POCT Ltd the future of diagnostic testing

POCT Ltd
the future of diagnostic testing
An evaluation of the mLabs D-Dimer point of care testing machine.
Julia Stephens
Colchester Hospital University NHS Foundation Trust
December 2012
Introduction
D-Dimers are fibrin degradation products (FDPs) formed from the breakdown of a clot during fibrinolysis. The
concentration of D-Dimer present can be used clinically in the diagnosis of deep vein thrombosis (DVT) and
pulmonary embolism (PE).
The current method used in the lab to test for D-Dimers is the ACL TOP machine. This is a negative predictor
method whereby a negative result can exclude the possibility of DVT or PE completely. A positive result in
not indicative of DVT or PE however, as high D-Dimer concentrations can be attributable to other factors
such as pregnancy, malignancy, liver disease etc. Due to other causes resulting in a high D-Dimer
concentration, the specificity will always be low. The specificity of the ACL TOP is ≈46.8%. The sensitivity
nevertheless needs to be as close to 100% as possible to ensure the method is accurate and so no false
negatives are reported. The ACL top has a sensitivity of 99.8%. Principally, the method involves a latex
enhanced immunoassay technique. The D-Dimer latex reagent is coated with the monoclonal antibody highly
specific for the D-Dimer. When a plasma containing D-Dimer is mixed with the latex reagent and the reaction
buffer, the D-Dimer latex particles agglutinate. The degree of agglutination is directly proportional to the
concentration of D-Dimer in the sample and is measured by the decrease transmittance of light caused by
the aggregates. The light transmittance is compared to a pre run reference curve as shown in figure 4. Low
and high D-Dimer controls are run against this reference curve as shown in figure 5 and 6. The controls need
to pass the quality control checks put in place before any tests can be run.
Point of care testing (POCT) is where medical testing can be done at or near the patient site of care. POCT
has many advantages over the current laboratory techniques used. The major advantages are the
convenience for the patients and that most tests provide immediate results. This makes it quick to manage
dosing or any other clinical requirements needed. POCT may be performed by a nurse, specialist or
alternatively by patients themselves which is of huge benefit for them. An example of where patients carry
out their own tests is in blood glucose testing for use in diabetics. Like most POCT methods this uses blood
from the fingertip rather than venous blood so less blood needs to be drawn. The disadvantage of POCT is
adequate training must be provided for clinical staff or patients using POCT in order to perform the tests
accurately, interpret results and in performing quality control.
The mLabs D-Dimer is a type of POCT analyser for use by health care professionals only. It uses venous
blood, unlike most POCT methods, as a large volume of blood is required for the test. The concentration of
D-Dimer present in the sample is correlated to the ability of a chosen antibody, with good specificity and
sensitivity, to bind to a specific structure of a molecule using an immunoassay technique. The sample placed
on the cartridge flows through, and the D-Dimer present binds to the reporter antibody in the reaction zone
forming a complex. As the complex flows through the detection zone it binds to the capture antibody which is
immobilised in this zone. This complex forms a sandwich structure remaining in this zone.
POCT Ltd
Unit 18 Arbroath Business Centre
Dens Road
Arbroath
Angus
DD11 1RS
Tel: 01241 439 020
Fax: 01241 439 190
Email: [email protected]
Website: www.poct.co.uk
POCT Ltd
the future of diagnostic testing
Where no D-Dimer is present the complex formed is washed away allowing the sandwich complex to be
detected by means of a fluorescent signal due to the attachment of a fluorescent dye on the reporter
antibody. A data drive is provided with cartridges which contains reference values. The specificity and
sensitivity of the method are not given and the negative predictive value is not stated.
The current ACL TOP analyser method will be tested using human samples against the mLabs D-Dimer
POCT method and then compared and evaluated.
Method
A sample requesting a D-Dimer is first tested using the ACL TOP. The sample received is spun down using a
centrifuge and placed in the machine in a rack with the relevant test requested. Refer to SOP CT0013v1.0 for
the complete method.
Once completed, the mLabs D-Dimer method is tested using the same sample. The pouched test cartridge is
removed from the fridge and left for ≈20/30 minutes to allow it to reach room temperature. As whole blood
needs to be employed for this method, the sample needs to be thoroughly mixed via gentle inversions. After
the cartridge has reached room temperature, 250μL of the sample is transferred drop wise into the cartridge
inlet. The cartridge is inserted into the holder, and so the test is run. The results from both methods were
subsequently recorded and repeated with 39 other samples.
Other experiments were also carried out on the mLabs D-Dimer. The penultimate experiment took a single
sample with an abnormal D-Dimer concentration result, as recorded by the ACL TOP, and tested this using
the same method as previous. The same sample was repeated 10 times. The last test again used a single
sample in order to test the D-Dimer concentration when using a range of volumes of blood. Volumes of
blood included 150, 200, 250 and 300μL.
Results
The first experiment tested the results of ACL TOP in comparison to the POCT method. The results of both
methods have to be interpreted differently since they have different cut off points for what is seen as a
positive or negative result. For the ACL TOP there is a cut off point of 250ng/ml where below that is classified
as negative and equal to or above is given as positive. The mLabs D-Dimer method however has a cut off
point of 500ng/ml. The samples that had disparities in the two methods were checked for DVT and PE. The
results are shown in Figure 1.
POCT Ltd
Unit 18 Arbroath Business Centre
Dens Road
Arbroath
Angus
DD11 1RS
Tel: 01241 439 020
Fax: 01241 439 190
Email: [email protected]
Website: www.poct.co.uk
POCT Ltd
the future of diagnostic testing
Figure 1:
Patient ID
HH343675G
HH343623P
Carol
HH345761S
HH346150K
HH346098X
HH346131R
HH344477E
HH344990K
HH344665B
HH347348J
HH347545N
HH347799A
HH347014R
HH348443D
HH348762G
HH349821V
HH349280F
HH349486W
HH349484H
HH351730T
HH351885Q
HH351872N
HH352048E
HH353051A
HH353221M
HH353313A
HH354604T
HH354562P
HH354414D
HH354304E
HH356135T
HH355583D
HH355508K
HH357647C
HH360391R
HH360564R
ACL TOP
(ng/ml)
256
185
173
363
993
138
143
239
32
160
79
73
47776
356
124
155
286
399
84
78
359
120
269
225
106
108
110
2834
45
1244
832
114
348
437
310
84
130
Interpretation
POSITIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
Patient ID
HH343675G
HH343623P
Carol
HH345761S
HH346150K
HH346098X
HH346131R
HH344477E
HH344990K
HH344665B
HH347348J
HH347545N
HH347799A
HH347014R
HH348443D
HH348762G
HH349821V
HH349280F
HH349486W
HH349484H
HH351730T
HH351885Q
HH351872N
HH352048E
HH353051A
HH353221M
HH353313A
HH354604T
HH354562P
HH354414D
HH354304E
HH356135T
HH355583D
HH355508K
HH357647C
HH360391R
HH360564R
POCT (ng/ml)
965.77
235.07
<50.00
226.9
995.13
<50.00
59.69
386.52
<50.00
<50.00
<50.00
<50.00
>10000.00
408.06
<50.00
<50.00
440.59
540.82
<50.00
477.91
720.7
138.93
297.36
111.6
<50.00
<50.00
<50.00
4281.55
<50.00
1964.87
840.67
55.4
780.6
601.93
398.72
271.26
<50.00
Interpretation
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
Comments
DVT excluded
No PE
DVT excluded
No PE
Figure 1. Table showing the concentration of D-Dimer (ng/ml) of 40 patients using the mLabs D-Dimer
and ACL TOP.
POCT Ltd
Unit 18 Arbroath Business Centre
Dens Road
Arbroath
Angus
DD11 1RS
Tel: 01241 439 020
Fax: 01241 439 190
Email: [email protected]
Website: www.poct.co.uk
POCT Ltd
the future of diagnostic testing
The next experiment involved repeating one sample in order to analyse the repeatability and precision of the
POCT method. The sample was repeated 10 times and from that the mean and standard deviation were
devised as shown in Figure 2.
Figure 2:
Patient ID: HH361734
Results using POCT (ng/ml)
1
2
3
4
5
6
7
8
9
10
Mean
SD
749.83
638.81
712.31
518.48
457.09
755.41
621.42
737.09
472.13
512.06
617.463
113.171078
Figure 2. Table showing the concentrations, when repeated, of D-Dimer in ng/ml in a single sample.
The last experiment tested the effect of various volumes on a sample in order to evaluate whether this had
an effect on the overall result. The results are shown in Figure 3.
Figure 3:
Volume of sample (μL)
Result on mLabs D-Dimer (ng/ml)
150
200
250
300
955.71
950.84
Invalid
920.05
Figure 3. Table showing the concentration of D-Dimer in ng/ml produced from the mLabs D-Dimer
when using a volume of 150, 200, 250 and 300μL.
Discussion
Based on the results received from figure 1, which tested the mLabs D-Dimer POCT method over the ACL
TOP current method, I am able to evaluate both methods. As both methods had separate cut of points in the
interpretation of a positive and negative value the concentration itself can’t be used. However there are
inconsistencies between the methods that were evident.
POCT Ltd
Unit 18 Arbroath Business Centre
Dens Road
Arbroath
Angus
DD11 1RS
Tel: 01241 439 020
Fax: 01241 439 190
Email: [email protected]
Website: www.poct.co.uk
POCT Ltd
the future of diagnostic testing
Five results which came out positive on the ACL TOP had a negative result on the mLabs D-Dimer using
their own individual cut off values. These patients were checked for DVT and PE to determine if false
negatives were reported. All of the results were checked and able to exclude DVT and PE. Concluding,
although there were five results that gave negative results as opposed to the ACL TOP they were proved to
not have DVT or PE so the method was still accurate for its purpose.
The second experiment allowed for the mLabs D-Dimer to be tested for its repeatability and precision. From
the results shown in figure 2 the mean came out abnormal, reflective of the result that should have been
produced having been tested previously on the ACL TOP. Results ranged from 457.09 to 749.83 so although
the mean was reported as abnormal there were two D-Dimer concentrations that came up as normal with
results of 457.09 and 472.13. The standard deviation was devised from the results. Standard deviation is a
statistical measure of the spread of dispersion from the mean calculated by taking the square root of the
variance. A low standard deviation indicates all the results are very close to the mean whereas a high
standard deviation indicates they are far from the mean. A standard deviation of approximately 113 resulted.
The last experiment, with results as shown in figure 3, was done to test whether the volume of blood used
had an effect on the results produced. Volumes used in the experiment ranged from 150 to 300μL. All the
results produced were positive with them ranging from 920.05 to 955.71. This shows that volume is not
critical within the range tested. The experiment could have been repeated numerous times to verify these
findings.
The samples tested using the ACL TOP method were done using protocols followed in the laboratory so
there were no discrepancies in the results produced from this. The samples tested using the mLabs D-Dimer
however, could have been improved in order to authenticate the conclusions drawn from the experiments.
One problem first encountered was the pipettes that were provided. They were difficult to use in that the
volume of blood couldn’t be drawn accurately. This was resolved using micropipettes to accurately draw the
volume of blood needed. However, from evaluating the use of blood volumes between 150-300μL it proved
that the volume didn’t need to be accurate so didn’t have a major effect on the overall results. Another
problem encountered that couldn’t be resolved was the use of samples that weren’t fresh. According to the
instruction book, the samples should have been tested within 60 minutes of the blood being taken but as the
samples received had to be tested on the ACL TOP beforehand this couldn’t have been avoided. This also
meant that the blood was first centrifuged as plasma is needed for use on the ACL TOP. Once testing was
complete on the current analyser the sample was mixed to recover the whole blood. Another problem
encountered was that no quality control was carried out throughout all the samples processed. Quality
control needs to be done in order to ensure a level of quality in every sample processed ensuring the
instrument functions correctly. No quality control was provided although it is available on request. Ultimately,
the correct quality control measures needs to have been in place to draw conclusions based on the findings
from the results generated.
Overall, the mLabs D-Dimer gave favourable results. The device was easy to use with step-by-step
instructions provided and is suitable for healthcare professionals to be trained. Quality control would need to
be provided and it would have to be registered with NEQAS before use on patients. Venous blood is still
required and as it is a manual method gloves along with the correct protective clothing would need to be
worn.
POCT Ltd
Unit 18 Arbroath Business Centre
Dens Road
Arbroath
Angus
DD11 1RS
Tel: 01241 439 020
Fax: 01241 439 190
Email: [email protected]
Website: www.poct.co.uk
POCT Ltd
the future of diagnostic testing
MANUFACTURER’S COMMENTS
Generally speaking, the clinical data looks good, although there were some discrepancies compared to the
lab analyser. The final clinical diagnosis indicates that mLabs D-dimer results match the clinical diagnosis.
Normally, the CV of mLabs D-dimer test is about 20% around LLD and is about 5-6% at cut off.
We noticed that the evaluation used patient whole blood samples for CV testing, which is different from many
manufacturers' common methods using control material.
The storage conditions for D-dimer test are 12 months at room temperature (from manufacturing). It was 28°C but now we have real time data to support room temperature storage.
POCT Ltd
Unit 18 Arbroath Business Centre
Dens Road
Arbroath
Angus
DD11 1RS
Tel: 01241 439 020
Fax: 01241 439 190
Email: [email protected]
Website: www.poct.co.uk