتويدي هو من أمراض المناعة الذاتية والتهاب مزمن يتميز باعراضه المفصلية الدم. وفقر بالضيق، والشعور والتعب، الوزن، كفقدان المفصلية وخارج األهداف :الهدف من هذه الدراسة تتمثل في العثور على انتشار أنواع مختلفة من فقر الدم وعالقته مع نشاط المرض بين المرضى الذين يعانون من التهاب المفاصل الروماتويدي في محافظة السليمانية وتحديد عوامل الخطر المرتبطة بها. المرضى والطرق :تم اختيار دراسة مقطعية أجريت في مستشفى عام السليمانية للفترة من أكتوبر 2114إلى نهاية يونيو .2115-عينة مالئمة من 111مريض يعانون من التهاب المفاصل الروماتويدي من المرضى الذين تم فحصهم في عيادة الروماتيزم .وقد تم اختيار مائة من الضوابط الطوعية الصحية من الفئات العمرية نفسها ،استخدمت نفس المجموعتين. كال في الدم فقر لتشخيص المعايير النتائج :كان معدل انتشار جميع أنواع فقر الدم بين المرضى المصابين بالتهاب المفاصل الروماتيزمي ( )%41أكثر من تلك الموجودة في المجموعة الضابطة .وكانت األنواع الشائعة من فقر الدم فقر الدم الناجم عن األمراض المزمنة ( ،)٪22ونقص الحديد ( ،)٪11الثالسيميا الصغرى ( ،)٪1وفقر الدم الوبيل( .)٪1لقد وجدنا ارتباط كبير بين الروماتيز الرثوي مع كل من الهيموغلوبين المنخفض ،وانخفاض الهيماتوكريت ،وارتفاع عدد كريات بيضاء وارتفاع راسب الدم .كان فقر الدم بين مرضى التهاب المفاصل الروماتويدي في دراستنا بشكل ملحوظ أكثر انتشارا بين مرضى انخفاض الوضع االجتما عي واالقتصادي .وكانت الغالبية العظمى من مرضى التهاب المفاصل الروماتويدي لديهم متوسط الى شديد النشاط ،والتي كشفت عن عدم وجود عالقة بين نشاط المرض وفقر الدم (.)1.3 = p الخالصة :مدى انتشار وأنواع فقر الدم بين مرضى التهاب المفاصل الروماتويدي في السليمانية كان مماثلة لتلك الموجودة في دراسات أخرى ،وكان مرتين اكثر من الناس االصحاء .في دراستنا خلصنا إلى أن القليل من األدلة الدم. وفقر المرض نشاط بين وجدت كلمات البحث :التهاب المفاصل الروماتويدي ،فقر الدم ،السليمانية 128 Iraqi J. Hematology, May 2016, vol.5, Issue 1 Original article A Clinical-Hematological Study of Pancytopenia Patients Attending Nanakaly Hospital in Erbil City Alan Isaac Isho1 , Nawsherwan Sadiq Mohammad2, Saran Abdulqadir Nooruldin3 1 MBChB-MSc Hematology/ Erbil Teaching Hospital 2 MBChB, FIBMS Hematology/ Pathology Department, College of Medicine/ Hawler Medical University/ Email: [email protected] Mob: 00964(0)7504384101 3 MBChB-MSc Hematology/ Rizgary Teaching Hospital Received: 16/3/2016 accepted: 18/4/2016 Abstract Background: Pancytopenia is a triad of low hemoglobin, white blood cells and platelets. Although it is a common clinical problem with an extensive differential diagnosis, there is a relatively little discussion of this abnormality in major textbooks of internal medicine and hematology. Objectives: This study aimed to determines the etiology and clinical profile of pancytopenic patients attending Nanakaly hospital. Materials and Methods: During a period of 6 months, 60 pancytopenic patients attended Nanakaly hospital, their ages ranged between one-81years. A control group of 50 age-matched apparently healthy person were tested for complete blood picture and reticulocyte count. History, physical examination and hematological parameters at presentation were recorded. Hematological profile included hemoglobin, total and differential leukocyte count, platelet count, reticulocyte count, peripheral blood and marrow smears together with marrow biopsy were assessed. Pancytopenic cancer patients on chemotherapy were excluded. Pancytopenia was defined as hemoglobin less than 10g/dl, WBC less than 4 x109/L and platelet count less than 150 x 109/L. Results: The mean Hb concentration, WBC count and platelet count in studied group were significantly lower than in control group. Hematological malignancies were the commonest cause of pancytopenia and accounted for (51.7%), they included: Acute leukemia (35%), myelodysplastic syndrome (11.7%), hairy cell leukemia (3.3%) and myelofibrosis (1.7%). Iraqi J. Hematology, May 2016, vol.5, Issue 1 129 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. Aplastic and megaloblastic anemia each of them accounted for (16.7%), hypersplenism was responsible for (10%). Other less common causes included enteric fever, kalaazar and secondary metastasis each of them accounted for (1.7%). Pallor was present in every case. Fever was present in (63.3%) and (25%) had bleeding manifestations at the time of presentation . Conclusions: The most common causes of pancytopenia were acute Leukemia, aplastic anemia and Megaloblastic anaemia, but rare causes like myelofibrosis, enteric fever, kala azar and secondary metastasis should also be kept in mind. Keywords: Pancytopenia, Bone marrow, malignant hematology. Introduction The term pancytopenia denotes Bone simultaneous reduction in all the formed examination elements of the blood i.e, erythrocytes, performed to every pancytopenic patient leukocytes and platelets. Pancytopenia is is of high diagnostic value (3). The center not a disease entity but a triad of of hematological diseases in Erbil, findings that may arise from a number of Nanakaly disease processes (1) . It is, therefore marrow aspirate/biopsy which is Hospital, routinely receives many patients whose complete blood count exists when hemoglobin level is below show 10 g/dl, leukocyte count below 4 x 109 /l However, no statistical figures are and platelet count below 150 x 109/l available here regarding number of cases Hence, a patient of pancytopenia may as well as their underlying causes. have anaemia, Therefore, we deemed necessary to do weakness and dyspnoea on exertion; our work on this group of patients to bleeding manifestation, like skin and determine these figures and to compare mucosal them with results of studies done in the symptoms due bleeding, to due to thrombocytopenia; and fever, ulceration of mouth and recurrent chest infections due to neutropenia.(2) pancytopenic parameters. other parts of Iraq and nearby countries. The aims of this study were to determine the spectrum of pancytopenia with it is frequency, common clinical presentation and etiology on the basis of Iraqi J. Hematology, May 2016, vol.5, Issue 1 130 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. full clinical and laboratory examination count, was done for every patient using specially bone marrow examination in automated blood counter our locality. Coulter® model AcT diff 2), Reticulocyte (Beckman preparations and Leishman’s stained peripheral blood were prepared freshly Materials and methods was and examined for each patient. Bone conducted during the period extending marrow aspiration and biopsy were from 13th Dec.2008 to 15th July 2009, at performed at Nanakaly hospital for all Nanakaly Hospital for Blood Diseases. the patients. Marrow aspirate smears A total of 60 patients with pancytopenia were prepared, stained with Leishman’s were included in this study. All patients stain and examined. Marrow trephine presenting with pancytopenia during the biopsy specimens were fixed in Bown’s study For solution and were sent to Rizgary purposes of this work, pancytopenia was Teaching Hospital for processing and defined as a hemoglobin less than interpretation. This prospective period were study included. 10g/dl, WBC less than 4 x109/L and Data were tabulated and platelet count less than 150 x 109/L. statistically analyzed using statistical Patients who were already diagnosed package for cases of pancytopenia due to different version 15). Chi square and tests of causes receiving association were chemotherapy or therapeutic radiation, applicable, analysis were excluded from this study. (ANOVA) was used to compare between and those social sciences (SPSS used of whenever variance Diagnostic work up of cases means of 3 groups or more of patient. A included three basic investigations i.e. a p-value of equal or less than 0.05 was complete considered as statistically significant. blood count, analysis of stained blood, marrow aspirate smears and bone marrow trephine biopsy. Results Laboratory tests were performed at the During the period of this study, 60 Nanakaly Hospital's laboratory. Two ml patients attending Nanakaly hospital venous blood were collected into an fulfilled the criteria of pancytopenia EDTA anticoagulated tube; a full blood according to the study protocol. There Iraqi J. Hematology, May 2016, vol.5, Issue 1 131 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. were 31 males and 29 females patients. acute lymphoblastic leukaemia and 9 Their ages ranged between one and 81 cases of acute myeloblastic leukemia. years with a mean age of (33.3 years). Comparing the various The commonest presenting features, in parameters among the three leading order of frequency were: Pallor (100% causes of pancytopenia (malignant blood of cases), fever in (63.3%), bleeding in diseases, (25%) and infections in (8.3%) of cases. megaloblastic anemia) Organomegaly was noted in aplastic significant anemia and there differences were regarding the (33.4%) of cases. These cases included WBC count, where patients with aplastic (16.7%) with splenomegaly, (11.6%) anaemia had the lowest figures ( p < had hepatosplenomegaly, (3.3%) had 0.04), hepatomegaly alone and one case had significantly lower in aplastic anaemia lymphadenopathy. Table (1) represents a than the other two conditions ( p< statistical 0.001).There summary of the routine the platelets, were no too, were significant hematological parameters of studied differences among the three groups patients. regarding the Hb and reticulocyte counts Majority of patients showed ( p = 0.11 and p = 0.97 ) respectively. normal red cell morphology (68.3%), Other hematological parameters showed macrocytosis was reported in (26.7%) of in table 4. cases and hypochromia was found in (5%) of cases. All patients had marrow aspirate and trephine biopsy Discussion Pancytopenia is commonly examinations and according to marrow encountered in hematological practice, it findings the causes of pancytopenia were usually indicates a serious condition and determined, the frequency of various it necessitates prompt action. The pattern causes of pancytopenia in this series is of diseases leading to pancytopenia may shown in table (2). Types of malignant vary in different population groups blood diseases are illustrated in table 3. depending on racial factors, nutritional Leukemic patients included 12 status and prevalence of infection (4) ; the males and 9 females, their mean age was frequency of various causes varies 18 years, and there were 12 cases of among different age groups too. Iraqi J. Hematology, May 2016, vol.5, Issue 1 132 A Clinical-Hematological Study of Pancytopenia In this study we had 60 cases of Alan I.I, Nawsherwan S.M, Saran A. N. patients ( 71.4%) and only in (40% ) of pancytopenia. The mean age of studied aplastic anemia patients was 33.3 years which is very fever with organomegaly at presentation close to that reported from other favors the diagnosis of acute leukemia, (1, 5, 6, 7, 8) developing countries . The while patients, accordingly bleeding unassociated with clinical presentation of our patients was organomegaly is likely to be due to classical and the relative frequencies of aplastic anemia. In this work the mean various Hb concentration was 8.14 g/dl, this presenting features were comparable to the finding of other (1, 3, 6, 7, 8, 9, 10) workers . Organomegaly figure is higher than that reported by 10) (8, this discrepancy is due to the lower was reported in 33.4% of studied frequency of aplastic anemia, in which patients, had anaemia is more severe, in this series had compared to their studies. The mean hepatosplenomegaly and only 3.3% had W.B.C and platelet counts (2.19 and hepatomegaly alone. The frequency of 55.35 X 109/L respectively) were similar splenomegaly in this study was similar to results observed by other workers to that reported by Ishtiaq et al from 10) of these splenomegaly, Pakistan (7) 16.7% 11.6% (8, . Pancytopenia has multiple causes and . Abdul Hamid reported a the prognosis is dependent on the cause. frequency of 48% in a series of The frequency of these causes has been pancytopenic patients in Yemen, he reported in a limited number of studies attributed this relatively high frequency (8, 9) to the high prevalence of malaria, malignancies as a cause of pancytopenia kalaazar and other infectious diseases Among studied (8) .In the present study hematological . was accounted for (51.7%) of cases. patients Acute leukemia was the most common presentation varied according to the cause underlying bleeding responsible for 35% of cases. Acute occurred most frequently among aplastic lymphoblastic type constituted 20% of anemia patients (60%), while none of pancytopenic megaloblastic had myelogenous leukemia formed 15% of most cases. In a study done by Jalaee and leukaemia Keihani in Tehran, acute leukemia was condition, thus anemia patients bleeding. Fever occurred frequently among acute Iraqi J. Hematology, May 2016, vol.5, Issue 1 of pancytopenia; cases, while it was acute 133 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. the commonest cause of pancytopenia reported a frequency of (18%) of (11) myelodysplastic syndrome , similar results were reported from (1) , Iqbal et al Sweden too, where neoplastic diseases reported an incidence of (2.4%) in the and radiation related marrow damage same series reported a similar frequency accounted for 32 % of cases while for myelofibrosis aplastic anemia accounted for 19% of . The second major causes of (4, 13) pancytopenia in this study were a plastic acute leukemia was the second most anemia and megaloblastic anemia, each common cause of pancytopenia, In many of them accounted for 16.7% of cases. A Asian countries acute leukemia ranked detailed history and thorough clinical the 3rd or even the 4th cause of examination did not help in establishing pancytopenia, cases (12) (16) . In studies from India being next to the cause of marrow hypoplasia, one of megaloblastic, Aplastic anemia or aplastic patients was Fanconi anemia after (1, 2, 3, 5, 6, 8, 10, 14, 15) malaria . The relatively high frequency of acute leukaemia as a cause of diagnosed as chromosomal study. The relative frequency of aplastic anaemia in this study is in agreement (9, pancytopenia in this study may be due to with findings of many other workers the fact that our locality has been the 10, 12, 17) battle field for a series of wars since the aplastic anaemia was the commonest 1960s and various weapons, including cause of pancytopenia (1, 3, 15, 17, 18). chemicals with potential leukaemogenic Epidemiologically, , however in many other studies aplastic effects have been used, another reason anemia has a pattern of geographic may be the blooming economy of variation opposite to that of leukemia, Kurdistan over the last decade may have with higher frequency in the developing reduced world than in the industrialized West the relative frequency of nutritional anemias. (19) . Large prospective studies indicate an The frequency of other blood annual incidence of two new cases per malignancies included were as follow; million populations in Europe and Israel myelodysplastic 20 hairy cell myelofibrosis syndrome leukemia (1.7%). (11.7%), (3.3%) Devi and et al Iraqi J. Hematology, May 2016, vol.5, Issue 1 . Its exact incidence in Kurdistan is not known due to lack of population – based studies. Studies from Thai land (21) and 134 A Clinical-Hematological Study of Pancytopenia China (22) Alan I.I, Nawsherwan S.M, Saran A. N. showed the incidence to be Megaloblastic anemia due to about three folds that in the west. Its vitamin B12 or folic acid deficiency is exact etiology still not known but an now a well –recognized and established autoimmune cause of pancytopenia mechanism has been (25) . It can either inferred from positive responses to non- present as bicytopenia or pancytopenia, transplant or rarely with thrombocytopenia only data (3) . therapies and laboratory Megaloblastic anemia in this (26) . The frequency of pancytopenia series was as common as Aplastic among anaemia, it accounted for (16.7%) of anaemia has range of 11% to 47% cases. Similar results were reported from almost all these studies, pancytopenia Pakistan by Naeem et al and Memon et was the main presentation and so was al (3, 15) . In Yemen Abdul Hamid and Shukry showed that megaloblastic (6) in the case in this study. Conclusion pancytopenia and accounted for (14.7%) of cases with megaloblastic anemia is the third commonest cause of (8) patients We concluded that physical . Studies from India, had examination and peripheral blood picture shown megaloblastic anemia to be the play an important role in planning first investigations in pancytopenic patients. most common cause of pancytopenia and accounted for 44% to 72% (9, 17) . Increased incidence of The most common aplastic anemia perhaps anaemia, but with the high of pancytopenia were acute Leukemia, megaloblastic anemia in those studies correlates causes and rare Megaloblastic causes like prevalence of nutritional anemia due to myelofibrosis, enteric fever, kala azar religious and socio-economical reasons. and secondary metastasis should also be In Europe in a study on 213 cases of kept in mind. pancytopenia, carried out by Imbert et al, (7.5%) of cases were due to megaloblastic anemia (23) . Mosso et al, showed the incidence of megaloblastic anemia to be (7.41%) (24). Iraqi J. Hematology, May 2016, vol.5, Issue 1 135 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. References An experience of 89 cases. J P M I 2004; 1. Devi PM, Laishram RS, Sharma PS, 18 (1): 76 –79. Singh AM, Singh MK, Singh YM et al. Clinico- hematological profile of 7. Ishtiaq O, Baqai B Z, Anwer F, pancytopenia in Manipur, India. Kuwait Hussain N. Patterns of pancytopenia Medical Journal 2008; 40 (3): 221- 224. patients in a general medical ward and a 2. Jha A, Sayami G, Adhikari RC, Panta AD, Jha R. Bone marrow examination in cases of pancytopenia. J Nepal med Assoc 2008; 47(169): 12 – 17. proposed diagnostic approach. J Ayub med coll abbottabsd 2004; 16: 8 -13. 8. Abdul Hamid G and Shukry SA. Patterns of pancytopenia in Yemen. Turk J Hematol 2008; 25: 71 -74. 3. Memon SH, Shaikh S, Akbar M, Nizamani A. Etiological Spectrum of pancytopenia based on bone marrow examination in children. Journal of the college of physicians and surgeons. Pakistan 2008; 18 (3): 163 – 167. 4. Gupta V, Tripathi S, Tilak V, Bhatia BD. A study of clinic-haematological profiles of pancytopenia in children. Tropical doctor 2008; 38: 241–243. 5. David S, Robert A, Innocent G, Lorraine L, Christine G, Boniface M et al. Pancytopenia in Zimbabwe. Am J med Sci 1999; 317(1): 22 - 32. 6. Niazi M and Raziq F.The incidence of 9. Khodke K , Marwah S, Buxi G, Yadav marrow RB, Chaturvedi NK. examination in Bone cases of pancytopenia. Journal Indian academy of clinical medicine 2001; 2: 55 – 59. 10. Ahmad N and Sajid N. Pancytopenia in paediatric patients. Pakistan paediatric journal 2008; 32: 15966. 11. Jalaee -Khoo H and Keihani M. The causes of pancytopenia. TUM J 2006; 64 (2): 1- 2. 12. Keisu M and Ost A. Diagnosis in patients with severe pancytopenia underlying pathology in pancytopenia. Iraqi J. Hematology, May 2016, vol.5, Issue 1 136 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. suspected of having aplastic anaemia. 18. Rehman HU, Fazil M, Khan FM. Eur J Haematol 1990; 45: 11- 14. The etiology pattern of pancytopenia in children up to 15 years. Pak Armed 13. Bhatnagar SK, Chandra J, Naryan forces. Med J 2003; 53 (2): 183 – 187. SH, Sharma S, Singh V, Dutta AK. Pancytopenia in children: Aetiological 19. Issaragrisil S, Leaverton PE, profile. Journal of Tropical Paediatrics Chansung K, Thamprasit T, Porapakham 2005; 51 (4): 236 – 239. Y, Vannasaeng S et al. Regional patterns in the incidence of aplastic anemia in 14. Varma N and Dash S. A reappraisal Thailand. Am J Hematol 1999; of underlying pathology in adult patients 164- 8. presenting with pancytopenia. 61: Trop Geogr med 1992; 44(4): 322 – 327. 20. Young NS. Hematopoietic cell destruction by immune Mechanisms In 15. Naeem Khan M ,Ayyub M, Nawaz acquired KH, Naeem N, Hussain T, Shujaat H et Hematol 2000; 37: 3 - 14. al. Pancytopenia: aplastic anaemia. Semin clinicopathological study of 30 cases at military Hospital, 21. Issaragrisil S, Chansung K, Kaufman Rawalpindi. Pak J pathol 2001; 12 (2): DW, Sirijirachai J, Thamprasit T, Young 37- 41. NS et al. Aplastic anemia in rural Thailand: it is association grain farming 16. Iqbal W, Hassan KH, Ikram N, Nur and pesticide exposure. Am J Public S. Aetiological break up in 208 cases of Health 1997; 87: 1551- 1554. pancytopenia. J Rawal Med Coll 2001; 5 (1): 7-10. 22. Yang C and Zhang X. Incidence survey of aplastic anemia in China. Chin 17. Khunger JM, Arulselvi S, Sharma U, Med Sci J 1991; 6: 203-207. Ranga S, Talib VH. Pancytopenia – a clinic haematological study of 200 cases. 23. Imbert M, Scoazec JY, Mdry JY, Indian J Pathol Microbiol 2002; 45(3): Jouzult H, Rochant H, Sultan C. Adult 375 – 379. patients presenting with pancytopenia: A Iraqi J. Hematology, May 2016, vol.5, Issue 1 137 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. reappraisal of underlying pathology and megaloblastic diagnostic procedures in 213 cases. Indian Pediatr 2008; 39: 453-457. Haematologic pathology 1989; 26. 3: 159-167. Jan Anaemia in children. MA.Thrombocytopenia in children. J Postgrad Med Inst 2004; 18: 353-358. 24. Mosso M, Cassata A, Prieto S, Osay L, salomon S, Carena J. Clinical impact Correspondence to: of pancytopenia in hospitalized patients. Nawsherwan Sadiq Mohammad Revista Medica universitaria 2008; Pathology 4 (4): 17. Department, College of Medicine/ Hawler Medical University Email: [email protected] 25. Chandra J, Jain V, Narayan S, Mob: 00964(0)7504384101 Sharma S, Singh V, Kapoor AK et al. Folate and cobalamin deficiency in Iraqi J. Hematology, May 2016, vol.5, Issue 1 138 Alan I.I, Nawsherwan S.M, Saran A. N. A Clinical-Hematological Study of Pancytopenia دراسة الخصائص السريرية والدموية من قلة كريات الدم الشاملة للمرضى الذين يراجعون مستشفى نانه كه لي في مدينة أربيل آالن إسحاق ايشو ،1نوشيروان صادق محمد, 2ساران عبد القادر نورالدين 3 / MBChB 1ماجستير أمراض الدم /مستشفى أربيل التعليمي / FIBMS ،MBChB 2أمر اض الدم /قسم علم األمراض ،كلية الطب /جامعة هولير الطبية /البريد اإللكتروني: [email protected] / MBChB 3ماجستير أمراض الدم /مستشفى رزكاري التعليمي الملخص: الخلفية :قلة الكريات الشاملة هو ثالوث الهيموغلوبين المنخفض ،وقلة خاليا الدم البيضاء والصفائح الدموية .على الرغم من أنها مشكلة سريرية شائعة مع وجود التشخيص التفريقي الواسع ،هناك مناقشة قليلة نسبيا لهذه الحاالت في الدم. وأمراض الباطني للطب الرئيسية الكتب األهداف :تهدف هذه الدراسة إلى تحديد المسببات والتعريف السريري للمرضى الذين يعانون من قلة الكريات الشاملة لي. كه نانه لمستشفى حضروا الذين المواد والطرق :خالل فترة 6أشهر ،حضر 66مريضا يعانون من قلة الكريات الشاملة الى مستشفى نانه كه لي ، تراوحت أعمارهم بين 11-1سنة .تم اختبار مجموعة السيطرة من 06شخصا من االصحاء المتطابقين بالعمرو تم اخذ صورة الدم ا لكاملة وتعداد الشبكيات .وقد تم تسجيل تاريخ المرض ،والفحص السريري ومعلومات فحص الدم . وتضمن الملف الدموي الهيموغلوبين ،وعدد وتفاصيل الكريات البيض ،عدد الصفائح الدموية ،عد الخاليا الشبكية، الدم المحيطي ومسحات نخاع جنبا إلى جنب مع خزعة نخاع .تم استبعاد مرضى السرطان على العالج الكيميائي مع قلة الكريات الشاملة .وقد عرفت قلة الكريات الشاملة كاالتي :الهيموجلوبين أقل من 16غم/د ل ،كرات الدم البيضاء أقل من /169 * 4لتر وعدد الصفيحات أقل من / 169* 106لتروكانت متوسط تركيز الهيموغلوبين ،وعدد كرات الدم البيضاء و الصفائح الدموية في المجموعة المدروسة أقل بكثير مما كانت عليه في المجموعة الضابطة. النتائج : .كانت األورام الخبيثة الدموية السبب األكثر شيوعا لقلة الكريات الشاملة وتمثل ( ،)٪01.5وشملت: سرطان الدم الحاد ( ،)٪30ومتالزمة اعتالل نخاع العظم ( ،)٪11.5سرطان الدم الشعيري ( )٪3.3وتليف نخاع العظم ( .)٪1.5فقر الدم الالتنسجي وفقر الدم الوبيل كل منها تمثل ( ،)٪16.5وفرط نشاط الطحال مسؤولة عن ( .)٪ 16وتضمنت أسباب أخرى أقل شيوعا مثل حمى المعوية ،والحمى السوداء واالنتشار الثانوي للسرطان كل منها تمثل ( .)٪1.5كان الشحوب موجود في كل حالة .كانت الحمى موجودة في ( )٪63.3و مظاهر النزيف في المراجعة. وقت ()٪20 االستنتاجات :كانت األسباب األكثر شيوعا لقلة الكريات الشاملة هي سرطان الدم الحاد وفقر الدم الالتنسجي وفقر الدم الوبيل ،ولكن األسباب النادرة مثل تليف نخاع العظم ،حمى المعوية ،الحمى السوداء واالنتشار الثانوي للسرطان االعتبار. في يوضع أن أيضا ينبغي كلمات البحث :قلة الكريات الشاملة ،نخاع العظام ،أمراض الدم الخبيثة. 139 Iraqi J. Hematology, May 2016, vol.5, Issue 1 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. Table 1: Statistical summary of the routine haematological parameters of studied patients: Haematological parameters Mean ± SD Patients (n = 60) Hb ( g/dl) 8.14 ±1.78 WBC×109/L 2.19 ± 0.91 Platelets×109/L Reticulocyte count (%) 55.35 ± 39.82 0.82 ±0.87 MCV (fl) 90.12 ± 12.5 MCH(pg) 31.1 ±4.98 MCHC( g/dl) 34.7 ± 2.51 Iraqi J. Hematology, May 2016, vol.5, Issue 1 140 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. Table 2: Causes of pancytopenia Causes of pancytopenia Frequency Percent Malignant blood diseases 31 51.7 Aplastic anaemia 10 16.7 Megaloblastic anaemia 10 16.7 Hypersplenism 6 10 Enteric fever 1 1.7 Kalazar 1 1.7 Secondary metastasis 1 1.7 Total 60 100 Table 3: Malignant blood diseases causing pancytopenia: Malignant blood diseases Frequency Percent from(60) studied patients Acute leukaemia 21 (67.7%) 35 Myelodysplastic syndrome 7 (22.6%) 11.7 Hairy cell leukaemia 2 (6.5%) 3.3 Myelofibrosis 1 (3.22%) 1.7 Total 31(100%) 51.7 Iraqi J. Hematology, May 2016, vol.5, Issue 1 141 A Clinical-Hematological Study of Pancytopenia Alan I.I, Nawsherwan S.M, Saran A. N. Table 4: comparison of hematological parameters between the three major causes of pancytopenia: Age HB ( g/dl) WBC×109/L 9 Platelets×10 /L Reticulocyte Count (%) Diagnosis No A. Acute leukemia 21 18.14±17.4 B. Aplastic anemia 10 26±21.20 C. Megaloblastic anemia 10 55.8±14.85 A. Acute leukemia 21 8.21±1.67 B. Aplastic anemia 10 6.85±1.57 C. Megaloblastic anemia 10 8.15± 1.92 A. Acute leukemia 21 2.1 ± 0.9 B. Aplastic anemia 10 2 ± 0.89 C. Megaloblastic anemia 10 2.89 ± 0.54 A. Acute leukemia 21 50 ± 34.3 B. Aplastic anemia 10 22.1± 17.6 C. Megaloblastic anemia 10 92.1±41.32 A. Acute leukemia 21 0.74± 0.62 B. Aplastic anemia 10 0.71± 0.28 C. Megaloblastic anemia Mean ±SD P-value < 0.001 Significance AXC BXC 0.11 0.04 AXC < 0.001 AXC 0.97 0.71±0.30 10 A. Acute leukaemia 21 78.18±8.73 B. Aplastic anemia 10 29.5 ± 6.4 C. Megaloblastic anemia 10 90±4.08 A. Acute leukaemia 21 62.42±23.8 Bone marrow B. Aplastic anemia 10 2.7±0.94 Blast cell (%) C. Megaloblastic 10 2.5±0.97 Bone marrow cellularity AXB < 0.001 AXC BXC < 0.001 AXB AXC anemia Iraqi J. Hematology, May 2016, vol.5, Issue 1 142 المجلة العراقية ألمراض الدم مجلــــة علميــــــة محكمـــــــة تصــــدر مرتيــــن في السنــــة عــــــــــن المركز الوطني لبحوث وعالج امراض الدم -الجامعة المستنصرية -بغداد -العراق المجلد 5العدد2 ايار 1026 رئيس التحرير مدير التحرير أ.د عالء فاضل علوان أ.د علي محمد جواد المظفر سكرتير المجلة د .نضال كريم الرحال هيئة التحرير الهيئة االستشارية (جامعة النهرين) أ.د.نصير عالوي(جامعة دهوك) أ.د .رعد جابرموسى أ.د.خالد نافع(جامعة الموصل) أ.د .بان عباس أ.د .علي مسلم(امريكا) ا.د سلمى عباس الحداد(جامعة بغداد) أ.د .احمد ابراهيم(لبنان) ا.م.د عالء الدين مظفر(الجامعة المستنصرية) أ.د .انور شيخه(جامعة السليمانية) ا.م.د عالءالدين سهام ناجي( جامعة بغداد) (جامعة النهرين) ا.د .وسيم فاضل التميمي(جامعة النهرين) أ.د .ميعاد كاظم(جامعة البصرة) أ.د .صبح المدلل(جامعة النهرين) أ.م.د نبيل سلمان(مصر) أ.م.د عالء صادق(جامعة بابل) أ.م.د احمد خضير(جامعة هولير) أ.م.د .رحيم مهدي(جامعة الكوفة) د .جعفر الغبان (م.الطفل المركزي) أ.م.د .اديب عباس(الجامعة المستنصرية) أ.م.د.مازن فيصل(جامعة بغداد) أ.م.د هيثم الربيعي(جامعة بغداد) د .فاتن ال ياسين(جامعة بغداد) د .ابراهيم خليل ابراهيم (م.الكاظمية ) د .بسام فرنسيس(م.بغداد التعليمي) د.اسعد عبد االمير ( م .البصرة التعليمي) د .عبد المجيد علوان(م.اليرموك التعليمي) صدر العدد االول في 1122 اول رئيس تحرير د.نبيل سلمان مراد اول مدير تحرير د.اديب الشامي