Study type: Blood collected in a lavender-top [EDTA] tube from maternal or post-partum patients. In general, a sample of maternal blood can be collected at any point in the pregnancy but within 5 days of delivery to be acceptable for evaluation. Consult the laboratory for specimen collection requirements specific to the test being requested as some procedures may have stricter requirements; related body system: . Freshly prepared blood smears are also acceptable. Cord blood may be requested for use as a positive control.
Individuals who are RhD-negative do not have the Rh antigen on the surface of their RBCs and will develop alloantibodies to the D antigen if exposed to RhD-positive RBCs. An Rh negative mother becomes sensitized when RhD-positive fetal cells cross the placental barrier. The mother will produce antibodies against RhD-positive RBCs resulting in hemolysis of the in utero fetus’s RBCs. The level of hemolysis can run along a spectrum of severity, which can be significant. The outcomes range from neonatal anemia that requires transfusion, to hyperbilirubinemia/kernicterus, to fetal demise. For additional information regarding hyperbilirubinemia, refer to the study titled “Bilirubin and Bilirubin Fractions Core Lab Study.” A part of ensuring that obstetric patients, especially those who are who are Rh negative, receive immunological protection from RhD alloimmunization begins with the prenatal screen. Part of prenatal testing includes an antibody screen to confirm the patient’s Rh type as positive or negative. In this way potential alloimmunization can be anticipated and a healthy pregnancy can be planned. Patients who do not receive prenatal care and present at a medical facility for delivery of their baby will be screened upon admission. Ensuring that Rh negative patients receive Rh(D) immune globulin (RhoGAM intramuscular [IM] or Rhophylac IM or IV), is the primary objective in order to prevent inadvertent alloimmunization by an RhD positive fetus. There are several studies used to identify and quantitate FMH. The quantitative methods report the size of the FMH, which is used to determine the Rh immune globulin (RhIG) dosage required to neutralize the bleed.
Rosette Test(Qualitative Screening)
Identification of a significant FMH is commonly carried out by first performing a qualitative screen called the Rosette test. A number of facilities use the rosette test because the relatively quick and simple procedure can be offered on any shift by laboratory personnel trained to perform the test. For the test to be valid and to distinguish maternal from fetal RBCs, it cannot be performed on blood from either a D positive mother or a D positive mother carrying a D negative fetus. A sample of maternal D negative blood is incubated with anti-D reagent, during which time any fetal D positive cells will bind to the anti-D reagent. The mixture is washed to remove any unbound anti-D reagent. Next, a reagent containing enzyme-indicator cDE/cDE (R2R2) RBCs is added. Any anti-D reagent bound fetal RBCs will agglutinate with the D positive indicator cells to form rosettes. Several drops of the mixture are added to a slide and reviewed microscopically. The number of rosettes defining a positive test may vary by specific assay (e.g., 3 or more in 10 fields or 7 or more in 5 fields). A positive test suggests a significant FMH (greater than 30 mL of fetal blood) and should be confirmed using a quantitative test. Note: False positives may be seen if the mother or fetus is weak-D positive. Sensitivity: the rosette test can qualitatively detect 10 mL of fetal whole blood in the maternal circulation.
Kleihauer-Betke (KB or Fetal Hemoglobin) Test (Quantitative Confirmation)
The Kleihauer-Betke acid-elution test is the most widely used confirmatory test for quantifying FMH. The KB relies on the principle that fetal RBCs contain mostly fetal hemoglobin (HbF), which is resistant to acid-elution whereas adult hemoglobin is acid-sensitive. Although the KB test is inexpensive and requires no special equipment, it lacks standardization and precision and may not be accurate in conditions with elevated F-cells. It is sometimes performed initially instead of beginning with the Rosette screen. The KB is more technically difficult to perform and interpret due to variables such as stain quality, oddities in the specimen cell size or shape, and subjectiveness of human interpretation. Also the frequency of test orders is relatively low, making it difficult to offer the test on all shifts; laboratories may only maintain a small percentage of staff qualified to perform the procedure. Scientific studies and proficiency challenges by regulatory agencies have identified significant variations in precision (repeatability) and accuracy (degree of agreement between measured and true value in a sample) between labs performing the KB. However, studies also show that the KB remains a commonly used method and it compares relatively well to flow cytometry in clinical sensitivity and specificity.
Once the maternal blood sample has been collected, a blood film of maternal RBCs is prepared, treated with an acid buffer, and stained. The acid solution causes hemoglobin to be leached from the maternal cells, giving them a ghostlike appearance. Fetal cells containing hemoglobin F retain their hemoglobin and are stained bright red. Approximately 2,000 cells are examined microscopically and counted. A percentage of fetal cells is reported. Enumeration of a total of 2,000 or more cells is important to achieve the accuracy and precision to detect an FMH of 15 mL of fetal RBCs or 30 mL of fetal whole blood, which is the amount of FMH that corresponds to a 300-mcg dose of RhIG. Calculation of RhIG dosage is based on the calculated size of FMH and should be done according to standard operating procedures and protocols, which may involve consultation between the requesting HCP, a pathologist, and a pharmacist. Some protocols allow for automatic dosing recommendations as part of the Fetomaternal Determination report.
Flow Cytometry (Quantitative Confirmation)
Most laboratories do not have the luxury of a dedicated flow cytometer for FMH testing. Enumeration of 50,000 or more cells allows for more accurate and precise determinations than a subjective manual count of 2,000 cells. Anti-HbF flow cytometry is a promising alternative to the KB, although its use is limited by equipment and staffing costs. Hematology analyzers with flow cytometry capabilities may be adapted for fetal cell detection, thus giving clinical laboratories a potentially attractive automated alternative for quantifying FMH. Some general laboratory instruments that use flow cytometry to perform routine blood cell counts (e.g., RBC, WBC, platelets, etc.) are being experimentally adapted to identify and enumerate fetal RBCs in maternal blood. However, FDA approved automated cell counters using flow cytometry currently involve body fluid cell counts or applications related to oncology. In pure flow cytometry, the percentage of fetal RBCs is directly measured by the flow cytometer.
Calculation of FMH in relation to volume of fetal blood is: 
- FMH (mL) = % of fetal cells × 50where:
- % of fetal cells = (absolute number of fetal cells counted/total cells counted)and
- the assumption is that maternal blood volume is 5000 mL and
- the conversion of absolute number of cells to percentage changes the blood volume factor from 5000 to 50
- Note: Some facilities may calculate maternal blood volume using the formula:(0.75 ([maternal height (inches) × 50] + [maternal weight in pounds × 25])
- Example: If 12 fetal cells were counted out of a total of 2,000 total RBCs using the KB method, then 12/2000 = 0.006 fetal cellsor 0.006 × 100 = 0.6% fetal cellsThen FMH = 0.6 × 50 = 30 mL(this is the same as multiplying the number of cells 0.006 by 5000 mL = 30 mL).
Calculation of FMH in relation to % fetal RBCs is:
- FMH fetal RBCs mL = fetal whole blood volume/2based on the assumption that the hematocrit of fetal whole blood is 50%.
- For example, if the quantity of fetal bleed is 30 mL of fetal whole blood, then FMH = (30/2) = 15 mL fetal RBCs.Postpartum RhIG should be given within 72 hr of delivery.
Examples of RhIG Dosage Calculations
- One 300 mcg vial will cover 30 mL of fetal blood. For the examples below assume the unit dosage (vial) is 300 mcg.
(Example 1) Number of vials needed:Vials needed = (% fetal cells × 50)/30For example, if 0.6% fetal cells counted then: (0.6 × 50)/30 = 30/30 = 1 vial.
(Example 2) Number of vials needed:Vials needed = (% fetal cells × 50)/30For example, if 1.2% fetal cells counted then: (1.4 × 50)/30 = 70/30 = 2.3 vials? No. If the decimal is less than 5, round down, and increase by one vial. The dose would be 3 vials.
(Example 3) Number of vials needed:Vials needed = (% fetal cells × 50)/30For example, if 2.8% fetal cells counted then: (5 × 50)/30 = 4.7 vials? No. If the decimal is equal to or greater than 5, round up, and increase by one vial. The dose would be 6 vials.