The blood samples were transported immediately to the laboratory where the D dimer test was conducted by the researcher using the COBAS® Roche cardiac D-dimer portable reader using the blood sample collected in a Lithium heparin sample bottle. The remaining blood sample was transported to the chemical pathology laboratory for analysis of Urea/electrolytes/Creatinine and lipid profile. The blood sample for blood glucose was transported in the Flouride Oxalate bottle to the laboratory where it was analysed immediately.
The ABO and Rh blood group was determined using tile technique that was performed by the researcher using the sample in the plain bottle. The remaining sample in the plain bottle was used for HIV screening and then centrifuged, separated, and the serum stored at a temperature of between -200C until all samples have been collected for the measurement of the serum Von Willebrands Factor (vWF) level.
47 3.5.6 LABORATORY TESTS:
1. D-dimer.
Test principles:
It is a quantitative immunological test for the detection of D-dimer in heparinised venous blood for use with the Cobas h 232 instrument. The test contains two monoclonal antibodies against fibrin degradation products which contain the D-dimer structure element. One of the antibodies is gold-labeled, the other biotynlated. The antibodies form a sandwich complex with D-dimer in the blood. Following removal of erythrocytes from the sample, plasma passes through the detection zone in which the gold labeled D-dimer sandwich complexes accumulate and the positive signal is displayed as a reddish line (the signal line). Excess gold labelled antibodies accumulate along the control line, signaling that the test was valid. The intensity of the signal line increases in proportion to the D-dimer concentration.
The optical system of the instrument detects the two lines and measures the intensity to a quantitative result and shows it in the display. It uses 150µL of venous blood, and measuring range of 0.1-4µg/mL.
Test procedure:
The researcher activated the COBAS® Roche cardiac D-dimer portable reader.
The patients initials, date and time were then be properly imputed.
The Roche D-dimer test strip was inserted into the D-dimer portable reader when the machine prompted.
A few minutes after this, the machine prompted the researcher to apply the heparinised venous blood sample on a specific point on the test strip.
After the application of the blood sample, the D-dimer portable reader processed the
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test and the result automatically within 8 minutes.
The reference level of D-dimer is less than 0.5 µg/mL.
COBAS® Roche cardiac D-dimer portable reader and D-dimer testing Kit.
2. vWF assay (ELISA method) Principle of the method:
The Thermo Scientific™ Pierce™ Human vWF ELISA is a solid-phase, sandwich-type enzyme-linked immunosorbent assay (ELISA). An antibody specific for von Willebrand factor (VWF) has been coated onto the wells of the microtiter strips provided. Samples, including standards of known von Willebrand factor (VWF) content, control specimens, and unknowns, are pipetted into these wells, followed by the addition of a biotinylated second antibody. During the first incubation, the von Willebrand factor (vWF) antigen binds simultaneously to the immobilized (capture) antibody on one site, and to the solution-phase biotinylated antibody on a second site. After removal of excess second antibody,
streptavidin-49
peroxidase (enzyme) is added. It binds to the biotinylated antibody to complete the four-member sandwich. After a second incubation and washing to remove all unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of von Willebrand factor (vWF) present in the original specimen.
Assay Procedure:
The test was performed by a qualified Chemical Pathologist with the active participation of the researcher.
All reagents and samples were brought to room temperature (18 - 25ºC) before use.
All standards and samples were run at least in duplicate.
100μL of each standard and sample were added into appropriate wells, which was then covered and incubated for 2.5 hours at room temperature or over night at 4ºC with gentle shaking.
The solution was then discarded and washed (by filling each well with Wash Buffer (300μL) using a multi-channel Pipette) 4 times with 1X Wash Buffer. Complete removal of liquid at each step was ensured for good performance.
After the last wash, any remaining Wash Buffer was removed by decanting.
The plate was inverted and blotted against clean paper towels.
100μL of 1X prepared biotinylated antibody was then be added to each well, and then incubated for 1 hour at room temperature with gentle shaking.
The solution was then discarded and the wash step was then repeated.
100μL of prepared Streptavidin-HRP solution was then added to each well and incubated for 45 minutes at room temperature with gentle shaking.
The solution was then discarded and wash step repeated. 100μL of TMB Substrate
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was then added to each well, incubated for 30 minutes at room temperature in the dark with gentle shaking.
The plate was evaluated within 30 minutes of stopping the reaction.
The absorbance is then measured on an ELISA plate reader set at 450nm and 550nm.
To correct for optical imperfections in the microplate, 550nm values was substracted from 450nm values.
Calculation of Results:
The standard curve was generated by plotting the average absorbance (450nm minus 550nm) obtained for each Standard concentration on the vertical (Y) axis vs. the corresponding vWF concentration on the horizontal (X) axis.
Results was calculated manually using graph paper.
The amount of vWF in each sample was determined by interpolating the vWF concentration (X axis) to the absorbance value (Y axis).
When the sample was diluted, the interpolated value obtained was multiplied by the dilution factor to determine the amount of vWF in the sample.
3. Blood grouping:
ABO cell grouping using a tile technique:
The researcher prepared a 20-30% of the patients’ red cell suspension by adding 5 drops of the patients packed red cells to 20 drops of saline.
A white ceramic tile was divided into four (4) zones, labeled 1-4, and named A, B, D, and control respectively.
One volume of patients 20-30% red cells was pippetted into zones 1,2 and 3 on the tile.
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One volume of 20-30% red cell suspension of a known blood type was pippetted onto zone 4, as control and mixed with the appropriate antibody.
One volume of anti-A and anti-B was pippetted onto zones 1 and 2 respectively.
One volume of anti-D was pippetted onto zone 3.
The contents on each zone were then mixed using a small clean piece of applicator stick for each.
The tile was then rocked gently from side to side, looking for agglutination.
Agglutination if present, and confirmed by viewing under the microscope, represented a positive result.
The results were then recorded after about two minutes.
4. Full Blood Count:
This was performed by the researcher using a Mindray 2800 Haematology autoanalyser in the Clinical haematology research laboratory of the department of medicine, AKTH.