Kleihauer Betke Test

Kleihauer Betke Test

Article Author:
Diann Krywko
Article Author:
Siva Naga Yarrarapu
Article Editor:
Sara Shunkwiler
11/14/2020 7:18:56 PM
For CME on this topic:
Kleihauer Betke Test CME
PubMed Link:
Kleihauer Betke Test


Fetomaternal hemorrhage (FMH) occurs when there is a break in the placental barrier, allowing blood from the fetal circulation to enter the maternal circulation. This disruption in the placental barrier may occur for many reasons, including intrauterine fetal demise and trauma. Trauma is the number one cause of pregnancy-associated maternal deaths in the United States.[1] It occurs in as many as 40% of traumas, increasing in frequency and amount with high-force trauma, blunt force trauma, abdominal trauma, and anterior placental placement in the uterus.

When FMH occurs, fetal hemoglobin (HbF) is mixed with maternal blood. In response to this exposure, the maternal immune system is activated, and isoimmunization (formation of anti-RhD antibodies) may occur if the mother is Rhesus-D protein (RhD) negative and the blood type of the fetus is RhD positive. It takes only 0.01 ml to 0.03 ml of FMH for the isoimmunization of the motherFuture pregnancies may be at risk for RhD disease if the fetus is RhD positive. The maternal antibodies bind to fetal RhD positive erythrocytes, leading to hemolysis, anemia, hydrops fetalis, and possibly fetal death.  

To prevent the formation of anti-RhD antibodies, Rho(D) immune globulin is indicated. Before 12-weeks gestational age, in the setting of an RhD negative mother and FMH, a mini-dose of 150 mcg Rho(D) immune globulin is given. This dose will suppress the immune response to 2.5 mL of Rh-positive red blood cells. After 12-weeks gestational age, a dose of 300 mcg is recommended. This standard dose of Rho(D) immune globulin (300 mcg) covers FMH up to 15 mL of fetal red cells (30 mL of whole fetal blood). However, there are times when the additional dose is necessary due to massive red blood cell FMH and subsequent maternal immune response. This is when the Kleihauer-Betke (KB) test is essential. (See Potential Diagnosis section for preliminary rosette testing).

Specimen Collection

The specimen is collected from the maternal patient through peripheral venous phlebotomy.


Korber noted in 1864 that HbF was resistant to alkali denaturation with NaOH much more so than adult hemoglobin (HbA). It was further determined that, when immersed in a citrate buffer pH of 3.3, the HbF remained intact while the HbA leaked out. Utilizing this property, the KB test was first described in 1957 by Enno Kleihauer and Klaus Betker.[2] The KB test is an acid-elution assay performed on maternal blood to determine the amount of HbF that has passed into the maternal circulation. The process exposes maternal blood smear to an acid solution. HbF, being resistant to the acid, remains intact, whereas HbA is removed. Following this, the smear is stained via Shepard’s method*. The fetal red blood cells are left rose-pink in color, and the maternal cells appear “ghost-like” due to the absence of staining. Though manual detection and quantification had been widely used, flow cytometry was more precise* and now may be utilized.  A total of 2000 cells is counted. Calculation of the percentage of fetal to maternal cells is used to estimate the total amount of FMH.[3]

Percentage of fetal cells = Number of fetal cells X 100 / Total number of RBCs


KB testing has obstetrical implications in the diagnosis and prognosis of preterm labor, fetal demise, and other conditions. 

There is some controversy on KB testing in the setting of trauma in pregnancy. This test has been historically only recommended for the Rh-negative pregnant patient with major trauma. However, the term ‘major’ is not specifically defined by most authors. Intuitively, however, the risk of FMH would increase with higher magnitude blunt force, anterior placental location, and coagulopathies, among other factors. Some advocate its use in all pregnant trauma patients, including those who are RhD negative. It has been shown that a positive KB test accurately predicts the risk of preterm labor following trauma, whereas clinical assessment does not.[4] Because this is an independent risk factor, many authors recommended routine use in the setting of trauma, regardless of Rh status and the mechanism or force of the trauma. The result then is used to guide management and education on prognosis.[5][6][7][6][5]

A study found that the Kleihauer Betke test was not useful to screen for fetal anemia, but instead, it could be used to explain fetal anemia.[8] Research has also shown decreased usefulness of systemic KB test after an external cephalic version for breech presentation and in the case of reduced fetal movement during pregnancy.[9][10][9]

Potential Diagnosis

The rosette test is a qualitative screening test performed on a maternal blood sample to determine if FMH has occurred between an Rh-positive fetus and an Rh-negative mother and serves as a useful screening test. Qualitative testing may be utilized before quantitative testing, i.e., if the rosette test is positive, a KB test should be performed to confirm and then subsequently quantify the amount of FMH. In massive trauma, the KB test may be utilized primarily, without the preliminary use of the screening rosette test. The Kleihauer Betke test is utilized to determine if there is fetal blood in maternal circulation, with a threshold of 5 mL.

The rosette test is performed by incubating the Rh-negative maternal venous whole blood sample with anti-Rho(D) immune globulin. The maternal cells are left unbound to the anti-Rho(D) as they are Rho(D) negative. During this incubation period, any Rh-positive fetal cells in the maternal sample are sensitized to the anti-Rho(D) immune globulin and bound. Enzyme treated indicator cells are added, only binding to the fetal cells that were present and sensitized, resulting in a process called erythrocyte rosetting or E-rosetting. The indicator cells will be at the center of the rosette, while the fetal RBCs will be clustered around the edges, like petals on a flower. This rosetting pattern may then be viewed under microscopy.

Kleihauer Betke test is utilized to determine if there is fetal blood in maternal circulation, with a threshold of 5 mL.

Normal and Critical Findings

Any value not reported as zero is abnormal and indicates FMH.

Interfering Factors

In the case of maternal persistence of fetal hemoglobin or other maternal hemoglobinopathies that result in elevated HbF, the KB test will be falsely positive, and flow cytometry must be used to quantitate the amount of fetal hemorrhage in maternal circulation.


The KB test is highly specific, with low sensitivity, having a threshold of 5 mL of FMH to be positive. Keep in mind that the amount of FMH to cause isoimmunization is only 0.01 mL to 0.03 mL. As such, it is not a test to determine if there is FMH, but rather to better estimate the amount of FMH. If positive, it is used to determine additional Rho(D) immune globulin dosing over the standard 150 or 300 mcg dosage that should be administered.

Clinical Significance

Calculating the RhIg Dosage

The following calculations should be used to determine an additional number of vials of Rho(D) immune globulin needed if the KB test is positive, indicating a large amount of FMH. One vial contains 300 mcg and will protect against 30 mL of fetal blood.

Volume (mL) of fetal blood = Percentage of fetal cells x 50

Number of Vials of 300 mcg RhIG required= Volume of fetal blood/30 mL

Combining equations: Number of vials* = Percentage of fetal cells x 50 / 30

* If the number to the right of the decimal point is less than 5, round down and add one vial.

* If the number to the right of the decimal point is greater than or equal to 5, round up and add one vial.

Example 1:  If 2.9% of fetal cells are reported on KB, what dose of RhIG would be required?

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

Example 2:  If 2.6% of fetal cells are reported on KB, what dose of RhIG would be required?

  • Volume of fetal blood: 2.6% x 50 = 130 mL
  • Number of vials = 130 mL/30 = 4.33
  • The decimal is less than 5, therefore round down and add 1 vial (4+1).
  • Answer: 5 vials


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[2] [Demonstration of fetal hemoglobin in erythrocytes of a blood smear]., KLEIHAUER E,BRAUN H,BETKE K,, Klinische Wochenschrift, 1957 Jun 15     [PubMed PMID: 13450287]
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[9] Lemaitre J,Planche L,Ducarme G, Systematic Kleihauer-Betke Test after External Cephalic Version for Breech Presentation: Is It Useful? Journal of clinical medicine. 2020 Jun 30;     [PubMed PMID: 32629792]
[10] Athiel Y,Maisonneuve E,Bléas C,Maurice P,Cortey A,Toly-Ndour C,Huguet-Jacquot S,Mailloux A,Jouannic JM, Reduced fetal movement during pregnancy: Is the Kleihauer-Betke test really useful? Journal of gynecology obstetrics and human reproduction. 2020 May 11;     [PubMed PMID: 32438135]