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