Kleihauer Betke Test

Specimen Collection

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

Procedures

In 1864, Korber noted 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 smears to an acid solution. HbF, 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. Calculating the fetal to maternal cells percentage is used to estimate the total amount of FMH.[3] The calculation is: percentage of fetal cells = number of fetal cells X 100 / total number of RBCs.

Indications

Kleihauer Betke 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 Rh-negative pregnant patients 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 KB test was not useful to screen for fetal anemia, but instead, it could be used to explain fetal anemia.[8] Research has also shown the 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, ie, 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 are at the center of the rosette, while the fetal RBCs are 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. This value also 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 is falsely positive. Also, flow cytometry must be used to quantify the amount of fetal hemorrhage in maternal circulation.

Complications

The KB test is highly specific, with low sensitivity, having a threshold of 5 mL of FMH to be positive. Remember 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 protects 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 1 vial.
  • * If the number to the right of the decimal point is greater than or equal to 5, round up and add 1 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