Evaluation of Flow Cytometry and Kleihauer Techniques for Quantification of Fetomaternal Hemorrhage: A Prospective Cohort Study in Southwestern Iran

Zeinab Keshavarz,Leili Moezzi,Abbas Behzad-Behbahani,Reza Ranjbaran,Sedigheh Sharifzadeh,Mahdokht Mahmoodi,Masooma Abdullahi

doi:10.15171/hpr.2018.26

Zeinab Keshavarz, Leili Moezzi + Show 5 more

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https://doi.org/10.15171/hpr.2018.26

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Abstract

Background: Quantification of fetal red blood cells (RBCs) in maternal blood is of great importance to calculate appropriate dose of post-deliver anti D immunoglobulin in a rhesus D (RhD)-negative woman. Objective: The aim of this study is to evaluate a direct immunofluorescence flow cytometry technique in artificial and clinical samples and compared it to the Kleihauer-Betke test (KBT). Methods: This study was a prospective cohort design. Blood samples from 26 pregnant women who gave birth to RhD positive babies were tested using direct immunofluorescence flow cytometry and KBT techniques to determine the amount of FMH in the maternal circulation. The zone of D-positive cells was identified employing artificial samples including 0.3%, 0.6%, 1%, 1.5%, 2%, 5%, 10%, and 50% of D-positive fetal cells in D-negative maternal cells. Results: Analysis of 26 clinical samples for FMH showed consistent quantification with the flow cytometry and Kleihauer techniques. Although a good correlation was found between the KBT and flow cytometry results, in artificial samples containing more than 2% of fetal RhD positive cells, the flow cytometry results were closer to theoretical percentages. In a patient with FMH >4 mL, the FMH and consequently the required vial of Ig were overestimated using KBT. Conclusion: Most of the FMH calculated could have been neutralized by doses less than 625 IU, whereas the routine dose in Iran is more than double that amount (1500 IU). This achievement demonstrates that adjusting between the RhD immune globulin (RhDIg) dose and FMH size is inevitable.

Highlights

Quantification of fetal red blood cells (RBCs) in maternal blood is of great importance to calculate appropriate dose of post-deliver anti D immunoglobulin in a rhesus D (RhD)-negative woman
A good correlation was found between the Kleihauer-Betke test (KBT) and flow cytometry results, in artificial samples containing more than 2% of fetal RhD positive cells, the fetomaternal hemorrhage (FMH) detected using flow cytometry was lower than that estimated by the KBT
These results are in agreement with those of Bayliss et al They showed that there is a poor correlation between KBT and Hospital Practices and Research 2018;3(4):[118-122] flow cytometry results in women having an FMH greater than 4 mL.[16]

Summary

Introduction

Quantification of fetal red blood cells (RBCs) in maternal blood is of great importance to calculate appropriate dose of post-deliver anti D immunoglobulin in a rhesus D (RhD)-negative woman. These antibodies cross the placenta into the fetal circulation and destroy the fetal RBCs, leading to hemolytic disease in the fetus and newborn (HDFN).[1,8,9,10] RhD immune globulin (RhDIg) administration can conceal the antigenic sites of fetal RBCs and prevent the subsequent responses of the maternal immune system.[11,12] Determining the fetomaternal hemorrhage (FMH) is an important factor in adjusting the dosage of RhDIg. The routine policy on RhDIg administration and prescription in Iran is that all RhD negative women bearing RhD positive fetuses receive 1500 international units (IU) (300 μg) of RhDIg, which is adequate for concealing up to 12 mL of fetal RBCs.[13] The Rosette test is a traditional test that confirms the presence of fetal RBCs in maternal circulation, but gives no information about the size of the FMH.[14,15,16] the Kleihauer-Betke test (KBT) quantifies the FMH, it has some disadvantages. KBT is inexpensive and performed with basic laboratory equipment, but the sensitivity of the test is affected by many factors during all steps, including film preparation, staining, elution, and interpretation of the stained blood films.[17,18,19,20,21] The flow cytometric (FC) technique is the most recent method that can evaluate fetal RhD RBCs in maternal blood by means of monoclonal antibodies against fetal markers like hemoglobin F (HbF) or surface RhD antigens.[22,23]

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