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Year : 2016  |  Volume : 1  |  Issue : 2  |  Page : 85-87

Cellular mimicry in kleihauer–betke assay

Department of Immunohematology and Blood Transfusion, Kasturba Medical College, Manipal University, Manipal, Karnataka, India

Date of Web Publication6-Sep-2016

Correspondence Address:
Shamee Shastry
Department of Immunohematology and Blood Transfusion, Kasturba Medical College, Manipal University, Manipal, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2455-8893.189852

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The Kleihauer–Betke (KB) test is a time-consuming test with a lot of variables which affect the calculation of the extent of fetomaternal hemorrhage (FMH). There have been several formulae proposed by various authors to calculate FMH using KB test. In this article, we propose a simple mathematical alteration to accommodate the correction for some of the factors that lead to falsely high estimation of FMH.

Keywords: Acid elution, hemoglobin F, Kleihauer–Betke assay

How to cite this article:
Doshi K, Shastry S, Shivhare A, Raturi M. Cellular mimicry in kleihauer–betke assay. Glob J Transfus Med 2016;1:85-7

How to cite this URL:
Doshi K, Shastry S, Shivhare A, Raturi M. Cellular mimicry in kleihauer–betke assay. Glob J Transfus Med [serial online] 2016 [cited 2023 Feb 4];1:85-7. Available from: https://www.gjtmonline.com/text.asp?2016/1/2/85/189852

  Introduction Top

Kleihauer–Betke (KB) test is an acid elution test which is based on the principle that fetal hemoglobin is resistant to acid elution in contrast to adult hemoglobin. These uneluted fetal red cells therefore stain bright pink with erythrosine against a faintly stained background of eluted adult red cells. The high inter- and intra-observer variability is due to several factors such as area covered in each high power and low power field, density of cells in the smear, and nature of the sample used such as packed cells, whole blood, or diluted whole blood.

The challenges that we face in interpreting a slide of KB test are due to cells which mimic fetal red blood cells (RBC). Uneluted cells of maternal origin such as maternal RBC with fetal hemoglobin and reticulocytes which are resistant to elution and are bigger than mature adult RBC, thus closely resembling fetal RBC, to mention a few. There have also been cases where lymphocytes were miscounted as fetal RBC.[1] The number of these mimicking cells is not significant in nondisease states, while in conditions such as hemoglobinopathies and autoimmune hemolytic anemias, they may interfere and result in inaccurate reporting of the KB test.

A distinction between fetal RBCs and fetal hemoglobin in maternal RBC or reticulocytes may not be entirely possible by KB test, and a circumscribed approach with other laboratorical and clinical details would assist in a more accurate reporting of the extent of fetomaternal hemorrhage (FMH). We hereby report two such cases in our clinical experience where we applied a custom-made mathematical formula for Indian population incorporating correction factors for uneluted maternal cells which may either be reticulocytes or be maternal RBC with fetal hemoglobin.

  Case Reports Top

Case 1

A 29-year-old antenatal female patient G3P2 L1A1D1 was admitted for an elective lower segment cesarean section at 35 weeks of gestation. KB test was performed in view of Rh negative pregnancy. A field of the slide examined is shown in [Figure 1]. As evident in the image, the total number of uneluted cells was 155 and total cells counted were 2085. FMH applying Mollison's formula was estimated to be 186 ml. At our center, we follow the modified KB test as given in the Technical Manual of American Association of Blood Banking.
Figure 1: Smear showing the result of Kleihauer–Betke test in Case 1

Click here to view

As in up to 98% of cases, the volume of FMH is <2 mL, and this high estimate made us re-examine the slide at ×100 magnification and review other hematological parameters which may be responsible for a falsely high FMH.[2]

Clinical details revealed history of weakness and headache at 25 weeks of gestation and was a previously diagnosed case of autoimmune hemolytic anemia. She was medically managed with steroids. On reviewing her hematological parameters, reticulocyte counts were elevated (7.75%), immature reticulocyte fraction was 0.7%, hemoglobin was 7 g%, and total bilirubin was mildly elevated.

The knowledge of her reticulocyte count made us slightly modify the existing formula, with correction for the reticulocytes and maternal blood volume. The corrected estimate of FMH in the final report was given as 5.1 mL.

Case 2

A 30-year-old primigravida with Rh negative pregnancy was admitted with labor pains and underwent normal vaginal delivery at term. KB test was performed and the estimated FMH applying Mollison's formula was 250 mL. This high volume of FMH though theoretically possible would mean fetal exsanguination. A clinically asymptomatic newborn made us review maternal hematological parameters which were within normal limits. After reviewing her hemogram, we advised hemoglobin electrophoresis on the mother's sample which revealed 15% fetal hemoglobin. Maternal or adult RBCs with fetal hemoglobin have been traditionally called F cells. During pregnancy, there is a physiological increase in F cells. This increase is prominent during the second trimester of pregnancy in response to stress erythropoiesis as circulating blood volume increases by 40%. Normal levels of hemoglobin F (HbF) range tenfold from 0.3% to 4.4% and several factors influence the levels of HbF. F cells differ from fetal cells in their volume and are also variable with respect to the membrane antigens and enzymes. High levels of F cells may be seen in a small percentage of healthy population, but every such observation requires further evaluation. While other pathological conditions associated with increased HbF were ruled out in this particular case, this posed a difficulty in interpreting the results of the KB test. As shown in [Figure 2], distinguishing uneluted cells of fetal origin from those of maternal origin was practically impossible.
Figure 2: Smear showing the result of Kleihauer–Betke test in Case 2

Click here to view

  Discussion Top

The KB test is a time-consuming test with a lot of variables which affect the calculation of the extent of FMH. This often leads to both over- and under-estimation of the extent of FMH. Although an overestimation rather than an underestimation is preferred due to the risk of underdosing of Rh immunoglobulin, an overdosing is not free of risks associated with plasma-derived products.[3] Despite its shortcomings, the KB test remains an easy to perform screening tool in centers with scarce resources. Careful attention to critical test requirements such as diluting test samples, pH of McIlvaine buffer, known positive and negative controls, improves test accurecy.

There have been several formulae proposed by various authors with assumptions of maternal blood volume.[4],[5],[6],[7] These formulae consider the average value of red cell volume of a pregnant woman at term as 1800 mL and applying the same to an Indian population with a much lower average body weight, and among whom, the prevalence of anemia is high and leads to inappropriate high estimation of the actual extent of FMH.

Diagnosing a falsely high FMH begins with the suspicion of the same coupled with the knowledge of all the conditions associated with a falsely high FMH. Women with a high reticulocyte count, hereditary persistence of fetal hemoglobin, and other hemoglobinopathies are among the several of the listed causes of a falsely high FMH.

Formula (Mollison, 1972):[4]

In the formula for calculating the volume of FMH, the three considerations are that only 92% of fetal cells stain darkly and that a fetal cell is 22% larger compared to an adult RBC and that the total volume of maternal red cells is 1800 mL.

We suggest a simple mathematical alteration to accommodate the correction in cases of either high maternal reticulocyte counts or high fetal hemoglobin with or without severe anemia. If the reticulocyte percentage or percentage of fetal hemoglobin in mother is considered as “a%” and her hematocrit as “b%,” then the proposed formula for fetal bleed as volume of packed fetal RBCs in mL could be (assumptions-only 92% of fetal RBCs are stained and fetal RBCs are 22% larger than adult RBC).

For example, in case of a 50 kg woman with a hematocrit of 21% and a reticulocyte count of 7%, if the number of eluted cells is 1850 and uneluted cells is 150, then applying the conventional formula as mentioned above gives us a volume of 194.5 mL (which is falsely high). On applying the customized formula, the volume is calculated to be 4.9 mL. In Case 1, we made the correction for the high reticulocyte count and maternal blood volume using the above formula. The corrected estimate of FMH in the final report was given as 5.1 mL. However, in Case 2, the high value obtained could be due to the elevated fetal hemoglobin level in the mother. An alternative method adopted for samples with high reticulocyte count is treating them with brilliant cresyl blue before preparing and fixing smears as suggested by KB in their original publication, which has been omitted in all the modifications of the acid elution assay.[8]

Having said that we agree and believe that the KB test may be used as a screening test alone and in all cases where the calculated dose of Rh immunoglobulin exceeds two vials, additional testing with flow cytometry may be warranted.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Shen R, Sandler SG. Transfusion medicine illustrated. Miscounting even one lymphocyte in the Kleihauer-Betke (acid-elution) assay can result in overdosing Rh immune globulin. Transfusion 2015;55:2069.  Back to cited text no. 1
Sebring ES, Polesky HF. Fetomaternal hemorrhage: Incidence, risk factors, time of occurrence, and clinical effects. Transfusion 1990;30:344-57.  Back to cited text no. 2
Kim YA, Makar RS. Detection of fetomaternal hemorrhage. Am J Hematol 2012;87:417-23.  Back to cited text no. 3
Mollison PL. Quantitation of transplacental haemorrhage. Br Med J 1972;3:115.  Back to cited text no. 4
Cunningham FG, Williams JW. Williams Obstetrics, 20th ed. Stamford, CT:Appleton & Lange.viii; 1997. p. 1448.  Back to cited text no. 5
American Association of Blood Banks. Technical Manual of the American Association of Blood Banks. 16th ed. Washington, DC: AABB; 2008. p. 985.  Back to cited text no. 6
Foley MR, Strong TH, Garite TJ. Obstetric Intensive Care Manual. 2nd ed. New York: McGraw-Hill Medical; 2004.  Back to cited text no. 7
Betke K, Kleihauer E. The acid elution technique and the question of the influence of membrane qualities on its results. Ann Soc Belges Med Trop Parasitol Mycol 1969;49:151-6.  Back to cited text no. 8


  [Figure 1], [Figure 2]

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