Kleihauer-Betke test

Background

• The Kleihauer-Betke (KB) test is the standard method for detecting and quantifying fetomaternal hemorrhage (FMH)
• First described in 1957 by Enno Kleihauer and Klaus Betke
• Based on the principle that fetal hemoglobin (HbF) is resistant to acid elution, whereas adult hemoglobin (HbA) is not
• Mechanism: A maternal blood smear is exposed to an acid bath (citric acid–phosphate buffer), which removes HbA from adult red blood cells while HbF-containing fetal cells remain intact; subsequent staining causes fetal cells to appear dark/rose-pink while adult "ghost" cells appear pale
• Primary clinical purpose: Quantify the volume of FMH to guide dosing of Rho(D) immune globulin (RhoGAM) in Rh-negative mothers
• FMH occurs in up to 40% of trauma cases involving pregnant patients and can also occur during placental abruption, intrauterine fetal demise, and invasive obstetric procedures
• As little as 0.01–0.03 mL of fetal blood is sufficient to trigger maternal Rh isoimmunization

Clinical Features

• Fetomaternal hemorrhage is often clinically silent; the KB test is a laboratory screening/quantification tool rather than one prompted by specific symptoms
• Clinical scenarios in which FMH should be considered:
  • Decreased fetal movement
  • Abnormal or nonreassuring fetal heart rate tracing (sinusoidal pattern is classic for fetal anemia)
  • fetal hydrops on ultrasound
  • Unexplained intrauterine fetal demise
  • Unexplained neonatal anemia at birth
  • Maternal trauma (blunt abdominal injury, motor vehicle collision)
  • Antepartum hemorrhage of uncertain etiology
• Massive FMH (>30 mL of fetal blood) can cause:
  • Fetal tachycardia or bradycardia
  • Sinusoidal fetal heart rate pattern
  • Fetal anemia, hydrops, and death

Differential Diagnosis

Causes of a positive KB test / elevated fetal cells in maternal circulation:

• True fetomaternal hemorrhage (trauma, abruption, procedures, spontaneous)
• Hereditary persistence of fetal hemoglobin (HPFH) — false positive
• Sickle cell disease — false positive
• Thalassemia — false positive
• Maternal malignancy (rare cause of elevated HbF)

Conditions that may prompt KB testing:

• Placental Abruption
• Trauma in Pregnancy
• Intrauterine Fetal Demise
• Antepartum hemorrhage of unknown origin
• Post-procedure (amniocentesis, external cephalic version, cordocentesis)

Evaluation

Indications for KB Testing

• Rh-negative mothers:
  • All Rh-negative mothers after a potentially sensitizing event to determine if additional RhoGAM doses (beyond the standard 300 mcg) are needed
  • ACOG recommends KB testing in all Rh-negative pregnant trauma patients
• Additional indications (regardless of Rh status):
  • Maternal trauma (the KB test predicts preterm labor risk after trauma more accurately than clinical assessment alone)
  • Unexplained elevated maternal serum alpha-fetoprotein
  • Unexplained fetal distress or abnormal heart rate tracings
  • Intrauterine fetal death
  • Unexplained neonatal anemia
• Not recommended for:
  • Diagnosis or exclusion of placental abruption — the KB test has no diagnostic utility for abruption

Test Procedure

• Specimen: maternal peripheral venous blood (EDTA tube)
• Timing: should be obtained as soon as possible after the suspected event; fetal cells are cleared from maternal circulation over hours to days
• A blood smear is made, exposed to acid buffer, then stained
• 2000 cells are counted; fetal cells (dark/pink) are distinguished from maternal ghost cells (pale)
• Result is reported as a percentage of fetal cells

RhoGAM Dosing Calculation

• One standard vial of RhoGAM = 300 mcg, which covers 30 mL of fetal whole blood (15 mL of fetal RBCs)
• Formulas:
  • Volume of fetal blood (mL) = % fetal cells × 50
  • Number of vials needed = volume of fetal blood ÷ 30
• Rounding rule:
  • If the decimal is < 0.5 → round down and add 1 vial
  • If the decimal is ≥ 0.5 → round up and add 1 vial
• Example: KB test reports 1.5% fetal cells
  • Volume = 1.5% × 50 = 75 mL fetal blood
  • Vials = 75 ÷ 30 = 2.5 → round up to 3, add 1 = 4 vials

Screening Algorithm

• Many institutions use a two-step approach:
  • Step 1: Rosette test (qualitative screen) — if negative, standard 300 mcg RhoGAM dose is sufficient
  • Step 2: If rosette test is positive → KB test (quantitative) to determine additional RhoGAM dosing
• In major trauma, many centers proceed directly to KB testing

Limitations

• Sensitivity: Poor sensitivity for small hemorrhages; interobserver variability is significant
• False positives: Maternal conditions with elevated HbF (HPFH, sickle cell disease, thalassemias) cause false elevations — one study found 32% of maternal samples had high HbF-containing cells, and 69% of those yielded clinically significant false positives
• Poor prognostic value: Larger retrospective studies have demonstrated limited correlation between KB test positivity and adverse pregnancy outcomes; its primary value is in quantifying FMH for Rh immune prophylaxis rather than predicting outcomes
• Not useful for diagnosing abruption: A retrospective cohort study found no positive KB tests among placentas later confirmed to have abruption on pathologic review
• Operator-dependent: Manual counting introduces subjectivity; results can vary between technicians
• Flow cytometry is a more precise alternative that uses anti-HbF antibodies to quantify fetal cells, with improved sensitivity and reproducibility, but is limited by higher cost and availability

Management

• Rh-negative mothers:
  • Administer standard 300 mcg RhoGAM for all potentially sensitizing events
  • If KB test indicates FMH > 30 mL fetal whole blood, administer additional vials per calculation above
  • RhoGAM should ideally be given within 72 hours of the sensitizing event
  • Before 12 weeks GA: mini-dose (150 mcg) is appropriate
• Trauma:
  • All pregnant trauma patients > 20 weeks GA should have continuous fetal monitoring (minimum 4–6 hours; extended to 24 hours if KB-positive or other concerning features)
  • A positive KB test after trauma predicts increased risk of preterm labor and warrants extended monitoring
  • Rh-negative trauma patients should receive RhoGAM regardless of KB result; KB test guides additional dosing
• Massive FMH (fetal compromise):
  • Immediate OB consultation
  • Continuous fetal monitoring
  • Consider emergent delivery if fetal heart rate tracing is nonreassuring
  • Intrauterine transfusion may be considered in select preterm cases in consultation with maternal-fetal medicine

Disposition

• All Rh-negative patients with a potentially sensitizing event should receive RhoGAM prior to discharge from the ED
• Patients with a positive KB test should be admitted for continuous fetal monitoring and serial labs
• Patients with a negative KB test after minor trauma and reassuring fetal heart rate monitoring for ≥ 4–6 hours may be considered for discharge with close OB follow-up
• OB consultation should be obtained for all cases of suspected significant FMH, abnormal fetal monitoring, or intrauterine fetal demise
• Transfer to a facility with obstetric and neonatal intensive care capabilities if not available on site

See Also

• Trauma in Pregnancy
• Placental Abruption
• Intrauterine fetal demise
• Third trimester bleeding
• Rh Incompatibility
• Vaginal Bleeding (Pregnant)

External Links

• Kleihauer Betke Test - StatPearls
• Kleihauer-Betke testing is important in all cases of maternal trauma - J Trauma 2004
• The Kleihauer-Betke test: clinical utility, indication, and correlation - Arch Pathol Lab Med 1995
• Diagnostic accuracy of KB testing to predict fetal outcomes - J Perinatology 2021