Use of Fetal Hemoglobin Quantitation for Rh-Positive Pregnant Females: A National Survey and Review of the Literature

The College of American Pathologists proficiency testing program offers samples for fetal red blood cell detection to laboratories that perform this test. Twice each year, 2 specimens are provided with each proficiency test. Participants can screen the specimens with a fetal rosette test and also determine the percentage of fetal red blood cells present by either a staining (KB test) or a flow cytometric technique.

The College of American Pathologists Transfusion, Apheresis, and Cellular Therapy Committee (formerly known as the Transfusion Medicine Resource Committee) sought to determine to what extent participating laboratories performed these tests specifically for Rh+ pregnant females. To this end, the College of American Pathologists committee added 9 supplemental questions to the 2016 Hemoglobin F, mailing B, proficiency test.¹  These added questions were as follows: (1) Does your laboratory perform fetal bleed quantitation testing in Rh+ women with risk for fetomaternal hemorrhage? (2) In what time frame does your laboratory provide the test results? (3) How many deliveries does your center do per year? (4) How many fetal bleed quantification tests does your laboratory do per year for the alternative (Rh+) indication? (5) How many fetal bleed quantification tests does your laboratory do per year for RhIg dosing (standard indication)? (6) What is the turnaround time published by your laboratory to complete an urgent (or “STAT”) fetal bleed quantification test after specimen arrival in the laboratory? (7) What department orders the fetal bleed quantification test for the alternative indication the most? (8) What type of providers order the fetal bleed quantitation test for the alternative indication? (9) When the fetal bleed quantitation is ordered for the alternative indication, how do the results of the testing guide the management of the mother and fetus? These additional survey questions could be answered by any appropriately trained laboratory staff member, per the proficiency test instructions. Respondents were allowed to involve their medical director, as needed, to assist with the more clinical questions.

There were 1696 survey responses received, and 118 were excluded prior to analyzing the data for the following reasons: duplicate survey submissions (n = 6), partial survey submissions (n = 20), and reference laboratory designation (n = 92). Reference laboratories were specifically excluded from the analysis because of a lack of applicability for the majority of survey questions. Responses that did not follow the survey's skip sequence directions were also adjusted for data validity. A total of 1578 surveys were included in the summary, for a 76% response rate from the 2067 laboratories surveyed.

Statistical analyses using Pearson χ² tests were performed to test for practice characteristics significantly associated with KB testing in Rh+ women. These characteristics included laboratory location, annual number of deliveries, and annual volume of KB testing for the standard indication. Only one practice characteristic was significantly associated with KB testing practice, so no multivariate models were evaluated. A P value <.05 was considered statistically significant. The survey results summary and analyses were generated with SAS 9.4 (SAS Institute, Cary, North Carolina).

A total of 1578 laboratory surveys were analyzed, and the majority of respondents (93.5%; 1476) were from the United States. Most of the 1149 laboratories that reported a fetal red blood cell count result performed fetal hemoglobin quantitation using a modified KB test (acid-elution test; 1099; 95.6%), and 50 laboratories (4.4%) reported using flow cytometry. More than half of the evaluated participants (824; 52.2%) noted that they performed fetal hemoglobin quantitation for Rh+ women, and about 39% (308 of 789) of those reporting sites performed more than 20 tests a year (Table 1). The evaluated laboratories represented a diverse group of clinical practices; hospitals with more than 500 and up to 10 000 deliveries were represented, and institutions with higher-volume delivery centers performed KB testing for Rh+ females significantly more frequently than lower-volume centers (Table 2). Requests for this testing primarily came from obstetricians or midwives (445 of 815; 54.6%), followed by emergency department providers (266 of 815; 32.6%). Accordingly, the majority of 816 participants noted that requests for alternative fetal hemoglobin quantitation testing came from obstetrical/birthing departments (461; 56.5%), followed by emergency departments (280; 34.3%). More than half of the 819 participants (440; 53.8%) noted the results of this testing were reported as always or often being treated as very important information for patient care, and the majority of 823 participants noted that turnaround time for this testing was expected to be completed in less than 6 hours (623; 75.7%). Importantly, 44.2% (362 of 819) of participants stated they were unsure how the results of the test were used by the ordering provider.

Supplemental questions appended to College of American Pathologists proficiency test surveys have been a valuable source of information on national laboratory and medical practices.^4–8  The current survey demonstrates that KB testing for Rh+ women is commonly ordered, especially in larger hospitals, and is generally treated as important clinical information, but the reason for the order is unclear for a significant proportion of laboratories.

The noted confusion among laboratory professionals is understandable, as the use of KB testing for Rh+ pregnant women has been rarely a focus of study, and much of what we know regarding the sensitivity and specificity of KB methods is for Rh-negative women.²  Although it has been validated for RhIg dosing, proficiency surveys and other evaluations of the acid-elution assay consistently find it lacking precision, with a wide range of interoperator and intraoperator variation.²  Accuracy has been shown to be very operator dependent and has a reported error rate of up to 50% when positive.⁹ 

A number of technical and patient factors impact the accuracy and reproducibility of the KB test.^10,11  First, the KB test can underestimate the amount of fetomaternal hemorrhage if fetal cells fail to stain.^12,13  The KB protocol itself is notoriously complicated and time intensive,¹³  and relatively minor variations in test performance can lead to unexpectedly large variations in results. Anecdotally, blood smears that are too thick, slides that are not allowed to adequately dry, and inappropriately short buffering times can all lead to fetal cell understaining. Second, underestimation may also occur because of the known decreasing hemoglobin F (HbF) content of fetal erythrocytes with advancing gestational age.¹³  By term, HbF may represent only 70% of the hemoglobin of the fetal red blood cell, and may be missed because of light staining.^13,14  Lastly, false-negative results could occur in situations where preformed antibodies, such as in ABO-incompatible pregnancies, result in the removal of fetal red cells by the maternal reticuloendothelial system before detection by a KB test.¹³ 

In contrast, overestimation may occur because maternal HbF can mimic the appearance of true fetal red cells and can lead to false-positive results. Among the medical conditions associated with an increased number of HbF-containing cells in adults are hereditary persistence of HbF, sickle cell anemia (with or without treatment with hydroxyurea), β-thalassemia, aplastic anemia, and certain other hematopoietic diseases associated with erythropoietic stress.^15–18  Maternal HbF-containing red cells are also known to increase naturally during pregnancy, compounding the problem.^16–18  In approximately 25% of pregnant women, HbF starts to increase after 8 weeks' gestation and may reach 7% by 32 weeks.^18,19  Lastly, 40% to 50% of all pregnancies late in gestation have some fetal red cells in circulation.²⁰  It is known that some fetal red cells normally enter the maternal circulation (usually less than 15 mL in most cases) during all pregnancies and circulate over a number of weeks without apparent clinical significance.²¹  As a consequence, neither positive nor negative KB test results may be accurate, and positive KB tests, especially late in pregnancy, may not be a result of acute fetal injury or harm, even though injury may have been reported clinically.

As noted by our survey, the KB test is often urgently recommended for Rh+ pregnant women in trauma based on the assumption that the injury might be great enough to cause a transfer of fetal erythrocytes that would lead to actionable fetal morbidity from abruption or uterine injury. Previous studies have supported the notion that significant fetomaternal bleeding events are predictive of fetal mortality.¹³  However, the evidence in support of the KB test result's being predictive of adverse fetal outcomes without other signs of fetal distress is lacking. We identified 16 observational studies evaluating 19 to 394 pregnant females that used the KB test as a predictive biomarker for fetal morbidity and mortality (Table 3). The majority of these studies likely used a combination of Rh+ and Rh-negative females, as only 3 (19%) of these studies actually defined the number of Rh+ females evaluated.^22–24  These studies also predominantly used fetal cell positivity (ie, any fetal cells detected) as the predictor of interest (only 1 of 16 used the predicted percentage of fetal bleed).²³  Given these caveats, we found that only 3 (19%) of the 16 studies identified the KB test as predictive of adverse fetal outcomes.^9,23,25  Trivedi et al²⁵  defined any positive KB test (regardless of degree of positivity) to be predictive of adverse fetal outcomes when paired with the occurrence of maternal trauma to the abdomen and a hospital stay greater than 2 days. Huissoud et al,²³  in a case-control study, identified a 2.5% or greater fetal bleed as defined by the KB test to be predictive of fetal death, postnatal death, or neonatal anemia. Lastly, Muench et al⁹  identified a positive KB test as predictive for preterm labor. Of the remaining studies, 8 found no association between the KB test and preterm delivery or labor,^24,26–32  8 found no association with placental abruption,^26,28–34  5 found no association with fetal mortality,^{24,30–32,35}  and 3 found no association with neonatal anemia.^22,35,36  Taken together, the current literature is at best not conclusive, and the significance of the KB test result and its utility as a predictor for fetal outcomes is questionable.

Because of the lack of primary evidence, expert guidelines also remain somewhat mixed regarding how the KB test should be used for Rh+ pregnant women in acute situations. In one guideline, Brown³⁷  documents that there is little evidence that KB testing predicts adverse outcomes, and that abnormal heart findings would be more predictive of fetal jeopardy. In contrast, Muench and Canterino³  state that a KB test should be considered in all trauma patients, regardless of Rh status, because it may be an indicator of the severity of uterine trauma and an indicator of those patients at risk of preterm labor. Murphy and Quinlan³⁸  also assert that the KB test should be performed after major trauma to determine the degree of fetomaternal hemorrhage in all pregnant women, regardless of Rh status. Even major medical textbooks such as Pediatric Emergency Medicine by Baren et al³⁹  recommend the use of KB testing for all pregnant trauma patients, and cite the study of Muench et al⁹  as evidence for this practice. Lastly, Wylie and D'Alton¹³  assert that, despite known limitations, the KB test should be used any time fetomaternal hemorrhage is suspected antenatally. These authors, however, admit that the turnaround time of this test may not be helpful in emergencies, and peripheral blood flow cytometry testing might be more helpful given the possible need for rapid intervention.

Unlike the rapid prognostic indications noted above, the KB test remains a gold-standard tool for all pregnant females when used to evaluate the presence of fetomaternal hemorrhage as a contributor to unexplained fetal demise. Fetomaternal hemorrhage is a cause of fetal death in an estimated 1.6% to 11% of reported cases,^40,41  and the definitive diagnosis of a lethal fetomaternal hemorrhage requires confirmation of significant fetal blood volume loss.⁴¹  Although the critical volume required for death remains unknown, some studies suggest that as little as 20 mL/kg blood volume transfused to the mother's circulation may be sufficient.¹³  Compared with the KB test, however, other available clinical and laboratory findings are even less diagnostic for this indication: maternal risk factors do not appear to predict an increased likelihood of massive fetomaternal hemorrhage,⁴⁰  and postmortem autopsy findings, such as nucleated red cells or placental pallor, have been shown in a number of studies to be suggestive but not pathognomonic for fetomaternal hemorrhage.^42,43  Consequently, the KB test can be diagnostically helpful in Rh+ females with unexplained fetal death, and can be useful even when drawn days after the event.⁴¹ 

The dosing of RhIg in Rh-negative pregnant females is an, albeit flawed, established and accepted use of the KB test. However, our survey of 1578 laboratories revealed that this test is often being used for rapid diagnostic or prognostic purposes in Rh+ pregnant females. Although only a minority of studies demonstrated any predictive value of the KB test in these patients, expert guidelines still cite this test as potentially useful, and these guidelines are likely driving the observed clinical practice. Clearly, larger, well-designed, adequately powered, and prospective studies will be needed to clarify whether KB testing has any prognostic role. That said, the KB test currently cannot meet the needs of a trauma patient: it cannot predict fetomaternal bleeds with reproducibility because of known technical and biological complexities, and it is not a test that can be done rapidly. Consequently, outside of its potential use as a diagnostic tool post–fetal demise, the KB test's utility to guide urgent clinical decision-making outside of RhIg dosing (which has a 72-hour window after exposure) is limited.

Laboratorians should work to educate providers regarding the limitations of current KB testing methodologies, and should urge providers to limit clinical decision-making based solely on results of this test outside of RhIg dosing.

We would like to acknowledge Meghan Delany, DO, MPH, for her initial work on helping to develop the additional College of American Pathologists survey questions.