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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation
  5. Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation
  6. Summary
  7. Acknowledgements
  8. References

The prevention of Rhesus D alloimmunization through Rh immune globulin (RhIg) administration is the major indication for the accurate detection and quantification of fetomaternal hemorrhage (FMH). In the setting of D incompatibility, D-positive fetal cells can sensitize the D-negative mother, resulting in maternal anti-D alloantibody production. These anti-D alloantibodies may lead to undesirable sequelae such as hemolytic disease of the newborn (HDN). Since the widespread adoption of FMH screening and RhIg immunoprophylaxis, the overall risk of Rh alloimmunization and infant mortality from HDN has substantially decreased. The rosette screen, the initial test of choice, is highly sensitive in qualitatively detecting 10 mL of fetal whole blood in the maternal circulation. As the screen is reliant on the presence of the D antigen to distinguish fetal from maternal cells, it cannot be used to detect FMH in D-positive mothers or in D-negative mothers carrying a D-negative fetus. The Kleihauer-Betke acid-elution test, the most widely used confirmatory test for quantifying FMH, relies on the principle that fetal RBCs contain mostly fetal hemoglobin (HbF), which is resistant to acid-elution whereas adult hemoglobin is acid-sensitive. Although the Kleihauer-Betke test is inexpensive and requires no special equipment, it lacks standardization and precision, and may not be accurate in conditions with elevated F-cells. Anti-HbF flow cytometry is a promising alternative, although its use is limited by equipment and staffing costs. Hematology analyzers with flow cytometry capabilities may be adapted for fetal cell detection, thus giving clinical laboratories a potentially attractive automated alternative for quantifying FMH. Am. J. Hematol., 2012. © 2011 Wiley Periodicals, Inc.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation
  5. Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation
  6. Summary
  7. Acknowledgements
  8. References

The accurate detection and quantification of fetal red blood cells (RBCs) in the maternal circulation are necessary for the prevention of Rhesus D alloimmunization among D-negative women because of fetomaternal hemorrhage (FMH). In the setting of D incompatibility, D-positive fetal cells may result in sensitization of the D-negative mother and subsequent maternal anti-D alloantibody production. These anti-D alloantibodies may be clinically silent or lead to severe consequences such as hemolysis, fetal anemia, hydrops fetalis, or even death for the current and/or future pregnancies. Although this paper focuses specifically on the detection of FMH for the prevention of D-alloimmunization, FMH testing is also used in the obstetric-gynecological setting for determining the presence of occult bleeding associated with various obstetrical events and conditions [ 1]. Commercially available Rh immune globulins (RhIg), which are human immunoglobulin preparations from plasma of donors with high-titer anti-D antibodies, have been shown to be effective in reducing D-sensitization and subsequent hemolytic disease of the newborn (HDN). Before the widespread adoption of FMH screening and immunoprophylaxis with anti-D immunoglobulin, the incidence of infant mortality from HDN in England and Wales in 1970 was 1.2 per 1,000 births. By 1989, this figure dropped to 0.02 per 1,000 births [ 2]. As appropriate dosing is calculated based on the volume of FMH, the prompt and accurate laboratory assessment of FMH is highly desirable.

One vial (300 μg) of RhIg is sufficient to protect against 30 mL of fetal whole blood or 15 mL of fetal RBCs. At 20 weeks gestational age, the fetoplacental blood volume is estimated to be 30 mL [ 1]. Since transplacental hemorrhage exceeding 1 mL of fetal RBCs during the antenatal period is extremely unlikely [ 3, 4], it is thought that a single vial is sufficient for antenatal prophylaxis at 28 weeks gestational age. In contrast, FMH during term delivery can exceed 30 mL of fetal whole blood, albeit at a low frequency (0.3% of pregnancies), requiring accurate quantification and more than one vial of RhIg to prevent alloimmunization [ 5]. Ramsey, on behalf of the College of American Pathologists Transfusion Medicine Resource Committee, evaluated the results of the College of American Pathologists' (CAP) proficiency testing for fetal RBC detection submitted by nearly 1,600 laboratories. He found that 20%–30% of laboratories underestimated the necessary dose of RhIg and concluded that laboratories should re-examine their protocols and training for calculating RhIg dosage [ 6]. The purpose of this brief review article is to provide a targeted overview of the current methods available for the detection and quantification of FMH and their diagnostic limitations, as well as special circumstances which may influence test selection and the interpretation of results.

Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation
  5. Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation
  6. Summary
  7. Acknowledgements
  8. References

Rosette screen

The rosette screen is a highly sensitive method to qualitatively detect 10 mL or more of fetal whole blood, or 0.2% fetal cells (volume/volume) in the maternal circulation. It has largely replaced the microscopic weak D test for this purpose because of its superior sensitivity [ 7–9] and is currently the only screening test that is FDA-approved for clinical use in the United States. In 1982, Sebring and Polesky first described the formation of microscopic fetal D+ aggregates upon incubation of fetal cells with enzyme-treated group O D-positive indicator RBCs and reagent anti-D serum [ 10]. The fetal cells must be D-positive and the maternal cells D-negative for the test to be valid. In the rosette screen, a maternal blood sample is first incubated with anti-D and then washed. The indicator D-positive RBCs are added and the sample is examined under a light microscope. In the presence of fetal D-positive cells, the indicator cells will form aggregates (or rosettes) around the fetal cells (Fig. 1). The rosette test may be falsely positive if the mother has a variant of the D antigen known as weak D, and falsely negative if the fetus/neonate is weak D (variant D antigens are discussed at greater length below in the section describing flow cytometry to detect D-positive RBCs). In addition, if the mother has a positive direct antiglobulin test (DAT) such as in the presence of an RBC autoantibody, the screening test may be false positive because of crosslinking and agglutination of the mother's antibody coated cells.

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Figure 1. Positive rosette test: Positive control from the ImmucorGamma® fetal bleed screening test using a 2%–4% suspension of red blood cells comprised of 99.4% group O D-negative cells and 0.6% group O D-positive cells. The image is at 50× magnification using a light microscope. A positive test consists of ≥3 clumps of agglutinated red blood cells upon examination of nine low-power fields. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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If the rosette test is negative, one vial (300 μg) of RhIg is sufficient to prevent immunization in 99% of patients [ 11]. A positive rosette test, which indicates a FMH exceeding 10 mL, requires quantification of the FMH by either the Kleihauer-Betke acid elution test or flow cytometry to determine the dose of RhIg required for prophylaxis. In the case of a known weak D phenotype of the fetus/neonate, a quantitative test that does not rely on the level of expression of the D antigen, such as the Kleihauer-Betke test or flow cytometry using anti-fetal hemoglobin (anti-HbF) antibodies, should be conducted rather than the rosette test. Among mothers with the O blood group carrying fetuses with the A, B, or AB blood group, naturally occurring isohemagglutinins will expedite the clearance of fetal cells in the maternal circulation prior to testing. Therefore, in the setting of anticipated ABO incompatibility, prompt testing around the time of suspected FMH is recommended.

The rosette test is inexpensive, straightforward and easy to perform, and does not require special equipment beyond a waterbath, centrifuge, and light microscope. Hence, it can be conducted around-the-clock with a turnaround time of 1–2 hr. FDA-approved, commercially available kits include the Fetal Bleed Screening Test (ImmucorGamma, Norcross, GA), which is the most widely used kit in the United States, and the FetalScreen II/Fetal Maternal Screening Test (Ortho-Clinical Diagnostics, Raritan, NJ) [ 12]. As the rosette test is reliant on the presence of the D antigen to distinguish fetal from maternal cells, it cannot be used to detect FMH in D-positive mothers, or D-negative mothers carrying a D-negative fetus.

Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation
  5. Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation
  6. Summary
  7. Acknowledgements
  8. References

Kleihauer-Betke acid-elution test

The Kleihauer-Betke acid-elution test, originally described by Kleihauer, Braun, and Betke in 1957 [ 13], relies on the principle that fetal RBCs containing mostly fetal hemoglobin (HbF) are resistant to acid elution whereas adult hemoglobin is acid-sensitive. Hence, when a thin peripheral smear prepared from a maternal blood sample is exposed to an acid buffer, hemoglobin from adult red cells is eluted while HbF is retained. Subsequent staining of the slide with hematoxylin results in dark pink-staining of fetal cells and very pale “ghost” outlines of maternal RBCs (Fig. 2). Calculating the percentage of fetal cells identified from the Kleihauer-Betke test (or flow cytometry) into the equivalent volume of FMH used to determine the correct RhIg dosing depends on which formula is employed. A commonly employed formula described in one obstetrics and gynecology textbook [ 14] as well as the American Association of Blood Banks' (AABB) Technical Manual is below:

  • equation image
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Figure 2. Positive Kleihauer-Betke test: Fetal hemoglobin (HbF) in RBCs is resistant to acid elution. Cells containing HbF stain bright red with eosin, whereas those without HbF appear as colorless ghosts. This image was originally published in the American Society of Hematology (ASH) Image Bank. John Lazarchick Kleihauer-Betke Hemoglobin F Acid Resistance-1 ASH Image Bank. 2004; 00002370. © the American Society of Hematology. Image bank by American Society of Hematology. Copyright 2004. Reproduced with permission from the American Society of Hematology in the format Journal via Copyright Clearance Center. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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For example: Kleihauer-Betke reported as 0.3% (6 fetal cells counted out of 2,000 total RBCs), (0.3/100) × 5,000 mL = 15 mL fetal whole blood, 15 mL/30 mL per vial of RhIg = 0.5 vials, Dose (as per AABB Technical Manual) = 2 vials (i.e., round + 1)

It is noted that the maternal volume is assumed to be 5,000 mL, which can result in the underestimation of the volume of calculated FMH in pregnant females with larger circulating volumes (females > 70 kg, with circulating volume of 70 mL/kg), and hence also the recommended RhIg dose. The College of American Pathologists has published a tool for calculating RhIg dose (accessible online at www.cap.org) that allows users to enter the maternal height and weight to better estimate blood volume. Mollison proposed an alternative formula for quantifying FMH noting that as fetal RBCs are 30% larger than adult RBCs and only 90% of fetal RBCs stain darkly, simply using the proportion of darkly-staining cells can underestimate the volume of FMH by a third [ 15]. The formula assumes that the maternal circulating red cell volume is 1,800 mL at term and applies a correction factor by multiplying the proportion of darkly-staining cells by 4/3 to obtain the volume of fetal RBCs:

  • equation image

Mollison also suggested doubling the dose of RhIg to 50 μg per 1 mL of fetal RBCs to allow for the possibility of a twofold underestimation. Table I displays variations in the estimated volume of FMH by using five published formulas, which reflects different assumptions regarding maternal blood volume, maternal hematocrit, fetal hematocrit, fetal mean corpuscular volume (MCV) relative to maternal MCV, and % of fetal cells that stain positive on acid elution. With 0.3% fetal cells observed in the maternal circulation, the estimated fetal whole blood volume ranges from 10.8 to 31.6 mL. A modified version of the Kleihauer-Betke test was proposed by Clayton et al., who found that preparing the citric acid-phosphate buffer at pH 3.2 resulted in optimal detection of FMH as small as 0.5 mL [ 19–21]. Subsequently, this modified version has been widely adopted.

Table I. Variation in Estimated Volume of Fetomaternal Hemorrhage for Kleihauer-Betke Test Result of 0.3% Using Published Formulas (Adapted from Wylie and D'Alton [1])
AuthorFormula
FMH in mL of fetal whole blood
  • a

    Assuming fetal HCT of 50%.

  • Abbreviations: FMH, fetomaternal hemorrhage; HCT, hematocrit; MBV, maternal blood volume; MCV, mean corpuscular volume; MRBCV, maternal red blood cell volume; and RBC, red blood cell.

Cunningham et al [16](HCTmaternal/HCTnewborn) × %fetal RBCs × MBV=(0.36/0.50) × 0.003 × 5,000=10.8 mL
Assumptions:
 HCTmaternal = 36%
 HCTnewborn = 50%
 MBV = 5,000 mL
Kleihauer(HCTmaternal/HCTnewborn) × %fetal RBCs × MBV=(0.35/0.45) × 0.003 × 5,800=13.5 mL
Assumptions:
 HCTmaternal = 35%
 HCTnewborn = 45%
 MBV = 5,800 mL
Creasy et al [14] and AABB Technical Manual [17]%fetal RBCs × MBV=0.003 × 5,000=15.0 mL
Assumptions:
MBV = 5,000 mL
FMH in mL of fetal RBCs
Mollison [15](1,800/ratio of unstained:darkly-stained RBCs) × (4/3)=[1,800/(1,994/6)] × (4/3)=7.2 mL or 14.2 mL of fetal whole blooda
Assumptions:
 MRBCV = 1,800 mL
 MCV of fetal RBCs is 30% larger than that of adult RBCs
 Only 90% of fetal RBCs stain dark pink on acid-elution
Foley et al [18](# Fetal RBCs/# adult RBCs) × MRBCV=(6/1,994) × 75 × 70=15.8 mL or 31.6 mL of fetal whole blooda
Assumptions:
 MRBCV = 75 mL/kg
 Average term woman weighs 70 kg

Although the manual Kleihauer-Betke test is the most widely used test to quantify the volume of FMH (95% of laboratories participating in the 2009 CAP proficiency testing for fetal RBC detection used the Kleihauer-Betke test for fetal RBC quantification) [ 12], it is not without its limitations. Aside from being laborious to perform (counting a minimum of 2,000 RBCs is recommended), the accuracy and precision of the test can be suboptimal because of lack of standardization leading to slight variations in test characteristics (i.e. thickness of blood smear, pH variations in the buffer used), interobserver and interhospital variations in result interpretation, and statistical imprecision associated with rare event analysis. In addition, differences in methods for calculating the volume of FMH from % fetal cells identified by the Kleihauer-Betke test as well as the recommended RhIg dose further add to the variability. Citing statistical imprecision of the Kleihauer-Betke test in determining the dose of RhIg, the AABB Technical Manual recommends rounding up and adding one vial if the calculated dose to the right of the decimal point is ≥0.5 or rounding down and adding one vial if the calculated dose to the right of the decimal point is <0.5.

Both over- and underestimation of FMH have been reported [ 15, 22–25], but most of the studies report the tendency of the Kleihauer-Betke test to overestimate FMH. Variables potentially contributing to both under- and overestimation are listed in Table II. Overestimation is preferred rather than underestimation, as the latter can result in inadequate RhIg dosing and subsequent sensitization. However, overestimation itself is not desirable, as it carries risks related to plasma-derived products and results in excess costs. Lafferty et al., in reporting the results of the External Quality Assessment conducted in Ontario in 2000 and 2001, observed that the poor reliability of the Kleihauer-Betke test may have resulted in underdosing RhIg in 19.4% of patients with FMH >10 mL [ 25]. If there is truth to these reports, the underestimation of large FMH by the Kleihauer-Betke test undermines its function in quantitating large volume FMH that may necessitate additional RhIg. Raafat et al. reported that standardizing the Kleihauer-Betke test, including recommendations for calculating the RhIg dose, resulted in an improvement in FMH quality assurance testing in Scotland [ 26]. In the end, the best estimate of maternal blood volume should probably be used to calculate the volume of FMH.

Table II. Variables Potentially Contributing to Over- or Underestimation of Fetomaternal Hemorrhage by the Kleihauer-Betke Test
Factors leading to overestimationFactors leading to underestimation
  1. Abbreviations: MCV, mean corpuscular volume; RBC, red blood cell.

Presence of F-cells that also stain dark pinkFailure to adjust for larger maternal circulating volume (for women weighing > 70 kg)
“Ghost” outlines of acid elution-sensitive adult RBCs which may be difficult to pick up and increase the ratio of fetal RBCs:adult RBCsIncomplete staining of fetal cells (only 90% stain dark pink)
Failure to correct for differences in MCVs of fetal and adult RBCs (MCV of fetal RBCs are 30% greater than that of adult RBCs)

Variations of the modified Kleihauer-Betke test have been proposed, including the substitution of the original 37°C elution for a convenient room temperature elution and automated detection. Various commercial kits and reagents are available. Pelikan et al. developed an automated readout of the Kleihauer-Betke test using an automated microscope equipped with a scanning stage and an image analysis system (Applied Imaging Corporation, Santa Clara, California) connected to a personal computer. By scanning 1,517 low power (10× objective) fields, the automated KBT showed strong correlation between theoretical and detected concentrations of fetal cells (R2 = 0.999) and improved accuracy of fetal cell detection in the range of 0.0001%–1% FHM compared to standard manual evaluation [ 27].

As the Kleihauer-Betke test is reliant on the visual discrimination of fetal and adult RBCs by staining intensity (dark pink fetal vs. minimally stained adult RBCs), the presence of F-cells containing intermediate concentrations of HbF can complicate the interpretation. F-cells are RBCs containing 20%–25% HbF and are present in normal adults at a range of 0.5%–7.0% of circulating RBCs [ 28–30]. F-cells are elevated in inherited hemoglobinopathies including sickle cell disease and β-thalassemia, hereditary persistence of fetal hemoglobin (HPFH), acute stress erythropoiesis, and pregnancy. In 25% of pregnant women, HbF begins to increase at 8 weeks' gestation and peaks at 18–22 weeks, and may reach levels as high as 7% by 32 weeks [ 31, 32]. Hence, the presence of F-cells may give either a false-positive test result or may overestimate the magnitude of a true FMH. Such cases of elevated F-cell levels presenting as FMH on Kleihauer-Betke testing have occurred in the setting of maternal HPFH, HPFH/sickle trait, and β-thalassemia [ 33–38]. In cases of known maternal history of elevated HbF and/or the presence of associated conditions (such as thalassemia or HPFH), it is advised that an alternative method to quantitate FMH such as flow cytometry be used.

Flow cytometry

Acknowledging the limitations of the Kleihauer-Betke test, flow cytometry-based methods for the quantification of fetal cells in the maternal circulation have been developed. These methods fall into two main categories depending on the target antigen of interest: HbF and RhD. FDA-approved reagents for both methods are commercially available.

Flow cytometry using anti-fetal hemoglobin antibodies.

Flow cytometry using monoclonal antibodies directed against HbF has some important advantages over the Kleihauer-Betke test in the quantitation of FMH: (1) cytometric methods can accurately distinguish adult F-cells from fetal RBCs (Fig. 3); (2) flow cytometry rapidly analyzes a greater number of cells (≥50,000), improving quantitative accuracy; (3) as flow cytometry is automated, it has greater reproducibility. Hence flow cytometry has broader clinical and research applicability, including the quantification of F-cells in conditions such as sickle cell disease which may be used to guide prognosis and monitor treatment. Although there are reports of elevated adult F-cells falsely increasing the fetal cell estimate by flow cytometry, the extent of the increase is mild (contributing <0.1% to the fetal RBC percentage) and has not been shown to contribute to clinically relevant false-positive results [ 23]. Currently there is only one FDA-approved commercially available kit consisting of purified murine monoclonal anti-human HbF IgG1 antibody (Invitrogen, Camarillo, CA), as well as a separate fetal red cell control kit (Fetaltrol, Trillium Diagnostics, Portland, ME). The antibodies are conjugated to either fluorescein isothiocyanate (FITC), R-phycoerythrin (R-PE), or indodicarbocyanine dye (tri-color); however, FITC-conjugated antibodies have been used in the majority of published studies.4

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Figure 3. Positive anti-fetal hemoglobin (anti-HbF) flow cytometry: Fetomaternal hemorrhage detection using anti-HbF antibody by flow cytometry (FMH QuikQuant Assay, Trillium Diagnostics, Bangor, Maine). Depending on anti-HbF fluorescence intensity, HbF-negative cells, F-cells, and fetal red blood cells (RBCs) can be distinguished. Various samples showing increased fetal RBCs of 1.7% and 6.5% (panels A and C, respectively), increased adult F cells (panel B), and no fetal red cells (panel D). Flow histograms courtesy of Dr. Bruce Davis, Trillium Diagnostics. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 4. Suggested testing algorithm for detection of fetomaternal hemorrhage. *In case of known maternal elevation of fetal hemoglobin (i.e., HPFH, HPFH/sickle cell trait, β-thalassemia), proceed to either anti-HbF or anti-D flow cytometry.

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For analysis by flow cytometry, an EDTA anticoagulated maternal blood sample is obtained from which the erythrocyte count is determined. In a typical protocol, 2.5 × 107 red cells are briefly fixed in 0.05% glutaraldehyde and then permeabilized with a detergent (Triton X-100) that enables the antibodies to penetrate the cellular membranes and bind to the intracellular HbF [ 23]. Davis and colleagues first described this permeabilization technique using brief glutaraldehyde and Triton X-100 treatments, which allowed for a total assay time of 1 hr for five to six samples. This was in contrast to other reported permeabilization methods involving acetone and alcohol washes that required either overnight incubations or numerous temperature-dependent steps with a tendency for cell aggregate formation [ 23]. Antibody stained cells are subsequently analyzed by a flow cytometer. Positive and negative controls are concurrently run and help differentiate fluorescence attributed to fetal RBCs from non-specific background staining of autofluorescent leukocytes and cellular debris. The positive control is also instrumental in setting the fetal cell gating parameters.

Prior CAP proficiency surveys have repeatedly shown that flow cytometry using anti-HbF antibodies is more precise (coefficient of variation (CV) <20%) than the Kleihauer-Betke methods (CV, 32%–80%) and has greater accuracy. The 1999 and 2001 CAP surveys included samples flanking the threshold of 0.6% fetal cells or 30 mL of fetal whole blood (15 mL of fetal RBCs), which is the amount of fetal whole blood that one vial (300 μg) of RhIg is purportedly sufficient to protect against. For the 0.4% fetal cell sample, laboratories using either the Kleihauer-Betke test or anti-HbF flow cytometry performed similarly, in that 50% of the laboratories overestimated FMH, reporting values >0.6%. For the 0.8% fetal cell sample, more than 10% of the labs using the Kleihauer-Betke test underestimated FMH, reporting results <0.6%, but laboratories using anti-HbF flow cytometry, all correctly determined the FMH volume [ 39].

Available data indicate that anti-HbF flow cytometry is better than the Kleihauer-Betke test as a confirmatory test for FMH. Although good correlation has been reported between the Kleihauer-Betke test and anti-HbF flow cytometry for both small and large FMH, published studies are in agreement with the CAP proficiency surveys that anti-HbF flow cytometry displays greater test and interlaboratory precision than the Kleihauer-Betke method [ 23, 40-42]. There is interest in using anti-HbF flow cytometry also as an initial screening test for FMH, especially because of its potential broader clinical application; however, data regarding whether flow cytometry has sufficient sensitivity to detect low concentrations of fetal cells (<0.1%) is mixed. Most studies report that flow cytometry accurately and precisely quantifies FMH volumes ≥0.1% [ 23, 41, 43]. At present, flow cytometry is not used for screening primarily because of the fact that most hospitals do not have access to a flow cytometer or resources to staff a flow cytometer around-the-clock. However, anti-HbF flow cytometry appears to be an appropriate confirmatory test for hospitals with adequate resources and staffing.

Flow cytometry using anti-D antibodies.

Anti-D antibodies can also be used in quantitating FMH, but their applicability is limited to the clinical scenario of D antigen incompatibility. Initial studies demonstrating the feasibility of flow cytometry for fetal cell quantification targeted the D antigen. Using this method, FMH as small as 0.1–0.2% can be quantitated [ 9, 40, 44]. FDA approved monoclonal anti-D antibodies are commercially available (Quant-Rho, Alba Bioscience, Edinburgh, UK). The advantages of using anti-D over anti-HbF antibodies are two-fold: the permeabilization step used in the anti-HbF method is omitted as the D antigen is expressed on the RBC surface, and F-cells do not interfere with the calculation of fetal cells. Importantly, flow cytometry cannot reliably detect the Rh variants, weak D and partial D, from D-negative cells, resulting in false-negative results [ 45]. The Rh blood group system is highly polymorphic and its variants are categorized into two groups: partial D and weak D phenotypes. Partial D variants are characterized by mutations in the extracellular domains leading to altered epitopes. In comparison, weak D variants are due to mutations in the intracellular or transmembrane domains resulting in a decreased number of qualitatively normal D antigens on the RBC surface. Although it is thought that weak D RBCs are highly unlikely to cause primary anti-D alloimmunization [ 46, 47], a case report of alloimmunization by partial D antigen in pregnancy has been reported [ 48]. Administration of prophylactic RhIg to the mother prior to obtaining the blood specimen can also complicate cytometric analysis using anti-D antibodies because the RhIg may bind to and block antibody binding sites on fetal RBCs, preventing their detection. Kumpel et al proposed a double staining procedure with FITC-conjugated anti-D antibodies followed by a FITC-conjugated anti-human IgG to ensure optimal labeling of D+ cells, regardless of the blocking effects by in vivo RhIg binding [ 49]. The FITC-conjugated anti-human IgG binds to the RhIg that is present on the D+ RBC surface. However, the labor involved in the extra incubation step would negate the advantage of the one-step anti-D method.

There is a paucity of data directly comparing cytometric methods using anti-HbF or anti-D, but one study showed that anti-HbF labeling significantly underestimates the volume of massive FMH (≥1% fetal cells) in comparison to anti-D labeling. In samples containing ≤0.6% fetal cells, no significant difference between the two methods was seen [ 50]. The reason for the difference is unknown, but the authors postulated that this may be because of potential variability in HbF concentration within fetal erythrocytes. The fetal-to-adult hemoglobin switch starts several weeks before birth [ 50], in contrast to the D antigen, which is detected on all erythrocytes in D-positive individuals by 6 weeks gestation [ 51]. Additional studies are needed to verify the potential of anti-HbF flow cytometry to underestimate massive FMH, as well as to directly compare the performance of the anti-HbF and anti-D methods with the Kleihauer-Betke test.

Combined flow cytometric methods.

A combined flow cytometric method using both anti-D and anti-HbF antibodies is available and has been shown to correlate well with the Kleihauer-Betke test [ 52]. The strength of this assay is its capacity to distinguish fetal cells from maternal cells for both D+ and D− women, although this is also true of the HbF method. However, in clinical situations where the mother may have increased circulating F-cells, the dual antibody method may still allow quantitation of fetal cells if there is a detectable disparity in the maternal and fetal D phenotypes. As the dual method can be broadly applied to all cases of FMH and the laboratory does not have to know the fetus/neonate's D status a priori, only one quantitative test can be offered. However, the use of multiple antibodies results in additional costs. The precise clinical circumstance in which the dual platform makes sense, such as a pregnant woman who has sickle cell disease and is D-negative, is fairly uncommon and probably does not justify the added complexity of testing (which may result in additional errors and cost). Other combined flow cytometric methods have been described, including the use of anti-HbF and anti-carbonic anhydrase (anti-CA) antibodies. Since fetal cells have little or no carbonic anhydrase (CA) compared to adult cells which show abundant expression of the enzyme, fetal RBCs (HbF+, CA−) can be easily distinguished from F-cells (HbFlow+, CA+) and normal adult RBCs (HbF−, CA+) [ 24]. A similar approach can be applied for the i and I antigens, which are expressed by fetal and adult RBCs, respectively.

Alternative antibody-based methods to flow cytometry.

Although flow cytometry has been shown to have greater precision and broader clinical applicability than the Kleihauer-Betke test, fewer than 5% of participating laboratories in CAP's 2009 proficiency testing for fetal red blood cell detection reported using it to quantify fetal cells [ 12]. The reasons for the low utilization of flow cytometry include staffing and equipment costs. The requirement for flow cytometry services to be offered around-the-clock is prohibitive for most hospitals. Alternative antibody-based methods to conventional flow cytometry include immunofluorescence microscopy and the use of a hematology analyzer for automated immunofluorescent enumeration of antibody labeled fetal cells. Ochsenbein-Imhof et al. found that flow cytometry and immunofluorescence microscopy were comparably accurate and precise in enumerating fetal cells stained with anti-D and phycoerythrin-conjugated anti-human IgG [ 44]. However, the microscopic method would still entail manual counting. Little et al. evaluated the use of a hematology analyzer (Abbott Cell-Dyn CD4000) for the quantification of FMH by immunofluorescence using a FITC-labeled monoclonal anti-D antibody. They analyzed a range of samples consisting of 0.04% to 1.50% fetal cells. They reported excellent agreement and correlation (R2=0.99) between observed and expected fetal cell percentages. Additionally, the predicted lower limit for the quantitation of FMH was 1.6 mL with a maximum CV of 15%, which is well-below the 15 mL threshold for additional RhIg dosing [ 53]. The application of a hematology analyzer for the quantitation of FMH is especially appealing, as it is fully automated, precise, and has a wide range of linearity. Additional studies to see if other antibodies, such as anti-HbF, are suitable for this platform are needed.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation
  5. Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation
  6. Summary
  7. Acknowledgements
  8. References

RhIg prophylaxis has reduced the overall risk of Rh immunization from 13.2% to 0.2%, and testing for large FMH has further decreased the risk to 0.14%. The few cases of Rh immunization have been mostly attributed to the occurrence of events prior to 28 weeks gestation [ 54]. The rosette screen has been shown to be a highly sensitive method to qualitatively detect large FMHs of ≥10 mL (0.2% fetal cells), with 15 mL serving as the threshold for increased RhIg dosing. As the test is designed to give a negative result when the amount of FMH is small (i.e. <2 mL or 0.04% fetal cells), which is the case in the vast majority of pregnancies [ 4], only few cases will necessitate confirmatory quantitative testing.

The two well-established confirmatory tests are the Kleihauer-Betke acid-elution assay and flow cytometry. Although the Kleihauer-Betke test is inexpensive and requires no special equipment, allowing it to be performed around-the-clock, it has several disadvantages: it is laborious to perform, lacks standardization, is imprecise, and may not be accurate in conditions with elevated F-cells. Flow cytometry has been shown to have greater precision and accuracy than the Kleihauer-Betke test. In addition, fetal cells can be distinguished from F-cells in conditions with elevated HbF. Despite these advantages, it has not been broadly implemented as it requires a flow cytometer, a trained technician, and is more expensive and difficult to offer around-the-clock. Flow cytometry is not without its limitations, as the anti-D method is limited to the clinical scenario of D antigen incompatibility and may not reliably detect weak and partial D. Although anti-HbF flow cytometry can better distinguish F-cells from fetal cells compared to the Kleihauer-Betke test, concerns have been raised whether anti-HbF labeling underestimates massive FMH [ 50]. The limitations of the one-antibody approach can be addressed by using two antibodies (i.e. anti-D and anti-HbF, or antibodies to some other antigen expressed differentially on fetal and adult cells, such as i or CA). This way, one test can be ordered for all patients, regardless of D status or history of F-cell elevations. However, the use of an additional antibody increases the cost of testing, and the relatively infrequent need to quantify FMH may make it cumbersome to maintain quality control and proficiency. From both resource allocation and cost-effectiveness standpoints as well as questionable sensitivity to serve as a screening test, flow cytometry seems best suited as a confirmatory test for FMH. However, it (or the Kleihauer-Betke test) may be useful as the first-line test in the case of a known weak D phenotype of the fetus/neonate or in lieu of the Kleihauer-Betke test in the setting of elevated HbF.The application of hematology analyzers that have features similar to multiparameter flow cytometers may enable labs to adopt a fully automated and precise antibody-based method for quantifying FMH. With around-the-clock availability and a short 30-minute turn-around time, confirmatory FMH testing can be integrated into routine hematology testing menu. Equivalent results to flow cytometry have been shown for both the anti-HbF and anti-D antigen methods using one model [ 53]. With the trend of integrating extended applications into routine hematology analyzers, the automated reticulocyte count being a notable example, additional platform options and broader clinical use is predicted to follow.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation
  5. Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation
  6. Summary
  7. Acknowledgements
  8. References

The authors are indebted to Drs. Michael Greene and Elizabeth Van Cott for critical review of the manuscript, to Dr. Bruce Davis for supplying the anti-HbF flow histograms, and Amy Slater for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation
  5. Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation
  6. Summary
  7. Acknowledgements
  8. References