The Kleihauer Technique: an accurate method of quantifying fetomaternal haemorrhage?

Abstract

Blood was taken from 100 consecutive asymptomatic women at 17 to 18 weeks of pregnancy for Kleihauer testing. When a proportion of these slides were assessed at a different hospital there was agreement in only 46%. When the number of fetal cells were quantified there were differences of over 500%. These results show a large inter-observer and inter-hospital variation in interpreting Kleihauer slides. If these investigations are to be performed, it is essential that clinicians can rely on the results. There is a need for quality control measures and audit so that laboratories can rectify any deficiencies in their techniques.Acid elution techniques quantifying fetomaternal haemorrhage have traditionally been used in obstetrics for rhesus negative women to determine the required dose of anti-D gammaglobulin needed after a possible sensitising event. These tests have also been used as a diagnostic aid in pregnant women with abdominal pain or vaginal bleeding and after stillbirth. Whilst using the Kleihauer technique to quantify fetomaternal haemorrhage at the time of amniocentesis, a high proportion of positive results were detected. Initially the percentage of positive tests was determined. We then went on to see if the same slides would be reported as positive for fetal cells at a different laboratory. Having discovered a wide variation in the reporting from different laboratories, we set up this study to quantify the difference in reporting slides between different technicians. MethodsBlood was taken from 100 consecutive women attending at 17 to 18 weeks of gestation for an alpha-fetoprotein test. Verbal consent was obtained from each woman. None of the women had abdominal pain or vaginal bleeding. The acid elution technique described by Kleihauer was used to demonstrate fetal cells'. Blood films were fixed in 80% ethanol and slides were then allowed to stand at 37OC in a citric phosphate buffer for 5 min. This had the effect of lysing the maternal haemoglobin A and leaving behind haemoglobin F. When the slides are stained with haematoxylin and eosin, adult cells show as faint ghosts whilst fetal cells remain darkly stained. The slides were examined in three different ways: 1. 2. 3.All 100 slides were read by unspecified medical laboratory scientific officers at Laboratory A. Then the first batch of 28 slides were read by unspecified medical laboratory scientific officers at Laboratory B. Both positive and negative slides were sent to Laboratory B as it was not known whether slides read as negative by Laboratory A would also be read as negative by Laboratory B. The results from each laboratory were compared. Finally, the number of fetal cells was quantified in 18 slides, by eight specified medical laboratory scientific officers (four at each laboratory).The number of cells per 50 low power fields was counted. The slides were reported as positive when any fetal cells were seen. Bleeds of 1 5 mL were quantified using the formula: ResultsOf the 100 slides screened at Laboratory A, 63 stained positively for fetal c...

Evaluation of FMH QuikQuant for the detection and quantification of fetomaternal hemorrhage

“…FCM exhibited a high reproducibility and a good correlation with KBT, which is the most widely used assay in clinical laboratory. We found a coefficient of variation lower than 20% for values higher than 10 fetal RBCs per 10,000 maternal RBCs (0.1%) which is better than the KBT and in accordance with previous studies (4,13). Additionally, the kit design includes a positive marker of nucleated cells, thus facilitating exclusion of these interfering cells with autofluorescence levels similar in fluorescein isothiocyanate (FITC) fluorescence intensity as anti-HbF labeled fetal RBC (12,27).…”

“…Hemoglobin F (HbF) resists to acid elution and fetal RBCs are stained in bright pink, while hemoglobin is eluted from adult RBCs that appear as ghost cells (3). Nevertheless, the KBT is labor-intensive and inaccurate, theoretically leading to inappropriate dose of anti-RhD immunoglobulin (4). This lack of precision is in part due to the subjective identification of adult cells with increased content of HbF, also called F cells, physiologically increased during pregnancy (5).…”

“…However, such strategy is limited to the determination of anti-RhD immunoglobulin dose, as it cannot distinguish fetal RBCs from adult cells in cases of RhD compatibility. In the last decade, FCM approaches using intracellular detection of HbF have been developed (4,(12)(13)(14)(15)(16)(17). Monoclonal anti-HbF antibody allows discrimination of three distinct populations: fetal RBCs, F cells, and adult RBCs (12,14) The quantification of F cells provided by FCM eliminates a major drawback of KBT.…”

Assessment of Fetomaternal Hemorrhage by Flow Cytometry and Kleihauer-Betke Test in Rh-Negative Pregnancies

“…However, it is often difficult to standardize. Further, it is laborious, difficult to reproduce and suffers from subjectivity and imprecision [3,4] . Flow cytometry (FC) for fetal cell detection is an evolving technology [3,5] .…”

Fetal RBCs Potential in Obstetric Protocols to Minimize Fetomaternal Hemorrhage

“…The value of FCM in detection of FMH is non-invasive technique used to adjust the dose of anti D, also can detect fetal genetic diseases as it detects fetal cells, so PCR and DNA studies can be done [31]. FCM at first used surface antigen to detect FMH, nowadays FCM approaches use intra-cytoplasmic Ag detection of Hb F [32] [33] [34]. Recently monoclonal anti-Hb F antibody allows discrimination of three different populations: fetal RBCs, F cells, and adult RBCs [11]- [34].…”