Transfusion Medicine Case Study: Fetal Maternal Hemorrhage in a Trauma Patient

A 31 year old woman, gravida 1 para 0, 35 weeks pregnant, arrived in the emergency room via ambulance following a fall down the stairs. The ER ordered a CBC, Type and Screen and a Kleihauer-Betke (KB) test and sent blood to the lab. The KB result was positive with 1.1 % fetal cells. Hypothetically, if this was an exam question, you might be asked, “How many doses of Rhogam should be administered?” But, before you grab your calculators, let’s explore that a bit.

Hemolytic Disease of the Fetus and Newborn (HDFN) has been described since the early 1600s, before blood groups were recognized. In the early 1900s, pioneers in blood banking, Landsteiner and Weiner, discovered the ABO and Rh blood groups, and, later, the Rh system became associated with HDFN. However, the antibody related etiology and pathogenesis of HDFN was not recognized until the late 1930s. Thus, the disease was written about in memoirs of midwives and physicians as early as 1609, but the mechanism involved was not described for another 300 years. The KB test was developed in 1957 by Enno Kleihauer and Klaus Betke to quantitate fetal maternal hemorrhage (FMH). The KB test allows physicians to diagnose and monitor and to initiate therapy to prevent the effects of HDFN. Finally, considered one of the most significant successes in medicine, prophylaxis for Rh HDFN, Rh immune globulin (RhIg), became available in 1968. The KB test is used to quantitate FMH in RhD negative mothers and the results can be used to calculate dosage for RhIg to prevent immunization. The KB test became one of the earliest examples of using a laboratory test to determine the appropriate dosage of a drug.1

KB testing has traditionally been used for RhD negative women to detect FMH and to determine the appropriate dose of RhIg to prevent immunization. In an RhD negative woman, we are concerned with immunization if the baby and mother are not antigenically similar. An RhD negative mother is given a prophylactic dose of RhIg at 28 weeks gestation. After delivery, when a newborn has a positive DAT and the fetal screen is positive, a quantitative test is needed to determine the appropriate dose of RhIg. In prenatal maternal trauma, there can also be a fetal bleed. Much as in childbirth, in a trauma, the baby’s blood can enter the mother’s circulation. This indicates placental hemorrhage and can be a prediction of preterm labor. In prenatal maternal trauma, the KB test has been used as aid in diagnosis and prognosis of HDFN, preterm labor and fetal demise. It can be used to determine if there has been a fetal bleed, and if so, to determine how much RhIg should be administered.

But, did you know that the KB test can also be used to determine FMH in RhD positive mothers? This is considered an alternative usage of the test. In the labs where I did KB tests, most fetal screens in Blood Bank were held until the following morning and performed on day shift. So, any KB tests on postpartum patients were also mostly done on day shift. I worked 2nd shift, and it was not uncommon to see KB tests ordered on RhD positive women. In fact, most of the KB tests ordered on 2nd and 3rd shift were from the ER and on RhD positive mothers. With RhD positive mothers, providers are not concerned with the mother producing anti-D, so RhIg is not a concern. Therefore, the answer to the hypothetical question posed above, is that this mother did not need any RhIg because, by checking the lab results it would be noted that this woman was Rh positive with a negative antibody screen.

A study performed in 2004 at the Shock Trauma Center, University of Maryland in Baltimore, reported that pregnant trauma patients with positive KB tests often had pre term contractions All patients in their study who experienced preterm contractions had positive KB tests. None of the patients with negative KB tests had uterine contractions. The conclusion was that “Kleihauer-Betke testing accurately predicts the risk of preterm labor after maternal trauma. Clinical assessment does not.” 2 They additionally concluded that, with a negative KB test, electronic fetal monitoring could safely be reduced. The major statement of the study, which has been incorporated into practice guidelines was that KB testing is important for all pregnant trauma patients, regardless of Rh status.2,3

In 2019 the College of American Pathologists Transfusion, Apheresis and Cellular Therapy Committee sent a survey with their proficiency testing program to determine how many participating laboratories perform KB tests on Rh positive pregnant females. 52% of the labs who responded noted that they performed quantitative fetal hemoglobin testing for RhD positive women, and about 39% reported performing more than 20 tests a year. The CAP group also reviewed literature detailing 16 observational studies and concluded that the literature supporting relying on the KB as a predictor of fetal distress was lacking evidence and nonconclusive. Despite the fact that doctors are ordering these and many laboratories are still performing this test STAT on RhD positive mothers, different guidelines for practice are mixed regarding if and how the KB should be used in these RhD positive trauma patients. Furthermore, many labs responded on the survey that doctors considered these results very important but that the labs were not sure how the results helped guide management of the mother or fetus.4

One of the problems some of these guidelines cite is that the KB test may not be rapid enough to use in trauma situations. Now, I have to start by saying that KB tests are probably no tech’s favorite test. The last hospital I worked at did KB tests in Hematology. Before that I worked at a hospital where we did KB tests in Blood Bank. There seems to be no way to avoid them! I would have to agree that a KB is not at all rapid. The test is both time sensitive, always ordered STAT, and very time consuming. Hands on time is considerable. I’ve gotten 2 in one night, on 2nd shift with only 4 or 5 techs manning the whole lab, and that makes for a busy night! Add a trauma or 2 to the mix, or a few units to wash for the NICU and you know why “Kleihaur-Betke” are not our favorite words.

Another concern is that the KB test is marketed as a quantitative test. The problem with this is that it is not very precise due to technical difficulty. In the KB acid elution test the mother’s blood is treated with acid and then stained and counterstained. Fetal cells contain HbF which is resistant to acid and these cells will remain bright pink. The mother’s cells, which are primarily HbA, will appear as faint ‘ghost’ cells. 2000 cells are counted and the percent of fetal cells is determined. The test is complicated and needs precision in staining, counting and calculations. A slide that’s too thick, poor timing of steps, slides that are not adequately dried, or fetal cells that fail to stain can all affect results and cause false negative results. In pregnant women HbF may be increased, and in women with hemoglobinopathies such as sickle cell anemia and thalassemia Hb-F can be increased, leading to false positive results. As well, late in pregnancy it would be considered normal to have some fetal cells in the mother’s circulation. Thus, both false negative and false positive KB results are not uncommon, and a positive report on a KB test may not accurately predict fetal distress.

Image 1. Kleihauer-Betke stain showing dark ink fetal cells and ‘ghost’ like pale maternal cells

In the CAP survey article, it was noted that, of participating labs, about 96% did KB tests and 4% use flow cytometry.4 Flow cytometry is accurate, sensitive and reliable for HbF determination. Flow cytometry uses antibodies directed against fetal hemoglobin and antibodies directed against adult RBCs. A clear separation of populations can be identified and quantitated. Despite the fact that it is well known that flow cytometry is a much more precise test for FMH, many laboratories continue to do KB testing. This is likely due to the fact that only a small percentage of labs have flow cytometers. If, in trauma situations, physicians want HbF determination with a “fast” turnaround time, KB testing can be done in house with no equipment necessary. This is not fast, but would, in most circumstances, be faster than sending a test to a reference lab.

The KB test has historically been validated and used to estimate the total amount of FMH, and the results used to calculate if additional doses of RhIg are indicated. The test has high specificity for HbF but can be subjective. Precision between techs and even with the same tech repeating the test can be relatively low. Because of this, the formula used to calculate RhIg dosage has a factor built in to make up for any imprecision. An alternate usage of the test, and the one used in this case example, is to predict outcomes and guide treatment in maternal trauma victims, regardless of Rh status.

While there is some controversy on using the KB test in these cases, it is none the less still recommended by many authors and included in medical guidelines.5 Providers are using the KB test more and more for assessing placental hemorrhage in cases of trauma and premature labor. Though immunophenotyping by flow cytometry has a greater accuracy, the KB test can give reliable results at a lower cost and with a faster turnaround time.

As always, this blog led me off on several tangents while writing. When I have an idea for a blog, I start with a case study or an interesting sample I have seen in the lab. The case study itself is the easy part, then I start researching and reading articles about the disorder, test or phenomenon that I am writing about. Often, when I read one article, I ask myself another question and say, “what if…?” and that leads to another article and another and another. Days later I can still find myself reading articles and chasing after more information. I love my job, I love being a Medical Laboratory Scientist and educator, and in true form of the curious MLS, I always want to investigate and never want to stop learning. Thus, this simple case about an alternative usage of Kleihauer-Betke (KB) test kept developing as I wrote. As a side note, it was interesting to see that the studies have had different conclusions and the guidelines for this use of the KB test have swayed over the years. It will be interesting to see what the future will bring. I have seen some articles about adding the HbF determination to hematology analyzers—wouldn’t that be nice!


  1. Reali G. Forty years of anti-D immunoprophylaxis. Blood Transfus. 2007;5(1):3-6. doi:10.2450/2007.0b18-06
  2. Muench MV, Baschat AA, Reddy UM, Mighty HE, Weiner CP, Scalea TM, et al. Kleinhauer-betke testing is important in all cases of maternal trauma. J Trauma 2004;57(5):1094-8.
  3. Michael V. Muench, Joseph C. Canterino, Trauma in Pregnancy, Obstetrics and Gynecology Clinics of North America, Volume 34, Issue 3, 2007, Pages 555-583.
  4. Matthew S. Karafin, Chad Glisch, et al, for the College of American Pathologists, Transfusion, Apheresis, and Cellular Therapy Committee; Use of Fetal Hemoglobin Quantitation for Rh-Positive Pregnant Females: A National Survey and Review of the Literature. Arch Pathol Lab Med 1 December 2019; 143 (12): 1539–1544.
  5. Krywko DM, Yarrarapu SNS, Shunkwiler SM. Kleihauer Betke Test. [Updated 2020 Sep 11]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan.

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

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