Using Flow Cytometry to Detect Fetal Red Blood Cells in Maternal Circulation
Introduction: The laboratory determination of the level of fetal red blood cells (FRBC) in maternal circulation remains an important assay in the obstetrical management of women with suspected fetomaternal hemorrhage (FMH) that can occur as part of delivery or due to uterine trauma, maternal hemorrhage, or other intrauterine complications. Rh D-negative mothers are routinely given one dose of Rh-immune globulin (RhIG) to prevent maternal alloimmunization. One vial of RhIg is enough to eliminate up to 30 ml of fetal whole blood. In most situations the amount of FMH is small or even undetectable, so this dose is adequate. However, in others, bleeding may be increased and will sensitize the mother. Therefore, accurately knowing the quantity of the FRBC in maternal circulation is imperative to prescribing the right amount of RhIG.
Three test methods are widely used to detect fetal red blood cells in maternal circulation:
- Qualitative Screening: the Rosette Anti-D assay (FETALScreenTM ,FMH RapidScreenTM )
- Semi-quantitative: Kleihauer-Betke (KB) cytochemical slide test, and
- Quantitative: Flow cytometry anti-Hemoglobin F assay.
Both the KB stain and the flow anti-HbF assay can be used to calculate the number of RhIg doses required to clear the Rh D-positive FRBCs from the mother's circulation. Even though each test can detect fetal cells there can be severe limitations to both sensitivity and accuracy when choosing the most widely used method, the KB stain. Despite its wide-spread use, the KB stain lacks standardization and is imprecise due to the high variability of the results and subjective interpretation1. The test can be labor intensive since the results require a 2000 RBC differential for each slide including controls. Identifying the FRBCs on a KB slide are not difficult. However, the background adult RBCs are often quite faint and washed out which makes counting them accurately a challenge. The Rosette Anti-D assay has greater sensitivity than the KB test, but it is best and most often used as a qualitative screening test for the presence of FRBCs versus providing quantitative results. This is a limitation since the % FRBC is used to calculate the RhIg dosage for the Rh D-negative mother.
Let's begin by describing the principle of each test beginning with the KB stain.
Kleihauer-Betke Slide Test
Rosette Anti-D Assay (FetalScreenTM / FMH RapidscreenTM)
Flow Cytometry anti-Hb F Assay
How do the two quantitative assays compare?
When comparing the positive results for both the KB stain and the Flow anti-F assay of CAP surveys for FRBC assays between 2017 and 2018 participant test precision is measured by the coefficient of variation (CV). The Flow Cytometry method demonstrated considerably higher precision than the KB method. See Figure 3 and Figure 4 below of result comparisons between the 2 methods.
Figure 3: Comparison of the Coefficient of Variation (CV) of Positive FRBC Results of the Flow Cytometric Anti-HbF method vs the KB Slide test. KB results based on combined scores for the 2 most widely used commercial kits, Simmler Fetal Cell Stain Kit and Sure-Tech Diagnostic Associates Kleihauer-Betke Fetal Hemoglobin Kit. (Source: CAP FRBC Detection Survey, 2017-2018).
A closer look at one* of the positive results using both methods demonstrates just how different the two assays are from a standpoint of precision. See Figure 4: CAP Survey of FRBC Results, Flow Method vs KB Stain.
*CAP HBF-B, specimen HBF-03, 2018
Figure 4: CAP Survey Fetal RBC Results, Flow Method vs KB Stain.Precision differences of up to 3x are seen between the two methods used for enumerating FRBC in maternal circulation making accurate, reproducible results more likely using the Flow cytometry method.
Participants were also asked to report the number of RhIG vials recommended for the patient with positive FRBC results. Using the comparison of results shown in Figure 4, some labs reported 0 vials using the KB stain while other labs reported values as high as 10 or more vials compared to the Flow method which reported between 2 and 4 vials respectively. By this comparison, the flow cytometry method is 3 times more likely on average to calculate correctly the % FRBC's present and thus provide a more accurate determination of Rh-Ig doses. Why is this difference so important and how might this impact the patient? Because the Rh-Ig dose is based on the quantitative findings of the FRBC assay and if undercalculated the risk of maternal alloimmunization increases while over-prescribing may not only be expensive but, in some resource-poor areas this practice may strain the available inventory.
What about turn-around time? Not all flow labs are open 24/7 so in the event of a sample arriving after hours how is it handled? First, some facts. The American College of Obstetricians and Gynecologists (ACOG) recommends that all Rh- mothers whose infants are Rh+ should be given RhIg at 28 weeks gestation and a minimum dose of 1 vial (300ug) post-partum within 72 hours of delivery. This window is easily satisfied in most institutions that offer flow cytometry testing. If the test is requested for determining discharging the patient the single vial dose is adequate in most instances. If the test is required more urgently such as a trauma, the Blood Bank or Stat lab can still perform the Rosette qualitative screening test to ascertain positive vs negative results of FRBCs. A positive screen should then be tested for quantity the next time the flow cytometry lab is open. Here is the protocol used by our institution:
Vanderbilt FRBC Testing Protocol
- Monday-Saturday, 7am-6pm, maternal blood sample of Rh- mother is routinely sent to the flow cytometry Lab for quantitative testing and determination of Rh-Ig doses.
- At other times, the initial request and sample is sent to the Blood Bank.
- Blood Bank Lab performs Rosette FRBC screening test.
- Negative samples: reported by the Blood Bank and includes Rh-Ig dose of 1 vial. No further testing performed.
- Positive samples are sent for flow cytometry for confirmation and quantitation of FRBC and Rh-Ig dose calculation the next time that lab is open.
- All flow quantitative testing will be provided within 48 hours of collection.
The flow cytometry anti-HbF assay demonstrates much higher precision and accuracy compared to the widely used KB stain and is therefore considered (by both the AABB and the ACOG) the gold standard for the quantitation of FRBC's for Rh negative mothers who give birth to Rh+ babies. Not every hospital has a flow cytometry laboratory but those that do or have access to a laboratory that performs flow immunophenotyping services should strongly consider adding the highly accurate and precise flow FRBC detection assay as a replacement for the older KB stain.
- Clinical and Laboratory Standards Institute (CLSI), H52-A2 Red Blood Cell Diagnostic Testing Using Flow Cytometry, Approved Guideline, Second Edition, 2014.
- Sandler SG, Gottschall JL. Postpartum Rh immunoprophylaxis. Obstet Gynecol. 120(6)1428-38. Dec 2012.
- Prevention of Rh D Alloimmunization. Clinical Management Guidelines for Obstetrician-Gynecologists, ACOG Practice Bulletin, Number 181, August 2017.
- College of American Pathologists Surveys and Anatomic Pathology Education Programs. Fetal RBC Detection, CAP HBF-B, 2018 Participant Summary.
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- Monoclonal antibodies directed to human fetal hemoglobin (HBF), package insert, Caltag Laboratories, Cat. #MHFH04, 01/03.
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- Porra, et al. Identification and quantification of fetal red blood cells in maternal blood by a dual-color flow cytometric method: evaluation of the Fetal Cell Count Kit. Transfusion 2007; 47: 1281-1289.
- Fetal Hemoglobin Test Kit (FCM) insert and personal communication, Sure-Tech Diagnostic Associates, 11012 Lin Valle, Suite D. St. Louis, MO 63123.FETALtrol package insert, Trillium Diagnostics, LLC, PO Box 67, Brewer, ME 04412.
- Paxton A, Bringing new rigor to RhIG Calculations, CAP Today, May 2008.
- Breacher ME, RhIG dose calculator, Technical Manual, 18th Edition, AABB Press, 2014
- FETALSCREENTM II package insert, ver. 2.0, Ortho Clinical Diagnostics, 2009.
- Immunocor Technical Communication CC-13-001-02, Norcross, GA 4/22/2014
Bruce Greig, MT(ASCP)SCYM