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MLO201608-COVER-nolabel.indd COVERI
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FROM THE EDITOR
By A lan L enhof f, Edi tor
A day in the life of clinical
laboratory science
ONE NICE THING ABOUT MY JOB IS THAT, as part of it, I
have the opportunity—and professional obligation—to read
news releases about advances in laboratory medicine or the
science that underlies it, and to follow the continuous flow
of important advancements. One day shortly before this
August 2016 issue of MLO went to press, there was an especially impressive group of news releases that came across my
desk. Lab Week is in April, but July 6 was a kind of unofficial
lab day, I guess, because I had the pleasure of learning that
day about all of the following:
• New screening test using blood biomarkers may
identify risk of colon cancer recurrence. Ludwig Institute
for Cancer Research scientists, working in collaboration with
colleagues in Australia and the United States, have shown
that fragments of tumor DNA circulating in the blood can be used to gauge the
risk of colorectal cancer recurrence and the efficacy of chemotherapy following
surgery. The finding, published in Science Translational Medicine, is an important
step toward the development of a noninvasive and more effective test for the
detection, monitoring, and treatment of cancer.
• “Omics” data improves breast cancer survival prediction. Precise predictions of whether a tumor is likely to spread would help clinicians and patients
choose the best course of treatment. New research reveals that profiling primary
tumor samples using genomic technologies can improve the accuracy of breast
cancer survival predictions compared to clinical information alone. Although this
method is not ready for use in the clinic, the proof-of-principle study, published in
GENETICS, shows that survival predictions improve when they incorporate comprehensive data on which genes are active in tumor samples compared to noncancerous tissues from the same patient
• Blood test to detect DNA fragments shed from colon cancers predicts
disease’s recurrence. Scientists at the Johns Hopkins Kimmel Cancer Center and
University of Melbourne report they have used a genetic test that spots bits of
cancer-related DNA circulating in the blood to accurately predict the likelihood of
the disease’s return in some—but not all—of a small group of patients with earlystage colon cancer. The DNA-based test, described in the July 6 issue of Science
Translational Medicine, if confirmed by further research, could eventually help clinicians decide which patients need additional treatment at the time of their initial
diagnosis of stage 2 cancer.
• Inexpensive blood test can discriminate between bacterial, viral infections,
study finds. Researchers at the Stanford University School of Medicine have made
an important breakthrough in their ongoing efforts to develop a diagnostic test
that can tell healthcare providers whether a patient has a bacterial infection and
will benefit from antibiotics. The team used publicly available patient gene expression data to pinpoint just seven human genes whose activity changes during an
infection; their pattern of activity can distinguish whether an infection is bacterial
or viral.
• Genetic testing can help deliver precision medicine to men with advanced
prostate cancer. Genetic testing in men with advanced prostate cancer could pick
up a significant proportion whose disease may be caused by inherited mutations
in genes involved in repairing DNA damage, a major new study reveals. Testing
prostate cancer patients for mutations in key DNA repair genes could identify
those who may benefit from precision treatments that specifically target DNA
repair weaknesses in cancer cells.
“Although this method is not ready for use in the clinic…” is the caveat that can
be applied to all of these fascinating examples of the breakthroughs that molecular
approaches may one day provide for clinical diagnostics—but that makes them
no less exciting. I remember how the late Paul Harvey sometimes used to include
medical research news in his daily radio commentaries, leading with “Today’s
news of perhaps the most lasting significance is….” Who knows which of these
items may prove to be the news of most lasting significance for July 6, 2016?
4
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AUGUST 2016
MEDICAL LABORATORY OBSERVER Vol.48, No.8
Publisher/Executive Editor/President
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John Brunstein, PhD, Biochemistry
(Molecular Virology)
President & CSO
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Laboratory Director
Michigan State University, East Lansing, MI
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Director, Supply Chain Analytics
University of Virginia Health System, Charlottesville, VA
Jeffrey D. Klausner, MD, MPH
Associate Clinical Professor of Medicine
Divisions of AIDS and Infectious Diseases
University of California, San Francisco, CA
Susan McQuiston, JD, MT(ASCP)
Instructor, Biomedical Laboratory Diagnostics Program
Michigan State University, East Lansing, MI
Donna Beasley, DLM(ASCP)
Manager
Huron Healthcare, Chicago, IL
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Clinical Associate Professor
SUNY Upstate Medical University, Syracuse, NY
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Service, University of Michigan Health System Department
of Pathology, Ann Arbor, MI
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7/12/2016 9:13:04 AM
ADVERTORIAL
A new diagnostic approach to Mycoplasma pneumoniae
A conversation with Ken Kozak, Chief Technical Officer, Meridian Bioscience, Inc., and Donna Mayne, BSMT, ASCP,
(CLS)NCA, Microbiology, Serology, Molecular Lab Manager, Sacred Heart Hospital, Pensacola, FL
Q: Why was a better test for Mycoplasma pneumoniae needed?
A (Kozak): Traditional methods of diagnosis (including serology) have low sensitivities, are dependent on a patient’s immune
response, and/or do not provide diagnostic results within a clinically relevant time frame. Specifically, serology is dependent on
a patient’s immune response, which takes time to develop, so in
the first week of testing you may only detect 25 percent of the
positives. X-rays are the other widely used method, which cannot definitively identify the cause of infection.
illumigene Mycoplasma Direct would provide physicians a definitive diagnosis and allow them to provide targeted treatment,
therefore lessening the much larger issue of antibiotic resistance.
respectively, which makes a rapid, targeted detection for
specific treatment difficult.
illumigene Mycoplasma Direct accurately detects infection
on the first day of symptoms and provides the ability to avoid
treating patients empirically to reduce the administration of
broad spectrum antibiotics and the likelihood of antimicrobial resistance. More importantly, the increased performance of
this innovative test enables a more rapid diagnosis, which in
turn, provides for earlier identification of outbreaks and prevention of secondary cases through implementation of control
measures.
Q: What are the main barriers to diagnosing Mycoplasma
Q: How does illumigene Mycoplasma Direct differ from other
pneumoniae effectively, and how does illumigene Mycoplasma
Direct overcome them?
molecular tests?
A (Mayne): Until now, Mycoplasma respiratory infections have
A (Kozak): illumigene Mycoplasma Direct is the only FDAcleared standalone molecular assay for the detection of Mycoplasma pneumoniae. This new test offers a simplified procedure which
lends itself for use in moderately complex laboratories. Our team
worked very hard to make process improvements without compromising performance. illumigene Mycoplasma Direct features
a simple three-step procedure that takes less than two minutes
of hands-on time and provides final patient results in under one
hour.
Because illumigene Mycoplasma Direct is a single analyte test,
our data has shown that it is more sensitive than the FDA-cleared
multi-analyte molecular panels that include Mycoplasma pneumoniae. This allows for better patient management because it is
able to detect more positives.
The illumigene test utilizes our proven loop-mediated isothermal amplification (LAMP) technology, which provides a simple
procedure, in conjunction with our cost-effective diagnostic platform that has the flexibility to run one or up to 10 patient samples
at a time and has a small 8½-by-11 inch footprint. Since it requires
no expensive capital equipment or service contracts, this innovative test is ideal for enabling a more rapid diagnosis and can be
widely employed into any moderately complex laboratory.
Q: How was illumigene Mycoplasma Direct tested? Who was
involved in developing the clinical trials?
A (Kozak): The clinical trial compared illumigene Mycoplasma
Direct to illumigene Mycoplasma on 456 prospective throat swab
samples from patients with upper respiratory illness at three different clinical sites in Missouri, Texas, and Florida. The overall
positive percent agreement was 96.0 percent and the negative
percent agreement was 97.7 percent, prior to discrepant analysis
being performed. A closer look at this data showed that illumigene Mycoplasma Direct detected 10 more positives that were
missed by the other assays. Prevalence was highest in the 3 to
12 year olds and 13 to 21 year olds, 15 percent and 13.2 percent,
respectively.
Q: How much better is your test as compared with the other
testing methods for Mycoplasma pneumoniae (serology, x-ray)?
A (Kozak): Current testing methodologies have low sensitivities and specificities when detecting Mycoplasma pneumoniae.
Traditional testing methods, such as serology and x-ray, have
documented sensitivities as low as 25 percent and 41 percent,
primarily been diagnosed using clinical signs and symptoms
and/or chest x-ray. These are subjective and non-specific criteria
that frequently result in misdiagnosis when used alone. Until recently, we offered Mycoplasma IgM testing as an additional tool
for physicians to use when considering Mycoplasma respiratory
infections; however, we discontinued this test earlier this year
due to lack of clinical correlation. The lack of a fast and accurate
test for Mycoplasma respiratory infections had been a significant
roadblock in Mycoplasma diagnosis.
With the availability of molecular tests for Mycoplasma, physicians are able to rule out or diagnose Mycoplasma with confidence; however, if these tests are not able to be performed and resulted quickly, empiric therapy must be considered. If this testing
is provided in a timely fashion, empiric therapy can be avoided.
The illumigene Mycoplasma Direct will provide physicians with
such a timely/accurate result, allowing proper diagnosis and
timely treatment while practicing good antibiotic stewardship.
It has been our experience that fast and accurate lab tests are
both a physician and patient satisfier. This type of test builds
the physician’s confidence in the laboratory and builds the patient’s confidence in both the physician and laboratory. Additionally, fast/accurate diagnosis leads to fast/accurate treatment and
better patient outcomes.
Q: How will illumigene Mycoplasma Direct be used in your
facility?
A (Mayne): At Sacred Heart, we will be offering this assay 24/7
and testing samples as they arrive in our laboratory. This “realtime” approach has been very successful for us for the GAS and
Pertussis illumigene assays. Our physicians and patients benefit
from these rapid and accurate results no matter what time of day
the sample is received in the lab.
Q: What other disease areas is illumigene involved in?
A (Kozak): The illumigene expanded molecular menu includes
tests in the following areas where they have proved highly
accurate and are used in nearly 1,500 institutions worldwide:
• Hospital Acquired Infections (Clostridium difficile)
• Tropical Diseases (Malaria)
• Sexual Health (Chlamydia [CT], Gonorrhoeae [NG], Group B
Streptococcus, HSV 1 &2)
• Respiratory (Group A Streptococcus, Pertussis)
AUGUST 2016
MLO201608_AD ADVERTORIAL-Meridian Final.indd 5
MLO - ONLINE.COM
5
8/25/2016 10:38:41 AM
TAGRISSO:
in patients with metastatic EGFR T790M mutation–positive NSCLC, as detected by an FDA-approved
test, at progression on or after EGFR TKI therapy
Identify EGFR T790M mutation in patients who progress on or after EGFR TKI therapy
• Nearly 2 out of 3 cases of progression with first-generation EGFR TKIs are related to the acquired
T790M mutation1
• Testing at progression provides the opportunity to identify mechanisms of resistance, including the
T790M mutation
TAGRISSO demonstrated efficacy and safety in two clinical trials
• TAGRISSO was researched in two separate, global, Phase II, single-arm, open-label clinical trials in
patients with EGFR T790M mutation–positive NSCLC who had progressed on or after EGFR TKI therapy2
• A 59% objective response rate (95% CI: 54–64) observed in patients (N=411) who progressed with previous
EGFR TKI therapy2
• The most common adverse reactions (>20%) observed in TAGRISSO patients were diarrhea (42%),
rash (41%), dry skin (31%), and nail toxicity (25%)2
• Interstitial lung disease (ILD) was reported in 3.3% of patients and was fatal in 0.5% of TAGRISSO patients2
Visit TAGRISSOhcp.com for more information
MLO201608_AD Astra-Zeneca-Spread.indd 6
7/8/2016 12:50:51 PM
IDENTIFY PATIENTS
APPROPRIATE FOR TAGRISSO
Visit cobasEGFRtest.com today to learn how.
IMPORTANT SAFETY INFORMATION
• There are no contraindications for TAGRISSO
• Interstitial Lung Disease (ILD)/Pneumonitis occurred in 3.3% and was fatal in 0.5% of 813 TAGRISSO
patients. Withhold TAGRISSO and promptly investigate for ILD in any patient presenting with worsening
of respiratory symptoms indicative of ILD (e.g., dyspnea, cough and fever). Permanently discontinue
TAGRISSO if ILD is confirmed
• QTc interval prolongation occurred in TAGRISSO patients. Of the 411 patients in two Phase II studies,
0.2% were found to have a QTc greater than 500 msec, and 2.7% had an increase from baseline QTc
greater than 60 msec. Conduct periodic monitoring with ECGs and electrolytes in patients with congenital
long QTc syndrome, congestive heart failure, electrolyte abnormalities, or those who are taking medications
known to prolong the QTc interval. Permanently discontinue TAGRISSO in patients who develop QTc interval
prolongation with signs/symptoms of life threatening arrhythmia
• Cardiomyopathy occurred in 1.4% and was fatal in 0.2% of 813 TAGRISSO patients. Left Ventricular Ejection
Fraction (LVEF) decline >10% and a drop to <50% occurred in 2.4% of (9/375) TAGRISSO patients. Assess LVEF
before initiation and then at 3 month intervals of TAGRISSO treatment. Withhold TAGRISSO if ejection fraction
decreases by 10% from pretreatment values and is less than 50%. For symptomatic congestive heart failure or
persistent asymptomatic LV dysfunction that does not resolve within 4 weeks, permanently discontinue TAGRISSO
• Advise pregnant women of the potential risk to a fetus. Advise females of reproductive potential to use
effective contraception during TAGRISSO treatment and for 6 weeks after the final dose. Advise males with
female partners of reproductive potential to use effective contraception for 4 months after the final dose
• The most common adverse reactions (>20%) observed in TAGRISSO patients were diarrhea (42%), rash (41%),
dry skin (31%) and nail toxicity (25%)
INDICATION
TAGRISSO is indicated for the treatment of patients with metastatic epidermal growth factor receptor (EGFR)
T790M mutation-positive non-small cell lung cancer (NSCLC), as detected by an FDA-approved test, who have
progressed on or after EGFR tyrosine kinase inhibitor therapy.
This indication is approved under accelerated approval based on tumor response rate and duration of response.
Continued approval for this indication may be contingent upon verification and description of clinical benefit
in confirmatory trials.
Please see Brief Summary of full Prescribing Information on adjacent pages.
References: 1. Yu HA, Arcila ME, Rekhtman N, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI
therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013;19:2240-2247. 2. TAGRISSO [package
insert]. Wilmington, DE: AstraZeneca Pharmaceuticals LP; 2015.
TAGRISSO is a trademark of the AstraZeneca group of companies.
©2016 AstraZeneca. All rights reserved. 3216302 4/16
MLO201608_AD Astra-Zeneca-Spread.indd 7
7/8/2016 12:51:01 PM
TAGRISSOTM (osimertinib) tablet, for oral use
Brief Summary of Prescribing Information.
For complete prescribing information consult official package insert
INDICATIONS AND USAGE
TAGRISSO is indicated for the treatment of patients with metastatic epidermal growth factor
receptor (EGFR) T790M mutation-positive non-small cell lung cancer (NSCLC), as detected by an
FDA-approved test, who have progressed on or after EGFR tyrosine kinase inhibitor (TKI) therapy.
This indication is approved under accelerated approval based on tumor response rate and duration
of response [see Clinical Studies (14) in the full Prescribing Information]. Continued approval
for this indication may be contingent upon verification and description of clinical benefit in
confirmatory trials.
DOSAGE AND ADMINISTRATION
Patient Selection
Confirm the presence of a T790M EGFR mutation in tumor specimens prior to initiation of treatment
with TAGRISSO [see Indications and Usage (1) and Clinical Studies (14) in the full Prescribing
Information]. Information on FDA-approved tests for the detection of T790M mutations is available
at http://www.fda.gov/companiondiagnostics.
Recommended Dosage Regimen
The recommended dose of TAGRISSO is 80 mg tablet once a day until disease progression or
unacceptable toxicity. TAGRISSO can be taken with or without food.
If a dose of TAGRISSO is missed, do not make up the missed dose and take the next dose as
scheduled.
Administration to Patients Who Have Difficulty Swallowing Solids
Disperse tablet in 4 tablespoons (approximately 50 mL) of non-carbonated water only. Stir until
tablet is completely dispersed and swallow or administer through naso-gastric tube immediately. Do
not crush, heat, or ultrasonicate during preparation. Rinse the container with 4 to 8 ounces of water
and immediately drink or administer through the naso-gastric tube [see Clinical Pharmacology
(12.3) in the full Prescribing Information].
Dose Modification for Adverse Reactions
Table 1
Recommended Dose Modifications for TAGRISSO
Target
Organ
Pulmonary
Cardiac
Other
Adverse Reactiona
Interstitial lung disease
(ILD)/Pneumonitis
QTc† interval greater than
500 msec on at least 2 separate ECGsb
b
c
†
Withhold TAGRISSO until QTc interval
is less than 481 msec or recovery to
baseline if baseline QTc is greater than
or equal to 481 msec, then resume at
40 mg dose.
Permanently discontinue TAGRISSO.
QTc interval prolongation with signs/
symptoms of life threatening arrhythmia
Asymptomatic, absolute decrease
Withhold TAGRISSO for up to 4 weeks.
in LVEFc of 10% from baseline and
• If improved to baseline LVEF, resume.
below 50%
• If not improved to baseline,
permanently discontinue.
Symptomatic congestive heart failure Permanently discontinue TAGRISSO.
Grade 3 or higher adverse reaction
Withhold TAGRISSO for up to 3 weeks.
If improvement to Grade 0-2 within
Resume at 80 mg or 40 mg daily.
3 weeks
If no improvement within 3 weeks
a
Dose Modification
TAGRISSO
N=411
Adverse reactions graded by the National Cancer Institute Common Terminology Criteria for Adverse Events
version 4.0 (NCI CTCAE v4.0).
ECGs = Electrocardiograms
LVEF = Left Ventricular Ejection Fraction
QTc = QT interval corrected for heart rate
CONTRAINDICATIONS
None.
WARNINGS AND PRECAUTIONS
Interstitial Lung Disease/Pneumonitis
Across clinical trials, interstitial lung disease (ILD)/pneumonitis occurred in 3.3% (n=27) of
TAGRISSO treated patients (n=813); 0.5% (n=4) were fatal.
Withhold TAGRISSO and promptly investigate for ILD in any patient who presents with worsening
of respiratory symptoms which may be indicative of ILD (e.g., dyspnea, cough and fever).
Permanently discontinue TAGRISSO if ILD is confirmed [see Dosage and Administration (2.4) and
Adverse Reactions (6) in the full Prescribing Information].
QTc Interval Prolongation
The heart rate-corrected QT (QTc) interval prolongation occurs in patients treated with TAGRISSO.
Of the 411 patients in Study 1 and Study 2, one patient (0.2%) was found to have a QTc greater than
500 msec, and 11 patients (2.7%) had an increase from baseline QTc greater than 60 msec [see
Clinical Pharmacology (12.2) in the full Prescribing Information].
In Study 1 and 2, patients with baseline QTc of 470 msec or greater were excluded. Conduct
periodic monitoring with ECGs and electrolytes in patients with congenital long QTc syndrome,
congestive heart failure, electrolyte abnormalities, or those who are taking medications known to
prolong the QTc interval. Permanently discontinue TAGRISSO in patients who develop QTc interval
prolongation with signs/symptoms of life threatening arrhythmia [see Dosage and Administration
(2.4) in the full Prescribing Information].
Cardiomyopathy
Across clinical trials, cardiomyopathy (defined as cardiac failure, pulmonary edema, ejection
fraction decreased or stress cardiomyopathy) occurred in 1.4% (n=11) of TAGRISSO treated
patients (n=813); 0.2% (n=2) were fatal.
In Study 1 and Study 2, Left Ventricular Ejection Fraction (LVEF) decline >10% and a drop to <50%
occurred in 2.4% (9/375) of patients who had baseline and at least one follow up LVEF assessment.
MLO201608_AD Astra-Zeneca-D1.indd 8
Assess LVEF by echocardiogram or multigated acquisition (MUGA) scan before initiation of
TAGRISSO and then at 3 month intervals while on treatment. Withhold treatment with TAGRISSO
if ejection fraction decreases by 10% from pretreatment values and is less than 50%. For
symptomatic congestive heart failure or persistent, asymptomatic LV dysfunction that does not
resolve within 4 weeks, permanently discontinue TAGRISSO [see Dosage and Administration (2.4)
in the full Prescribing Information].
Embryo-Fetal Toxicity
Based on data from animal studies and its mechanism of action, TAGRISSO can cause fetal harm
when administered to a pregnant woman. In animal reproduction studies, osimertinib caused postimplantation fetal loss when administered during early development at a dose exposure 1.5 times
the exposure at the recommended human dose. When males were treated prior to mating with
untreated females, there was an increase in preimplantation embryonic loss at plasma exposures of
approximately 0.5-times those observed in patients at the 80 mg dose level.
Advise pregnant women of the potential risk to a fetus.
Advise females of reproductive potential to use effective contraception during treatment with
TAGRISSO and for 6 weeks after the final dose. Advise males with female partners of reproductive
potential to use effective contraception for 4 months after the final dose [see Use in Specific
Populations (8.1), (8.3) and Clinical Pharmacology (12.3) in the full Prescribing Information].
ADVERSE REACTIONS
The following adverse reactions are discussed in greater detail in other sections of the labeling:
Interstitial Lung Disease/Pneumonitis [see Warnings and Precautions (5.1) in the full Prescribing
Information]
QTc Interval Prolongation [see Warnings and Precautions (5.2) in the full Prescribing Information]
Clinical Trials Experience
Because clinical trials are conducted under widely varying conditions, adverse reaction rates
observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of
another drug and may not reflect the rates observed in practice.
The data described below reflect exposure to TAGRISSO (80 mg daily) in 411 patients with EGFR
T790M mutation-positive non-small cell lung cancer who received prior EGFR TKI therapy, in two
single arm studies, Study 1 and Study 2. Patients with a past medical history of ILD or radiation
pneumonitis that required steroid treatment, serious arrhythmia or baseline QTc interval greater
than 470 ms were excluded from Study 1 and Study 2. Baseline patient and disease characteristics
were: median age 63 years, 13% of patients were ≥75 years old, female (68%), White (36%),
Asian (60%), metastatic (96%), sites of brain metastases (39%), World Health Organization (WHO)
performance status of 0 (37%) or 1 (63%), 1 prior line of therapy [EGFR-TKI treatment only, second
line, chemotherapy-naïve (31%)], 2 or more prior lines of therapy (69%). Of the 411 patients, 333
patients were exposed to TAGRISSO for at least 6 months; 97 patients were exposed for at least 9
months; however no patient was exposed to TAGRISSO for 12 months.
In Studies 1 and 2, the most common (>20%) adverse reactions (all grades) observed in TAGRISSOtreated patients were diarrhea (42%), rash (41%), dry skin (31%), and nail toxicity (25%). Dose
reductions occurred in 4.4% of patients treated with TAGRISSO. The most frequent adverse
reactions that led to dose reductions or interruptions were: electrocardiogram QTc prolonged
(2.2%) and neutropenia (1.9%). Serious adverse reactions reported in 2% or more patients were
pneumonia and pulmonary embolus. There were 4 patients (1%) treated with TAGRISSO who
developed fatal adverse reactions of ILD/pneumonitis. Other fatal adverse reactions occurring in
more than 1 patient included pneumonia (4 patients) and CVA/cerebral hemorrhage (2 patients).
Discontinuation of therapy due to adverse reactions occurred in 5.6% of patients treated with
TAGRISSO. The most frequent adverse reactions that led to discontinuation were ILD/pneumonitis
and cerebrovascular accidents/infarctions.
Tables 2 and 3 summarize the common adverse reactions and laboratory abnormalities observed
in TAGRISSO-treated patients.
Table 2
Adverse Reactions (>10% for all NCI CTCAE* Grades or >2% for Grades 3-4)
in Study 1 and Study 2
Adverse Reaction
Gastrointestinal disorders
Diarrhea
Nausea
Decreased appetite
Constipation
Stomatitis
Skin disorders
Rasha
Dry skinb
Nail toxicityc
Pruritus
Eye Disordersd
Respiratory
Cough
General
Fatigue
Musculoskeletal
Back pain
Central Nervous System
Headache
Infections
Pneumonia
Vascular events
Venous thromboembolisme
All Grades
%
Grade 3-4f
%
42
17
16
15
12
1.0
0.5
0.7
0.2
0
41
31
25
14
18
0.5
0
0
0
0.2
14
0.2
14
0.5
13
0.7
10
0.2
4
2.2
7
2.4
* NCI CTCAE v4.0.
7/8/2016 12:49:56 PM
TAGRISSOTM (osimertinib) tablet, for oral use
a
b
c
d
e
f
Includes cases reported within the clustered terms for rash adverse events: Rash, rash generalized, rash
erythematous, rash macular, rash maculo-papular, rash papular, rash pustular, erythema, folliculitis, acne,
dermatitis and acneform dermatitis.
Includes dry skin, eczema, skin fissures, xerosis.
Includes nail disorders, nail bed disorders, nail bed inflammation, nail bed tenderness, nail
discoloration, nail disorder, nail dystrophy, nail infection, nail ridging, onychoclasis, onycholysis,
onychomadesis, paronychia.
Includes dry eye, vision blurred, keratitis, cataract, eye irritation, blepharitis, eye pain, lacrimation
increased, vitreous floaters. Other ocular toxicities occurred in <1% of patients.
Includes deep vein thrombosis, jugular venous thrombosis, and pulmonary embolism.
No grade 4 events have been reported.
Additional clinically significant adverse reactions occurring in 2% or more of patients treated with
TAGRISSO included cerebrovascular accident (2.7%).
Table 3
Common Laboratory Abnormalities (>20% for all NCI CTCAE Grades)
in Study 1 and Study 2
Laboratory Abnormality
Clinical Chemistry
Hyponatremia
Hypermagnesemia
Hematologic
Lymphopenia
Thrombocytopenia
Anemia
Neutropenia
a
TAGRISSO
N=411
Change from Baseline
Change from Baseline to
Grade 3 or Grade 4 (%)a
All Grades (%)
26
20
3.4
0.7
63
54
44
33
3.3
1.2a
0.2
3.4
The only grade 4 laboratory abnormality was 1 patient with grade 4 thrombocytopenia.
DRUG INTERACTIONS
Drug interaction studies with inhibitors, inducers or substrates of CYP enzymes and transporters
have not been conducted with TAGRISSO.
Effect of Other Drugs on Osimertinib
Strong CYP3A Inhibitors
Avoid concomitant administration of TAGRISSO with strong CYP3A inhibitors, including macrolide
antibiotics (e.g., telithromycin), antifungals (e.g., itraconazole), antivirals (e.g., ritonavir),
nefazodone, as concomitant use of strong CYP3A inhibitors may increase osimertinib plasma
concentrations. If no other alternative exists, monitor patients more closely for adverse reactions
of TAGRISSO [see Dosage and Administrations (2.4) and Clinical Pharmacology (12.3) in the full
Prescribing Information].
Strong CYP3A Inducers
Avoid concomitant administration of TAGRISSO with strong CYP3A inducers (e.g., phenytoin,
rifampicin, carbamazepine, St. John’s Wort) as strong CYP3A inducers may decrease osimertinib
plasma concentrations [see Clinical Pharmacology (12.3) in the full Prescribing Information].
Effect of Osimertinib on Other Drugs
Avoid concomitant administration of TAGRISSO with drugs that are sensitive substrates of CYP3A,
breast cancer resistance protein (BCRP), or CYP1A2 with narrow therapeutic indices, including
but not limited to fentanyl, cyclosporine, quinidine, ergot alkaloids, phenytoin, carbamazepine,
as osimertinib may increase or decrease plasma concentrations of these drugs [see Clinical
Pharmacology (12.3) in the full Prescribing Information].
USE IN SPECIFIC POPULATIONS
Pregnancy
Risk Summary
Based on data from animal studies and its mechanism of action, TAGRISSO can cause fetal harm
when administered to a pregnant woman. There are no available data on TAGRISSO use in pregnant
women. Administration of osimertinib to pregnant rats was associated with embryolethality and
reduced fetal growth at plasma exposures 1.5 times the exposure at the recommended human dose
[see Data]. Advise pregnant women of the potential risk to a fetus.
In the U.S. general population, the estimated background risk of major birth defects and miscarriage
in clinically-recognized pregnancies is 2% to 4% and 15% to 20%, respectively.
Data
Animal Data
When administered to pregnant rats prior to embryonic implantation through the end of
organogenesis (gestation days 2-20) at a dose of 20 mg/kg/day, which produced plasma exposures
of approximately 1.5 times the clinical exposure, osimertinib caused post-implantation loss and
early embryonic death. When administered to pregnant rats from implantation through the closure
of the hard palate (gestation days 6 to 16) at doses of 1 mg/kg/day and above (0.1-times the AUC
observed in patients at the recommended dose of 80 mg), an equivocal increase in the rate of
fetal malformations and variations was observed in treated litters relative to those of concurrent
controls. When administered to pregnant dams at doses of 30 mg/kg/day during organogenesis
through lactation Day 6, osimertinib caused an increase in total litter loss and postnatal death. At
a dose of 20 mg/kg/day, osimertinib administration during the same period resulted in increased
postnatal death as well as a slight reduction in mean pup weight at birth that increased in magnitude
between lactation days 4 and 6.
Lactation
Risk Summary
There are no data on the presence of osimertinib in human milk, the effects of osimertinib on the
breastfed infant or on milk production. Administration to rats during gestation and early lactation
was associated with adverse effects, including reduced growth rates and neonatal death [see Use in
MLO201608_AD Astra-Zeneca-D2.indd 9
2
Specific Populations (8.1) in the full Prescribing Information]. Because of the potential for serious
adverse reactions in breastfed infants from osimertinib, advise a lactating woman not to breastfeed
during treatment with TAGRISSO and for 2 weeks after the final dose.
Females and Males of Reproductive Potential
Contraception
Females
Advise females of reproductive potential to use effective contraception during treatment with
TAGRISSO and for 6 weeks after the final dose [see Use in Specific Populations (8.1) in the full
Males
Advise male patients with female partners of reproductive potential to use effective contraception
during and for 4 months following the final dose of TAGRISSO [see Nonclinical Toxicology (13.1)
Infertility
Based on animal studies, TAGRISSO may impair fertility in females and males of reproductive
potential. It is not known if the effects on fertility are reversible [see Nonclinical Toxicology (13.1)
Pediatric Use
The safety and effectiveness of TAGRISSO in pediatric patients have not been established.
Geriatric Use
One hundred eighty-seven (45%) of the 411 patients in clinical trials of TAGRISSO were 65 years
of age and older, and 54 patients (13%) were 75 years of age and older. No overall differences
in effectiveness were observed based on age. Exploratory analysis suggest a higher incidence of
Grade 3 and 4 adverse reactions (32% versus 25%) and more frequent dose modifications for
adverse reactions (23% versus 17%) in patients 65 years or older as compared to those younger
than 65 years.
Renal Impairment
No dedicated clinical studies have been conducted to evaluate the effect of renal impairment on
the pharmacokinetics of osimertinib. Based on population pharmacokinetic analysis, no dose
adjustment is recommended in patients with mild [creatinine clearance (CLcr) 60-89 mL/min] or
moderate (CLcr 30-59 mL/min) renal impairment. There is no recommended dose of TAGRISSO for
patients with severe renal impairment (CLcr <30 mL/min) or end-stage-renal disease [see Clinical
Pharmacology (12.3) in the full Prescribing Information].
Hepatic Impairment
No dedicated clinical studies have been conducted to evaluate the effect of hepatic impairment on
the pharmacokinetics of osimertinib. Based on population pharmacokinetic (PK) analysis, no dose
adjustment is recommended in patients with mild hepatic impairment [total bilirubin <upper limit
of normal (ULN) and AST between 1 to 1.5 times ULN or total bilirubin between 1.0 to 1.5 times
ULN and any AST]. There is no recommended dose for TAGRISSO for patients with moderate or
severe hepatic impairment [see Clinical Pharmacology (12.3) in the full Prescribing Information].
17 PATIENT COUNSELING INFORMATION
Advise the patient to read the FDA-approved patient labeling (Patient Information).
Interstitial Lung Disease/Pneumonitis
Inform patients of the risks of severe or fatal ILD, including pneumonitis. Advise patients to contact
their healthcare provider immediately to report new or worsening respiratory symptoms [see
Warnings and Precautions (5.1) in the full Prescribing Information].
QTc Interval Prolongation
Inform patients of symptoms that may be indicative of significant QTc prolongation including
dizziness, lightheadedness, and syncope. Advise patients to report these symptoms and to inform
their physician about the use of any heart or blood pressure medications [see Warnings and
Precautions (5.2) in the full Prescribing Information].
Cardiomyopathy
% TAGRISSO can cause cardiomyopathy. Advise patients to immediately report any signs or
symptoms of heart failure to their healthcare provider [see Warnings and Precautions (5.3) in
the full Prescribing Information].
Embryo-Fetal Toxicity
% TAGRISSO can cause fetal harm if taken during pregnancy. Advise pregnant women of the
potential risk to a fetus.
% Advise females to inform their healthcare provider if they become pregnant or if pregnancy is
suspected, while taking TAGRISSO [see Warnings and Precautions (5.3) and Use in Specific
Populations (8.1) in the full Prescribing Information].
Females and Males of Reproductive Potential
% Advise females of reproductive potential to use effective contraception during treatment with
TAGRISSO and for 6 weeks after the final dose [see Use in Specific Populations (8.3) in the full
% Advise males to use effective contraception during treatment and for 4 months after the final
dose of TAGRISSO [see Use in Specific Populations (8.3) in the full Prescribing Information].
Lactation
Advise women not to breastfeed during treatment with TAGRISSO and for 2 weeks after the final
dose [see Use in Specific Populations (8.2) in the full Prescribing Information].
Distributed by:
AstraZeneca Pharmaceuticals LP
Wilmington, DE 19850
TAGRISSO is a trademark of the AstraZeneca group of companies
©
AstraZeneca 2015
3182206 11/15 Issued 11/15
7/8/2016 12:50:22 PM
NE W S
T R ENDS
Infectious Diseases
Monkey study shows Zika infection
prolonged in pregnancy. University of
Wisconsin–Madison researchers studying
monkeys have shown that one infection
with Zika virus protects against future infection, though pregnancy may drastically
prolong the time the virus stays in the
body. The researchers published a study
describing their work establishing rhesus
macaque monkeys as a model for studying the way Zika virus infections may
progress in people.
The researchers infected monkeys
with the type of Zika virus causing an epidemic that first became apparent in South
America in 2015, and found that those
monkeys resisted infection by the same
Zika strain 10 weeks later.
But their findings also uncovered a
stark contrast in the length of infection of
pregnant monkeys versus males and nonpregnant females. Though non-pregnant
animals in the study were found to be
free of Zika virus within 10 days of infection, the virus persisted in the blood of
pregnant monkeys for 30 days to 70 days.
The prolonged infection has implications
for the severe impacts of Zika virus during
pregnancy. One possible explanation for
the persistence of the virus in pregnancy
is that the immune systems of mothers-tobe are compromised, and they aren’t able
to clear the virus as fast.
Though the researchers have been
performing ultrasounds on Zika-infected
pregnant monkeys and collecting fluid
from their wombs, they can’t yet say
whether the still-growing fetuses themselves are infected or whether any of them
are developing microcephaly.
New study sheds light on how some
survive Ebola. A first-of-its-kind Ebola
study has yielded clues to how some
people are able to survive the deadly virus and suggests possible avenues for
treatments that could save more lives.
Researchers at the U.S. Centers for Disease
Control and Prevention (CDC), Emory University School of Medicine, and University
of Nebraska Medical Center analyzed the
immune responses of Ebola patients treated in the United States. Their study was
recently published online in the Clinical
Infectious Disease Journal.
This is the first time researchers have
been able to study Ebola virus disease
(EVD) using samples taken from patients
during both their illness and recovery.
In the seven U.S. patients, researchers
tracked 54 different markers of immunesystem activity from hospital admission
until the day of discharge. Among the
10
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A N A LYSIS
seven patients, five had moderate EVD
and two had severe EVD requiring
mechanical ventilation and dialysis.
“We were able to identify the particular
components of patients’ immune systems
that successfully fought off the virus,”
says lead author Anita McElroy, MD, PhD.
“These are the parts of the immune system that we need to tap into to develop
new therapies.”
The results show patients with severe
EVD had high levels of virus in their blood
and out-of-control immune responses,
leading to destruction of healthy tissues,
multisystem organ failure, shock, and, in
most cases, death. In contrast, patients
with moderate EVD had strong, healthy
immune responses that were able to control the virus. All patients with moderate
illness and one patient with severe illness
survived.
It is unclear why some people’s immune
systems respond more effectively to viruses. Possible contributing factors include
genetics and whether or not a person has
other illnesses or conditions. Identifying
which parts of the immune system malfunction in severe EVD cases as well as the
parts that function well in moderate cases
could lead to the design of therapies that
might theoretically inhibit the disease’s
progression.
Cancer
HPV vaccine reduced cervical abnormalities in young women. Young women who
received the human papillomavirus (HPV)
vaccine through a school-based program
had fewer cervical cell anomalies when
screened for cervical cancer, found a
new study in CMAJ (Canadian Medical
Association Journal).
“Eight years after a school-based HPV
vaccination program was initiated in
Alberta [Canada], three-dose HPV vaccination has demonstrated early benefits,
particularly against high-grade cervical
abnormalities, which are more likely to
progress to cervical cancer,” writes study
co-author Huiming Yang, MD.
Alberta has both a school-based HPV
vaccination program and a populationbased screening program for cervical
cancer. In 2008, the province introduced
HPV vaccination for Grade 5 girls (aged
10-11) and a three-year catch-up program
for Grade 9 girls (aged 14-15); in 2014, it
was expanded to include boys. The program provides three doses of the vaccine
that protects against two strains of HPV,
which account for 70 percent of all cases of
cervical cancer.
To determine whether HPV vaccination
had an impact on Papanicolaou (Pap) test
results, researchers looked at data on the
first cohort of women who participated in
both the school vaccination program and
cervical cancer screening. Of 10,204 women born between 1994 and 1997, 1,481 (14.5
percent) were cases—that is, they had cervical anomalies detected during screening
—and the remaining 8,723 (85.5 percent)
were controls—with no cervical abnormalities detected. Among cases, most (1,384,
93.5 percent) had low-grade cervical abnormalities, and the remaining 97 (6.5
percent) had high-grade abnormalities.
Fifty-six percent of study participants
were unvaccinated, and 44 percent had
received one or more doses of the HPV
vaccine before being screened for cervical
cancer. Of the women who had been vaccinated, 84 percent received three or more
doses. Among the unvaccinated women,
16.1 percent had cervical abnormalities,
compared with 11.8 percent in the fully
vaccinated group.
Coagulation
Blood coagulation detector may help in
monitoring stroke risk. Researchers at
Tokyo Medical and Dental University have
shown that an analyzer recently developed to measure blood coagulability has
the sensitivity to detect hypercoagulatibility associated with stroke risk in those without atrial fibrillation (AF), a common risk
factor for stroke.The study was reported in
PLOS ONE.
To estimate the risk in AF patients and
determine the requirement for anticoagulation therapy, the CHADS2 predictive
score was used. Because some parts of
this score are also associated with atherosclerosis risk and increased blood coagulability, a high score has been proposed as
linked to hypercoagulability (an increased
tendency for blood clotting) in both AF and
non-AF patients. However, this association has not been fully investigated, partly
owing to the lack of a sensitive means of
detection. The researchers used a highly
sensitive technique to measure small
changes in blood coagulation, and found
hypercoagulability in non-AF patients with
high CHADS2 scores.
Several physical and chemical factors affect blood clotting; some can be measured
over time to determine blood coagulability
and likelihood of clot formation. Dielectric
blood coagulometry (DBCM) is a recently
developed test that measures changes in
the dielectric permittivity of whole blood,
representing clumping of red blood cells.
The researchers used DBCM to detect
changes in the dielectric permittivity of
whole blood at 10 MHz. Comparisons between untreated blood and that with added heparin (a blood thinner) or tissue factor (a blood-clotting accelerator) enabled
derivation of a coagulability index.
“We calculated the end of acceleration
AUGUST 2016
7/12/2016 9:00:55 AM
NE W S
time (EAT) as an index of coagulability
from temporal changes in dielectric permittivity,” coauthor Satomi Hamada says.
“This value reduced when tissue factor
was added, and increased with heparin
present. It was sensitive enough to detect
small changes in coagulability, particularly
in hypercoagulability.”
The researchers found that patients receiving warfarin had a significantly longer
EAT than those without, confirming the
anticoagulation effect. They also showed
that patients with high CHADS2 scores
had a significantly shorter EAT that represented hypercoagulability compared with
patients with lower scores.
Genetics/Genomics
Women with BRCA1 gene mutation
at higher risk of deadly uterine cancer.
Women who carry the BRCA1 gene mutation that dramatically increases their risk
of breast and ovarian cancers are also
at higher risk for a lethal form of uterine
cancer, according to a study led by a Duke
Cancer Institute researcher.
This newly defined risk, the first to show
a conclusive link between the BRCA1
gene mutation and a small but significant
chance of developing an aggressive uterine cancer, could become a consideration
in weighing treatment options.
Currently, women with the BRCA1 mutation often have preventive surgeries to
remove both breasts, as well as their ovaries and fallopian tubes, based on studies
showing that the gene mutation elevates
their risk for cancers in those organs. But
conflicting evidence has created controversy over the need to remove the uterus.
Smaller studies identified a link between
the gene mutation and uterine cancer, but
a larger study had been lacking until now.
Says lead author Noah D. Kauff, MD,
“Our study presents the strongest evidence to date that women with this genetic mutation should at least discuss
with their doctors the option of having a
hysterectomy along with removal of their
ovaries and fallopian tubes.”
In the research, published in JAMA
Oncology, Kauff and colleagues from nine
other institutions analyzed data from 1,083
women. All had BRCA1 or BRCA2 genetic
mutations, had undergone removal of
their ovaries and fallopian tubes, and were
followed for a median 5.1 years. Incidences of uterine cancer in the BRCA-positive
women in the study were compared to the
rates that would be expected in the general population, based on data from the U.S.
government’s Surveillance, Epidemiology,
and End Results program.
Among the BRCA-positive women,
eight uterine cancers were reported within
the study period—a rate that was slightly
T R ENDS
higher but not statistically different than
the national norm. Of those cancers, however, five were of an uncommon, very
aggressive subtype called serous endometrial cancer. All but one of the serous
endometrial cancers occurred in women
with the BRCA1 genetic mutation.
Given the incidence of this cancer in
the wider population, only about .18 cases
would be expected among women with
the BRCA1 mutation over the time period
analyzed, meaning the women with the
BRCA1 trait were at significantly higher
risk.
New Assays
FDA clears test for genetic markers for
antibiotic-resistant bacteria. The FDA has
cleared for marketing the Xpert Carba-R
Assay, an infection control aid that tests
patient specimens to detect specific
genetic markers associated with bacteria that are resistant to Carbapenem
antibiotics.
Carbapenem antibiotics are widely
used in hospitals to treat severe infections. These resistant organisms are
commonly referred to as Carbapenemresistant Enterobacteriaceae, or CRE, and
have been reported in almost all states
within the United States.
Current methods to identify colonization with CRE or other resistant organisms rely on growing bacteria from fecal
material in cultures, which are then subjected to antimicrobial susceptibility testing to determine in vitro susceptibility to
antimicrobial agents.
The Xpert Carba-R Assay tests specimens directly taken from patients, which
are usually obtained by rectal swabs, for
the presence of five different genetic markers that are associated with carbapenemase, the enzyme produced by CRE.
The assay is intended as an aid in infection control and can be used in conjunction
with other clinical and laboratory findings.
Although it tests for the most prevalent
carbapenemase genes associated with resistance to carbapenem antibiotics, it does
not detect the bacteria, carbapenemase
activity, or other possible non-enzymatic
causes of carbapenem resistance. The
Xpert Carba-R Assay tests only for genetic
material.
The assay also does not detect all types
of carbapenemase genes, and it is important to recover bacteria for accurately
tracking the spread of carbapenem resistance. Labs should continue to perform
standard bacterial culture in conjunction
with it. In addition, concomitant cultures
are necessary to recover organisms for
epidemiological typing, antimicrobial susceptibility testing, and for confirmatory
bacterial identification.
A N A LYSIS
Lab-tested diagnosis needed when treating patients with persistent diarrhea.
Persistent diarrhea, which is diarrhea
that lasts at least 14 days, is an illness
typically caused by parasites or bacteria
and requires accurate diagnosis in order
to determine what treatment to give, according to Herbert L. DuPont, MD, director of the Center for Infectious Diseases
at The University of Texas Health Science
Center at Houston (UTHealth) School of
Public Health.
In a literature review published recently in JAMA, DuPont advises medical
practitioners to be alert when diagnosing persistent versus acute diarrhea in
patients. It is common for doctors not to
focus on how many days their patients
have had diarrhea, he adds.
“I’d like to educate doctors about the
importance of taking the history and
assessing duration of illness,” DuPont
says. For acute diarrhea, the lab has a
minimal role, restricted to patients passing bloody stools. If a patient has had
diarrhea for two weeks or more, the doctor should focus on the cause of the disease through laboratory testing, with an
emphasis on parasites.”
Acute diarrhea lasts less than two
weeks, and it is typically caused by viruses or toxins. Persistent diarrhea is most
commonly caused by bacteria or parasites, including Giardia, Cryptosporidium and Cyclospora. Less common parasites include Entamoeba Cystoisospora
belli, Dientamoeba fragilis, Strongloides
stercoralis, and microsporidia. These
parasites, all of which can be detected
in laboratory testing, can be contracted
through food or water or from other people, and are commonly picked up while
traveling.
A new testing method called multiplex
polymerase chain reaction (PCR) was
developed within the past year. This simultaneous, single test identifies unique
DNA sequences to detect a panel of
causes of diarrhea. Two platforms have
been approved by the U.S. Food and
Drug Administration for use.
Previously, researchers would culture
bacteria from a stool sample and examine it for isolated bacteria. For parasites,
they would either look under a microscope or, for three of the parasites, use
commercial enzyme immunoassays.
Both methods were only able to identify
a small number of parasites.
Clarification: The July 2016 Clinical Issues
article, “Automated analyzers add efficiency
to laboratory testing” by Maria Luz Rodriguez, PhD, incorrectly placed the subhead
that read “Microarray-based technologies”
on page 32. Liquid chromatography and
mass spectrometry as well as laboratory
automation for other fields are not under
this subhead.
AUGUST 2016
MLO201608-Observatory_FINAL.indd 11
THE OBSERVATORY
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11
7/13/2016 9:05:05 AM
By Liana F. Romero, PhD, MBA, MT(ASCP)
T
he Affordable Care Act and population management
have significantly changed how diabetes screening is
viewed. In today’s preventive care environment, healthcare providers understand that staying ahead of the progression of the disease and associated comorbidities, minimizing
the financial implications of disease progression, and improving patient outcomes and quality of life are dependent
on the proactive screening for and management of at-risk
patients. Laboratory and point-of-care testing (POCT), therefore, play a critical role and bring significant value in the
screening, monitoring, and management of these patients.
In this article, we will review the physiological implications
of diabetes, risk factors, and importance of population management. We will conclude with an overview of the current
Continuing Education
To earn CEUs, see test on page 20 or online at
www.mlo-online.com under the CE Tests tab.
LEARNING OBJECTIVES
Upon completion of this article, the reader will be
able to:
1. List factors that are important in controlling the
at-risk management of diabetes.
2. Describe symptoms, causes, and complications of
various types of diabetes.
3. Describe trends, statistics, and healthcare cost
dilemmas in regard to diabetes.
4. Discuss programs that have led to standardization
and improvement among a variety of HbA1c testing
methods and the outcomes.
12
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MLO201608-CE Story_MECH_AL.indd 12
AUGUST 2016
practices for screening, diagnosis, and monitoring and
how the laboratory and POCT add value in the context of
healthcare reform initiatives.
Physiologically, the condition of diabetes (diabetes mellitus) is characterized by elevated blood glucose or hyperglycemia, primarily driven by the deficiency of insulin production
in the beta cells of the pancreatic islets or the inability of cells
to respond to insulin that is produced.1,2 Elevated blood glucose is often accompanied by symptoms that include frequent
urination and increased thirst and hunger. If the diabetes
goes undiagnosed and untreated, short term, acute complications include diabetic ketoacidosis and hyperglycemic coma.
However, the severity, complexity, and mortality of long-term
complications of diabetes are of most concern from a chronic
disease or population management perspective. These conditions include chronic kidney failure, cardiovascular disease,
stroke, ulcerations and wounds in the lower extremities, and
eye diseases including cataracts, retinopathy, and glaucoma.3,4
The National Diabetes Statistics Report (2014) from the
Centers for Disease Control and Prevention (CDC) lasers in
on the ever-increasing burden of diabetes. A key statistic to
note is the estimated 9.3 percent of the population of the
United States—29.1 million individuals—who have diabetes.5 Of those individuals, 21 million have been diagnosed,
but more than eight million remain undiagnosed and therefore untreated. In 2012, diabetes and its related complications accounted for $245 billion in total medical costs and
lost work and wages. This figure is up from $174 billion in
2007.5
The estimated number of individuals 20 years of age or
older who are pre-diabetic is 86 million, versus 79 million in
2011, with these individuals being at increased risk of developing multiple chronic diseases and conditions over their
lifetime.5 Indeed, the prevalence of pre-diabetes, diabetes,
and associated chronic diseases has doubled over the past
two decades, primarily as a result of the burden of obesity
across the U.S. (Figure 1, pg. 14).6
continued on page 14
7/12/2016 4:01:25 PM
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HbA1c
continued from page 12
Types of diabetes
Type 1 diabetes was previously called insulindependent diabetes mellitus or juvenile-onset
diabetes. Although disease onset can occur at
any age, most cases are diagnosed in patients’
mid-teens. In a type 1 diabetic individual,
the body’s immune system has attacked and
destroyed the beta cells, incapacitating the
pancreas’s ability to make the amount of insulin needed to moderate glucose levels in
the body.
There is no way to prevent the onset of
type 1 diabetes, but the disease is manageable. Treatment for type 1 diabetes includes
administration of insulin by injection, pump,
or orally; appropriate diet and exercise; and
monitoring and control of blood pressure and
lipid levels.5,7
Type 2 diabetes, which used to be called
adult-onset diabetes, can affect individuals
of all ages, but onset is most often seen in
middle-aged and older individuals. The risk
for developing disease increases among individuals who are overweight and inactive.
Increased prevalence of diabetes is also seen
among specific ethnic groups such as African
Figure 1. Age-adjusted prevalence of obesity and diagnosed diabetes among U.S. adults
Americans, Hispanics, and Native Amerimonitoring and control of blood pressure and lipid levels.5,7
cans, as well as among lower socio-economic groups in
An alarming trend across the U.S. is the increasing incicertain geographical regions of the U.S. (Figure 2, pg. 16).5,7,8
dence of type 2 diabetes among individuals < 20 years of
Type 2 diabetes usually begins with insulin resistance—a
age (Figure 3). Additionally, the SEARCH for Diabetes in
condition that occurs when fat, muscle, and liver cells do not
Youth study (SEARCH) estimated that in 2001 about 154,000
use insulin to carry glucose into the body’s cells to use for
individuals in the U.S. aged < 20 years were living with diaenergy.1,6 As a result, the body needs more insulin to help
betes, and that each year approximately 15,000 youth aged
glucose enter cells. Initially, the beta cells of the pancreas
< 20 years were diagnosed with type 1 diabetes and 3,700
will produce more insulin to manage the added demand. But
with type 2 diabetes).11 The most recent update from the
eventually the pancreas is no longer able to produce suffiSEARCH researchers, released in 2012, shows data that type
cient insulin when blood sugar levels increase, such as after
2 diabetes in 10-to-19 year-olds had increased 21 percent
meals.6,9 At this point, type 2 diabetes has ensued.
between 2001 and 2009.8,12
Treatment for type 2 diabetes includes use of diabetes
Gestational diabetes’ cause is not known, but the Amerimedications, dietary changes, and increased physical accan Diabetes Association (ADA) speculates that hormones
tivity (as diet and lack of exercise are recognized as highsecreted by the placenta block the action of the mother’s
risk factors for developing type 2 diabetes), along with the
insulin in the body, resulting in
insulin resistance and subsequent
buildup of glucose levels. A 2014
analysis by the CDC indicates
that the prevalence of gestational
diabetes is as high as 9.2 percent.12
Rare types of diabetes include
latent autoimmune diabetes in
adults (LADA), maturity onset
diabetes in the young (MODY),
cystic fibrosis–related diabetes
(CFRD), Cushing’s syndrome, and
antiretroviral-associated diabetes.
These diabetes types account for
one percent to five percent of all
diagnosed cases.13
Diabetes and chronic health
conditions
Figure 3. Increase in type 2 diabetes in adolescents over select years5,7
14
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Diabetes is illustrative of the
chronic health conditions that
are currently at the source of our
healthcare challenges. This is pri-
AUGUST 2016
7/12/2016 4:01:42 PM
marily driven by the complexity and severity of the longterm complications of diabetes. In the undiagnosed or
poorly managed patient, diabetes complications may be
disabling and eventually life-threatening. Complications
from diabetes include the following:14
• Cardiovascular disease, including coronary artery disease with
chest pain (angina), heart attack, stroke, and narrowing of arteries
(atherosclerosis).
• Nerve damage (neuropathy) leading to tingling, numbness,
burning or pain that usually begins at the tips of the toes or fingers and gradually spreads upward, ultimately resulting in loss
of sensation. For men, it may lead to erectile dysfunction.
• Kidney damage (nephropathy) that can lead to kidney failure or irreversible end-stage kidney disease, which may require
dialysis or a kidney transplant.
• Eye damage (retinopathy) resulting in damage to the blood
vessels of the retina (diabetic retinopathy), potentially leading to
blindness. Diabetes also increases the risk of other serious vision
conditions, such as cataracts and glaucoma.
• Foot damage as a result of nerve damage leading to cuts and
blisters which may become seriously infected and may ultimately
lead to toe, foot, or leg amputation.
• Skin conditions as a result of heighted susceptibility to bacterial
and fungal infections.
• Hearing impairment
• Alzheimer’s disease. (Type 2 diabetes may increase the risk.)
A study published in the Journal of Medical Economics
estimated the 2012 U.S. patient costs per acute event for the
following major complications:15
• Cardiovascular disease: myocardial infarction $56,445; ischemic
stroke $42,119; congestive heart failure $23,758; ischemic heart
disease $21,406; and transient ischemic attack $7,388
• Kidney disease: $71,714 for end stage renal disease
• Eye disease: $2,862 blindness
• Nerve/foot damage: $9,041 for lower extremity amputations
and $2,147 for diabetic foot ulcers.
Consider that, since 2012, healthcare costs have continued
to escalate along with the number of individuals who have
developed or are at risk for developing diabetes and associated chronic disease conditions as listed above. Through
population management efforts, patients with diabetes or
predisposition for diabetes can be identified, monitored,
and managed in order to control disease, minimize the risk
of complications, and reduce healthcare costs. And, indeed,
these efforts begin with the laboratory.
If your
HbA1c results are
just a number…
HbA1c in diagnosis and monitoring of diabetes
Hemoglobin A1c (HbA1c) is made by the non-enzymatic attachment of glucose to the N-terminal valine of the ß-chain
of hemoglobin. During the normal process of red blood
cells dying and breaking down, the HbA1c is released. Consequently, the level of HbA1c is reflective of the average
glucose concentration to which red blood cells were exposed over their eight-to-twelve- week life span.16 HbA1c
had been customarily employed in the monitoring of diabetic patients and in guiding therapy. But with the ADA’s
2010 endorsement to use HbA1c as the main criterion for
the diagnosis of type 2 diabetes, HbA1c was propelled
to the limelight for its utility in both the screening for
pre-diabetes and the diagnosis of diabetes.9,19,20
The endorsement by ADA was founded on data demonstrating that patients with HbA1c levels of 6.5 percent or
greater had the same rate of retinopathy as those diagnosed
using the standard criteria of fasting blood glucose and
glucose tolerance testing 9 (Table 1, page 18). HbA1c testing
www.sebia-usa.com
[email protected]
1.800.835.6497
Because patients deserve more.
7/12/2016 4:01:50 PM
HbA1c
Figure 2. Diagnosed diabetes rates by race/ethnicity (2012)10
also provided key advantages over blood glucose testing
including no requirement for fasting, sample draw convenience, and less susceptibility to variations in levels due to
patient health, and analytical stability for laboratory testing.19 Some disadvantages of HbA1c were also considered,
including increased cost and the potential to miss about 20
percent of patients with diabetes when compared to using
fasting glucose and glucose tolerance testing. Additionally,
HbA1c levels may vary due to patients’ race or ethnicity,
and for conditions with abnormal red cell turnover, such
as pregnancy, recent blood loss or transfusion, and some
anemias, the diagnosis of diabetes must rely on blood
glucose and not HbA1c. 19
The International Experts Committee (IEC) had also
recommended the use of HbA1c. However, the IEC recognized the lack of standardization that existed among the
100-plus methods available for HbA1c testing. The IEC recommended that the testing methodologies must be certified
by the National Glycohemoglobin Standardization Program
(NGSP) or traceable to the Diabetes Control and Complications Trial (DCCT) or UK Prospective Diabetes Study (UKPDS). 16,17 The goal of the IEC was to ensure that the methods were able to generate highly similar results on a given
blood sample. The NGSP was established by the American
Association of Clinical Chemistry (AACC) to execute the
protocol and establish the standardization process. The
subsequent improvement in performance of HbA1c led to
an increase in laboratories reporting HbA1c, from 50 percent in 1993 to approximately 99 percent by 2004, as well
as simultaneous improvements in accuracy and reduced
variability among laboratories.16,17
Laboratory-based HbA1c testing is well established,
and numerous immunoassay, enzymatic, and ion exchange HPLC methods for HbA1c testing have been certified by NGSP. As of 2015, three POCT methods have
also been certified.21 A primary advantage of HbA1c
POCT, as well as glucose POCT, is expediting clinical decision by allowing providers to more quickly assess the
efficacy of diabetes treatment and influence on health
outcomes (Figure 4). This aids in the collective effort to
lower a patient’s future HbA1c levels by enabling timely
therapeutic modifications.21
The NGSP certification for POCT HbA1c tests does not
mandate or account for end-user proficiency testing. How-
Figure 4. Improvement in outcomes utilizing HbA1c for monitoring glycemic control23
16
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AUGUST 2016
7/12/2016 4:01:59 PM
ever, proficiency testing materials are available from the
College of American Pathologists (CAP). It is up to the specific healthcare setting to make the determination to participate in a proficiency program. Proficiency testing of POCT
devices is recommended to ensure the integrity of test
results and reduce testing errors.23
It’s time to take
another look.
Alternate markers of glycemia
Fructosamine, glycated albumin, and 1,5-anhydroglucitol
(1,5-AG) are three other available markers for glycemia.24
• Fructosamine reflects the average blood sugar concentration
over the prior two to three weeks.
• Glycated albumin reflects mean glucose levels over the prior
two to three weeks.
• 1,5-AG levels are associated with rapid glucose fluctuations
and response within 24 hours.
For purposes of diagnosis and monitoring of diabetes, the
alternate markers of glycemia are recommended to be used
in conjunction with HbA1c. Presently, these markers may be
best suited for testing in specific subsets of diabetic patients,
such as those with variant hemoglobin, iron deficiency
anemia, pregnancy, and chronic liver or kidney disease.24
Toward population management of diabetes
The burden of diagnosed and undiagnosed diabetes on
the health of the U.S. population, coupled with the everescalating costs associated with disease management, treatment, and co-occurring conditions, can hardly be overstated. The CDC views the growing prevalence and incidence
of diabetes as one of the most important public health issues of our time. Consequently, the CDC has published
the “Compendium of Effective Public Health Strategies to
Prevent and Control Diabetes” to educate the population on
the consequences of diabetes and spearhead a call to action
for screening and lifestyle improvements.25
Simultaneously, the Centers for Medicare and Medicaid
Services (CMS) aims to address and curtail the burden of
disease through population management initiatives, including pro-active screening, improved monitoring, and
patient engagement in lifestyle changes. Risk factors such
as obesity, high blood pressure, family history of diabetes,
elevated lipid levels, and individuals over the age of 65 are
eligible for screening. Medicare, Medicaid, and private payers advocate the pro-active screening of the general population for type 2 diabetes using such tests as HbA1c and
blood glucose levels.
And so the laboratory and providers performing POCT
play an integral part in facilitating the testing required to
achieve the goals of the CDC, CMS, and private payers
in reducing the burden of diabetes in the U.S. Screening
for undiagnosed diabetes, management of diabetes, management of disease progression, and monitoring for cooccurring conditions and therapeutic efficacy are contingent on the availability and accessibility of standardized
and accurate HbA1c and blood glucose testing.
REFERENCES
1. Sonksen P, Sonksen J. Insulin: understanding its action in health and disease.
British Journal of Anaesthesia. 2000;85(1): 69–79.
2. Shoback D, Gardner DG, eds. Greenspan’s basic & clinical endocrinology 9th
ed. Chapter 17. New York: McGraw-Hill Medical. 2011.
3. World Health Organization (WHO). Diabetes fact sheet N 312. 2016. http://www.
who.int/mediacentre/factsheets/fs312/en/.
4. National Eye Institute. Facts about diabetic eye disease. 2016. l https://nei.nih.
gov/health/diabetic/retinopathy.
www.sebia-usa.com
[email protected]
1.800.835.6497
7/12/2016 4:02:08 PM
HbA1c
Glucose Testing and Interpretation
Normal
High Risk for Diabetes
Diabetes
FPG <100 mg/dL
IFG
FPG ≥100-125 mg/dL
FPG ≥126 mg/dL
2-h PG <140 mg/dL
IGT
2-h PG≥140-100 mg/dL
2-h PG≥200 mg/dL
Random PG ≥200 mg/dL +
symptoms
AIC <5.5%
5.5 to 6.4%
For screening of prediabetesa
≥6.5%
Secondaryb
Abbreviations: AIC = hemoglobin AIC: FPG = fasting plasma glucose: IFG = impaired fasting glucose: IGT = impaired glucose
tolerance; PG = plasma glucose.
a
AIC should be used only for screening prediabetes. The diagnosis of prediabetes, which may manifest as either IFG or IGT, should
be confirmed with glucose testing.
b
Glucose criteria are preferred for the diagnosis of diabetes mellitus (DM). In all cases, the diagnosis should be confirmed on a
separate day by repeating glucose or AIC testing. When AIC is used for diagnosis, follow-up glucose testing should be done when
possible to help manage DM.
Table 1. Criteria for diagnosis of diabetes mellitus20
7/12/2016 4:02:18 PM
5. Centers for Disease Control and Prevention. National diabetes statistics report,
2014. 2015. http://www.cdc.gov/diabetes/data/statistics/2014StatisticsReport.html.
6. John Hopkins Bloomberg School of Public Health. Pre-diabetes and diabetes
nearly double over the past two decades. 2014. http://www.jhsph.edu/news/newsreleases/2014/pre-diabetes-and-diabetes-nearly-double-over-the-past-two-decades.html.
Move beyond that
number and see
the whole story...
7. National Institute of Diabetes and Digestive and Kidney Diseases. Your guide to
diabetes: Type 1 and 2. 2014. http://www.niddk.nih.gov/health-information/healthtopics/Diabetes/your-guide-diabetes/Pages/index.aspx.
8. Imperatore G, Boyle J, Thompson TJ, et al. Projections of type 1 and type 2
diabetes burden in the U.S. population aged <20 years through 2050. Diabetes Care.
2012;35(12):2515-2520.
9. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2004;27(suppl 1), s5-s10.
10. American Diabetes Association. Statistics about diabetes. 2012. http://www.
diabetes.org/diabetes-basics/statistics/?referrer=https://www.google.com/.
11. Liese AD, D’Agostino RB Jr, Hamman RF, et al., SEARCH for Diabetes in Youth
Study Group. The burden of diabetes mellitus among US youth: prevalence estimates from the SEARCH for Diabetes in Youth Study. Pediatrics. 2006;118:1510–1518.
12. American Diabetes Association. What is gestational diabetes? http://www.diabetes.org/diabetes-basics/gestational/what-is-gestational-diabetes.html.
13. Centers for Disease Control and Prevention (CDC). Basics: diabetes. 2015. http://
www.cdc.gov/diabetes/basics/index.html.
14. Mayo Clinic. Diabetes complications. http://www.mayoclinic.org/diseasesconditions/diabetes/basics/complications/con-20033091.
15. Ward A, Alvarez P, Vo L, Martin S. Direct medical costs of complications of diabetes in the United States: estimates for event-year and annual state costs (USD
2012). Journal of Medical Economics. 2014;17(3):176-183.
16. Sacks DB. Carbohydrates. In Tietz Textbook of Clinical Chemistry and Molecular
Diagnostics. 2006. St. Louis: Elsevier Saunders. p. 837–902.
17. Sacks DB. Measurement of Hemoglobin A1c: A new twist on the path to harmony. Diabetes Care. 2012:35(12): 2674-2680.
18. Sacks DB. A1C versus glucose testing: A comparison. Diabetes Care.
2011;34(2):518-523.
19. American Association of Clinical Endocrinologists (AACE). American Association of Clinical Endocrinologists / American College of Endocrinology Statement on
the use of hemoglobin A1c for the diagnosis of diabetes. 2010. https://www.aace.
com/files/position-statements/a1cpositionstatement.pdf.
20. American Association of Clinical Endocrinologists (AACE). American
Association of Clinical Endocrinologists / American College of Endocrinology—
Clinical practice guidelines for developing a diabetes mellitus comprehensive care
plan. 2015. https://www.aace.com/files/dm-guidelines-ccp.pdf.
21. Lenters-Westra E, Slingerland RJ, Three of seven hemoglobin A1c point-ofcare instruments do not meet generally accepted analytical performance criteria.
Clinical Chemistry. 2014;60(8):1062–1072.
22. Whitely HP, Yong EV, Rasinen C. Selecting an A1c point-of-care instrument.
Diabetes Spectrum. 2015;28(3):201-208.
23. Health and Medicine. Understanding the new IFCC HbA1c Numbers. 2009. http://
www.slideshare.net/PeninsulaEndocrine/new-hb-a1c-numbers-presentation.
24. Kalyani RR, Walker-Harris V. Alternative markers of glycemia: fructosamine,
glycated kalbumin, 1,5-AG. John Hopkins Diabetes Guide. 2014. http://www.hopkinsguides.com/hopkins/view/Johns_Hopkins_Diabetes_Guide/547055/all/Alternative_markers_of_glycemia:_fructosamine__glycated_albumin__1_5_AG.
25. Centers for Medicare and Medicaid Services (CDC). 2013. Effective Public
Health Strategies to Prevent and Control Diabetes: a compendium. http://www.cdc.
gov/diabetes/pubs/pdf/PublicHealthCompedium.pdf.
Liana F. Romero PhD, MBA, MT(ASCP),
serves as Senior Director, US Strategic
Marketing and Clinical Affairs, within
Siemens Healthcare US Laboratory
Diagnostics. Dr. Romero is focused on
connecting laboratory capabilities with
hospital administration to navigate
and thrive in the dynamic environment driven by healthcare reform, incentive programs,
quality measurements, evolving reimbursement models,
and hospital consolidation.
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Patients deserve more than just a number.
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Visual curve displays hemoglobinopathies when present
Insist on results that tell you more.
Insist on Sebia.
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[email protected]
1.800.835.6497
7/12/2016 4:02:29 PM
CON T INUING E DUC AT ION T E S T DIABETES: THE CURRENT STATE OF AFFAIRS FROM A
POPULATION MANAGEMENT VIEW
TEST QUESTIONS
MLO and Northern Illinois University (NIU), DeKalb, IL, are co-sponsors in
offering continuing education units (CEUs) for this issue’s article DIABETES:
THE CURRENT STATE OF AFFAIRS FROM A POPULATION MANAGEMENT
VIEW. CEUs or contact hours are granted by the College of Health and
Human Sciences at Northern Illinois University, which has been approved
as a provider of continuing education programs in the clinical laboratory
sciences by the ASCLS P.A.C.E.® program. Approval as a provider of
continuing education programs has been granted by the state of Florida
(Provider No. JP0000496). Continuing education credits awarded for
successful completion of this test are acceptable for the ASCP Board of
Registry Continuing Competence Recognition Program. Readers who
pass the test successfully (scoring 70% or higher) will receive a certificate
for 1 contact hour of P.A.C.E.® credit. Participants should allow three to five
weeks for receipt of certificate. The fee for this continuing education test
is $20. This test was prepared by Amanda Voelker, MPH, MT(ASCP), MLS
Clinical Education Coordinator, School of Allied Health and Communicative
Disorders, Northern Illinois University, DeKalb, IL.
TEST ANSWER FORM
DIABETES: THE CURRENT STATE OF AFFAIRS FROM A POPULATION
MANAGEMENT VIEW
1.
a. minimizing financial healthcare
implications
b. improving patient outcomes/
quality of life
c. staying ahead of the
progression of disease
d. all of the above
2.
B
C D
O OOO
O OOO
3. O O O O
4. O O O O
5. O O O O
6. O O O O
7. O O O O
8. O O O O
9. O O O O
10. O O O O
11. O O O O
12. O O O O
13. O O O O
14. O O O O
15. O O O O
16. O O O O
17.O O O O
18. O O O O
19.O O O O
20. O O O O
Circles must be filled in, or test will not be graded.
Shade circles like this: O Not like this: O
1.
2.
3.
P
4.
5.
E
FEE FOR THIS CE TEST IS $20.
FL: Your FL license number:____________________
(required for CE credit)
6.
FEE FOR THIS CE TEST IS $20
Make check payable to: Northern Illinois University
7.
8.
MAILING ADDRESS
HOME WORK
STATE
ZIP
INSTITUTION/FACILITY
WORK PHONE
E-MAIL ADDRESS
Send your check with this form to: University Outreach Services
Northern Illinois University, DeKalb, IL 60115-2860 Phone: 815-753-0031
20
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MLO201608-CETest_FINAL.indd 20
Type I diabetes cases are most
commonly diagnosed
during the mid-teenage years.
at birth.
in adulthood.
at age three to five years.
Increased prevalence of type II
diabetes is seen among which
ethnic groups?
a.
b.
c.
d.
9.
CITY
It has been reported that
in 2012 diabetes-related
complications accounted for
$235 billion in total medical
costs and lost work/wages.
a.
b.
c.
d.
PRINT CLEARLY
NAME
12.0%
5.3%
7.5%
9.3%
a. True
b. False
FEE NOT REFUNDABLE OR TRANSFERABLE
Tests can be taken online or by mail.
Easy registration and payment options
are available through NIU
by following the links found at
www.mlo-online.com/ce.
pneumonia.
eye diseases.
urinary tract infections.
none of the above
According to the CDC’s National
Diabetes Statistics Report
(2014), what percentage of the
U.S. population is estimated to
have diabetes?
a.
b.
c.
d.
CE Licensure Information for FL and CA:
CA: Accrediting Agency: 0001
(for use in submitting your CE credits to CA)
Long-term complication
of diabetes include kidney
failure, cardiovascular disease,
stroke, ulcerations/wounds in
extremities, and
a.
b.
c.
d.
E
P
The most common short-term
complications of diabetes are
ketoacidosis and hyperglycemic
coma.
a. True
b. False
P = Poor; E = Excellent
1. To what extent did the article focus 3. How will you use
on or clarify the objectives?
the CE units?
state license
recertification
2. To what extent was the article
employment
well-organized and readable?
other
Symptoms of diabetes mellitus
most commonly include
a. dizziness, fever, and fatigue.
b. increased thirst, joint pain, and
fatigue.
c. frequent urination, increased
thirst and hunger.
d. frequent urination, increased
thirst and fever.
August 2016 [This form may be photocopied. It is no longer valid for CEUs after February 28, 2017.)
A
In regard to population
management, what good
outcomes depend upon the
proactive screening for and
management of at-risk diabetes
patients?
African Americans
Hispanics
Native Americans
all of the above
The main difference in
treatment for type I versus type
II diabetes is
a. how much exercise is to be
performed each week.
b. a more restricted diet in type I
diabetes.
c. the way in which insulin/
medication is administered.
d. all of the above
10. What alarming trend has been
identified in the U.S. regarding
type II diabetes?
a. resistance of drugs in current
treatment options
b. increased incidence of type II
diabetes in individuals less than
20 years old
c. both a and b
d. none of the above
11. The ADA speculates that
gestational diabetes is caused by
placental hormones blocking the
actions of the mother’s insulin
in the body, creating insulin
resistance and buildup of glucose
levels.
a. True
b. False
12. Hemoglobin A1c (HbA1c) reflects
a patient’s average glucose
concentration to which blood
cells were exposed over
a.
b.
c.
d.
12 to 15 weeks.
5 to 10 weeks.
8 to 24 weeks.
8 to 12 weeks.
13. Currently, HbA1c is used in both
screening for prediabetes and for
the diagnosis of diabetes.
a. True
b. False
14. The key advantage(s) of using
HbA1c over blood glucose testing
are
a. no fasting requirement, sample
draw convenience, less variation
in levels, and analytical stability.
b. sample draw convenience and
less variation in levels.
c. no fasting requirement, less
variation in levels, and analytical
stability.
d. analytical stability only.
15. HbA1c levels vary in patients who
a. are pregnant.
b. have had recent blood loss/
transfusion.
c. suffer from anemias.
d. all of the above
16. What was established to ensure
that HbA1c methods are able to
generate highly similar results on
a given blood sample?
a. The International Experts
Committee
b. The Diabetes Control and
Complications Trial
c. The National Glycohemoglobin
Standardization Program
d. none of the above
17. The improvement in performance
from the answer to #16 led to an
increase in labs reporting HbA1c,
from
a.
b.
c.
d.
50% in 1993 to 89% in 2004.
50% in 1993 to 99% in 2004.
25% in 1993 to 50% in 2004.
25% in 1993 to 99% in 2004.
18. Currently, POCT users for HbA1c
must participate in a proficiency
testing program.
a. True
b. False
19. Alternate markers to determine
glycemia that are recommended
to be used in conjunction with
HbA1c are
a.
b.
c.
d.
1,5-AG levels.
fructosamine.
glycated albumin.
all of the above
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SPECIAL REPORT
HbA1c
The continuing case for
point-of-care testing for HbA1c
By Gavin Jones, BSc(Hons)
T
here is an ongoing conflict between traditional clinical
laboratories and the relative new kid on the block, pointof-care testing (POCT). Of course, the laboratory system
will likely always be king. But there is absolutely a place for
POCT, especially as the way in which we approach healthcare,
especially diagnostics, develops beyond the usual settings.
POCT ensures the rapid provision of diagnostic information, ideally during one consultation, to enable clinical decisions to be made at the earliest opportunity. Such rapid provision of information facilitates optimization of the care process.
The potential for any application of POCT can, therefore, be
judged in terms of its contribution to decision making and to
the process of care.
In the case of the management of diabetes patients, POCT
for glycated hemoglobin (HbA1c) may offer a number of advantages—as long as the performance characteristics of the
analyzers used are equivalent to those employed in the central
laboratory, and can be certified as such.
The use of HbA1c for management of diabetes
Glycated hemoglobin (HbA1c) is well-recognized as a reliable
measure for glycemic control. The role of HbA1c testing in the
management of patients with diabetes has been established
for several decades, while its role in the diagnosis of diabetes
has been documented more recently.
These applications are based on the fact that HbA1c levels
reflect the average circulating glucose concentration over the
lifespan of red blood cells (two to three months). This is because the glycated hemoglobin molecule in blood is highly
stable once it has formed after exposure to glucose, since glucose binds irreversibly in a non-enzymatic reaction to the Nterminal valine of hemoglobin A in red blood cells (Figure 1).
Therefore, use of HbA1c can offer greater clinical information
than a single glucose measurement taken at a particular time.
Furthermore, there is evidence that HbA1c is a good predictor
of an individual’s risk of developing long-term complications
of diabetes, e.g., cardiovascular disease.1-3
Serial measurements of HbA1c can show how an individual’s glucose control (and hence risk of complications) change
in response to alterations in management over time. It is recommended that HbA1c should be measured every two to six
months, with target HbA1c levels set for individual patients
and therapy adjusted accordingly.4 General targets of HbA1c
for diabetic individuals, depending on their risk of severe
hypoglycemia, cardiovascular status, and co-morbidities,
should be set between 6.5 percent and 7.5 percent (48-58
mmol/mol), with the non-diabetic reference range being 4.0
percent to 6.0 percent (20-42 mmol/mol). Within the United
Kingdom, for example, current National Institute for Health
and Clinical Excellence (NICE) guidelines stipulate that, providing there is no disabling hypoglycemia, the target HbA1c
concentration for children, young people, and adults with
type 1 diabetes is 7.5 percent, and that additional support
should be offered if HbA1c is consistently greater than 9.5
percent.
One consideration is that HbA1c results may be affected by
any condition that leads to a change in red blood cell survival;
however, even then, HbA1c can be used to detect trends in a
patient’s glycemic control rather than for target setting.
The growing case for HbA1c in diabetes diagnosis
The attributes of HbA1c measurement for the management
of diabetes are equally applicable for use in the diagnosis of
diabetes. Furthermore, the performance of HbA1c has been
shown to be equal to that of fasting blood glucose tests commonly used for type 2 diabetes screening.5
Unlike glucose levels, which are affected by what a patient has eaten and drunk in the previous two to three hours,
HbA1c levels do not require a patient to fast prior to the
test. Consequently, as a simple and immediate test for diabetes, POC HbA1c can support early identification of at-risk
individuals. This would then rapidly enable them to make
changes to their lifestyle, in order to significantly reduce the
risk of developing type 2 diabetes or eliminate it altogether.
The ability to rapidly assess and change risk outcomes has
significant health benefits for the patient and also reduces
overall healthcare costs related to the complications of type
2 diabetes. It is known that patients diagnosed with diabetes
Figure 1. Glycated hemoglobin (HbA1c) explained
22
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HbA1c
who maintain low blood HbA1c levels significantly reduce the
onset of complications after diagnosis.6
The World Health Organization has recommended the use
of HbA1c for the diagnosis of diabetes,7 and similar guidance
has followed in several countries.8,9 In the UK, for example,
NICE published guidelines in 2013 for diabetes prevention
which aim to identify people at high risk of type 2 diabetes
and to offer cost-effective and appropriate interventions to
prevent or delay onset.8 Used in conjunction with a lifestyle
health risk assessment, these guidelines advocate the use of
HbA1c levels to allow healthcare providers to advise individuals on treatment regimens, depending on their classification
as low, moderate, or high risk of developing type 2 diabetes.
NICE has since followed up with new guidelines in 2015 for
the management of type 2 diabetes in adults which suggests
HbA1c levels should be tested at three-to-six month intervals
and six-month intervals once a stable HbA1c level has been
obtained.9
Likewise in the United States, a recent American Diabetes
Association workgroup report concluded that the HbA1c
assay is an accurate, precise measure of long-term glycemic
levels that correlates well with diabetes complications and offers several advantages over laboratory measures of glucose.10
However, it should be noted that use of HbA1c for diabetes diagnosis is not appropriate for all individuals. The test should
not be used in children, young people, pregnant women, individuals in whom type 1 diabetes is suspected, individuals
whose symptoms have been of short duration, or patients who
are acutely ill.11,12
It has also been suggested that the cut-off value for diabetes
diagnosis generally quoted, 6.5 percent (48 mmol/mol), may
not be appropriate for all populations. Additionally, individuals within the range of 6.0 percent to 6.4 percent (42-47 mmol/
mol) should be considered at high risk of developing diabetes
and be advised appropriately and retested annually. Those
with values less than 6 percent (42 mmol/mol) should be tested
every three years.12
Current guidance, therefore, supports the employment of
HbA1c measurement in both screening for type 2 diabetes and
in the management of patients with diabetes. Use of POCT
could improve management of patients with established diabetes in both primary and secondary care settings and also
enable earlier type 2 diabetes diagnosis.
HbA1c in POCT-based diabetes monitoring
Early HbA1c determination was based on laboratory-based
methods including ion exchange and affinity chromatographic methods, with alternative affinity and immunological
methods following later, taking HbA1c into the point-of-care
environment.
There is certainly a strong case for employing POC-based
HbA1c testing for diabetes monitoring, since lying at the heart
of the concept of POCT is the principle that medical tests are
convenient and immediate to the patient. Typically, patients
with existing diabetes are monitored for HbA1c every three to
six months. This generally involves a nurse or phlebotomist
visit for venepuncture, with a follow-up appointment one to
two weeks later to discuss results once they are available from
the laboratory.
From a patient experience perspective, use of POCT for
HbA1c can enhance satisfaction levels. This is because use
of POCT means that after one visit they can leave with an
immediate action plan or relevant prescription, should results
indicate the need. Furthermore, enabling earlier therapeutic decisions may result in improved diabetic control, better patient
outcomes, and enhanced clinic efficiency, with fewer patient
visits and subsequent economic benefits. For example, a Swedish before-and-after study investigated the economic costs and
benefits of implementing HbA1c home testing.13,14 It confirmed
a reduction in costs due to fewer clinic visits, reduction in total treatment costs, time saved resulting from reduced labor
costs in both administration and sampling, and reduced travel
costs—as well as a reduction in mean HbA1c levels.
In addition, four observational studies of more than 5,700 patients with diabetes, in which there was immediate feedback of
results to patients, also showed significant reductions in HbA1c
levels.15-18 One of these studies demonstrated maintenance of
improved HbA1c levels for a period of four years.17 A recent
systematic review of quality improvement (QI) strategies in
the management of diabetes demonstrated that QIs involving
greater adherence to guidelines can help improve HbA1c levels.8 There is also good evidence to show that patient satisfaction is improved using POCT and that immediate knowledge of
a patient’s HbA1c levels is associated with better outcomes, as
judged by reduced HbA1c levels.16-24
What to look for in a POC HbA1c analyzer
Most POC HbA1c analyzers use a drop of blood (4 to10 μL) taken from a finger prick (capillary blood) or venous sample that is
applied to a reagent cartridge and then inserted into a desktop
device for analysis. Time to reporting of HbA1c results is generally between three and 10 minutes, depending on the analyzer.
Keys to the effectiveness of any HbA1c POC analyzer are simplicity, audit trails, certification, and methodology:
Simplicity. Ensuring that a POC analyzer is as easy to use as
possible will minimize the chance of user error and hence the
need for retesting, with subsequent time and cost implications.
An analyzer that is simple to use also ensures minimal training
requirements, again contributing to affordability.
Features that can support ease of use include the employment
of ready-to-use reagent cartridges that can be inserted straight
into the analyzer with the blood sample then added directly,
without the need for sample preparation such as premixing or
pipetting. Minimizing the number of steps in the procedure not
only reduces the opportunity for user error but also helps to
standardize results by eliminating any variation introduced by
different operators, particularly when pipetting and mixing.
Audit trails. For patient safety purposes, audit trails should
also be available. Barcode scanning for patient and user identification, as well as for confirmation of the batch of reagents
and controls used, ensures that an analyzer can provide such
trails. Quality control results on two levels that are recorded
and held within the analyzer’s memory are also ideal for
auditing purposes.
Certification. Certification of the analyzer in order to confirm
delivery of accurate, standardized results should also be a key
consideration. In an effort to standardize HbA1c results, the
American Association of Clinical Chemistry (AACC) set up the
National Glycohemoglobin Standardization Program (NGSP)
in 1996. In parallel, the International Federation of Clinical
Chemistry (IFCC) developed reference methods for glycated
hemoglobin. In 2006 and 2007, an international consensus
between IFCC and AACC was reached.25
The calibration and certification of laboratories and manufacturers to the same standards has improved the conformity
of results. However, in practice differences can still be observed
among technologies and among individual systems. These observed differences arise because of heterogeneity of hemoglobins, underlying differences in technologies (e.g., ion exchange,
boronate affinity, immunoassay), calibration drifts, or lot-to-lot
variability. However, if the manufacturer follows the recommendations of the IFCC and NGSP to ensure instruments and
AUGUST 2016
SPECIAL REPORT
MLO - ONLINE.COM
23
7/12/2016 4:26:29 PM
SPECIAL REPORT
HbA1c
reagents are accurately aligned and traceable to the reference
method, this should not prove problematic.
Methodology. There are POC HbA1c analyzers available
whose results are not affected by hemoglobin variants (which
do not result in reduced erythrocyte life span), labile glycated
hemoglobin, or hematocrit levels. Such analyzers use boronate
fluorescence quenching technology (BFQT),26 which is associated with multiple optical measurements. This methodology is
traceable to well-documented boronate affinity HPLC systems
used in reference laboratories. However, as BFQT does not require chromatographic separation, the methodology allows for
accurate POC measurement of HbA1c to deliver results comparable to chromatography-based techniques. Recent evidence
suggests that the latest POCT systems deal with hemoglobin
variants equally as well as HPLC systems.27 (Figure 2).
Figure 2.
Conclusion
There are strong arguments for the use of POCT for HbA1c
where the performance characteristics of the systems are equivalent to those employed in the central laboratory (or even better
in some cases) and are certified as such. POCT offers improved
access to testing, as well as enabling immediate clinical decision
making, discussion with the patient, and implementation of appropriate treatment and/or lifestyle advice. Further, as POCT
enables testing to be undertaken closer to patients, it affords
greater convenience for them, thereby improving the likelihood
of treatment compliance by empowering them. This should
ensure better glycemic control, which is the ultimate goal.
REFERENCES
1. Khaw KT, Wareham N, Bingham S, Luben R, Welch A, Day N. Association of hemoglobin A1c with cardiovascular disease and mortality in adults: the European prospective investigation into cancer in Norfolk. Ann Intern Med. 2004;141:413–420.
2. Selvin E, Marinopoulos S. Meta-analysis: glycosylated hemoglobin and cardiovascular disease in diabetes mellitus. Ann of Internal Medicine. 2004;141:421–431.
3. ten Brinke R, Dekker N, de Groot M, Ikkersheim D. Lowering HbA1c in type 2 diabetics results in reduced risk of coronary heart disease and all cause mortality. Prim Care
Diabetes. 2008;2:45–49.
4. Diabetes UK. HbA1c Standardisation: Information for Clinical Healthcare Professionals. 2009. http://www.diabetes.org.uk/Guide-to-diabetes/Monitoring/Blood_glucose/
Glycated_haemoglobin_HbA1c_and_fructosamine/HbA1c_Standardisation_Information_for_Clinical_Healthcare_Professionals. Accessed May 21, 2013.
5. Bennett CM, Guo M, Dharmage SC. HbA(1c) as a screening tool for detection of Type
2 diabetes: a systematic review. Diabet Med. 2007;24:333-343.
6. Diabetes Control and Complications Trial Research Group. The effect of intensive
treatment of diabetes on the development and progression of long-term complications
24
MLO - ONLINE.COM
in insulin-dependent diabetes mellitus. N Engl J Med. 1993;329:977-986.
7. World Health Organization. Use of glycated haemoglobin (HbA1c) in the diagnosis
of diabetes mellitus. 2011. www.who.int/diabetes/publications/report-hba1c_2011.pdf.
Accessed May 21, 2013.
8. National Institute for Health and Clinical Excellence. Preventing type 2 diabetes: risk
identification and interventions for individuals at high risk. 2012. www.nice.org.uk/nicemedia/live/13791/59951/59951.pdf. Accessed May 21, 2013.
9. NICE Guidelines: Type 2 diabetes in adults: management. 2015. https://www.nice.org.
uk/guidance/ng28.
10. American Diabetes Association. International Expert Committee Report on the role
of the A1C Assay in the diagnosis of diabetes. Diabetes Care. 2009;32(7):1327-1334.
11. Inzucchi SE. Clinical practice. Diagnosis of diabetes. N Engl J Med. 2012;367:542550.
12. Farmer A. Use of HbA1c in the diagnosis of diabetes. BMJ. 2012;345:e7293.
13. Snellman K, Eckerborn S. Possibilities and advantages of HbA1c: eight years experience. Diabet Med. 1997;14(5):401-403.
14. Plüddemann A, Price CP, Thomson M, Wolsteholme J, Heneghan C. British Journal
of General Practice. 2011;61:139-140.
15. Grieve R, Beech R, Vincent J, Mazurkiewcz J. Near patient testing in
diabetes clinics: appraising the cots and outcomes. Health Technol Assess. 1999;3:1-74.
16. Ferenczi A, Reddy K, Lorber DL. Effect of immediate hemoglobin A1c
results on treatment decisions in office practice. Endocr Pract. 2001;7:85–
88.
17. Petersen JR, Finley JB, Okorodudu AO, Mohammad AA, Grady JJ,
Bajaj M. Effect of point-of-care on maintenance of glycemic control as
measured by A1C. Diabetes Care. 2007;30:713–715.
18. Rust G, Gailor M, Daniels E, McMillan-Persaud B, Strothers H, Mayberry R. Point of care testing to improve glycemic control. Int J Health
Care Qual Assur. 2008;21:325–335.
19. Driskell OJ, Holland D, Hanna FW, et al. Inappropriate requesting of
glycated hemoglobin (Hb A1c) is widespread: assessment of prevalence,
impact of national guidance, and practice-to-practice variability. Clin
Chem. 2012;58:906-915.
20. Peterson KA, Radosevich DM, O’Connor PJ, et al. Improving Diabetes Care in Practice: findings from the TRANSLATE trial. Diabetes Care.
2008;31:2238-2243.
21. Laurence CO, Gialamas A, Bubner T, et al; Point of Care Testing in General Practice Trial Management Group. Patient satisfaction with point-ofcare testing in general practice. Br J Gen Pract. 2010;60:e98–104.
22. Cagilero E, Levina EV, Nathan DM. Immediate feedback of HbA1c levels improves glycemic control in type 1 and insulin-treated type 2 diabetic
patients. Diabetes Care. 1999;22(11):1785-1789.
23. Miller CD, Barnes CS, Phillips LS, et al. Rapid A1c availability improves clinical
decision-making in an urban primary care clinic. Diabetes Care. 2003;26(4):1158-1163.
24. Kennedy L, Herman WH, Strange P, et al; GOAL A1C Team. Impact of active versus
usual algorithmic titration of basal insulin and point-of-care versus laboratory measurement of HbA1c on glycemic control in patients with type 2 diabetes: the Glycemic Optimization with Algorithms and Labs at Point of Care (GOAL A1C) trial. Diabetes Care.
2006;29(1):1-8.
25. Geistanger A, Arends S, Berding C, et al; on behalf of the IFCC Working Group on
Standardization of Hemoglobin A1c. Statistical Methods for Monitoring the Relationship between the IFCC Reference Measurement Procedure for Hemoglobin A1c and
the Designated Comparison Methods in the United States, Japan, and Sweden. Clin
Chem. 2008;54(8):1379-1385.
26. Wilson DH, Bogacz JP, Forsythe CM, et al. Fully automated assay of glycohemoglobin with the Abbott IMx analyzer: novel approaches for separation and detection. Clin
Chem. 1993;39(10):2090-2097.
27. Lenters-Westra E. An evaluation of the Quo-Test® performance against NGSP criteria and sigma-metric. 2016.
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EDUCATION
P R EGN A NC Y/ P R E N ATA L
An update on
gestational
diabetes mellitus
By David Grenache, PhD
A
t first glance, screening pregnant women for gestational diabetes mellitus (GDM) seems like it should be straightforward. After all, the screening tests are designed to identify
pregnant woman with high concentrations of blood glucose, and
laboratory tests that measure glucose are accurate and precise. So
what’s the problem?
Before that question is tackled, consider a few facts about
GDM:
• It is defined as any degree of glucose intolerance with onset
or first recognition during pregnancy that isn’t overt diabetes
mellitus.
• Each year in the United States approximately four million women give birth, and 240,000 of these women, or six percent, develop
diabetes during their pregnancy.
• The incidence of GDM is a function of the screening test used to
diagnose the disorder (more about this below).
• Although GDM often resolves after pregnancy, it does place the
mother and fetus at risk of serious health issues. For the mother
these include preeclampsia and an increased chance for the development of type 2 diabetes after pregnancy. For the fetus these include macrosomia, shoulder dystocia, and increased risk of obesity
during childhood.
The question of how best to screen for GDM is one not easily
answered because experts don’t agree on how best to screen for
the disorder. While all agree that both mother and infant can experience adverse outcomes if GDM goes undetected and untreated, there is lack of consensus on the best way of identifying it.
Some diagnostic dilemmas
To better understand the current debate, consider how GDM
screening has been done in the U.S. since 1964. Most practitioners
follow a two-step process:
1. A screening test is done at 24 to 28 weeks of gestation by measuring blood glucose one hour after a non-fasting patient consumes a 50-gram dose of glucose. A result greater than 140 mg/
dL is usually used as the cutoff, although a lower cutoff of 130 mg/
dL may also be used. Those with a result greater than the cutoff
undergo diagnostic testing.
2. The diagnostic test is the three-hour oral glucose tolerance test
(OGTT). This test requires the collection of a fasting blood sample
followed by consumption of a 100-gram dose of glucose with additional blood samples obtained every hour for four hours. The
test is considered positive, and GDM confirmed, if two or more
of the four glucose test results are above designated cutoffs.
There are two sets of cutoffs currently used: those of the National
Diabetes Data Group or the Carpenter-Coustan cutoffs.
Importantly, these two cutoff sets are modifications of those
developed 52 years ago that were selected to identify women
who were more likely to have diabetes after pregnancy. Further, the requirement that two or more abnormal results are
required to define an abnormal result was arbitrarily selected
to avoid misclassifications due to “laboratory errors.” It should
also be noted that in much of the rest of the world, a one-step
approach is used to diagnose GDM using a two-hour, 75-gram
OGTT. GDM is diagnosed when any single glucose result
exceeds a diagnostic cutoff recommended by the World Health
Organization (WHO).
The lack of a universal protocol for diagnosing GDM has
made it impossible to compare different studies of GDM. In addition, because different investigators have used different protocols for identifying women with the disorder, its true prevalence
is not known. To further complicate matters, there has been ambiguity regarding the maternal and fetal morbidities associated
with varying degrees of maternal hyperglycemia. To address
these shortcomings, the Hyperglycemia and Adverse Pregnancy
Outcomes (HAPO) study was conducted.
Controversial criteria
HAPO was a multicenter international study that included more
than 23,000 women in nine countries who were given a 75-gram,
two-hour OGTT. Results clearly demonstrated strong and continuous associations between maternal glucose and adverse outcomes such as increased birth weight, C-section delivery, and neonatal hypoglycemia. The continuous associations between glucose
and outcome meant that translating the HAPO results into clinical
practice would be challenging. Recognizing that, the study investigators called for a re-defining of the criteria used to diagnose
GDM. As a result, the International Association of the Diabetes
and Pregnancy Study Groups (IADPSG) convened to establish
new diagnostic criteria for the diagnosis of GDM.
Ultimately, this group settled on diagnostic cutoffs that reflected an odds ratio of 1.75 times the mean for the outcomes
of increased neonatal body fat, large for gestational age infants, and an umbilical cord serum C-peptide concentration
greater than the 90th percentile. They also recommended that
a universal, one-step, two-hour, 75-gram OGTT be performed
during pregnancy and that the diagnosis of GDM be made
when any single cutoff on the two-hour OGTT was met or
exceeded.
It was immediately recognized that use of the IADPSG cutoffs
would likely double the incidence of women diagnosed with
GDM, and this alone lessened an enthusiastic adoption of the new
protocol. The loudest critic was the American College of Obstetricians and Gynecologists (ACOG), which continued to support the
two-step approach to screening and diagnosis and recommended
that additional studies be performed before changes to GDM diagnostic criteria were adopted. Notably, the Endocrine Society, the
WHO, and the American Diabetes Association (ADA) have accepted the one-step approach. (The ADA also accepts the two-step
approach and so is somewhat noncommittal on the controversy.)
26
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AUGUST 2016
MLO201608-Education-COMBINED_MECH_AL.indd 26
7/12/2016 2:34:01 PM
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EDUCATION
P R EGN A NC Y/ P R E N ATA L
Next-generation sequencing
and the future of IVF
By Lia Ribustello, BS, MS
M
edical laboratories play a vital role in helping
patients achieve success with assisted reproductive technologies (ART) such as in vitro fertilization
(IVF). Recent advances in genetic screening such as next
generation sequencing (NGS) are revolutionizing how IVF
will be performed in the future. NGS can sequence DNA
and RNA more quickly than ever before. New applications like these technologies are raising hopes for improved
IVF success rates to help patients achieve their dreams of
building a family.
Preimplantation genetic screening
According to Centers for Disease Control and Prevention
(CDC) estimates, one in eight couples have trouble achieving or sustaining a pregnancy,1 and approximately 7.4 million women in the United States have received help for infertility.2 The use of ART has doubled over the past decade.3
While these statistics provide a general overview of the
frequency of fertility struggles in the U.S., they don’t address the complexity that is added by the societal trend
of women waiting until later in life to start a family. As a
woman’s age increases, the success rates of IVF decrease.
This decline is due, in part, to the increase in chromosome
abnormalities, or aneuploidy, in the eggs and resulting embryos as a woman ages. For instance, women under 35 will
produce 40 percent of embryos with chromosome abnormalities; women from 35 to 39 about 70 percent; women 40
to 42 close to 80 percent; and older women 90 percent or
more. 4 Aneuploidy is responsible for the majority of miscarriages and serious genetic disorders. As a result, the ability
to screen for the chromosomal health of embryos is an exciting advancement in the field of ART and is the objective of
preimplantation genetic screening (PGS) technologies.
PGS may be recommended for women who have experienced recurrent miscarriage or are over age 35, or women
for whom multiple fertility treatments have failed.5 PGS
technologies have a single aim: identifying chromosomally
normal embryos for transfer to the womb. Euploid embryos have a higher chance of implantation, and the resulting
pregnancies have a lower chance of miscarriage. The viability of the embryo selected for transfer is one of the most
important factors influencing IVF success.
Over the years, genetic screening of embryos has become
instrumental in helping to identify aneuploid embryos,
and the field continues to advance rapidly. Fluorescence in
situ hybridization (FISH) was replaced by DNA microarray analysis or array comparative genomic hybridization
(aCGH) in the late 2000s, and these techniques, broadly
speaking, are now being replaced by NGS. Each advance
provides a more complete picture of the genetic structure
of an embryo and its overall health prior to implantation.
Deciphering DNA
Today, NGS is gaining in popularity as costs start to decline
and the technology improves. In particular, high-resolution
NGS (hr-NGS) enables even more detailed examination of
28
MLO - ONLINE.COM
chromosomes and thus better detection of abnormalities
through high-resolution sequencing.
To perform NGS for PGS, the first step is whole genome
amplification of the sample obtained from the embryo,
which is usually about five cells. Since every biopsy is different, the DNA is then quantified so the starting material
from each sample is at the correct concentration.
Next, the DNA is fragmented into smaller pieces with
specific adapter sequences added to each piece. A low-cycle
polymerase chain reaction (PCR) proceeds so that each sample can be individually barcoded with indexes. This is an
important step because it distinguishes each sample when
sequenced and is what allows attachment to the flow cell
during sequencing. Size selection steps are then performed
to remove unwanted library fragments and primers, and
then the library is normalized to ensure equal library representation. Afterward, all samples are pooled together to
create a single library that is then sequenced.
High-resolution NGS has the potential to provide the
whole genome sequence of an embryo, enabling detection
of chromosome count as well as inherited and de novo gene
defects. Also called “high throughput sequencing,” hr-NGS
is more scalable and cost-effective than previous technologies as laboratories can sequence an increased number
of samples simultaneously during a single experiment.6
Combined with laboratory automation technologies that
increase consistency and reduce human error, hr-NGS is a
significant advancement in the field of ART.
A key differentiator for hr-NGS is its ability to reveal a
wider range of mosaic embryos7—those that contain a mixture of normal and abnormal cells. Mosaicism is extremely
common in early human development, affecting 30 percent
of blastocyst-stage embryos. 8 Recent studies suggest mosaicism plays an important role in pregnancy loss, though
some mosaics do go on to become successful pregnancies.
Knowledge of the presence of mosaicism may be useful to
select the most viable embryos during IVF, thus increasing
the likelihood of pregnancy, reducing the chances of a pregnancy loss, and improving the odds of delivering a healthy
child.
hr-NGS in the future
High-resolution NGS may be the future, and in fact it is
already available. The first baby using hr-NGS was born
in 2013. Some studies suggest that there may be up to 60
percent fewer miscarriages using NGS than other PGS
AUGUST 2016
7/12/2016 2:34:13 PM
9
techniques. As hr-NGS is further validated through clinical studies, future applications may involve coupling this
technology with other types of tests.
For instance, today it is possible to examine the mitochondrial DNA (mtDNA)—the tiny organelles that generate energy for the cell. Elevated mtDNA is associated with
failure to implant.10 Women who use hr-NGS can add mtDNA testing to boost their chances even further of achieving
a successful pregnancy. PGS and mtDNA analysis can be
performed in parallel on a single blastocyst biopsy, providing a more thorough picture of embryo health without a
significant change in workflow for the clinic or patient.10
Due to the rapid growth and advancement of PGS techniques, PGS is not yet recommended by major professional
societies. That said, it is clear that the field of ART is changing, and changing in a way that favors use of these novel
technologies. The American Society for Reproductive Medicine recommends no more than two embryos be implanted
in women under 35—and doctors should consider using
just one. The U.S. may be moving toward a single embryo
transfer during IVF, similar to Europe, making the use of
PGS technologies such as NGS even more important to
increase the odds of a successful pregnancy.
REFERENCES
1. Chandra A, Copen CE, Stephen EH. Infertility and Impaired Fecundity in the
United States, 1982–2010: Data From the National Survey of Family Growth. National
Health Statistics Reports. Number 67, August 14, 2013. http://www.cdc.gov/nchs/
data/nhsr/nhsr067.pdf.
2. Centers for Disease Control and Prevention. Infertility. Key Statistics from the
National Survey of Family Growth, 2006-2010. http://www.cdc.gov/nchs/fastats/infertility.htm.
3. Centers for Disease Control and Prevention. National ART Surveillance System,
Preliminary Data, 2014. Available at: http://www.cdc.gov/art/reports/index.html.
4. Munne S, Ribustello L, Kolb B, et al. Blastocysts needed to transfer at least one
euploid embryo: data from 10,852 pre-implantation genetic screening (PGS) cycles,
Fertility and Sterility. 2015;104(3):e13-e14.
5. Fact Sheet: Preimplantation genetic testing. American Society of Reproductive
Medicine. http://www.asrm.org/uploadedFiles/ASRM_Content/Resources/Patient_
Resources/Fact_Sheets_and_Info_Booklets/PGT_2014.pdf.
6. Fiorentino F, Bono S, Biricik A, et al. Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical
preimplantation genetic screening cycles. Hum. Reprod. 2014 http://humrep.oxfordjournals.org/content/early/2014/10/21/humrep.deu277.long.
7. Behjati S, Tarpey PS. What is next-generation sequencing? Arch Dis Child Educ
Pract Ed. 2013 Dec; 98(6): 236–238. Published online 2013 Aug 28. doi: 10.1136/archdischild-2013-304340.
8. Fragouli E, Alfarawati S, Spath K, Tarozzi N, Borini A, Wells D. Fertility and Sterility. 2015; 104(3):Supplement, Page e96. Available at: http://www.fertstert.org/article/
S0015-0282(15)00799-2/abstract.
9. Munne, S. New methods for embryo selection: NGS and MitoGradeTM Available at: http://www.eivf.net/documents/sum_2015/Santiago_Munne.pdf.
10. Fragouli E, Cohen J, Munne S, Grifo J, McCaffrey C, Wells D. The biological
and clinical impact of mitochondrial genome variation in human embryos. ASRM
Scientific Program Prize Paper (unpublished).
Lia Ribustello BS, MS, serves as
Laboratory Supervisor in New Jersey
for Reprogenetics, Inc. She is a
certified scientist in Molecular Biology
through the American Society for
Clinical Pathology.
To be clear, all experts agree that screening pregnant women for GDM is an important part of obstetrical care. Indeed,
the United States Preventative Services Task Force has recommended screening all pregnant women for GDM. The
controversy centers on how to best screen for the condition.
Several studies have now been conducted that evaluate outcomes when GDM is identified via the one-step or two-step
approach. Perhaps not surprisingly, the conclusions of these
various studies are not in agreement with each other. As
expected, all studies report an increased number of women diagnosed with GDM using the one-step test. However, the outcomes
of these pregnancies are reported as improved in some studies,
while others describe no improvements compared to GDM cases
diagnosed by the two-step test. The debate, therefore, continues,
as it remains unclear if the increased number of women identified as having GDM by the one-step approach have truly benefited from treatments for GDM or if they have been subjected
to unnecessary (and potentially harmful) interventions. A uniform approach to identifying GDM is needed, but more data and
more studies will be necessary before a consensus opinion on
one that produces the greatest benefits with the least risks will
be achieved.
David Grenache, PhD, is a professor
of pathology at the University of Utah
School of Medicine and section chief
of clinical chemistry at ARUP
Laboratories. He is board-certified by
the American Board of Clinical
Chemistry and is a fellow in the
National Academy of Clinical
Biochemistry. He also maintains a blog, “The Pregnancy
Lab” (www.pregnancylab.net), focused on the laboratory
tests performed during pregnancy.
For further reading
American Diabetes Association. (2016). Classification
and Diagnosis of Diabetes. Diabetes Care. 39 Suppl 1,
S13–22. http://doi.org/10.2337/dc16-S005.
HAPO Study Cooperative Research Group, Metzger BE,
Lowe LP, Dyer AR, et al. Hyperglycemia and adverse
pregnancy outcomes. The New England Journal of Medicine. 2008;358(19). 1991–2002. http://doi.org/10.1056/
NEJMoa0707943.
International Association of Diabetes and Pregnancy
Study Groups Consensus Panel, Metzger BE, Gabbe
SG, Persson B, et al. International Association of Diabetes and Pregnancy Study Groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care. 2010:33(3):676–682.
http://doi.org/10.2337/dc09-1848.
ACOG. (2011). Committee opinion no. 504: screening and
diagnosis of gestational diabetes mellitus. Obstetrics
& Gynecology. 118(3):751–753. http://doi.org/10.1097/
AOG.0b013e3182310cc3.
Blumer I, Hadar E, Hadden DR, Jovanovic L, Mestman,
JH, Murad, MH, Yogev Y. Diabetes and pregnancy: an endocrine society clinical practice guideline. The Journal of
Clinical Endocrinology and Metabolism. 2013;98(11):4227–
4249. http://doi.org/10.1210/jc.2013-2465.
AUGUST 2016
MLO - ONLINE.COM
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7/12/2016 2:34:25 PM
SPECIAL FEATURE
G A S T ROEN T E ROL OGY
Individualized risk prediction in
Barrett’s esophagus
By Aaron D. DeWard, PhD, and Rebecca Critchley-Thorne, PhD
B
arrett’s esophagus (BE), which is a precursor to esophageal adenocarcinoma (EAC), is an increasing healthcare
challenge in the United States, but new tools are quickly
emerging to combat the uncertainty associated with the disease.
Barrett’s esophagus management decisions are often based on
subjective diagnoses, so better diagnostic and prognostic techniques are needed to augment BE patient care. Here, we summarize a number of tools available to clinical decision-makers
to aid diagnoses and predict the risk of disease progression,
which will optimize patient surveillance intervals and guide
therapeutic interventions.
The clinical problem
There are an estimated 12 million to 17 million people who have
BE in the United States.1 Although malignant progression is
rare in BE, a small subset of patients will develop EAC,2,3 which
has a five-year survival rate of less than 20 percent.4 Furthermore, the incidence of EAC continues to rise in the U.S. Treatment options for EAC are limited, so early detection is critical
for optimal patient management.
Currently, patients with BE are surveilled by endoscopic biopsies with the goal of detecting disease progression early. Diagnoses are not always clear-cut, and can be inconclusive even
after review by a specialized pathologist.5 Recent guidelines
published by the American College of Gastroenterology recommend endoscopic ablative therapy for patients diagnosed with
high-grade dysplasia, but there is uncertainty about the surveillance interval, true risk of progression, and treatment recommendations for patients with low-grade dysplasia or who are indefinite for dysplasia, and for patients with non-dysplastic BE.6
Therefore, it can be difficult to distinguish patients with BE who
are at high risk for progression to EAC from those whose disease will not progress, thus making surveillance and treatment
decisions challenging.
presence of columnar epithelium in pinch biopsies taken during endoscopy. While this approach is valuable, it is limited by
the random nature of the sampling and observer variability in
the histologic diagnosis. New sampling techniques have been
developed that aim to overcome some of these limitations. One
such technique is computer-assisted brush biopsy, which can be
used as an adjunct to standard biopsy to detect BE and to aid in
the identification of dysplasia.7,8 Brush biopsies allow for more
esophageal tissue to be sampled, thus improving tissue coverage and increasing the potential to detect BE and dysplasia in a
single endoscopy.
Volumetric laser endomicroscopy (VLE) is an endoscopic
technique that utilizes advanced imaging technology to generate three-dimensional images of tissue in vivo.9,10 Compared to
other in vivo imaging methods, VLE increases imaging depth
and decreases acquisition time and can be used to guide biopsy
samples to locations that potentially have abnormalities, and
to mark regions for therapeutic intervention. In addition, nonendoscopic tissue collection devices have been developed as a
minimally invasive option to increase patient compliance and
increase detection rates.11,12 The collection device is swallowed,
and then it collects cells as it is removed back out of the mouth
of the patient. Importantly, non-endoscopic devices have the
potential to identify patients with BE that might have been
overlooked if they were initially unwilling to undergo standard
endoscopy.
New risk prediction approaches
While timely identification of patients with BE is an essential
first step, subsequent monitoring and treatment recommendations are not always clearly defined. Clinical and pathologic
variables are inadequate to predict which patients will progress
to EAC , and over-surveillance of patients is common due to uncertainty in the diagnostic stage and anxiety relating to the unknown risk of developing EAC.13 Healthcare providers require
Enhanced diagnostic techniques
tools to accurately stratify patients based on their risk of disDiagnosis of BE relies on endoscopic recognition of salmease progression. Such tools will allow increased surveillance
on-pink colored esophageal lining and confirmation of the
and early therapeutic intervention for the subset of patients at
high risk for progression
to EAC, and permit longer
surveillance intervals for
patients at very low risk.
Several approaches have
been developed for risk
stratification in BE. One
approach, which has been
validated in clinical studies and is commercially
available as a laboratorydeveloped test (LDT), is a
tissue systems pathology
assay that quantifies multiple key biomarkers in
Figure 1. Tissue system biomarkers detected in BE biopsies. Sections from BE biopsy blocks were labeled by multiplexed
BE biopsies to produce an
immunofluorescence and imaged by whole slide fluorescence scanning to detect markers of epithelial abnormalities (Panel
individualized risk score
A: p16-green, alpha-methylacyl-CoA racemase (AMACR)-red, p53-yellow) and markers of inflammation and stromal processfor progression.14 The teches (Panel B: CD68-green, COX-2-red; Panel C: HIF-1alpha-green, CD45RO-red). Hoechst labeling of nuclei is shown in blue in
nology underlying this
each panel. Whole slide images are analyzed by software to segment subcellular compartments and tissue structures, and
assay quantifies not just
to extract multiple, quantitative biomarker and morphology measurements from the relevant compartments/structures.
30
MLO - ONLINE.COM
MLO201608-SpecFeature_MECH_AL.indd 30
AUGUST 2016
7/12/2016 9:11:36 AM
COBAS® EGFR MUTATION TEST V2
The only FDA-approved test to use plasma to
detect EGFR mutations in NSCLC
Companion diagnostic for
Tarceva® (erlotinib) and
TAGRISSO™ (osimertinib)
for patients with NSCLC
The cobas® EGFR Mutation Test v2 is the first and only companion diagnostic test to receive FDA
approval to use a liquid biopsy specimen for testing of patients with non-small cell lung cancer
(NSCLC). The test can use samples from plasma or from tumor tissue to identify patients who
are likely to benefit from first-line treatment with Tarceva® (erlotinib). The plasma option offers
physicians a way to make testing more accessible by using a simple blood draw instead of a
surgical biopsy to obtain a suitable sample.
With flexible sample requirement, clinically proven broad and sensitive mutation coverage, and
speed to result, the cobas® EGFR Mutation Detection Test v2 removes barriers to testing and
gives physicians the information needed to make confident treatment decisions for their critically ill
patients.
Talk to your Roche representative or visit www.cobasEGFRtest.com
for more information.
COBAS and LIFE NEEDS ANSWERS are trademarks of Roche.
TARCEVA is a trademark of OSI Pharmaceuticals, LLC, Farmingdale, NY, 11735, USA,
an affiliate of Astellas Pharma US, Inc.
TAGRISSO is a trademark of AstraZeneca AB.
All other product names and trademarks are the property of their respective owners.
© 2016 Roche. PP-US-07552-0616
MLO201608_AD Roche.indd 31
7/8/2016 1:58:18 PM
THE THINPREP SYSTEM
The first FDA-approved
liquid-based Pap test that is
significantly more effective
than conventional.1*
The first FDA
approval of
glandular disease
labeling.1
The first FDA approval
for an automated
imager, the ThinPrep
Imaging System.
The first and only
FDA-approved collection
media for use with all
FDA-approved HPV tests.
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MLO201608_AD Hologic-SPREAD.indd 32
7/8/2016 12:55:43 PM
We proudly celebrate the
20th Anniversary of the
ThinPrep® Pap test.
When the ThinPrep Pap test was
introduced in 1996, all Pap testing
was done via conventional “Pap
smears,” and HPV testing was not a
part of the cervical cancer screening
algorithm. Since then, there has been
a significant reduction in invasive
cervical cancer in the U.S.2 That’s
something to celebrate.
Today, more than 650 million
ThinPrep Pap tests have been
performed, with more than
6,000 ThinPrep processors
installed globally.3
The ThinPrep family has been a
leader in cervical cancer screening,
and it has remained the trusted choice
in Pap testing for the large majority
of top healthcare providers and
laboratories across the U.S.3,4
Today, Pap+HPV Together™ (cotesting) is supported as the best
strategy for detecting high-grade
cervicovaginal lesions in women ages
30-65, according to recent studies.5,6
Guidelines also recommend the use
of co-testing as the preferred
screening choice for women ages
30-65.7 Hologic continues to stand
behind this testing modality as the
best screening strategy for women.
While we celebrate our past,
we continue to look to the
future.
The ThinPrep Pap test is the most
trusted and widely used Pap test on
the market. With your partnership,
we have contributed to a decline in
cervical cancer rates. We’re looking
forward to delivering you innovative
tools and advancing cervical cancer
screening together for the next 20
years and beyond.
Re
Ref
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Learn why the world trusts the ThinPrep system at ThinPrep.com
MLO201608_AD Hologic-SPREAD.indd 33
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SPECIAL FEATURE
G A S T ROEN T E ROL OGY
epithelial abnormalities that are indicative of progression, but
also stromal changes, such as angiogenesis and infiltration of
specific immune cell subsets that play important roles in tumor development and progression.15 This approach utilizes
immunofluorescence labeling to detect a series of biomarkers
on formalin-fixed paraffin-embedded (FFPE) tissue sections
(Figure 1, pg. 30). After capturing whole slide digital images
of the tissue, specialized image analysis software automatically
segments specific subcellular compartments and tissue structural components and quantifies biomarker expression patterns in the context of the cellular and tissue architecture. This
imaging approach has the advantage of assessing multiple key
cell types, including immune cells, and multiple pathways of
malignant progression, while maintaining essential spatial and
contextual information. A multivariable classifier is then used
to integrate the quantitative biomarker and morphology data
into a risk score, which is used to estimate the individual patient’s risk of disease progression within the next five years, as
well as to identify patients who might already have prevalent
HGD or EAC.14,16
Other approaches have assessed individual biomarkers or
panels of biomarkers stained by immunohistochemistry on
FFPE tissue and found a modest benefit for risk prediction.17,18
However, these tools rely on manual interpretation of biomarkers on tissue slides and have not yet been implemented for risk
prediction in clinical practice. Other approaches have examined mutations using next generation sequencing and PCR and
found that patients who progressed to HGD or EAC had an increased mutational load.19,20 While not currently commercially
available for clinical testing, these methods have the advantage
of high-throughput detection of multiple mutations that may
indicate future malignant progression. The drawback of these
methods is the loss of spatial context of molecular changes.
Furthermore, these methods do not assess cellular changes
in the stroma and morphologic changes that indicate risk of
progression.
Individualized risk prediction methods will ease the unnecessary concern of patients whose BE is at low risk of progressing
while highlighting patients at high risk to ensure they receive
more aggressive care. Another important benefit is the potential
for cost savings by extending endoscopic surveillance intervals
in low-risk patients, and by early therapeutic intervention in
patients at high risk of progression, which is expected to reduce
the significant cost burden associated with cancer treatment
and end-of-life care.21,22
New tools and new optimism
In summary, accurate risk prediction provides an opportunity
to improve patient management by providing better outcomes
while improving the efficiency of healthcare spending in the
management of BE. Ultimately, the goal is to reduce the incidence and mortality of EAC in patients with BE, which can be
accomplished by improving methods of early detection and intervention. The new tools that improve diagnostic accuracy and
provide accurate risk stratification are an exciting step forward
in this process.
4. Cancer Facts & Figures 2016 American Cancer Society.
5. Yantiss RK. Diagnostic challenges in the pathologic evaluation of Barrett
esophagus. Arch Pathol Lab Med. 2010;134(11):1589-1600.
6. Shaheen NJ, Falk GW, Iyer PG, Gerson LB. ACG Clinical Guideline: Diagnosis and
Management of Barrett’s Esophagus. Am J Gastroenterol. 2015;111(1):30-50.
7. Johanson JF, Frakes J, Eisen D. Computer-assisted analysis of abrasive transepithelial brush biopsies increases the effectiveness of esophageal screening: a multicenter prospective clinical trial by the EndoCDx Collaborative Group. Dig Dis Sci.
2010;56(3):767-772.
8. Anandasabapathy S, Sontag S, Graham DY, et al. Computer-assisted brush-biopsy
analysis for the detection of dysplasia in a high-risk Barrett’s esophagus surveillance
population. Dig Dis Sci. 2010;56(3):761-766.
9. Swager A, Boerwinkel DF, de Bruin DM, et al. Volumetric laser endomicroscopy in
Barrett’s esophagus: a feasibility study on histological correlation. Dis Esophagus. May
8 2015, doi: 10.1111/dote.12371.
10. Leggett CL, Gorospe EC, Chan DK, et al. Comparative diagnostic performance of
volumetric laser endomicroscopy and confocal laser endomicroscopy in the detection
of dysplasia associated with Barrett’s esophagus. Gastrointest Endosc. 2016;83(5):880888 e882.
11. Ross-Innes CS, Debiram-Beecham I, O’Donovan M, et al. Evaluation of a minimally
invasive cell sampling device coupled with assessment of trefoil factor 3 expression
for diagnosing Barrett’s esophagus: a multi-center case-control study. PLoS Med.
2015;12(1):e1001780.
12. Iyer P, Johnson ML, Lansing R, et al. Discovery, Validation and Feasibility Testing
of Highly Discriminant DNA Methylation Markers for Detection of Barrett’s Esophagus
Using a Capsule Sponge Device. Gastroenterology. 2016;150(4):S66-67.
13. Cai JX, Campbell EJ, Richter JM. Concordance of Outpatient Esophagogastroduodenoscopy of the Upper Gastrointestinal Tract With Evidence-Based Guidelines. JAMA
Intern Med. 2015;175(9):1563-1564.
14. Critchley-Thorne RJ, Duits LC, Prichard JW, et al. A Tissue Systems Pathology Assay for High-Risk Barrett’s Esophagus. Cancer Epidemiol Biomarkers Prev.
2016;25(6):958-968.
15. Prichard JW, Davison JM, Campbell BB, et al. TissueCypher: A Systems Biology
Approach to Anatomic Pathology. Journal of Pathology Informatics. 2015;6:48.
16. Critchley-Thorne RJ, Davison JM, Prichard JW, et al. Sa1257 A Tissue Systems Pathology Test Detects a Field Effect Associated With High Grade Dysplasia and Esophageal Cancer in Barrett’s Esophagus Patients. Gastroenterology. 2016;150(4):S259.
17. Bird-Lieberman EL, Dunn JM, Coleman HG, et al. Population-based study reveals
new risk-stratification biomarker panel for Barrett’s esophagus. Gastroenterology.
2012;143(4):927-935 e923.
18. Horvath B, Singh P, Xie H, Thota PN, Sun X, Liu X. Expression of p53 predicts risk of
prevalent and incident advanced neoplasia in patients with Barrett’s esophagus and
epithelial changes indefinite for dysplasia. Gastroenterol Rep (Oxf). Oct 19 2015.
19. Del Portillo A, Lagana SM, Yao Y, et al. Evaluation of Mutational Testing of Preneoplastic Barrett’s Mucosa by Next-Generation Sequencing of Formalin-Fixed, ParaffinEmbedded Endoscopic Samples for Detection of Concurrent Dysplasia and Adenocarcinoma in Barrett’s Esophagus. J Mol Diagn. 2015;17(4):412-419.
20. Eluri S, Brugge WR, Daglilar ES, et al. The Presence of Genetic Mutations at Key
Loci Predicts Progression to Esophageal Adenocarcinoma in Barrett’s Esophagus. Am
J Gastroenterol. 2015;110(6):828-834.
21. Gordon LG, Mayne GC, Hirst NG, Bright T, Whiteman DC, Watson DI. Cost-effectiveness of endoscopic surveillance of non-dysplastic Barrett’s esophagus. Gastrointest
Endosc. 2014;79(2):242-256 e246.
22. Hao J, Snyder SR, Pitcavage JM, Critchley-Thorne RJ. A Cost-Effectiveness Analysis of A Test That Predicts Risk of Malignant Progression In Barrett’s Esophagus. Value
in Health. 2016;19(3):A6-7.
Aaron D. DeWard, PhD, serves as
Research Scientist for Cernostics, Inc.,
provider of the TissueCypher Barrett’s
esophagus assay.
REFERENCES
1. Hayeck TJ, Kong CY, Spechler SJ, Gazelle GS, Hur C. The prevalence of Barrett’s
esophagus in the US: estimates from a simulation model confirmed by SEER data.
Dis Esophagus. 2010;23(6):451-457.
2. Wani S, Falk G, Hall M, et al. Patients with nondysplastic Barrett’s esophagus have
low risks for developing dysplasia or esophageal adenocarcinoma. Clin Gastroenterol
Hepatol. 2011;9(3):220-227; quiz e226.
3. Thota PN, Lee HJ, Goldblum JR, et al. Risk stratification of patients with barrett’s
esophagus and low-grade dysplasia or indefinite for dysplasia. Clin Gastroenterol
Hepatol. 2014;13(3):459-465 e451.
34
MLO - ONLINE.COM
MLO201608-SpecFeature_MECH_AL.indd 34
Rebecca Critchley-Thorne, PhD, serves
as Vice President, Research and
Development, and co-founder of
Cernostics, Inc.
AUGUST 2016
7/12/2016 9:11:50 AM
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LAB MANAGEMENT
QC/Q A
External and internal QC for blood gases
By Al Jordan
Q
uality Control (QC) and Quality Assurance (QA) in the
clinical lab have changed. The Centers for Medicare and
Medicaid Services (CMS) has embraced a voluntary QC
option for meeting CLIA quality control standards called Individualized Quality Control Plan (IQCP), which was implemented in January 2016 for all labs that have been utilizing Equivalent
Quality Control (EQC). CLIA QC regulations will remain the
same as published in 2003. All of the pre-analytical, analytical,
and post-analytical systems requirements in the CLIA regulations will remain in effect.
There are many different aspects of the IQCP for laboratories
to consider. This article will cover the minimum requirements
for use of external quality controls with regard to blood gas QC.
Minimum guidelines
The minimum guidelines according to CLIA 88 (2003) require
laboratories to perform external quality control at least one time
per eight-hour shift.1 The IQCP does not change this requirement. In addition to the daily QC requirement, the laboratory
is also responsible for calibration verification for all non-waived
“moderate to high-complexity” test systems.
The laboratory must perform calibration verification at least
twice per year, for documentation purposes, as well as whenever
any of the following occurs: 1) after any major preventive maintenance; 2) when critical parts affecting an instrument’s performance are replaced; 3) after the laboratory switches lot numbers
on the reagents it used in conjunction with an instrument; and 4)
after the laboratory identifies an unusual trend or shift reflected
in its control material, or results that fall outside of established
acceptable limits.
External vs. internal
What qualifies as external quality controls vs. internal quality
controls?
Many of today’s automated blood gas (ABG) instruments
now have internal onboard QC. Since January 2016 there have
been questions on whether a laboratory could be cited on inspection for utilizing these internal controls to fulfill their
external control requirement as per CLIA 88. One of the questions is: At what point is the QC material introduced to the
ABG instrument for daily QC? If the internal QC is introduced
at the beginning of the sample flow path on the ABG instrument, does it constitute an external QC? On some ABG instruments with internal QC, the QC material is entered into the
sample flow path further down the line. Following the first
rule of the IQCP, the risk assessment (pre-analytic phase), contains five mandatory areas to include: 1) specimen handling; 2)
personnel handling the sample; 3) instrument; 4) reagent; and
5) environment.2
The laboratory runs the risk of error utilizing internal QC as
its external QC requirement. Since the ABG instrument is introducing the QC sample automatically, it eliminates the specimen
handling and personnel handling of the sample; the risk assessment for these two mandatory areas is eliminated by the ABG
instrument; the risk of error for these areas is not tested with the
use of an internal control. The pre-analytic phase, specimen/personnel handling the sample, is missed completely. Potential errors could be missed, particularly when time from patient draw
to introducing the sample is a critical factor in the testing of the
samples, as with blood gas testing.
When considering the blood gas pre-analytic phase, metabolic
changes occur rapidly after sample drawing. The most obvious
36
MLO - ONLINE.COM
step to counter the metabolic change is to analyze the sample
quickly. The International Federation of Clinical Chemistry
(IFCC) recommends keeping transportation times to a minimum,
as it will affect the values of the blood gas testing.3 Utilizing internal QC could create a potential error in the pre-analytic phase,
as it does not allow the laboratory to take into consideration the
sample transportation time, or that properly trained personnel
is handling the sample, as well as the QC material. Since these
are major factors in patient sample handling for blood gas, they
should also be considered in QC material for blood gas.
External QC material options
When evaluating external QC material for blood gas, it is recommended that laboratories use QC material that is most like patient sample. The majority of blood gas QC manufacturers use an
aqueous buffered material or perfluorocarbon emulsions solution. Although these are accepted QC matrices, they may not be
the best option for blood gas QC. The other option to consider is
reduced bovine hemoglobin solution (RBHS). An influential article published in 1993 established that there is a strong benefit to
using blood gas QC manufactured with RBHS as the external QC
material,4 and that has been widely accepted in the years since.
With the enforcement of the IQCP, laboratories should consider adopting a QC plan that includes the use of material that
most resembles patient samples. If labs eliminate the questions
of the internal QC material qualifying as an external control, and
have trained personnel introducing the QC sample, at least three
levels, once per each eight-hour shift, this most mimics a safe
pre-analytic practice.
In order to meet laboratory requirements for calibration verification on blood gas analyzers, lab directors should consider
the matrices of the material being used. RBHS-based calibration
verification material will most resemble that of human samples.
Using liquid stable, as opposed to lyophilized QC material,
will also assist in eliminating any pre-analytical errors while
reconstituting the freeze dried material.
These small adjustments in a QC plan for laboratories might
save the lab considerable stress at inspection time, as well as assist in meeting requirements as per CLIA 88 (2003).
REFERENCES
1. American College of Physicians. CLIA and your laboratory: a guide for physicians
and their staff. November 2014. https://www.acponline.org/system/files/documents/
running_practice/mle/clia-and-your-lab.pdf. 46.
2. The Joint Commission. Quality control option changing for clinical laboratories.
https://www.jointcommission.org/assets/1/6/JCP0314_QC_Clin_Labs.pdf
3. Baird G. Preanalytical considerations in blood gas analysis. Biochemia Medica.
2013;23(1):19-27.
4. Mahoney JJ, Wong RJ, Van Kessel AL. Reduced bovine hemoglobin solution evaluated for use as a blood gas quality-control material. Clin Chem.1993:39(5): 874-879.
Al Jordan serves as Vice President,
Sales and Marketing, for Massachusetts-based Phoenix Diagnostics, a
manufacturer of quality control and
calibration verification material for
blood gas, electrolyte, and chemistry
analyzers worldwide. He has more
than 30 years’ experience in the
laboratory equipment and clinical
diagnostics industry.
AUGUST 2016
MLO201608-LabMgmt-Phoenix_MECH_AL.indd 36
7/12/2016 2:47:59 PM
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Just because you’re meeting the regulatory minimum of once-a-day QC, doesn’t mean
you sleep well at night, or are confident that your laboratory has patient risk under control.
Daily QC may be adequate for some tests, but using the same approach for higher risk
tests could put some patients at risk for inappropriate treatment due to undetected
instrument failure. Can your lab afford that risk - or the delay and costs of repeating patient
samples? Bio-Rad Mission: Control is the first tool a laboratory can utilize to help
assess and manage those risks objectively: calculate your current risks, and help select
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MLO201608_AD BioRad-19509.indd 37
7/8/2016 12:52:48 PM
LAB MANAGEMENT
QC/Q A
Resolving QC failures
By W. Greg Cooper, CLS, CQA, MHA
M
ost medical technologists and technicians, responsible for outputting test results as quickly and reliably
as possible, hate it when their smooth flow of work is
abruptly interrupted by an out-of-control QC rule flag. Suddenly they are faced with delayed reports, the prospect of
a complicated technical investigation, and a litany of questions they must ask themselves, such as:
• “Is the out-of-control condition real?”
• “Do I really have a problem with the analytical system?”
• “If I do have a problem, when did it start and how many patient
samples are affected?”
• “What should I do first?”
• “What should I do next?”
The laboratory would have much less of a conundrum if it were using a new QC rule proposed in a 2012
Clinical Chemistry article.1 This rule allows, as part of
its process control, the possibility of a second set of QC
measurements when the first results are inconclusive.
The rule is called a Repeat 1:2s QC Rule, where 1:2s designates that the “run” is rejected when a single control is out 2
standard deviations (2s). This helps improve the rule’s ability to verify whether the process is out of control. If the rule
triggers a second set of QC results and the repeat results are
within their 2 standard deviation (SD) limits, then the laboratory can assume the condition was a random occurrence
and patient results can be reported. However, if any repeated
QC result is outside its 2 SD limits (regardless of the control
material) the run is rejected and patient results are held for
retest.
The test system and control system
Before troubleshooting begins in earnest, the laboratory
should consider the possibility that common failure modes
of the test system or the control system may be the cause of
the out-of-control condition.
The test system includes the reagents, hardware, and software. It may require more in-depth investigation and analy-
• Random instrument malfunction
• Power fluctuations
• Temperature fluctuations (environment and incubation chamber)
• Test operator mistake
• Technique (technique-sensitive testing)
• Reagent preparation (error in or inconsistent preparation of reagent,
failure to consistently follow manufacturer instructions)
• Control material (error in or inconsistent preparation of control
material, failure to consistently follow manufacturer instructions)
• Software malfunction
• Possible contamination
Table 1: Common causes of random error
sis, but the laboratory should at least first consider those few
things in the test system that are known to cause problems
or have a history in the lab of causing problems. These could
include such things as the reagent integrity, the tubing and
pumps used for sample and dispense, and the light source.
The control system includes the control materials,
the mean and standard deviation used to set the LeveyJennings chart, and the process control rules applied. Any of
these could be the cause of the out-of-control condition. The
technologist or technician should verify the control materials are in date, have been properly stored, and have been
properly prepared. Attention should be given to open vial
stabilities published in the product insert. Most control materials must be kept between 2-8oC, even after reconstitution,
so if the control material is kept at room temperature on the
analytical bench most of the day, open vial stabilities and
consequently control results may be compromised.
The mean and standard deviation are key to an effective
Levey-Jennings chart. Both must be calculated using sufficient data collected over a period of time, allowing for
multiple calibrations, multiple reagent lots, maintenance,
and multiple operators when technique may be an issue. If
either the mean or standard deviation is out of synchrony
with actual test performance, the Levey-Jennings chart will
frequently issue out-of-control alerts.
Recalibration is often the first but misguided reaction to an
out-of-control condition. Keep in mind that every time the
laboratory calibrates, it creates an opportunity for introduction of new or additional measurement error. Consequently,
if a laboratory calibrates more frequently than recommended by the manufacturer, it is one indicator that the quality
control system is likely out of synchrony with current test
performance.
Random error and systematic error
If the quality control system is found to be appropriate
and effective, then troubleshooting begins. First, the type
and approximate size of the error must be characterized,
because this will lead the investigator to possible specific
causes which are either random or systematic. This can be
accomplished using control data from previous QC events
(recent and historical QC), or the laboratory may choose to
take a more contemporary approach by testing multiples of
additional controls at different concentrations (Ns of 2 to
8 or more) and then examining the data for clues as to the
magnitude and cause of the out-of-control condition.
If the control pattern suggests random error, then the laboratory should investigate common contributors to random
error (Table 1). If a cause can be found, corrective action is
taken. If a cause cannot be found, then the laboratory should
perform comprehensive instrument maintenance followed
by recalibration. The control materials are retested, and if the
results are out of control, then the laboratory must continue
to sequester all patient results and undertake a root cause
analysis. If the control results are in control, then all patient
samples are retested and reported. Remember, random error
conditions occur, well, randomly, so all patient results prior
to the correction are suspect.
38
MLO - ONLINE.COM
AUGUST 2016
MLO201608-LabMgmt-COMBINED_MECH_LM.indd 38
7/12/2016 3:35:09 PM
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LAB MANAGEMENT
QC/Q A
The role of calibration verification/linearity
in the POCT market
By Glenn Mitchell
“I
s calibration verification/linearity required on my
POCT system—or not?”
If you are one of the many who have purchased an
instrument for point-of-care testing (POCT), this is a very
legitimate question. POCT user manuals will typically state
in one form or another that the linearity of the instrument
should be checked before the instrument is used for patients
for the first time. The user manual may also refer to calibration verification in lieu of linearity, or it may refer to both
terms. And in many instances, but not all, the manufacturer
may include a calibration verification/linearity kit with the
initial shipment of the test system to be used upon initial setup. The user manual will also generally state that subsequent
linearity tests are to be determined by the facility operating
the system.
So, the question above should perhaps be reformulated as
two questions. Once the test system is calibrated, controls are
set and run, and calibration has been verified and/or linearity has been established: “Is calibration verification/linearity
required on my POCT system moving forward? And, what
exactly is the difference between calibration verification and
linearity?” Let’s tackle the second question, first.
Calibration verification and linearity
Calibration verification is the process of assaying materials of
known concentration in the same manner as patient samples
to assure that the test system is accurately measuring samples throughout the reportable range of the instrument—
reportable range being the range of the test results for an
analyte, from minimum to maximum, which the instrument
test system can accurately measure. Per Clinical Laboratory
Improvement Amendment (CLIA) guidelines, at least three
samples (low, middle and high) spanning the reportable
range of the test are run as unknowns (performed in normal
test mode, not calibration mode), and the values obtained are
compared to the known values of the calibration verification
material. If the calibration verification confirms that calibration settings are valid for a specified method, no further calibration actions are necessary. Calibration verification is to
be performed every six months, or more often if specified in
the user’s test system instructions, the facility’s QC protocol,
and under circumstances outlined in CLIA 493.1255(b)(3).
Linearity, as defined by CLSI’s EP-06-A guideline, is as follows: “The ability (within a given range) to provide results
that are directly proportional to the concentration (amount)
of the analyte in the test sample.” Linearity studies are performed to determine the linear reportable range for an analyte. Linearity is achieved when measured results are directly proportional to the concentration of the analyte in the test
sample, within a given range.
One reason one may find the term linearity so frequently used in analyzer package inserts is that before the final
CLIA regulations were published in 2003, laboratories were
required to perform linearity studies every six months for
quantitative high complexity tests. Linearity studies, as
40
MLO - ONLINE.COM
such, are no longer required by CLIA, as calibration verification has basically replaced the term and process of linearity
studies. Please be aware, however, that linearity, as defined
above by CLSI’s EP06-A guideline, is still recommended and
is considered Good Laboratory Practice.
Moving forward, please note also that all testing, regardless of where performed, including POCT, is regulated by
CLIA. CLIA section 493.1255 requires, among other things,
that laboratories verify calibration. This section also requires
at a minimum that laboratories follow the manufacturer’s
protocol for calibration verification.
Waived and nonwaived testing
Which brings us to the first question: “Is calibration verification required on my POCT system?”
All laboratory clinical tests, POCT included, at a minimum
are regulated by CLIA and are classified as either waived
or nonwaived. Nonwaived tests are further classified as
moderately complex or high complexity.
The majority of POCT systems, defined as medical diagnostics testing that is performed outside of the clinical lab
in close proximity to where the patient is receiving care, are
classified by CLIA as waived. As defined by CLIA, waived
tests are simple, easy-to-run tests with a negligible risk for
an incorrect result. A CLIA-waived test system by definition
is cleared for home use by the U.S. Food and Drug Administration (FDA) and requires no training for the end user. Per
CLIA, COLA, The Joint Commission, and CAP, calibration
verification is not required for waived testing. The only requirement for waived tests is that the user follow, at a minimum, the manufacturer’s instructions. Be aware, however,
that state regulations can supersede CLIA guidelines. In
Massachusetts, for example, if a doctor’s office lab is statelicensed, it is required to perform calibration verification on
waived tests.
Test systems, including POCT, not categorized by the FDA
as waived are considered nonwaived. Per CLIA guidelines,
calibration verification is required on nonwaived tests. Nonwaived tests are further classified as moderately complex or
high complexity. Moderately complex nonwaived tests are
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LAB MANAGEMENT
QC/Q A
QC continued from page 38
Calibration continued from page 40
• Calibration (this is a primary cause of systematic measurement error
in the test system)
• Reagent integrity (new lot or newly prepared; second-most common
cause of systematic measurement error)
• Lack of or inconsistent instrument maintenance
• A LJ chart that is improperly set (target mean or standard deviation
not properly calculated)
• Instrument malfunction (reagent or sample pipette malfunction)
• Light source (deterioration)
• Incubation chamber temperature (shift in temperature or fluctuation)
• Failure to follow manufacturer instructions for performing the test
• Operator technique for technique-sensitive tests
• Software malfunction (recent updates can cause problems)
• Possible contamination (of reagents, tubing, water flushes, ancillary
equipment such as pipettes, etc.)
Table 2: Common causes of systematic error
If the control data pattern suggests systematic error, then
the laboratory should investigate common contributors to
systematic error (Table 2). Systematic measurement error is
much easier to find and identify than random error. The difficulty is in determining at what point in time the systematic
measurement error occurred, making the number of patient
samples between QC testing events really important. For
example, if a laboratory only tests quality control materials
every 500 patient samples, then it faces the difficult task of
deciding which and how many of the previously tested samples need to be retested. In some cases, time may be a factor
as well. It is possible that the lapse in time for 500 samples
may exceed the analyte stability, invalidating any chance to
retest. On the other hand, a laboratory that tests a QC material every 50 patient samples is in a much better position to
correct any bad patient results.
Once the cause for the systematic measurement error is
found and corrected, the test is recalibrated. Quality control
materials are retested. If the results are out of control, then
the laboratory must sequester all test results and undertake
root cause analysis. If the results are in control, then all patient samples that are believed to have been affected by the
out of control condition are retested and reported.
REFERENCE
1. Parvin C, Kuchipudi L, Yundt-Pacheco J: Should I repeat my 1:2s QC rejection? Clin
Chem, 2012;58(5):925-929.
W. Greg Cooper, CLS, CQA, MHA, is
a licensed clinical lab scientist in the
state of California. He is an ISO 15189
auditor and has a consulting business
focused on activities that improve
laboratory quality.
42
MLO - ONLINE.COM
performed in a laboratory setting on automated clinical lab
equipment with qualified personnel who possess skills in the
reading and interpretation of quantitative results. QA, QC,
calibration, and proficiency may be required as well. POC
tests that are classified as nonwaived fall under this moderately complex category and, as stated above, calibration
verification will be required.
High complexity nonwaived systems differ from moderately complex tests, as high complexity tests (such as cytology, flow cytometry, PCR, gel electrophoresis, MRI, and
CT scans) typically are not quantitative and as such may require substantial training and/or specialized knowledge in
the preparation, process, and interpretation of results. Even
though at this point POCT systems do not fall under the high
complexity heading, understanding this distinction is important. That is because, if a site develops its own procedure
or chooses to modify its waived (or moderately complex)
protocol, the test automatically bumps up from waived to
nonwaived high complexity status, and calibration verification, among other procedures, may be required in order to
be fully compliant by state and federal regulations. Modification examples could include not following manufacturers’
directions or performing tests on sample types not specified
in the manufacturers labeling.
Moving forward, how do end users determine the complexity designation of their POCT system? The manufacturer’s user manual or package insert should state whether the
test system is waived or nonwaived. If you still cannot locate
the necessary information, you can go online, call the manufacturer directly, or utilize one or more of these FDA and CMS
web-based resources:
• www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCLIA/search.cfm
• www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfclia/testswaived.cfm
• www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfclia/analyteswaived.cfm
To sum up: “POCT” is simply an acronym that is used to
describe the location where testing is to be performed, such
as at bedside or near the site of patient care. The complexity
designation of a POCT, waived or nonwaived, categorizes
the test so that proper quality control and regulatory procedures can be dictated and followed per the designation.
Calibration verification, though not required by CLIA on
waived tests systems, is required on nonwaived tests and under special circumstances with waived tests, including manufacturer’s directive and if states, municipalities, or other
regulatory and accreditation agencies require it. Linearity, as
defined above, is not required by CLIA but is recommended
and is considered Good Laboratory Practice.
While it is true that the vast majority of POCT systems
today are waived and thus typically do not require calibration verification, nonwaived designation of POCT is sure to
increase as current and future healthcare requirements and
advances in technology will allow for decreased instrument
footprints, an increase in turnaround time and throughput,
ease of use, expanded instrument quantitative data capabilities, and thus the requirement of calibration verification.
Glenn Mitchell serves as a National
Accounts Manager for AUDIT
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DRUGS OF A BUSE
CLINICAL ISSUES
Approaches to prescription opioid
abuse testing
Urgency drives new techniques
By Jenny Cybulski
T
he epidemic of prescription opioid abuse has been worsening
for decades in the United States, but it has broken through to
the public consciousness only in the past year or so. People
have been sensitized to the problem by the deaths of celebrities
from the misuse of opioid painkillers—most recently, of course,
the celebrated musician Prince. The misuse of these drugs has also
been discussed in the current presidential campaign, and federal
and state efforts to combat it have frequently been in the news. The
public attention is overdue; according to the Centers for Disease
Control and Prevention, more than 18,000 deaths related to opioid
overdose occurred in 2014.1
To combat this abuse crisis, certain states have legally mandated
that healthcare providers require tests for patients receiving pain
management drugs in an attempt to detect opioid misuse. This
is not only to protect the patients by screening for overuse, but
also to detect opioid levels below the dosage level, which could
indicate that patients are reselling their drug refills. Because of the
advent of these physician monitoring programs, laboratories must
be able to accurately detect low levels of these drugs, in order to
ultimately determine whether patients with chronic pain are
abusing prescription opioids.
Screening options
Laboratories have a diverse choice of sample matrices to screen
for opioid levels, but they ultimately need to choose the option
that will maximize workflow and keep costs low. Whole blood
is a unique matrix because it enables the analysis of the drug
compounds in parent rather than metabolized form. However,
it requires an invasive collection technique and can be challenging to pretreat due to the complexity. Oral fluid is a sample matrix that has had a lot of research focus recently. Although easy
to collect, the collection devices used in oral fluid analysis can
introduce their own unique challenges due to the presence of
surfactants.2 Urine is a dependable sample matrix that doesn’t
require the lengthy pretreatment steps that are necessary with
whole blood, or the use of a collection device, which is required
with oral fluid. It does, however, require a hydrolysis step to
calculate correct concentrations of drug compounds. Due to
the accuracy and precision associated with urine analysis and
the developed methods, it’s widely considered to be the preferred sample matrix choice. It is both simple and inexpensive to
perform opioid drug testing on urine.
Urine Sample
DILUTE & SHOOT
Dilute Sample
(10x-30x)
PPT & FILTER
SPE
Add 4:1 ACN or MeOH
Develop Method
BETA-GLUCURONIDASE
REMOVAL PRODUCT
Dilute Sample
in 40% MeOH
Vortex
Condition & Equiliberate
Spin Down (optional)
Filter (protein prec. plates)
or Centrifuge
Load
Transfer to Vial
Load Sample
Transfer
Wash
Collect Pass Through
Blow Down & Reconstitute
(~15-25 min)
Elude
Transfer to Vial
Transfer to Vial
Blow Down and Reconstitute
LC/MS Analysis
Figure 1. Comparative workflows
44
MLO - ONLINE.COM
MLO201608-ClinIssues_MECH_AL.indd 44
AUGUST 2016
7/12/2016 3:13:53 PM
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Toxicology laboratories have been using opiate tests to detect hydrocodone in the past
and have seen inadequate performance. Our newest addition to the drug testing menu, the
Thermo Scientific™ DRI® Hydrocodone Assay, provides accurate results and is specific to
hydrocodone and its major metabolites in human urine. This liquid, ready-to-use immunoassay
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CLINICAL ISSUES
DRUGS OF A BUSE
ELISA vs. LC/MS
Laboratories running urine drug tests have traditionally used
enzyme-linked immunosorbent assays (ELISA), but a case can
be made that this testing approach is outdated and can be unreliable. For example, false negatives can alter the dependability of results, and the inability to accurately quantify drug concentrations
leads to unreliable analysis.3 If ELISA tests are positive, the patient
method as their sample preparation technique. They take the
samples with residual ß-glucuronidase enzyme and “dilute” between 10 and 30 times the original volume. The diluted sample
is then injected onto the LC column. However, the large dilution factor reduces the concentration of proteins/enzymes in
the sample along with the concentration of analytes, which
can result in poor sensitivity and can amplify any ion suppression that may be
occurring in the analysis.2
Another deficiency of the “dilute-andshoot” method is that it compromises the LC
column. One way to help alleviate the enzymatic buildup during a “dilute-and-shoot”
procedure is to centrifuge the sample to
pellet the enzyme and collect the supernatant, but this does not completely remove ßglucuronidase from the matrix (Figure 2).
One new ß-glucuronidase removal method
is a sample preparation technique that removes
ß-glucuronidase after enzymatic hydrolysis.
The sorbent eliminates the ß-glucuronidase
from the sample but does not interact with
opioid analytes. While the technique requires
rougly the same amount of time as a “diluteand-shoot” procedure, about one minute, the
sensitivity of the analytes is increased significantly. In one study, the sensitivity of THCCOOH was compared using the two preparaFigure 2. HPLC/UHPLC Column Lifetime: Beta-Glucuronidase removal product vs. Dilute-and-Shoot
tion methods, and the new technique showed
more than three times the sensitivity.
samples still need to be run on a mass spectrometer (MS) to quanDue to the rapid demand for results, lab leaders have to evalutify the concentration of drugs present. The time it takes to run
ate methods for drug testing and choose the most efficient techan ELISA is comparable to other tests, but this method requires
niques to analyze samples. Sample matrices, types of analysis
a more hands-on approach for laboratory technicians as well as
modes, and sample preparation techniques need to be considered
separate tests for each drug analyte. It has been argued that liquid
when making this critical decision. Laboratories cannot afford any
chromatography with mass spectrometry analysis (LC/MS) offers
mistakes or lack of sensitivity when testing patients for opioid
an efficient and easy way to separate and quantify a large class
abuse. Inaccurate results could negatively affect the quality of life
of opioid compounds. Many labs are moving toward testing with
for patients using prescribed pain management drugs. Utilizing
LC/MS first as advancements make it easier to run the tests and
the most beneficial techniques for the laboratory helps to guarmethod development becomes simpler. They prefer chromatograantee that patients who are required to undergo drug testing for
phy over ELISA for the testing of many opioid drug compounds
their medications will not only get rapid answers but also accurate
because they think that it meets their needs for increased accuracy,
results as well.
improved workflow efficiency, and reduced analysis time.
REFERENCES
The metabolism of drugs must be understood in order to ana1. National Vital Statistics System, Mortality File. Number and age-adjusted rates of
lyze urine by LC/MS. Before drugs exit the body through urine
drug-poisoning deaths involving opioid analgesics and heroin: United States, 2000–
excretion, they are tagged with a glucuronic acid, which helps to
2014. Center of Disease and Control/ National Center for Health Statistics. 2015. http://
www.cdc.gov/nchs/data/health_policy/AADR_drug_poisoning_involving_OA_Heroin_
change the polarity of the drug compound and aids in the absorpUS_2000-2014.pdf.
tion into the kidneys. Before chromatography analysis can occur,
2. Edinboro LE, Backer RC, Poklis A. Direct analysis of opiates in urine by liquid chrothe glucuronide must be cleaved through hydrolysis. This can
matography-tandem mass spectrometry. Journal of Analytical Toxicology. 2005;29(7):
be done in two ways: acid hydrolysis and enzymatic hydrolysis.
704-710.
Some labs prefer enzymatic hydrolysis because it cleaves the bond
3. Allen K. Screening for drugs of abuse: which matrix, oral fluid or urine? Annals of
without introducing harsh solvents into the sample or altering
Clinical Biochemistry. 2011;48(6):531-541.
peaks.
4. Lynch K, Wu A Yang, H. Development and validation of a novel LC-MS/MS opioid
confirmation assay: evaluation of ß-glucuronidase enzymes and sample cleanup methIn enzymatic hydrolysis, ß-glucuronidase is introduced to
ods. Journal of Analytical Toxicology. 2016;40:323–329.
cleave the glucuronide bond on the drug metabolite, resulting in
5. Wang P, Stone JA, Chen KH, Gross SF, Haller CA, Wu AH. Incomplete recovery of
free drug compounds.4.5 While this is effective, ß-glucuronidase
prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites.
can precipitate out in the LC column during the run, which can
Journal of Analytical Toxicology. 2006;30(8):570-575.
negatively affect the column’s selectivity and lifetime and can result in buildup in the MS. Analysts employ a variety of methods
to prepare hydrolyzed urine samples for LC/MS analysis includJenny Cybulski graduated from
Vanguard University with a BS in
ing “dilute-and-shoot,” protein precipitation, and solid phase exbiology and a minor in chemistry.
traction (SPE). The workflows associated with each technique are
She now serves as a Product
outlined in Figure 1, page 44.
“Dilute-and-shoot” vs. ß-glucuronidase removal
Most analysts in toxicology and clinical laboratories who utilize
chromatography for urine analysis employ the “dilute-and-shoot”
46
MLO - ONLINE.COM
MLO201608-ClinIssues_MECH_AL.indd 46
Communications Manager for
Torrance, California-based
Phenomenex, Inc.
AUGUST 2016
7/12/2016 3:14:05 PM
Syndromic Testing: The right test, the first time.
Respiratory • Blood Culture ID • Gastrointestinal • Meningitis
MLO201608_AD BioFire.indd 47
7/8/2016 12:52:13 PM
A GUIDE T O MOL ECUL A R DI AGNOS T IC S
Direct molecular diagnostics for sepsis
By John Brunstein, PhD
D
espite great strides in medical practice over the last 150 years, sepsis
(bloodstream infections coupled
with an excessive inflammatory response) remains a very significant problem. In fact, it’s estimated that globally,
sepsis is more common than heart attack
and claims more lives than any one form
of cancer. In the United States alone, severe sepsis has an estimated incidence of
300 per 100,000 population (.3 percent),
with approximately half of these cases
occurring outside of ICU settings and
up to one-quarter of patients with severe
sepsis dying as a result.1 In worst cases,
the time of untreated disease progression from detection to fatality can be as
little as a few hours. As if these statistics
weren’t sobering enough, the incidence
of sepsis even in developed countries is
observed to be increasing.2
Clearly, this is a medical condition
for which more effective diagnosis is
needed with both rapid test speed (short
turnaround time or TAT) and extreme
sensitivity. (Sepsis doesn’t necessarily
equate to high bloodstream pathogen titers; more than half of adults with sepsis
have less than one cfu/ml of associated
pathogen in peripheral blood.3) Finally,
identification of the causative pathogen
is essential in selecting appropriate antibiotic therapy as part of the treatment.
These requirements make molecular diagnostics (MDx) an attractive approach
to consider for sepsis diagnostics.
The conventional non-MDx workflow
for sepsis diagnosis is blood culture (BC)
(with various approaches to number and
types of BC bottles to collect, in an effort
to not be misled by insignificant transient
bacteremias or sampling contamination),
followed by traditional microbiology
workup (plate culture for identification
and antibiotic susceptibility determination) on positive BCs. The major downside to this approach is that it can take
several days to perform.
There are three common approaches
to applying MDx to this workflow in
an effort to speed up the process: direct
MDx on peripheral blood (pre-BC); MDx
on samples from positive BC; and MDx
on individual colonies on plate culture
derived from a positive BC. Of these,
direct MDx on peripheral blood sample
offers by far the largest potential saving
in critical time to diagnosis and will be
our main focus.
48
MLO - ONLINE.COM
MLO201608-Primer_MECH_AL.indd 48
MDx on peripheral blood
At first glance, this might seem like a
simple approach. Get a blood sample,
extract nucleic acids, and run some sort
of PCR, and you’re done, right? Unfortunately it’s not that easy; let’s consider
what some of the technical challenges
are.
First, there’s the issue of low pathogen
titer. Most nucleic acid extraction methods take input volumes of 1 ml raw sample or less (in fact, many work on 0.5 ml
or less). Sampling statistics aren’t working in our favor here, either; if a specimen has 1 cfu/ml, we would actually
need to sample almost 3 ml to achieve a
95 percent probability of getting that one
colony. At 0.5 ml per sample, we’d need
to take six samples to get this confidence
level—and bear in mind that most adult
sepsis cases don’t even reach 1 cfu/ml.
The bottom line: sample size is an immediate hurdle to direct MDx sepsis diagnosis. Any method looking to do this will
probably need to sample on the order
of 10 ml of peripheral blood, and have
some sort of selective enrichment process to collect the bacteria into a workable portion for nucleic acid extraction.
Fortunately, this hurdle can be handled
by a variety of methods, such as selective
centrifugation or microfluidic sorting; if
you look to do direct sepsis MDx, expect
to have to do something like that.
Once we have a nucleic acid extract
from a suitably large peripheral blood
sample, our second challenge is “What
do we look for?” In terms of causative
organisms, the majority of sepsis cases
arise from a handful of organisms; however, it’s a very “long tail” distribution
with very many organisms contributing
small numbers of cases that we’d rather
not overlook. If we restrict ourselves to
looking for bacterial pathogens, there
is at least one molecular target in common to all of them, the 16S ribosomal
RNA (rRNA) gene. Genetic pressure
has maintained enough conservation
in this sequence to allow for the design
of PCR primer sets which can be very
nearly “pan-species,” so that a single
PCR reaction can confirm or deny the
presence of bacterial DNA in our extracted sample. This sounds at first to
be a good approach; and indeed, several
commercial systems take this approach
to at least rule out sepsis when bacterial DNA is not detected. In the event
that DNA is detected, however, a positive 16S rRNA result is not tremendously
helpful; without knowing the organism species (singular or in some cases,
plural), appropriate antibiotic therapy
choices cannot be informed and we have
limited benefit toward the initiation
of suitable therapy. Ideally then, we
also wish our MDx method to provide
pathogen species identification.
Our 16S rRNA gene can still be used
for this, if we’re willing to sequence internal “hypervariable” regions inside the
conserved flanking sequences and compare these sequences against known species 16S libraries. This approach is both
accurate and broad spectrum, although
it is neither fast nor readily amenable to
high throughput and automation. Despite these limitations, this method has
been in use in some clinical settings for
some time now, and it can be a powerful tool in the early detection and appropriate classification of sepsis cause and
treatment selection.
Peripheral blood, multiplex
methods
Other methods approach this by employing multiplex methods. The reader will
hopefully recall from earlier installments
of “The Primer” that this is the simultaneous use of multiple target-specific (in this
case, likely pathogen species) primer sets
within a single PCR reaction, with some
form of downstream analysis such as amplicon size, melt temperature, sequencespecific array capture, or distinctive fluorophore labelling used to identify which
species-specific amplicon (or amplicons)
are generated from the sample. Multiple
commercial methods exist which employ
variations on this approach, which generally has rapid TAT coupled with good
sensitivity and specificity, at the cost of
less than complete coverage of potential causal organisms. If such a method
is inexpensive and provides answers in
a sizeable proportion of cases, while the
remaining cases continue to be analyzed
by traditional microbiological methods,
then it has clear benefits to the “average”
patient even with these shortcomings.
One powerful bit of information from
the classical microbiological workflow in
sepsis is the antibiotic resistance profile
of the infecting organism. While empirical data on the likely antibiotic susceptibility of an identified organism in a
AUGUST 2016
7/12/2016 9:06:52 AM
A GUIDE T O MOL ECUL A R DI AGNOS T IC S
particular geographic setting is a good
start for selecting antibiotic therapy,
changes in antibiotic resistance profiles of various pathogen species do
occur, and, given the potential rapidity of disease progression, waiting to
see if a first-choice antibiotic is working may not be in the patient’s best
interest. By the time an outlier with
unexpected resistance is recognized, it
may be too late.
Some MDx methods for sepsis determination attempt to address this by including detection of common antibiotic
resistance markers such as MecA, VanA,
or various penicillin binding proteins
(PBPs). These approaches generally
share three common weakness, however.
• First, there are a large number of genetic
variants of these resistance genes, making
it essentially impossible to screen for all
of them. Compounding this, they tend to
have relatively quick genetic drift, meaning the common versions of one of these
genes today may not be the common
form in a year, leading to potential false
negative results. Addressing this through
constant surveillance and revision of the
suitable target primer sets for PCR-based
detection approaches is costly and cumbersome from an assay validation and
regulatory perspective.
• Second, even if a particular antibiotic
resistance marker is detected by MDx approaches, that is not strictly equivalent to
meeting clinical guidelines for antibiotic
resistance, which includes dose response
considerations (“breakpoints”). That is, an
organism may have the resistance gene,
yet express it poorly or at low levels, and
thus not display the associated resistance
as formally defined.
• Third, most MDx methods utilized in
the sepsis context don’t have a way to
unequivocally associate a detected antibiotic resistance marker with the bacterial
species if more than one species is present
(in blood culture terms, a mixed infection). This last problem is perhaps best
illustrated in the context of methicillin resistance associated with MecA; peripheral
blood collected by venipuncture is sometimes contaminated with coagulase-negative Staphylococcus such as S. epidermidis,
which in turn frequently carries the MecA
gene. If a sample is detected by MDX as
positive for S. epidermidis, S. aureus, and
MecA, does the patient have a MRSA infection—or an easily treatable non-MRSA,
with contamination?
albeit at a cost of lost TAT improvements.
Sampling of blood culture bottles either
before or after they have detected positive for bacterial growth by conventional
methods allows us to avoid the low target titer problem; rather than < 1 cfu/ml
of the original sample, a positive inoculated BC bottle will have large pathogen
titers suitable for reliable collection and
detection in more manageable sample
sizes (0.5 ml or less). Application of
MDx at this stage can still save 24 to 48
hours needed for traditional plating and
colony growth, enumeration, and antibiotic susceptibility testing. As such, this
is a viable compromise solution, and
several commercial systems take this
approach.
Finally, if molecular detection of specific antibiotic resistance markers is an
acceptable surrogate of phenotypic antibiotic resistance determination, but
there’s a desire to be able to definitively
assign detected markers to a specific species in a potentially mixed sample, then
MDx may be applied subsequent to a
positive BC being plated. Selection of
individual colonies from such a plate ensures that any detected resistance markers are associated with the identified
colony species, and may be done with
exceptionally rapid and crude nucleic
acid extraction techniques (such as heat
lysis without further purification) followed by rapid PCR methods, which can
thus still save a day or more compared
to allowing colony growth in presence
of antibiotic test materials required for
accurate determination of phenotypic
breakpoints. (Note this is also the point
THE PRIMER
in the traditional workflow where mass
spectrometry is most commonly applied,
as it can provide extremely rapid, accurate, and inexpensive species identification minutes from isolated colony to
identification, although currently lacking
antibiotic susceptibility data.)
Molecular diagnostics offers hope for
improved treatment of a common and
serious condition; however, this hope
is not without complexities in actual realization. Multiple MDx approaches to
sepsis diagnosis are available in various markets, and it is hoped that the
above consideration of the challenges
each of these approaches in general
face and the compromises they make
in providing a solution will help to
guide selection of the method(s) most
appropriate to the reader’s setting.
REFERENCES
1. Mayr FB, Yende S, Angus DC. Epidemiology of
severe sepsis. Virulence 2014;5(1):4–11.
2. Martin GS. Sepsis, severe sepsis and septic shock:
changes in incidence, pathogens and outcomes. Expert
Review of Anti-infective Therapy. 2012;10(6):701–706.
3. Towns ML, Jarvis WR, Hsueh PR. Guidelines on blood
cultures. J Microbiol Immunol Infect. 2010;43(4):347-349.
John Brunstein, PhD,
is a member of the
MLO Editorial
Advisory Board. He
serves as President
and Chief Science
Officer for British
Columbia-based
PathoID, Inc., which provides consulting
for development and validation of
molecular assays.
MDx further “down the path”
As opposed to doing MDX direct from
peripheral blood for sepsis, application
of MDx further down the diagnostic path
helps to resolve some of these issues,
AUGUST 2016
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FUTURE BUZZ
NE X T GENE R AT ION SEQ UENCING
The technology and clinical applications
of hybrid capture NGS
By John Havens, PhD
C
linicians are challenged with identifying variants of disease and determining appropriate treatment
plans on a daily basis. The more relevant
the information they have at their fingertips, the more likely a successful patient
outcome. Nucleic acid testing (NAT) is a
common method of detecting the presence of specific disease-associated bacteria and viruses, such as HIV, and is used
in routine blood screening. Though it is
inexpensive and reliable, NAT cannot
always provide clinicians with all the
information they need, however, which
is where a more detailed analysis of
samples becomes necessary.
Whole genome next generation sequencing (NGS) can provide a wealth of
information to direct treatment. However, it has been a costly option for clinicians, particularly for large numbers of
samples. A more cost-effective option is
targeted NGS, in which genomic regions
of interest are enriched prior to sequencing. Targeted NGS focuses resources on
acquiring relevant data and enables multiplexing, so many patient samples can
be analyzed simultaneously. As such,
certain applications can benefit from targeted NGS, from analyzing mutations
in cancer to viral integration sites and
variants.
While targeted NGS can be performed
cheaply via polymerase chain reaction
(PCR), processing the numbers of samples common in many clinical research
facilities at the required level of quality
and consistency is more suited to the approach of hybrid capture. This technology
uses labelled DNA probes complementary to the target region for enrichment
from fragmented genomic samples. Obtaining high efficiency and accuracy, as
well as minimizing cost, requires careful
consideration of probe design.
Optimized oligo design
Quality control
Oligonucleotides used for hybrid capture
NGS can be designed based on a variety
of technologies. One such technology,
which is seeing increased use in clinical
diagnostics, utilizes biotin-streptavidin
interaction for enrichment. 5’ biotinylated probes are designed to hybridize
to the target DNA, and isolated using
streptavidin beads (Figure 1).
Instead of many shorter overlapping
probes, this variation of the technology
uses longer 120 bp probes designed for
end-to-end tiling across the target region.
This maintains a high capture efficiency
while reducing the number of overall
probes required, saving cost. In addition,
this length of probe can tolerate several
mismatches and still pull down the targets,
affording the detection of novel variants.
The challenge with longer oligos, however, is that the coupling efficiency of each
additional base affects the final yield of the
full-length oligo. While obtaining a 99.6
percent efficiency results in 62 percent of
products being full-length for 120-mers,
losing just one percent efficiency reduces
full-length yield to as little as 15 percent.
Highly efficient synthesis in this variation
of hybrid capture is coupled with biotinylation only of full-length product, thereby
ensuring only the complete oligos are
enriched, improving sequencing reliability.
This method of hybrid capture also employs blocking oligos for library adaptors
to maximize targeting efficiency. These
blocking oligos are especially important in
reducing costs via multiplexing, as libraries carry unique barcodes to associate each
read with the appropriate sample, which
must be obscured during pull-down. Targeting may be improved by 60 percent
through this method, resulting in lower
cost and the generation of highly-focused
clinically relevant data.
High data quality, especially when
searching for specific variants of a target, is essential in targeted sequencing
panels.
Commercially synthesized hybrid capture oligos, such as the type described
here, are produced individually, allowing
more stringent and individualized QC.
Specifically, electrospray ionization mass
spectroscopy (ESI-MS) is used to confirm
the absence of truncated species. Those
samples found with truncated species can
undergo re-synthesis and be QC’d again,
saving waste.
Interestingly, looking in more detail
at quality-control data highlights the
stochastic nature of synthesis failures
(Figure 2, pg. 53). When analyzing an
exome research panel of approximately
430,000 probes covering 39Mb, for every
re-synthesis round, the number of failures
decreased exponentially. This indicates
that failure is random, rather than being
sequence-specific, and as such, a re-synthesis approach could be applied to all oligos.
This provides clinicians some assurance
that their diagnostics results can be consistent regardless of target.
These data suggest that when oligos undergo only a single synthesis, as is the case
with array-synthesis platforms, the probe
population will have varying levels of
truncation. This could lead to insufficient
reproducibility of enrichment and therefore inconsistent NGS results.
By synthesizing, quality controlling,
and normalizing every probe individually, the amount of each probe in the
final pool can be determined. This benefits clinical applications through identical performance across batches. Moreover,
commercial probe panels are available
that conform to FDA 21 CFR Part 820
certification for medical devices (GMP),
Figure 1. Biotin-based oligo design for hybrid capture NGS. This hybrid capture probe utilizes a 120 bp biotin-labelled oligo for isolation using streptavidin magnetic beads. Cot-1 DNA blocks for repetitive regions, and blocking oligos block library adaptors, increasing targeting. End-to-end tiling, coupled
with individual probe synthesis and QC, provides more even coverage than overlapping designs.
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FUTURE BUZZ
NE X T GENE R AT ION SEQ UENCING
The transformation of pathogen diagnostics
through next generation sequencing
By Crystal Icenhour, PhD
C
linical laboratories have multiple
methods for diagnosing infectious
disease, but the fact remains that
despite culture, microscopy, antibodybased testing, and molecular testing such
as PCR, up to 75 percent of infections are
not diagnosed in a timely manner, if at
all.1,2 One of the biggest issues is that a
target is needed to know which test(s)
can lead to an accurate diagnosis; the
laboratorian must know what pathogen
is suspected. And since many pathogens
exhibit common symptoms, it is often
difficult to determine an appropriate
target.
The diagnostic challenges have resulted in a reliance on differential diagnosis
and can result in spread of infectious diseases, longer hospital stays, more critical
illnesses, longer recoveries, and compromised outcomes related to delayed or
ineffective treatment. In addition, partly
due to ineffective diagnostics, antibiotics
are significantly overused in the United
States. Such misuse of antibiotics is creating drug-resistant pathogens. According
to the Centers for Disease Control and
Prevention (CDC), “Each year in the
United States, at least two million people
become infected with bacteria that are resistant to antibiotics, and at least 23,000
people die each year as a direct result of
these infections.”3
In the midst of these concerns is the reality that infectious disease outbreaks are
no longer “local.” As large populations
travel internationally, local authorities
are often under-resourced in coping with
identified outbreaks, and inadequate
diagnostics and treatments leave us all
more vulnerable. News headlines alert
us to the Lyme disease season and to the
risk of the Zika virus, while not so long
ago people in Africa were dying by the
thousands from Ebola. The confluence of
all these factors makes it more important
than ever to develop accurate and timely
diagnostics for infectious diseases.
19,000 known pathogen genomes. Now
a significant advancement in NGS is
capable of yielding the most accurate
and comprehensive pathogen diagnoses of any method available. In theory,
it is possible to diagnose all possible
pathogens utilizing the power of NGS.
From a single sample, it is possible
to simultaneously screen any clinical
sample for all pathogens (whether bacteria, virus, fungus or parasite). The
sample can be tissue, blood, swab, stool,
or any sample that contains the pathogen being sought. This method not only
can identify known pathogens, but is
also capable of revealing the presence
of unknown pathogens. In this new application of NGS, unbiased genetic sequencing provides a “genetic blueprint”
from all detectable microorganisms in
the sample. Sophisticated bioinformatics can make sense of the raw data by
matching it against all sequenced pathogen genomes to identify the pathogens
present in the sample. This system
can detect the presence of multiple
pathogens.
Other applications of NGS include
pathogen surveillance (such as in animal populations, water supplies, or
food processing) and can discover previously unrecognized or never-before
seen pathogens. Since all genetic information from the sample is sequenced,
it is not necessary to know which microbes are suspected; a target is not
necessary. Moreover, whole genome/
whole transcriptome sequencing methods can provide higher sensitivity and
specificity than other diagnostic technologies in use today. NGS also has the
ability to identify drug-resistance genes
in identified pathogens, study the interaction between host and pathogens,
and characterize the host’s immune
response through gene regulation
exploration.
NGS: consider the possibilities
Many experts see sequencing as the future for clinical laboratory diagnostics,
but while this breakthrough technology
holds exciting potential, there are some
important cautions to consider before
adopting NGS for pathogen diagnosis.
Current challenges for broad implemen-
Next generation sequencing (NGS) has
been effectively implemented in the
diagnosis of cancers and genetic disorders, including neonatal testing, but it
is much easier to evaluate the human
genome alone as opposed to more than
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NGS: be aware of the challenges
tation of NGS for pathogens include
turnaround time (TAT) and costs—not
inconsiderable real-world obstacles.
The outlook is improving considerably, however. The cost for sequencing
a single human genome has dropped
from ~$14 million in 2006 to ~$1,500 in
2016 (it should be noted that the fabled
“$1000 Genome” requires substantial
instrumentation and human capital,
which is not attainable by most clinical
laboratories). And, while TAT is currently slowed by the time required for
sample preparation and sequencing,
advanced protocols for sample preparation are being introduced that can
reduce it, as well as the cost of sequencing. One example of such improvements
is the use of barcoding to allow multiplexing of up to 96 samples in a single
sequencing run.
An additional challenge is in collecting samples that include the pathogen
being sought. Sample collection and
stabilization is crucial for identification
of pathogens; the pathogen can only be
identified if it is present in the sample.
Samples must be extracted and processed with robust protocols that should
be conducted under appropriate quality
guidelines. Further, when conducting
whole genome sequencing from humanderived samples (particularly tissue and
blood), it can be challenging to obtain
adequate pathogen DNA/RNA due to
the overabundance of host genetic material. Host cell depletion methods can
be utilized, but concentrations of the
remaining pathogens are often small,
and it is challenging to obtain adequate
sensitivity levels. Because it is crucial
to obtain adequate sequencing coverage of the pathogens to ensure the best
sensitivity and specificity, molecular biologists trained in NGS should be consulted with respect to sample collection,
sample extraction, library preparation
methods, and sequencing parameters.
Another issue: while clinical laboratories are often comfortable with the
wet lab techniques of extraction, library
prep, and/or whole genome sequencing, it is just as important to recognize
the critical role of data analysis of the
complex data sets generated by NGS.
Data analysis for pathogen detection
AUGUST 2016
7/12/2016 3:00:58 PM
Hybrid continued from page 50
is a highly complex process, as dozens
or even hundreds of microorganisms
must be analyzed in a single test. Further, pathogenic versus non-pathogenic
determinations must be made, as all
clinical samples will contain many nonpathogenic microorganisms. A few data
analysis tools for pathogen detection
have become available, but they have
not yet been validated for clinical use.
Further challenges for pathogen NGS
data analysis involve the generation,
safety, storage, transport, computation,
and archiving of these massive data files
(up to 2 terabytes).
Looking ahead
While these challenges may seem intimidating, engaging strong strategic partners with expertise in NGS and complex
data analysis can make adoption of NGS
pathogen diagnostics achievable today.
The best strategic partners will be able
to provide recommendations for sample
collection, extraction, and preparation,
as well as be able to handle the complex
data analysis phase of pathogen identification. Balancing the complexities, along
with engaging experts, will facilitate a
complete transformation of pathogen diagnostics through NGS. Today is an exciting time for clinical laboratories as this
important technology is being embraced.
Imagine a world where a single clinical
sample is able to yield all of the data needed for pathogen identification, pathogen drug response, and host-pathogen
immune response …this world is quickly
approaching!
REFERENCES
1. Lyme Disease Association, Inc. National Institutes
of Health study on Lyme disease reveals significant
chronic symptoms and common misdiagnosis. 2005.
http://www.lymediseaseassociation.org/index.php/
lda-press-releases/326-national-institutes-of-healthstudy-on-lyme-disease-reveals-significant-chronicsymptoms-and-common-misdiagnosis.
2. Tomas MY, Getman D, Donskey CJ, Hecker MT.
Over-diagnosis of urinary tract infection and under-diagnosis of sexually transmitted infection in adult women
presenting to an emergency department. J Clin Microbiol. 2015. http://jcm.asm.org/content/early/2015/06/05/
JCM.00670-15.abstract.
3. Centers for Disease Control and Prevention. Antibiotic resistance threats in the United States, 2013.
Executive summary. www.cdc.gov/drugresistance/pdf/
Crystal Icenhour, PhD,
is the founding Chief
Executive Officer of
Aperiomics, a Northern
Virginia company
currently offering the
Absolute*NGSSM
Pathogen Detection
suite of products.
further ensuring quality for clinical
applications.
For studies with large numbers of
samples, this approach becomes highly cost-effective, especially as it allows
a modular approach to probe panel
design, with individual targets mixed
to adapt the study for new regions of
interest.
Targeted NGS in cancer
provide insight into patterns in HPV
integration and its role in cancer.
In addition to oncogenic viruses, infectious agents such as Ebola and Zika virus
can be traced with targeted NGS. The ability of hybrid capture probes to tolerate
several mismatches enables novel variants
of rapidly mutating viruses to be detected.
Different isolates can then be tracked to
different geographic regions, potentially
allowing the source of the outbreak to be
identified.
Acute myeloid leukemia (AML) is characterized by faulty differentiation of heIntegrating NGS into the clinic
matopoietic stem cells, deriving from
By bringing the cost of sequencing down,
mutations arising at cell division. In one
targeted NGS using hybrid capture probes
case, on analysis of cells taken from bone
can complement NAT strategies when
marrow, a patient presented with four difmore detailed information is needed.
ferent subclones of AML cells with varyTargeted NGS presents the latest ining prevalence, as analyzed by targeted
novation in sequencing technology, with
NGS. Following chemotherapy, none of
cutting-edge probe and assay designs
the cancerous clones could be detected
that reduce costs and improve reliability.
by standard blood analysis, although targeted NGS detected
the clonal evolution
of leukemic cells.
The patient eventually relapsed as a
result of the least
prevalent subclone
at diagnosis, which
developed an additional mutation,
proliferating in the
absence of the others. NGS analysis
allowed detection
of this evolved clone
before it was seen
Figure 2. Oligo synthesis failures are stochastic, not sequence-specific.
by standard blood
The re-synthesis approach ensures a consistent probe population and
characterization.
therefore uniform NGS results.
This example inBringing the benefits of these new techdicates how improved sequencing could
nologies into the clinic requires an interincrease the chances of detecting rare canface between clinicians and genomics excerous clones. In clinical diagnostics, inperts. As these technologies proliferate, in
creasing sequencing depth to 500x would
addition to growing in-house expertise,
bring the level of sensitivity required to
it is now becoming a popular option to
detect clones down to five percent prevalook toward external partnerships for a
lence. Such a level of sequencing depth is
complete solution, from bioinformatics to
only feasible through targeted NGS, and
customized NGS panels.
in these types of cases helps clinicians
identify clues pointing to patient relapse.
REFERENCE
Interestingly, it has also been indicated
1. Klco JM et al. Association between mutation clearthat diminishing the level of cancerous
ance after induction therapy and outcomes in acute myclones by chemotherapy to a level below
eloid leukemia. JAMA. 2015;314(8): 811-822.
five percent correlates with improved pa1
* Figure 1 - Oligo refers to xGen Blocking Oligo and
tient outcome, and this can be directly
Probe refers to xGen Lockdown Probe, both from IDT.
tracked with sensitive NGS.
Viral research
John Havens, PhD,
serves as Vice President
of Business Development at Coralville,
Iowa-based Integrated
DNA Technologies.
Targeted NGS also has utility in virology.
For example, genomic integration of the
human papillomavirus (HPV) can cause
several cancers. Hybridization probes
designed against the 8Kb HPV genome
provide detection of the integration site.
Sequencing the flanking regions can also
AUGUST 2016
MLO - ONLINE.COM
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7/12/2016 3:01:07 PM
MANAGEMENT MATTERS
Continuous quality management in the laboratory
By Nancy Alers, MS, MT(ASCP)CM
I
t is estimated that about 60 percent to 70 percent of clinical
decisions are made based on laboratory results.1 Laboratory
quality, therefore, plays a vital role in the delivery of quality patient care. But quality can mean different things to different labs and different supervisors. This article will briefly
explore the concept of laboratory quality and how a closely
monitored quality assurance plan can help ensure efficient
laboratory operations.
Quality can be an intangible, hard-to-measure concept, and
it has been defined in multiple ways by different entities over
the years: from a product that is free of deficiencies (ASQ); to a
product whose inherent characteristics fulfill requirements (ISO
9000); to a system that gets the right care to the right patient, at
the right time (HHS.gov).
One recent consensus, from the Organization for Economic
Cooperation and Development, stresses “that quality involves
safety, effectiveness, appropriateness, responsiveness, or patient centered care, equity or access and efficiency.”2 In laboratory medicine, quality is often interpreted as adherence to
regulatory standards3 or compliance with CLIA regulations.
Where one might take issue with such formulations is that they
seem to imply that without requirements, there would be no
need for quality. In fact, there are situations in the laboratory for
which there is no set regulatory standard; situations that may
warrant careful evaluation and consideration from laboratory
management.
CLIA regulations apply to all laboratories the same,4 providing a roadmap to ensure laboratories can meet minimum quality standards. But not all laboratories are created equal, and
therefore quality is much more than laboratory compliance.
The World Health Organization (WHO) defines quality in
laboratory medicine as the ability to produce accurate, reliable,
and timely results. WHO also stresses that it is important for
a laboratory to have a good laboratory quality management
system (QMS) in order to achieve this goal.5
Quality management system
Laboratory continuous quality monitoring is not a novel idea.
Section 493.1200 (b) of the CLIA regulations states, “each laboratory’s quality system must include an assessment component
that ensures continuous improvement of the laboratory’s performance and services through ongoing monitoring that identifies, evaluates and resolves problems (USDHHS, 2003).6
So what is new about laboratory quality? The Patient Protection and Affordable Care Act of 2010 has changed the laboratory landscape, and as a result quality has become more relevant than ever. The new impetus is for laboratories to think
about value instead of volume, which translates into a need to
have leaner processes, work more efficiently, eliminate redundant testing, and work cooperatively with the rest of the clinical
patient management team.7
A QMS is an overarching system of specific policies and
procedures to address all the activities within a laboratory. A
QMS goes beyond Quality Control (QC) and Quality Assurance
(QA), and it entails looking at the laboratory as an all-inclusive
system, ensuring there are guidelines for addressing activities
in all areas. A comprehensive QMS can help a laboratory ensure
that all activities are monitored and evaluated, and results are
accurate and reliable.
There are different QMS models out there that can be used.
Whichever is selected, it is essential that the QMS addresses all
activities in the path of workflow from the pre-analytic to the
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post-analytic testing phase. To ensure the quality of the overall
system, lab leaders need to ensure the quality of the activities in
the different testing phases.
Quality assurance reviews
A good QMS will require ongoing assessments as part of the
QA plan. A QA plan is the backbone of the laboratory QMS.3
Quality assurance reviews should be tailored to the unique
needs of the laboratory. Factors such as staff competency, test
complexity, population served, documentation practice tendencies, test volume, etc., should be taken into consideration when
determining the focus and frequency of reviews.
Data obtained from QA reviews should be evaluated carefully by the laboratory director or manager, and opportunities for improvement should be realized. Data evaluation will
reveal the risks or tendencies specific to your system, which
can lead to errors. Once risks are identified, efforts should be
guided toward corrective and preventive measures. Error detection, correction, and prevention are the ultimate goals of a
well-structured QA plan.
A culture of quality
Promoting a culture of quality should not only be done during
the inspection window; it should be an everyday effort. In addition to having competent staff, ensuring all standard operating
procedures are up to date and followed, and monitoring processes everyday through quality control practices, the laboratory must have a good QA plan. A QA plan will look at all the
activities performed on the path of workflow from the pre- to
post-analytic phase and determine if there are opportunities for
improvement.
Laboratories must remember that quality is a lot more than
regulatory compliance, and it includes having competent staff,
streamlined processes, good communication, and constant
evaluation.
REFERENCES
1. The Lewin Group. The value of laboratory screening and diagnostic tests for prevention and health care improvement. http://www.chi.org/uploadedFiles/Industry_
at_a_glance/Lewin%20Report%20on%20Dx%20Tests%20(2009).pdf.
2. Anderson G, Hussey PS. Comparing health system performance in OECD countries. Organization for Economic Cooperation and Development. Health Aff (Milwood).
2001;20: 219-232 doi: 10.1377.
3. Dai SY. What defines a laboratory Quality System? www.foodsafetymagazine.com/
magazinearchive1/octobernovember-2013/what-defines-a-laboratory-quality-system
4. Harmening, D. Laboratory Management Principles and Processes. 2nd edition.
Philadelphia, PA. FA Davis; 2007.
5. World Health Organization. Laboratory Quality Management System Handbook.
http://apps.who.int/iris/bitstream/10665/44665/1/9789241548274_eng.pdf
Published: 2011. Accessed: March 1, 2016.
6. Clinical Laboratory Improvement Amendments of 1988:42 CFR [K(1)-493.1200 (b)].
7. Miles J, Weiss RL. The role of laboratory medicine in Accountable Care Organizations. https://www.aruplab.com/files/resources/suite/ACO%20Lab%20Strategy_whitepaper.pdf.
Nancy I. Alers, MS, MT(ASCP)CM,
serves as a Laboratory Manager at
Fort Washington Medical Center in
Fort Washington, MD.
AUGUST 2016
7/12/2016 9:09:16 AM
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WASHINGTON
REPORT
Precision medicine and the
cancer “moonshot”
What will be the impact of these initiatives on laboratory medicine?
By Larry Altshuler, MD
I
n his State of the Union address delivered in January 2015, President
Obama announced the launch of his
administration’s Precision Medicine Initiative. In his 2016 Address, the president
challenged medical science to commit to
a “Moonshot” to find a cure for cancer.
Both initiatives are extremely worthwhile, and it is invaluable for medical
science to have the “bully pulpit” of
the presidency behind critical research.
Of course, implementing the programs
will be difficult, and the impact they will
have on the healthcare system will be
significant. Also, the two initiatives are
best understood as overlapping to some
degree; cancer, after all, is one of the
diseases/disorders that is most amenable to a precision medicine approach.
Precision medicine and cancer
Precision medicine is often defined as the
process of finding a specific treatment
based on the individual characteristics of
each individual patient. In actuality, it is
not meant to treat each person uniquely,
but to find subgroups of people with the
same genetic characteristics for which
unique treatments are applicable.
Cancer is the one disease for which
precision medicine is already being applied. “Targeted” drugs have been developed based on genetic variations and
mutations found in particular tumors.
The president’s initiative wants to apply precision medicine to other diseases
as well, including Alzheimer’s disease,
obesity, diabetes, and mental illness. In
fact, genetic testing is currently available
for 2,000 medical conditions, although
most of these will not be included in the
initiatives, at least for now. So, what can
we expect with the conditions that will
be involved?
Although there have been successes
with cancer, there have also been many
failures. Part of the reason is that precision medicine is more than just genetics;
it also involves epigenetics, which relates
to lifestyle and environmental factors
that modify the expression of the genes.
So it is not as simple as identifying dysfunctional genes and finding a treatment
to block or modify them. With specific
cancers, we may only be dealing with a
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few genes and a few epigenetic factors;
other diseases may involve dozens of
genes and epigenetic factors, and thus
the process may be much more complex.
The “Moonshot” metaphor hearkens
back to President Kennedy’s 1961 challenge to the Space Program to put an
American on the moon by the end of the
1960s. (The goal was met with Apollo 11
in July 1969.) As such, it is a powerful
call to action. President Obama did not
set a timetable to “cure” cancer, and doing so would in fact be unrealistic. This
is because there are more than 100 different types of cancer, and even more subtypes of those cancers. Cancers also are
very “devious”; for example, some have
developed methods of preventing detection from the immune system as well
and mutating to become resistant to various treatments. The failures of precision
medicine in the cancer arena, and the fact
that successes have been relatively few
for only a handful of cancer subtypes,
demonstrate the difficulties. In fact, most
cancer experts doubt that cancer will
ever be eliminated totally.
It is also important to realize that
both initiatives will require enormous
amounts of money, research, and extensive collaboration among all medical
providers. They may also change the
way medicine is practiced and taught
and how healthcare is provided to each
person and reimbursed to each medical
provider. They will also require a significant change in the way research is
conducted and applied.
The role of laboratory medicine
The two initiatives will also profoundly
affect laboratory medicine, as labs will
play a significant role. Labs will be at
the forefront of both initiatives and may
reap financial benefits from their involvement. After all, both initiatives are
fundamentally based on testing. First,
they will involve genetic testing, which
has become less expensive year by year,
and in the near future may be feasibly
accomplished by virtually any clinical
lab. Such testing is not used only to determine individual characteristics of the
patient, but also of a tumor or the tissues
involved in other diseases. In addition,
the effect of epigenetic variables will
also have to be tested. This will involve
not only newly developed lab tests, but
also the correlation of present lab tests
that reflect epigenetic factors. Even basic
chemistries and tissue testing may eventually impact whether or not a specific
treatment can be applied successfully.
In addition, for the “Moonshot,” additional research will attempt to find elements in the body’s tissues (including
blood) that can detect cancer before it is
discovered by other means. Both initiatives will also require tests from body
tissues and tumor tissues that will determine whether a specific treatment will or
will not be effective. A present example
is the K-ras determination for metastatic
colon cancer; studies show that patients
whose tumors express the mutated version of the KRAS gene will not respond
to two specific chemotherapy agents. On
the other hand, if a breast or esophageal
cancer has a mutation in the Her2Neu
gene, there are two chemotherapies that
can “target” and slow or destroy the
growth of the cancer. A great effort will
be made to find similar gene mutations
and correlate them to various treatments;
this applies not just to cancer but to other
chronic diseases as well.
In sum, these two initiatives will significantly impact the way medicine is
practiced. They both hold promise to
stimulate new discoveries, but such discoveries most likely will be incremental.
The process will also take a long time;
we should think in terms of decades, not
years. We should also realize that they
are enormous undertakings and will require substantial resources, which may
wax and wane depending on the progress observed and the priorities of political leadership. And, certainly, laboratory
medicine will be integrally involved.
Larry Altshuler, MD, is the author of
DOCTOR, SAY WHAT? The Inside Scoop
to Getting the Best Health Care and
DOCTOR, SAY WHAT? The Guides: What
Works and What Doesn’t for Over 90
Medical Conditions. He is a practicing
internist, hospitalist, and integrative
practitioner at a major cancer center in
the Midwest.
AUGUST 2016
7/12/2016 9:16:36 AM
SCC
Soft Computer
www.softcomputer.com
An authorized IBM Business
Partner for 24 years, SCC Soft
Computer offers systems that run
on the IBM Power Systems – Power
Architecture-based server line. SCC
Soft Computer’s robust laboratory
and genetics information system
software solutions are powered by
IBM’s AIX Power Series Platform.
SCC’s laboratory and genetics information
management systems:
Personalized Solutions
for Personalized Medicine
The understanding of disease as a multifaceted
entity—combined with the correlation of test results—
has resulted in more accurate disease identification,
prognosis, and treatment. This, in turn, has led to the
concept of personalized medicine, where understanding
a given patient’s unique disease presentation on all
levels enables a physician to select the most effective
treatment for that patient.
Genetic research and testing can produce large amounts
of data that must be analyzed, stored, and correlated—
not only with drug reactions, but also with numerous
patient characteristics. Attempting to accomplish this
using manual methods or basic electronic applications
is neither cost-effective nor efficient.
Customizable and robust software must be employed
to support the analysis, storage, and correlation efforts,
and to establish the large number of possible test
protocols. Likewise, this software should be capable
of providing physicians and testing labs online
availability of the most current test interpretations and
recommendations, which is crucial for the successful
practice of personalized medicine.
At SCC Soft Computer, we’re building a foundation for
personalized medicine with SCC’s Genetics Information
Systems Suite®, which provides a full range of genetics
information management tools designed to automate
workflow. This suite can be integrated with hospital
information systems and offers a selection of highly
configurable modules that allow for predefined or
unique, user-defined protocols.
SCC’s integrated laboratory and genetics software
solutions assist healthcare providers in personalizing
patient disease treatments based on each individual’s
unique history and genetic composition, as derived from
laboratory and clinical data. Clinical evidence can be
corroborated with genetic profiles, giving doctors faster
turnaround times in patient treatment. With test results
and genetically derived information from manual and
automated sources at your fingertips, SCC’s powerful
integrated information management solutions can help
narrow the gap between diseases and cures.
To learn more, visit us at www.softcomputer.com.
To schedule a demonstration with one of our experts,
please contact Ellie Vahman at [email protected].
Visit SCC Soft Computer at AACC 2016
Booth 1349 | August 2 – 4 | Philadelphia, Pennsylvania
Visit SCC Soft Computer at CAP 2016
September 26 – 28 | Las Vegas, Nevada
SCC’s Genetics Information Systems Suite®:
®
SoftCytogenetics t4PGU.PMFDVMBS t4PGU'MPX$ZUPNFUSZ®t4PGU1BUI%Y®t4PGU)-"®t4PGU#JPDIFNJTUSZ®t4PGU(FOF1PSUBM™
MLO201608_AD SCC.indd 57
®
7/8/2016 2:01:29 PM
PRODUCT FOCUS
AU T OM AT ION
Laboratory automation solution
Combined with state-of-the-art
analyzers and intelligent informatics, Power Express provides
a total laboratory automation
solution that maximizes uptime,
minimizes errors, and allows
laboratories to achieve optimal
workflow without the work. Labs can achieve both operational and clinical improvements with 1) LEAN automation that
eliminates bottlenecks for consistent workflow, improves FTE
utilization, and expedites delivery of results; 2) consolidated
multidisciplinary workflow that employs single point of entry
for all core laboratory disciplines; 3) scalable automation that
provides freedom to grow menu and capacity; and 4) fast and
reliable results that consistently deliver TAT <30 minutes, helping to increase physician satisfaction and speed to treatment.
Beckman Coulter, www.rsleads.com/608ml-151
Automated immunohematology system
The TANGO infinity automated immunohematology system maximizes workflow efficiency with minimal pre- and
post-analytic operator steps. The instrument offers confidence in result accuracy by monitoring critical processes
including verification of all liquid pipetting for every sample, reagent, and assay. Its intuitive software
is easy to learn and use, even for intermittent operators. The
proven Erytype S and Solidscreen II reagent lines minimize
false positive results yet provide the appropriate sensitivity to detect clinically significant antibodies and weak antigen
types. The lean and secure TANGO infinity allows generalists
to confidently perform testing, freeing valuable immunohematology experts to perform complex serological tasks.
Bio-Rad, www.rsleads.com/608ml-152
Simultaneous detection and identification of
multiple analytes
The BioPlex 2200 System combines
proprietary random access multiplex
testing with innovative software and
QC features to maximize workflow
and ensure results accuracy. The fully
automated BioPlex 2200 eliminates
batch testing and provides reproducible results with fast turnaround times. This system supports
a variety of autoimmune, infectious disease, and specialty
assays, such as the fifth-generation HIV Ag-Ab Assay. BioPlex immunoassays use magnetic beads infused with fluorescent dyes and coated with antigen and antibody ligands,
allowing simultaneous detection and identification of multiple analytes from a single sample. BioPlex 2200 can process a minimum of 150 individual bead results for each assay.
HIV 1/2 Supplemental Assay
The Geenius HIV 1/2 Supplemental Assay differentiates antibodies to HIV Types 1 and 2 in serum, plasma and whole blood.
A reader and proprietary software provides automated reading and interpretation with clear, accurate onscreen
results in 30 minutes with full traceability. Geenius is faster and easier than
Western Blot and a walk-away alternative to the Multispot HIV-1/HIV-2 Rapid
Test. It covers the second step of the HIV testing algorithm recommended by the CDC. Bio-Rad, www.rsleads.com/608ml-154
HbA1c results and hemoglobin variants
The D-100 System is the future of HbA1c
testing. With innovative solutions to maximize workflow efficiency, the D-100 System allows high volume laboratories to
quickly and easily report HbA1c results
while also detecting hemoglobin variants.
With one-touch operation and streamlined
result review, the D-100 delivers gold standard HbA1c results. The D-100 is a unique HbA1c testing system with no interference from common hemoglobin variants.
With an impressive throughput and dramatically reduced operator hands-on time, the D-100 produces HbA1c results that
are automatically reviewed and flagged. The software interface
is easy to use, and designed to maintain continuous operation.
Barcode driven microbiology specimen
processor and work-up system
COPAN’s WASPLab brings microbiology
out of the manual era and straight into
full automation. WASPLab is a barcodedriven Microbiology specimen processor and work-up system which connects
to WASP DT using a conveyor track.
WASPLab moves samples from front-end
processing to full specimen management, automated incubation, and digital Microbiology. With its modular design and
small footprint, WASPLab can be customized to the unique
needs of the lab, including robotic plate management system,
smart incubators, and state-of-the-art image acquisition technology. With the revolutionary algorithms paired with sophisticated image analysis software, WASPLab is changing the way
labs work and opening the door for groundbreaking digital
Microbiology. Copan, www.rsleads.com/608ml-156
Blood bank automation
Hemo Bioscience’s Hemo-QC is a whole
blood quality control kit intended for
the daily quality control of routine blood
bank reagents used in manual, semiautomated and automated blood typing
and antibody screening test systems. Features include 7 ml containers compatible with all instrument
systems currently available in the United States; three tubes
for daily quality control of ABO/Rh(D) and antibody detection;
42-day closed vial stability and 10-day open vial stability; cost
effectiveness, as fewer reagent sets are used monthly; monthly shipments on standing order; and FDA 510(k) clearance.
Hemo Bioscience, www.rsleads.com/608ml-157
58
MLO - ONLINE.COM
MLO201608-ProductFocus_MECH_GH.indd 58
AUGUST 2016
7/12/2016 4:58:05 PM
KapSafe™-mini
An Automated
bench-Top Recapper
Also available
as a
multi-rack system
Safety & efficiency
are important
laboratory issues.
Eliminate potential repetitive stress problems, even with
smaller tube volumes, during manual recapping by using
an automated and affordable recapper
from the makers of the
Pluggo™ Decapper.
You have used us
as a decapping
solution, now we have a
recapping solution.
PROTECT
YOUR
HANDS
Visit our website for
additional information
www.lgpconsulting.com
Laboratory Growth & Productivity
WWW.LGPCONSULTING.COM
1.877.251.9246
Accommodates all major tube sizes
and a variety of analyzer racks
Serving laboratories since 2002 | Contact us for literature and sales information
MLO201608_AD LGP.indd 59
7/8/2016 12:57:57 PM
PRODUCT FOCUS
AU T OM AT ION
Automated bench-top recapping
For safe and affordable automated benchtop recapping, the KapSafe Recapper has
three models to fit both small and large
volume facilities: KapSafe, KapSafe-LS,
and the KapSafe-mini. Manual recapping
and decapping can cause many challenges, and laboratory staff will appreciate the
protection offered with automated equipment and affordable solutions. Safe recapping is essential regardless of volume. All
KapSafe models have no pneumatics, no
noise, small footprint, and throughput up to 1,200 tubes per
hour. LPG Consulting, www.rsleads.com/608ml-158
High-volume automation solution
Designed for high-throughput
laboratories, cobas connection
modules (CCM) offer the ability
to consolidate the vast majority
of a laboratory’s testing volume,
at high throughputs—up to 2,000
tubes per hour, while providing
predictable workflow and turnaround times. The CCM provides
customers the ability to connect to the Roche standalone automation portfolio directly to Roche analytics without compromising the flexibility of the standalone automation concept.
Based on its design, this high-volume automation solution provides convenient sample loading, quality and quantity checks,
workflow flexibility, and multidisciplinary connectivity, such as
hematology, coagulation, and the first IVD vendor to connect
molecular testing solutions validated for cross-contamination
compliance.*Expected Q4/16 availability in the U.S. Roche,
www.rsleads.com/608ml-159
Automating primary vial preprocessing steps
Improving laboratory efficiency,
the cobas p 480 instrument accepts PreservCyt, SurePath liquidbased cytology vials as well as
cobas PCR Media tubes; processes
four vials simultaneously; and requires minimal training with intuitive interface. It reduces hands-on
time and repetitive motions with four unique workflows: Decapping; Recapping; Aliquoting; and Reagent addition and
heating. It improves sample reproducibility and process reliability: a) Sample chain of custody is assured with primary and
secondary vial barcode matching; b) All vials are spun prior to
opening to remove potentially contaminating droplets from
sample caps; c) Precision pipetting using CO-RE tip, Total Aspirate and Dispense Monitoring (TADM) and AntiDroplet Control
(ADC) technologies reduces opportunities for contamination
and ensures sample integrity; d) No LIS or data connection is
required; and e) Printable reports capture all sample IDs, sample error, and reagent lot and expiration information. Roche,
60
MLO - ONLINE.COM
Capillary separation for proteins, hemoglobins,
and HbA1c
Sebia’s
CAPILLARYS
2 Flex Piercing provides high resolution
capillary
separation
for proteins, hemoglobins, and HbA1c. The
CAPILLARYS 2 Flex
Piercing offers proven
technology, and high
throughpu, maximizing walk-away capabilities. The CAPPILLARYS 2 Flex Piercing
utilizes proprietary software, PHORESIS, allowing pathology
ease of use and interpretation as well as networking seamlessly proven at installations in the United States and worldwide.
Sebia, www.rsleads.com/608ml-161
Automated sample processing
Aptio Automation combines intelligent technologies with workflow expertise from
Siemens Healthineers
to deliver flexible solutions that advance
laboratory productivity.
Labs can leverage enhancements introduced
in 2016—including more diagnostic analyzers, pre-analytical
modules, and process management tools—to simplify multidisciplinary testing. Highly adaptable, needs-based Aptio
Automation solutions will enable lab leaders to accelerate
sample processing, achieve consistent turnaround times, and
proactively control operations for better patient outcomes.
Siemens Healthineers, www.rsleads.com/608ml-162
Free-standing and TLA ready automation
system
Sysmex XN-9000 provides comprehensive multi-discipline
testing modules for hematology and HbA1c testing. The system is configurable to the level of efficiencies desired, with
a combination of the following modules: advanced hematology testing analyzers (XN-10), reflexive slidemaker/stainer (SP10), automated digital cell image (DI-60), pre / post-analytical
EDTA tube management (TS-10), HPLC-HbA1c testing (Bio-Rad
VARIANT II TURBO Link), and integrated decision logic software (Sysmex WAM). The design flexibility of the system offers a free standing, lavender top management island, with
integration to various TLA tracks available in the market today.
Sysmex, www.rsleads.com/608ml-163
AUGUST 2016
7/12/2016 4:58:19 PM
B E YO N D A B E T T ER B OX ™
ADVANCED HEMATOLOGY ANALYZERS
TO IMPROVE YOUR LAB
NEXT GENERATION
DIAGNOSTICS
ADVANCED TOOLS
& TECHNOLOGIES
PROCESS
OPTIMIZATION
HARMONIZED
SUPPORT
Sysmex has a decades-long legacy of developing better analyzers. Today, we’ve moved well beyond
“building better boxes” into four key areas to create a more holistic, intuitive ecosystem that improves
lab operations, promotes better care and enhances patient management practices.
THEY ALL LINK TOGETHER
• NEXT GENERATION DIAGNOSTICS – continuing to pioneer the future of diagnostic performance
• ADVANCED TOOLS & TECHNOLOGIES – proprietary tools that drive greater insight into your lab
• PROCESS OPTIMIZATION – moving beyond simple automation to a fully integrated environment
• HARMONIZED SUPPORT – combining truly personalized service with a revolutionary technology
platform
Go Beyond a Better Box™ at W W W.SYSMEX.COM/BEYOND_MLO to see how Sysmex
improves hematology and your entire lab.
Join us in Philadelphia at AACC 2016, booth number 1727.
© Sysmex America, Inc. 2016. All rights reserved.
MLO201608_AD Sysmex.indd 61
7/8/2016 2:02:50 PM
NEW PRODUCTS
Pre-filled specimen jars
2.0 mL blood collection tube
Surgipath AffirmSeal Pre-Filled Specimen Jars include
four new sizes; 40mL, 60mL, 90mL and 120mL, and are offered in multiple packaging configurations. Pre-filled with
10 percent neutral buffered formalin, AffirmSeal jars are
specifically designed for safe collection and transport of
various sized specimens. The jars feature a plastic mold
design that creates two clearly audible and tactile “clicks”
when the lid is properly secured. This provides confirmation that the specimen is secure, helps prevent leakage,
and offers an added level of safety from formalin fumes for couriers and technicians.
AffirmSeal jars are made of durable plastic to prevent cracking when inadvertently
dropped, and comply with 95 kPa pressure testing for safe air shipment and use in pneumatic transport systems. The packaging is made of chipboard material, making it safe for
operating room use. In terms of configurations, Mini-Pak sizes allow for easy breakdown
of the cases for distribution to other labs and physician offices. Alternatively, flat packs
save storage space and provide added durability during transit.
Cell-Free DNA BCT, a direct-draw tube for the collection and preservation of whole blood samples,
is now available as a 2.0 mL draw tube. Cell-Free
DNA BCT is a direct-draw blood collection tube with
a formaldehyde-free preservative stabilizing nucleated blood cells. This unique stabilization prevents
the release of genomic DNA, allowing isolation
of high-quality cell-free DNA which can be further
used for a wide range of downstream applications
commonly used in clinical research studies, drug
discovery, and diagnostic assay development.
Cell-Free DNA BCT reduces the need for immediate
plasma preparation due to its stabilization properties. Cell-Free DNA is stable for up to 14 days, while
circulating tumor cells are stable for up to four
days, at room temperature, allowing convenient
sample collection, transport, and storage. The new
2.0 mL tube, and the 10.0 mL tube, are available
in multiple kit configurations. Cell-Free DNA BCT
is For Research Use Only, not for use in diagnostic
procedures in the U.S.
Leica Biosystems
CLASSIFIEDS
Streck
Energy saving laboratory fume hood
The UniFlow CE AireStream is a full duty fume
hood in a compact size, which offers 50 percent
energy savings over conventional hoods. The
CE’s low flow constant volume by-pass design
maintains consistent face velocity. CE fume
hoods are offered in 30”, 36”, 48”, and 72” widths
and can be equipped with a wide selection of
accessories to meet specific process needs. CE
fume hoods are constructed totally of composite
resin for superior chemical resistance, no rust,
and can be supplied with or without an exhaust
blower in standard or explosion proof models.
HEMCO is an ISO 9001:2008 Certified Company.
HEMCO
MEDICAL/TECHNICAL
SALES REPRESENTATIVE
Helena Laboratories has four
entry-level positions available
as a Medical/Technical Sales
Representative for medical laboratory products. The positions are
based in New York, New Jersey,
Pennsylvania and North Carolina.
Each position has responsibility for a multi-state territory. The
candidate must have a degree In
Biological Sciences or Chemistry,
Medical Technologist preferred.
The position provides base salary,
commissions, bonuses, company
automobile and expenses.
Contact Human Resources at
[email protected]
EEO/AA Employer
ADVERTISER
INDEX OF ADVERTISERS
WEB
PAGE
Advanced Instruments, Inc. .........................................www.aicompanies.com ............................................................. 51
American Medical Technologists .................................www.americanmedtech.org...................................................... 18
American Proficiency Institute .....................................www.api-pt.com ........................................................................... 3
ARKRAY ..........................................................................www.arkrayusa.com .................................................................. 27
AstraZeneca ...................................................................www.TAGRISSOhcp.com .........................................................6-7
AstraZeneca ...................................................................cobasEGFRtest.com ..................................................................8-9
AUDIT MicroControls Inc..............................................www.auditmicro.com ................................................................ 39
Bio-Rad Laboratories ....................................................www.bio-rad.com/diabetes ....................................................... 13
Bio-Rad Laboratories ....................................................www.qcnet.com/mc-mlo ........................................................... 37
BioFire Diagnostics .......................................................biofiredx.com ............................................................................. 47
CellaVision .....................................................................www.cellavision.com................................................................. 35
CompuGroup Medical ..................................................www.cgm.com/us ........................................................................ 1
Hologic - HIV ..................................................................USAptimaVirology.com........................................................... IBC
Hologic Inc .....................................................................www.hologic.com .....................................................................IFC
Hologic, Women’s Health..............................................ThinPrep.com ........................................................................32-33
ITL BioMedical ...............................................................www.itlbiomedical.com/scu...................................................... 55
Kamiya Biomedical .......................................................www.k-assay.com/MLO.php ..................................................... 43
LGP Consulting..............................................................www.lgpconsulting.com ........................................................... 59
Meridian Bioscience Inc................................................http://goo.gl/57ZNiz ...................................................................... 5
Michigan State University ............................................www.bld.natsci.msu.edu/online-education............................. 63
Millipore Sigma .............................................................sigma-aldrich.com/clinical........................................................ BC
Owen Mumford Inc .......................................................www.owenmumfordinc.com .................................................... 25
Quantimetrix Corp ........................................................quantimetrix.com ....................................................................... 21
Randox Laboratories Ltd ..............................................www.randox.com/keepyourinstrumentincheck ...................... 41
Roche Diagnostics .........................................................www.cobasEGFRtest.com ......................................................... 31
SCC Soft Computer .......................................................www.softcomputer.com ............................................................ 57
Sebia Inc .........................................................................www.sebia-usa.com .................................................................. 15
Sysmex America Inc .....................................................www.sysmex.com/beyond_mlo ............................................... 61
Thermo Fisher Scientific - Clinical Diagnostics..........www.thermoscientific.com ....................................................... 45
This index is provided as a service. The publisher does not assume liability for errors or omissions.
62
MLO - ONLINE.COM
AUGUST 2016
7/12/2016 4:58:33 PM
MLO201608_AD MichiganState.indd 63
7/8/2016 12:58:32 PM
EXECUTIVE SNAPSHOT
By A lan L enhof f, Edi tor
Diagnostics for infectious diseases, cancer
screening, and safeguarding the blood supply
If you were explaining Hologic to
someone who is not familiar with the
organization, how would you characterize its primary areas of expertise?
At the highest level, Hologic is focused on
enabling healthier lives, everywhere, every
day, by pursuing what we call “the Science
of Sure.” This means we strive to provide
healthcare providers and patients progressive certainty and ever-greater peace of
mind in their clinical decisions. We do this
by developing, manufacturing, and commercializing premium diagnostic, medical
imaging, and surgical products, many of
which serve the healthcare needs of women.
How does diagnostics fit in with the
company’s overall business? What
are the synergies between the company’s business divisions? Diagnostics
is Hologic’s largest business, represent-
STEPHEN P. MACMILLAN
Chairman of the Board
Hologic
Professional
I joined Hologic in December 2013 as
President and CEO, and was elected
Chairman of the Board in June 2015. From
2005 to 2012, I served as President and CEO
of Stryker. Before that, I held a variety of
roles with Pharmacia, Johnson & Johnson,
and Procter and Gamble.
Education
I earned a BA in Economics from Davidson
College, and graduated from Harvard
Business School’s Advanced Management
Program.
Personal
I serve on the boards of Boston Scientific
and AdvaMed, the medical device industry’s
trade association. In terms of philanthropy,
I’m proud that the company has pledged to
make 30 donations of up to $30,000 each
to non-profit groups focused on healthcare
and STEM education, to commemorate
Hologic’s 30th anniversary.
64
MLO - ONLINE.COM
MLO201608-ExecSnap_MECH_AL.indd 64
ing about 45 percent of revenue last year.
Many of our diagnostic products, including the ThinPrep Pap test, our Aptima assays for sexually transmitted diseases, and
our fFN test for preterm labor risk, address
important issues in women’s health, as do
our mammography systems and gynecologic surgical products. There are important synergies across our diagnostics franchises. For example, several of our Aptima
molecular tests can be performed from a
ThinPrep sample. Across the businesses,
we call on different kinds of customers, so
synergies are fairly limited, although we
are exploring a few areas. For example,
our diagnostics sales reps are beginning
to share information about our Genius
3D mammograms with their OB/GYN
customers.
On your watch, Hologic has accomplished a significant corporate turnaround during the last few years.
How has this enhanced the company’s ability to serve its customers?
Both Hologic and our diagnostics division
have returned to growth, and our recent
addition to the S&P500 stock index reflects
this dramatic turnaround. To make it happen, we attracted an entirely new leadership team to drive long-term, sustainable,
organic growth, rather than the debt-laden
acquisitions from the company’s past.
Based on these acquisitions, Hologic paid
more in interest expense than it invested
in research and development in the year
before I arrived. But as results have improved, we’ve used strong cash flows to
pay down debt, and consistently increased
R&D spending. And we’re beginning to
reap the rewards. For example, we recently
launched our viral load tests for HIV, hepatitis C, and hepatitis B in Europe, and have
begun to file regulatory applications in the
United States. That’s a great way to serve
our customers—by introducing innovative
new products.
Getting back to the Aptima family of
products—how do they advance diagnostics for HIV, hepatitis, and other
sexually transmitted diseases? Our
Aptima products include assays for sexually-transmitted infections (STIs), including chlamydia, gonorrhea, the human papillomavirus (HPV), and trichomonas. In
addition to providing accurate and reliable
performance, the Aptima assays run on
our fully automated, random access Panther system. Panther builds on the success
of our first generation automated instrument, Tigris, and incorporates customer
requests for greater flexibility and walk-
away time, with a small footprint. And
with the addition of the viral load assays,
we are actively expanding the Panther
menu. Finally, we are developing a new
system called Panther Fusion, which will
be a “sidecar” adding PCR capabilities and
a new assay format to Panther. That product will launch with our next generation
of respiratory virus tests, and over time
give labs the opportunity to run most of
their major molecular volume on one fully
automated, compact instrument.
Recently there have been proposed
changes to cervical cancer screening guidelines. How is that changing testing? With reference again to our
ThinPrep Pap test and the Aptima HPV
technology, Pap testing has contributed
to a significant reduction in cervical cancer deaths over several decades, and has
proven to be one of the most successful
screening programs of all time. The results
are even better when Pap testing is combined with a test for HPV, an approach
commonly referred to as co-testing. An
HPV test was approved as a primary
screen for cervical cancer in 2014, but
published studies support the superior
performance of co-testing. A study of 8.6
million women conducted by Quest and
published last year in Cancer Cytopathology demonstrated that nearly one in five
cervical cancers were missed by HPV primary screening, and a recent study led by
investigators at Houston Methodist Hospital supported these findings. We firmly
believe that women are best served when
physicians adhere to the recommendation
of the American College of Obstetricians
and Gynecologists (ACOG), which prefers co-testing for women between the
ages of 30 and 65.
How have your Procleix assays increased the safety of the blood supply by detecting the genetic material
of HIV-1, hepatitis, and other viruses? Our Procleix assays, sold by our partner Grifols, were the first molecular tests
approved by the FDA to screen donated
blood. These tests, when combined with
other screening measures, have helped
reduce the risk of contracting potentially
deadly viruses like HIV-1 and hepatitis
C from a blood transfusion to less than
one in a million. We also are developing a
molecular assay to screen donated blood
for the Zika virus. This demonstrates
Hologic’s ability to quickly address new
and emerging threats, as well as our commitment to help safeguard the donated
blood supply.
AUGUST 2016
7/12/2016 9:15:15 AM
In viral load testing
A change in direction
is within sight.
COMING SOON
Additional Aptima target assays in development:
E?SpblXeeiX\΋[ihƄlgXnbih• HBV viral load • HSV 1 & 2 • M. genitalium • BV • Candida
Aptima assays now available:
CT/NG • Trichomonas vaginalis • HPV • HPV GT
To get the full picture, visit USAptimaVirology.com
The Aptima HIV-1 Quant assay is not available for sale in the U.S.
ADS-01484-001 Rev. 001 ©2016 Hologic, Inc. All rights reserved. Hologic, The Science of Sure, Aptima and associated logos are trademarks and/or registered trademarks of
Hologic, Inc. and/or its subsidiaries in the United States and/or other countries. This information is intended for medical professionals in the U.S. and is not intended as a product
solicitation or promotion where such activities are prohibited. Because Hologic materials are distributed through websites, eBroadcasts and tradeshows, it is not always possible
ni[ihnlieqa]l]mo[agXn]lbXemXjj]XlΥBilmj][bƄ[bhîlgXnbihihqaXnjli\o[nmXl]XpXbeXZe]îlmXe]bhXjXlnb[oeXl[iohnlsΨje]Xm][ihnX[nsiolei[XeEiei`b[l]jl]m]hnXnbp]il
write to [email protected].
MLO201608_AD Hologic-19998.indd COVERIII
7/8/2016 12:55:10 PM
The integrity and accuracy of genetic analysis relies
on the quality of the reagents and components
utilized in your workflow. Every laboratory – every
doctor – and every patient relies on the quality of our
products to deliver certainty in their results.
Learn More about our complete offering for the
Genetic Analysis Workflow:
Sample Preparation
• GenElute™ DNA & RNA Purification Kits
• Extract-N-Amp™ Kits
Amplification
• SeqPlex™ DNA & RNA Amplification Kits
• JumpStart™ Taq Polymerase and PCR Reagents
Sequencing
• ISO13485 Adapters and Probes
Available in package sizes to fit your needs.
WE ARE QUALITY.
sigma-aldrich.com/clinical
84269
1125
©2015 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA and SIGMA-ALDRICH are
trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries.
GenElute, Extract-N-Amp, SeqPlex and JumpStart are trademarks of Sigma-Aldrich Co.
LLC. Sigma-Aldrich Corp. is a subsidiary of Merck KGaA, Darmstadt, Germany.
MLO201608_AD Sigma-Aldrich.indd COVERIV
7/8/2016 2:02:18 PM

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