Comparison of Methodology for the Identification of Foetal Cells in Kleihauer Testing
University of Wales Institute Cardiff
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Introduction Fetomaternal Haemorrhage (FMH) occurs during pregnancy due to trauma or obstetric causes that lead to the transfer of foetal red cells into the maternal circulation.’1 FMH detection is an important aspect during pregnancy, as a consequence of FMH is haemolytic disease of the foetus and newborn (HDFN).1 ‘HDFN is the result of red cell alloimmunisation in which immunoglobulin G (IgG) antibodies passage from the maternal circulation across the placenta into the circulation of the foetus’ 2, ‘attaching to the foetal red cells which are destroyed in the foetal reticuloendothelial system.’ 3 The rhesus antibody, anti-D, is the most frequently described antibody in HDFN. 4 Quantification of FMH can be accomplished using an acid-elution test or by flow cytometry. The aim of this study is to compare three commercially purchased acid elution methods, with the current in-house method used at Royal Glamorgan Hospital, Llantrisant, South Wales. The National External Quality Assessment Service (NEQAS) results within the laboratory are unsatisfactory at calculating smaller FMH volumes. This study is being conducted to determine whether any of the commercially purchased methods are more effective at detecting the smaller volume FMHs than the current in-house Kleihauer-Betke method. Method The commercially purchased methods compared included the Clin-Tech Limited Shepard’s Stain Kit (Method 1), the Guest Medical Diagnostic Foetal Red Cell Detection Kit (Method 2), the Inverclyde Biologicals Foetal Red Cell Detection Kit (Method 3) and the in-house method (Method 4). Quality controls were stained with each batch of slides. Samples of known FMH volume, quantified using flow cytometry, were produced by the Welsh Blood Service. The nominal volumes of these known samples were 1ml, 2ml, 4ml, 6ml, 8ml and 20ml. The actual volumes for the samples were 1ml, 2.2ml, 4.1ml, 6.2ml, 8.4ml and 20.5ml respectively. The actual results and the nominal results differ as these were the closest results that could be obtained using the flow cytometry. The known FMH volume samples then used to prepare blood films for staining. Each FMH volume for the four methods required 10 blood films. Following slide analysis, Mollison’s formula was used to calculate the FMH volume. Results Comparison of the mean FMH volumes to the flow cytometry volumes was conducted. A linearity plot was constructed of mean FMH volume against known FMH volume. This was carried out for the four methods being compared. Discussion The gradient of a graph measures a proportional bias, whereas, the intercept of a graph measures a constant bias. The gradient p value for the linear plots constructed, indicated that there was a statistically significant difference between the flow cytometer results and the measured results. However, the intercept p value indicated that there was not a statistically significant difference. Comparison of methodology, staining and cost effectiveness was conducted and it was determined that either the Guest Medical method or the Inverclyde Biologicals method would satisfy these variables. Conclusion On the basis of statistical analysis there was no statistical difference between any of the methods compared. The study carried out was a useful pilot study but further investigations could be performed.
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