Kleihauer-Betke Test

Specimen Collection

The specimen is collected from the maternal patient. This collection is performed via peripheral venous phlebotomy.

Procedures

In 1864, Korber noted that HbF was resistant to alkali denaturation with sodium hydroxide, much more so than adult hemoglobin (HbA). When immersed in a citrate buffer at pH 3.3, HbF remained intact, whereas HbA leaked. Using this property, the Kleihauer-Betke test was first described in 1957 by Enno Kleihauer and Klaus Betke.[3] The Kleihauer-Betke test is an acid-elution assay performed on maternal blood to quantify the amount of HbF that has entered the maternal circulation. The process exposes maternal blood smears to an acid solution. HbF, which is resistant to the acid, remains intact, whereas HbA is removed.

Subsequently, the smear is stained using the Shepard method. The fetal red blood cells appear rose pink, while maternal cells appear ghost-like due to the absence of staining. Manual counting of approximately 2000 cells has traditionally been used to quantify fetomaternal hemorrhage, though flow cytometry is now recognized as a more precise alternative.[4] The calculation is as follows:

% fetal cells = (number of fetal cells × 100) ÷ total number of red blood cells

Although the core principle of the Kleihauer-Betke test remains the same, modern practice includes a few nuances. Laboratories may use slightly different acid buffers or staining protocols, but all rely on HbF's resistance to acid to differentiate fetal from maternal red blood cells.[3] While the initial calculation of fetal cells as a percentage of total cells provides a basic estimate of fetomaternal hemorrhage, subsequent calculations to determine the actual volume often rely on assumptions about maternal blood volume and fetal red blood cell size. These refinements help guide more accurate dosing of Rho(D) immune globulin. 

A new staining protocol for the Kleihauer-Betke test was recently developed and evaluated. As discussed, the traditional Kleihauer-Betke test has several limitations and can be difficult to interpret, particularly in patients with hemoglobinopathies.[5] In such cases, staining can appear ambiguous, leading to false-positive results or overestimation of fetal cells, ultimately reducing the accuracy and reliability of the test. To address this, investigators introduced a modified staining protocol that uses an alternative acid elution solution and a different stain, rather than the traditional hematoxylin method.[6] The goal was to improve slide clarity and make interpretation more straightforward and reproducible. Compared with the standard technique, the modified protocol demonstrated improved overall accuracy (from 78% to 85%), reduced operator variability, increased specificity (from 56% to 70%), and approximately a 30% reduction in false-positive results. Overall, the new staining method made it easier to distinguish fetal red blood cells from adult cells and other morphologically similar cells, particularly in challenging cases.

Indications

Kleihauer-Betke testing has obstetrical implications in the diagnosis and prognosis of preterm labor, fetal demise, and other conditions. However, its use in the setting of trauma during pregnancy remains controversial. Historically, Kleihauer-Betke testing has been recommended primarily for Rh-negative pregnant individuals after major trauma to quantify fetomaternal hemorrhage and guide anti-D immunoglobulin dosing. The risk of fetomaternal hemorrhage is expected to increase with a greater magnitude of blunt force, anterior placental location, and maternal coagulopathies, among other factors. Results from earlier studies suggested that a positive Kleihauer-Betke test may independently predict the risk of preterm labor following trauma, leading some to advocate for routine testing in all pregnant patients with trauma, regardless of Rh status, with results used to guide management and counseling regarding prognosis.[7]

More recent evidence, however, has questioned the utility of Kleihauer-Betke testing as a prognostic or screening tool in trauma. Results from larger retrospective studies have demonstrated limited sensitivity and poor correlation between Kleihauer-Betke test positivity and adverse pregnancy outcomes, suggesting that its primary value remains in quantifying fetomaternal hemorrhage or Rh immune prophylaxis rather than in predicting outcomes.[8] Technical limitations of the Kleihauer-Betke test, including interobserver variability, low sensitivity for small hemorrhages, and false-positive results in conditions associated with elevated maternal HbF, further limit its clinical usefulness, particularly when more precise methods such as flow cytometry are available. Similarly, subsequent research has shown decreased utility of routine Kleihauer-Betke testing after external cephalic version for evaluating reduced fetal movement and as a screening test for fetal anemia, where it may be more useful as an explanatory rather than diagnostic tool.[9][10]

Potential Diagnosis

The rosette test is a qualitative screening assay performed on maternal blood to determine whether fetomaternal hemorrhage has occurred between an Rh-positive fetus and an Rh-negative mother. This test serves as a useful initial test before quantitative testing. If the rosette test is positive, a Kleihauer-Betke test should be performed to confirm the presence of fetomaternal hemorrhage and quantify its volume. In cases of major trauma, the Kleihauer-Betke test may be used directly without prior screening. The Kleihauer-Betke test is generally interpreted using a threshold of approximately 5 mL of fetal blood in maternal circulation.

The rosette test is performed by incubating the Rh-negative maternal venous whole blood sample with anti-Rho(D) immune globulin. Maternal cells, lacking the D antigen, remain unbound, whereas any Rh-positive fetal cells in the maternal circulation are sensitized and bound by the anti-Rho(D). Enzyme-treated indicator cells are then added; they bind only to sensitized fetal cells, forming erythrocyte rosettes (E-rosettes). Under microscopy, the indicator cells occupy the center of the rosette, with fetal red blood cells clustered around them like petals of a flower.

In modern practice, several important considerations arise regarding the rosette and Kleihauer-Betke tests. The sensitivity of the rosette test can vary with the testing kit used, and some methods reliably detect only larger fetomaternal hemorrhage, typically greater than 10 to 15 mL. The test only works if the fetus is truly RH positive, since weak D variants may affect detection. Although the Kleihauer-Betke test often uses a practical threshold of 5 mL, actual dosing decisions are based on the percentage of fetal cells and the maternal blood volume. Flow cytometry is increasingly used as a more precise alternative to the Kleihauer-Betke test, although cost and availability can limit its routine use. Overall, the basic algorithm remains the same: begin with qualitative rosette screening, followed by quantitative testing if the result is positive, or proceed directly to Kleihauer-Betke testing or flow cytometry in cases of major trauma.

Normal and Critical Findings

Any value other than zero is considered abnormal and suggests the presence of fetomaternal hemorrhage.

Interfering Factors

Although the Kleihauer-Betke test remains widely used, it has recognized limitations, particularly in conditions associated with elevated maternal HbF (more than 5% of total red blood cells), such as sickle cell disease, thalassemias, and persistence of fetal hemoglobin, where it may yield false-positive results.[11][12] In these instances, maternal red blood cells and fetal cells resist acid elution, leading to false-positive results. One study reported that 32% of maternal samples contained high levels of HbF-containing cells, and 69% of those yielded clinically significant false positives on Kleihauer-Betke testing.[13] This overestimation can lead to the unnecessary administration of high doses of Rho(D) immune globulin. Flow cytometry provides a more precise alternative by quantifying fetal hemorrhage in maternal circulation, but its use is limited by cost. Since 2017, cell-free DNA testing for fetal RhD status has become increasingly available and may help reduce unnecessary administration of Rho(D) immune globulin.[11]

Complications

The Kleihauer-Betke test is highly specific but has low sensitivity, with a positivity threshold of approximately 5 mL of fetomaternal hemorrhage. By comparison, only 0.01 to 0.03 mL of fetal blood is sufficient to cause isoimmunization. Therefore, the Kleihauer-Betke test is not meant to detect the presence of fetomaternal hemorrhage, but rather to provide a more accurate estimate of its volume. When positive, it guides whether additional doses of Rho(D) immune globulin are required beyond the standard 150 or 300 mcg.

Clinical Significance

The Kleihauer-Betke test is clinically significant because it quantifies fetomaternal hemorrhage volume, which guides appropriate dosing of Rho(D) immune globulin to prevent alloimmunization. Accurate calculation of fetal blood volume in maternal circulation is essential, as underestimation can lead to inadequate prophylaxis, whereas overestimation may result in unnecessary treatment.

Calculating the Rho(D) Immune Globulin Dosage

The following calculations should be used to determine the additional number of vials of Rho(D) immune globulin required when the Kleihauer-Betke test is positive, indicating a high level of fetomaternal hemorrhage. One vial contains 300 mcg and protects against 30 mL of fetal blood.

• Volume (mL) of fetal blood = % of fetal cells × 50
• Number of vials of 300 mcg Rho(D) immune globulin required = volume of fetal blood ÷ 30 mL
• Combining equations: Number of vials = (% of fetal cells × 50) ÷ 30
  • If the number to the right of the decimal point is <5, round down and add 1 vial.
  • If the number to the right of the decimal point is ≥5, round up and add 1 vial.

Example 1: If 2.9% of fetal cells are reported on the Kleihauer-Betke test, what dose of Rho(D) immune globulin is required?

• Volume of fetal blood: 2.9 × 50 = 145 mL
• Number of vials = 145 mL ÷ 30 = 4.83
• The decimal is ≥5; therefore, round up and add 1 vial (5 + 1).
• Answer: 6 vials

Example 2: If 2.6% of fetal cells are reported on the Kleihauer-Betke test, what dose of Rho(D) immune globulin is required?

• Volume of fetal blood: 2.6 × 50 = 130 mL
• Number of vials = 130 mL ÷ 30 = 4.33
• The decimal is <5; therefore, round down and add 1 vial (4 + 1).
• Answer: 5 vials

These calculation examples demonstrate how the Kleihauer-Betke test results translate into clinically actionable decisions regarding Rho(D) immune globulin dosing and patient management. Despite its utility, clinicians should interpret Kleihauer-Betke test results in light of known limitations, including false-positive results from maternal HbF and variability in manual slide interpretation.