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

Copyright © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Background In the Rh (rhesus)blood group system, the RhD antigen is the most immunogenic antigen on the surface of human red blood cells (RBCs). It is an approximately 30 KD integral RBC membrane protein encoded by the RHD gene. This gene is located on chromosome 1p34-p36.1-6 Although most of the Iranian population carry the RHD gene (RhD positive individuals), about 10.08% of them have complete deletion of this gene (RhD negative individuals), and the immune systems of this group could develop specific antibodies against the RhD antigen if exposed to RhD positive RBCs.7 This special situation could occur in pregnancy. Fetal RBCs can enter maternal circulation and RhD incompatibility with maternal blood could stimulate the production of anti-RhD antibodies in RhD-negative women who carry RhD-positive fetuses. In the next pregnancy, these antibodies cross the placenta into the fetal circulation and destroy the fetal RBCs, leading to hemolytic disease in the fetus and newborn (HDFN).1,8-10 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 Abstract

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. 135][16] Although the Kleihauer-Betke test (KBT) quantifies the FMH, it has some disadvantages.This test is based on the resistance of fetal hemoglobin to acid elution.8][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

Objective
In this study, an EC method for quantitating fetal RhD RBCs in comparison with the Kleihauer technique was assessed in both artificial and clinical samples.Fluorescein isothiocyanate (FITC) labeled monoclonal anti-D was used to calculate fetal RhD positive cells in maternal blood by FC.

Sample Preparation 3.1.1. Clinical Samples
In this prospective cohort study, 28 RhD negative pregnant women were enrolled during their most recent prenatal obstetric visit at Hafez Hospital, Shiraz, Iran.The minimum sample size was calculated using MedCalc Statistical Software version 14.8.1 (MedCalc, Ostend, Belgium) based on the equation N = ([Zα + Zβ]/C) 2 + 3, α=0.05,β=0.20 and the expected correlation coefficient (r) = 0.5.Women included in the study were RhD negative and had husbands who were RhD positive.Those women who had a history of any hemoglobinopathy disorders were excluded.Up to 12 hours after delivery, the peripheral blood of mothers who delivered RhD positive babies (5 mL) was collected in tubes with ethylenediaminetetraacetic acid (EDTA, INTERLAB Laboratory Products, Turkey), and the samples were stored at 4°C until processing.

Spiked Samples
Artificial samples were prepared by adding varying amounts of RhD positive cord blood to blood samples from RhD negative non-pregnant women.Different dilutions (0.3%, 0.6%, 1%, 1.5%, 2%, 5%, 10%, and 50%) of fetal RBCs in maternal RBCs were used in this study.Previously, to calculate the exact amount of cord blood to be added to adult blood, the RBC counts of both cord and adult blood were determined by an automated cell counter (Sysmex KX-21N, Japan), and the required volume of the cord RBCs to be added was calculated using the formula of the Scientific Subcommittee of the Australian & New Zealand Society of Blood Transfusion. 24

Kleihauer-Betke Acid-Elution Test
The KBT test was performed according to the description given by Kleihauer, Braun, and Betke in 1957 with slight modification. 25amples were diluted with normal saline (N/S).Thin layers of diluted blood were smeared on slides.After drying, the slides were fixed in 80% ethanol for 5 minutes and then incubated in citric-phosphate buffer (pH: 1.5) for 5 minutes at 37°C.Staining was done by incubation in erythrosine (Merck, Germany) and hematoxylin acid (Merck, Germany) for 5 minutes, respectively.The slides were rinsed with water and dried between all steps.The fetal cells were counted microscopically using a high objective lens (40х).Malison's formula was employed to calculate the volume of FMH. 26,273.Flow Cytometry First, the direct flow cytometry protocol was conducted by adding different percentages of RhD positive cells to RhD negative blood using FITC-BRAD-3 monoclonal anti-D (Research American product, USA).Whole blood was collected in EDTA and washed twice with phosphatebuffered saline-bovine serum albumin (PBS-BSA).10 6 RBCs were separated and incubated with 2 μL of FITC monoclonal anti-D for 30 minutes at 37°C.As a negative control, unstained RhD positive and RhD negative RBCs were used.Flow cytometry was performed on a BD FACS Calibur (BD Bioscience, USA), and a minimum of >50 000 events were analyzed in each experiment.RBCs were gated using logarithmic forward and side scatter (FSC/SSC), and the gated RBCs were evaluated in the FL1 channel.Data was analyzed using FlowJo 7.6 software, and the FMH was calculated as mentioned for KBT.

Statistical Analysis
Clinical samples were collected using the simple random sampling (SRS) method.The Pearson correlation coefficient (r) analysis was used to compare the results of KBT and flow cytometry.Statistical tests were performed at the 0.05 significance level and results of P < 0.05 were considered significant.All statistical analyses were carried out by SPSS, version 16.0 (Ltd, Hong Kong).

Results
This study included 28 RhD negative pregnant women.Two mothers gave birth to RhD negative babies and were excluded from the study.The KBT was performed at the two incubation times of 25°C for 10 minutes and 37°C for 5 minutes.The results showed more resolution between fetal and ghost adult cells at 37°C.Thus, the clinical samples were incubated at 37°C for 5 minutes (Figure 1).The detection thresholds of the two methods were assessed by analyzing the artificial samples ranges between 0.3% and 50% of RhD positive cord blood in RhD negative maternal blood (Figure 2).Although the FMH greater than 2% were slightly overestimated by KBT (Table 1), the correlation between the results of various suspensions of FMH using both methods, the KBT and flow cytometry, and theoretical percentages showed that the two methods were accurate (r KBT = 1.000, r f = 1.000, and P < 0.05).The KBT and flow cytometry were performed on 26 clinical blood samples.Nine samples had no detectable FMH, 17 had <4 mL, and 2 samples had an FMH >4 mL.According to the results of both methods, the required vial of RhIG (625 IU) in 24 patients was the same.Although the 2 methods predicted the FMH of both high and low RhD positive cell concentrations accurately (r = 0.999, P < 0.05), patients with FMH >4 mL, the FMH, and consequently the required vial of Ig were overestimated using KBT (Table 2).

Discussion
Measuring the size of the FMH after delivery, abortion, or any invasive procedures in the pregnancies of RhD negative women is an essential step to estimating the adequate dosage of RhDIg.In this study, the FMH volume was estimated in clinical and artificial samples by KBT and flow cytometry.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 FMH detected using flow cytometry was lower than that estimated by the KBT.In fact, the flow cytometry results were closer to the theoretical percentages.These results are in agreement with those of Bayliss et al.They showed that there is a poor correlation between KBT and   flow cytometry results in women having an FMH greater than 4 mL. 16loyd-Eva indicated that, although flow cytometry more accurately estimated large FMHs (1-7 mL), FMHs less than 1 mL were quantified more accurately by KBT. 28Pelikan et al showed that flow cytometry is not sensitive enough to detect an FMH less than 0.1%.Data from the clinical and artificial samples in this study did not confirm these results. 8lthough most studies have used monoclonal anti-D to estimate FMH size, 28,29 others 30,31 have employed monoclonal anti-HbF (fetal hemoglobin) to detect fetal RBCs in maternal circulation.In addition to extra steps, including cells fixation and permeabilization, the quantification of FMH using anti-HbF is less accurate compared to anti-D.Kennedy et al pointed out that large What Is Already Known?RhD incompatibility between the RhD negative pregnant women and their RhD positive fetuses leads to hemolytic disease of the fetus and newborn (HDFN).In the hospitals of Iran, all these women routinely receive 300 μg (1500 IU) of RhDIg.

What This Study Adds?
The current study highlights the real need for reassessment of the routine policy of RhDIg administration and prescription.Besides the cost of RhDIg, determining the FMH decreases some concerns about the risk of viral and prion transmission by this blood product.According to the current findings, measuring FMH should be an essential part of RhD negative women prenatal care.

Research Highlights
FMHs were underestimated using anti-HbF.According to Kennedy's article, the probable reason for this finding is related to fetal-to-adult hemoglobin switching.Although the expression of the D antigen on all erythrocytes was completed and stabilized by 6 weeks gestation, fetal-toadult hemoglobin switching begins several weeks before birth and only approximately 70% of hemoglobin in cord blood is fetal hemoglobin. 32

Conclusion
Based on this study, 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 adjustment between the RhDIg dose and FMH size is inevitable.In fact, besides the cost of RhDIg, there are some concerns about the risk of viral and prion transmission through this blood product.Although the flow cytometry method using anti D reagent is the most reliable technique for measuring FMH and determining the sufficient dose of RhDIg, the KBT method could be established and employed in local hospitals.

Figure 1 .
Figure 1.Comparison of the KBT Results in 2 Different Elution Temperatures: A. Elution at 25°C for 11 min; B. Elution at 37°C for 5 min.

Figure 2 .
Figure 2. Fetomaternal Hemorrhage Detection Using Anti-RhD Antibody by Flow Cytometry in 3 Different Dilutions of RhD Positive RBCs in RhD Negative blood.M2 region shows the population of RhD positive cells.

Table 1 .
Estimated FMH (Volume of Fetal RBCs) and Number of RhDIg Vials Required in Clinical Samples

Table 2 .
Results of Prepared Dilutions of RhD Positive Cells in RhD Negative Red Cells