Developement of a new staining protocol for the Kleihauer-Betke test to facilitate the reading of dificult cases

Author:

Serban Adrian1,Tholance Yannick1,Aanei Carmen2,Campos Lydia3,Iobagiu Cristina4

Affiliation:

1. Laboratory of Biochemistry, University hospital of Saint Etienne

2. Institute NeuroMyoGene, University Claude Bernard Lyon 1

3. Laboratory of Hematology, University hospital of Saint Etienne

4. Immunohematology laboratory, Etablisement Francais du Sang

Abstract

Abstract Background The Kleihauer–Betke (KB) test allows the detection of fetal red blood cells (containing fetal hemoglobin, HbF) in the maternal blood to identify and quantify potential fetal-maternal hemorrhages. In certain cases, detecting fetal red blood cells with conventional staining is difficult. False-positive results or overestimation of the quantity of fetal red blood cells may occur in cases of maternal hemoglobinopathy. In this study, we developed a new staining protocol to facilitate the reading of difficult smears and improve the precision of the quantification of fetal red blood cells; we also analyzed the performance of this new method. This study assessed blood samples with and without hemoglobin abnormalities, which present difficulties when interpreting the KB test. Methods The new staining formula is based on an improved elution technique and the use of a different stain instead of hematoxylin. To test this staining method, 16 samples from patients with abnormal hemoglobin electrophoresis and 14 samples from patients with normal hemoglobin electrophoresis were analyzed using the KB test with the classical staining method and the new staining method. In addition, a second series was prepared using the same samples spiked with fetal red blood cells from newborn blood, to compare the accuracy of the two methods in identifying fetal red blood cells. Results In the 60 slides analyzed with both staining methods, we found that the new technique improved the accuracy from 78–85%; lowered the coefficient of variation between the operators, which decreased from 20.7–12.7%; increased the specificity in our population from 56–70%; and decreased the number of false-positive cases by 30%. Conclusions We successfully developed a new staining technique that facilitates the reading of difficult slides and improves the specificity of the detection of fetal red blood cells. This technique is recommended as a secondary method to use before sending the sample for additional exploration.

Publisher

Research Square Platform LLC

Reference22 articles.

1. Kleihauer E, Braun H. K. Betke ; Demonstration von fetalem hamoglobin in den erythrocyten eines blutausstrichs; Jg 35, IIeft 12, 1957.

2. Garty BZ, Ludomirsky A, Danon YL, Peter JB, Douglas SD. ; Placental transfer of immunoglobulin G subclasses; 1994; 1(6): 667–9, Clin Diagn Lab Immunol.

3. Makar; Detection of fetomaternal hemorrhage;Yeowon A;Am J Hematol,2012

4. BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn;Qureshi H,2014

5. Muench MV, Baschat A, Reddy UM, Mighty HE, Weiner CP, Scalea TM. C. R. Harman; Kleihauer-Betke Testing Is Important in All Cases of Maternal Trauma; The Journal of TRAUMA Injury, Infection, and Critical Care; 2004, volume 57 number 5, pag 1094–1098; 10.1097/01.TA.0000096654.37009.B7.

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