a. Detection of HDN can be done through prenatal and postnatal test.
b. Prenatal test is performed to identify those women at risk of having babies affected with HDN. Specimens used are clotted blood sample from both mother and father.
c. Similar to prenatal test, postnatal also used clotted blood sample, but taken from mother and infant.
d. Cord blood sample is taken through needle aspiration, and should be delivered and refrigerated for minimum of 7 days.
e. The severity of HDN can be access through in vitro assays, that is:
• Antibody titration
• Quantifying anti-D
• Maternal history
• IgG subclass distribution
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Figure 1: Summary of diagnosis test flow. |
Prenatal Laboratory Test
- ABO and rhesus (Rh) typing
ABO and Rh typing is done both on father and mother sample. In addition, weak D is also done to confirm the true Rh-negative, if the result shows Rh-negative. In Rh-HDN, paternal Rh phenotyping can be very helpful in predicting future pregnancies outcome. It’s not a problem if both of the parents are rhesus-positive. But, rhesus-negative mother and the rhesus-positive father who are getting a baby, there are possibilities for the baby to suffered from HDN. Other HDN-type used this test in determining whether the father’s RBC is homozygous or heterozygous for the gene producing immunizing antigen. It should be borne by testing with anti-D, -C, -c, -E and -e, in determining the infant’s degree of risk of exposure. This should be done in all cases involving clinically significant antibody. At about 12 weeks of gestation, Rh-negative women should be tested for anti-D. Usually, Rh typing of maternal sample is done especially in second or subsequent pregnancy. Titration of anti-D is done to find out the strength and rising trend of titre – done by two-weekly/monthly anti-D titre determination
- Antibody screening
Antibody screening test is done after ABO and Rh typing to detect if any unexpected antibodies present within mother’s blood. It is repeated at week 28 prior to Rhogam (RhIg) administration in Rh-negative women. Once detected, antibody identification is done in order to identify the antibody present, either significant or insignificant antibodies. The problem comes when the unexpected antibodies are the significant one. Titration is done if the antibodies detected are the significant ones.
- Titration
Because other factors vary, such as the Rh-antibody–binding constant, the amount of D antigen on the RBC membrane, and the ability of the fetus to maintain reasonable circulating RBC hemoglobin without compromising hepatocellular function and umbilical portal venous circulation, Rh-antibody titrations predict only the fetus at risk of suffering from erythroblastosis erythromatosis. Beside, titration can be done for two purposes: 1) distinguishing between mildly affected infants with others, and 2) detecting increase antibody level on serial testing. Due to its sensitiveness, many laboratories used indirect antiglobulin test compare to albumin test. The indirect antiglobulin titer that puts the fetus at risk may be 1:32 to 1:64 but must be determined individually for each laboratory. Regular antibody titrations must be carried out during pregnancy because the antibody titer is the basis for selecting the mother and her fetus whose are at risk and require further investigation. Methods for titration vary from one laboratory to another. The first sample is freeze if subsequent titer is requested, so that a comparison between first titer results with second titer can be done. After run both titers in parallel, the end point is compare and if it shows rising of two-fold or greater, it is consider clinically significant. A fourfold increase in titer is significant and implies that that the fetus is Rh positive and is potentially affected. A more sensitive test is done, that is amniocentesis.
- Amniocentesis
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| Figure 3: Liley graph |
Figure 3 shows Liley graph plotted in reading amniocentesis result. Basically, this graph is divided into 3 parts: 1) lower zone - indicates either no disease or only a mildly affected fetus. 10% require exchange transfusion; 2) mid zone - indicate a moderately affected fetus, which is a candidate for an early delivery & possible exchange transfusion; and 3) upperzone - fetus require immediate attention as severed disease is predicted.
For those who can't imagine how the amniocentesis is done, lets look at the video to make things clearer:
- Percutaneous umbilical cord blood sampling (PUBS)
With the development of sophisticated ultrasound equipment and the availability of perinatologists skilled in its use, percutaneous fetal umbilical blood sampling became feasible in the mid-1980s. This procedure allows the direct measurement of all blood parameters that can be measured after birth (hemoglobin, hematocrit, blood groups, direct antiglobulin testing, serum bilirubin levels, platelet and leukocyte counts, serum protein levels, and fetal blood gases). In addition, this technique also allows for direct fetal transfusion. Fetal blood sampling is by far the most accurate means of determining the degree of severity of fetal hemolytic disease (in the absence of hydrops) and the need for fetal treatment measures. Fetal blood sampling is a relatively benign procedure, carrying with it a traumatic fetal mortality rate of a fraction of 1%. Because it carries a high risk of fetomaternal TPH, its use is recommended only when serial amniotic fluid ΔOD 450 readings rise into the upper 65% to 75% level of zone 2 or when an anterior placenta cannot be avoided at amniocentesis and maternal pregnancy history or maternal alloantibody titers place the fetus at risk. Fetal blood sampling may be possible as early as 18 weeks' gestation; it usually is feasible by 20 to 21 weeks' gestation. The preferred sampling site is from the umbilical vessel (preferably the vein) at its insertion into the placenta. For this reason, the procedure is technically easier if the placenta is implanted on the anterior uterine wall. The risks of PUBS include amnionitis, premature rupture of membranes, fetal loss, puncture of the umbilical artery and umbilical cord hematoma.
Postnatal Laboratory Test
Postnatal test can be done by using both newborn and maternal blood sample.
As antibodies had not fully develops in newborn, only the ABO forward grouping is done on newborn sample. Rh typing is done in order to identify the baby’s Rh group. Weak D test is also performed if the baby is Rh negative. Positive Rh result obtain is not solely indicates that the newborn is Rh-positive, but it may also indicate the presence of maternal antibody that heavily coated on newborn’s RBC. Furthermore, newborns who were transfused while intrauterine often type Rh negative or weakly Rh positive. Therefore, it is impossible to get a proper Rh type without heating fetal RBC first to eluate off the antibody. ABO and Rh typing for maternal sample is done similarly to prenatal test.
DAT test should be performs on cord blood RBCs in identifying in vivo sensitization by maternal antibody. There are varies factors that contribute to the strength of DAT, which include maternal antibody strength, number of antigenic site directed by maternal antibody and total amount of blood infant receive from transfusion. Elution is done when the DAT outcome is positive. In addition, elution is also performed if the baby is symptomatic even if the DAT test shows negative result. It is unnecessary to perform elution on infant’s RBC if there is available of maternal blood and a single antibody is identified. If however, the maternal serum is negative for unexpected antibodies, so it is suggested of having ABO antibodies or an antibody to a low-frequency antigen. Diagnosing a low-frequency antigen can be aids by testing the eluate with paternal RBCs. Even though the antibody screening tests and panels give negative result, testing the maternal serum against paternal serum confirms the presence of antibody. As stated above, elution is done after getting the result of DAT. Type of cells used to test eluate depends on antibody found on mother. Below are 2 examples of situations that shows usage of different types of elute cells.
Antibody screening only performed on maternal sample, as the newborn did not fully develop their own antibodies. It is done similar to those procedure perform for prenatal test.
Protection of mother against the immunizing effects of Rh-positive fetal RBCs is vital, therefore it is important for the Rh-negative women to receive appropriate amount of RhIG. The half-life of the RhIG in the absence of significant mixing of fetal with maternal circulation, called fetal-maternal hemorrhage, is approximately 21 days. Two tests are commonly used to detect and quantify FMH: Rosette test and Kleihauer-Betke test.
i. Rosette test
- ABO and Rh typing
As antibodies had not fully develops in newborn, only the ABO forward grouping is done on newborn sample. Rh typing is done in order to identify the baby’s Rh group. Weak D test is also performed if the baby is Rh negative. Positive Rh result obtain is not solely indicates that the newborn is Rh-positive, but it may also indicate the presence of maternal antibody that heavily coated on newborn’s RBC. Furthermore, newborns who were transfused while intrauterine often type Rh negative or weakly Rh positive. Therefore, it is impossible to get a proper Rh type without heating fetal RBC first to eluate off the antibody. ABO and Rh typing for maternal sample is done similarly to prenatal test.
- Direct antiglobulin test (DAT) and elution.
DAT test should be performs on cord blood RBCs in identifying in vivo sensitization by maternal antibody. There are varies factors that contribute to the strength of DAT, which include maternal antibody strength, number of antigenic site directed by maternal antibody and total amount of blood infant receive from transfusion. Elution is done when the DAT outcome is positive. In addition, elution is also performed if the baby is symptomatic even if the DAT test shows negative result. It is unnecessary to perform elution on infant’s RBC if there is available of maternal blood and a single antibody is identified. If however, the maternal serum is negative for unexpected antibodies, so it is suggested of having ABO antibodies or an antibody to a low-frequency antigen. Diagnosing a low-frequency antigen can be aids by testing the eluate with paternal RBCs. Even though the antibody screening tests and panels give negative result, testing the maternal serum against paternal serum confirms the presence of antibody. As stated above, elution is done after getting the result of DAT. Type of cells used to test eluate depends on antibody found on mother. Below are 2 examples of situations that shows usage of different types of elute cells.
· Mom: anti-A, anti-B and/or anti-AB; negative antibody screening
Baby: A or B blood group
o Test eluate against screening cells, A1 and B cells.
o May performed Lui freeze-thaw or acid elution.
o Strength of DAT varies depends on maternal antibody strength, no of antigenic sites for antigen to which maternal antibody is directed, how much blood the infants has received from transfusion.
· Mom: positive antibody screen
o Perform acid elution.
o Against the same cells that react in mother ab identification panel.
o Include reagent A1 and B cells if mother is group O, baby is A or B.
- Antibody screening and identification
Antibody screening only performed on maternal sample, as the newborn did not fully develop their own antibodies. It is done similar to those procedure perform for prenatal test.
- Fetal-maternal hemorrhage (FMH).
Protection of mother against the immunizing effects of Rh-positive fetal RBCs is vital, therefore it is important for the Rh-negative women to receive appropriate amount of RhIG. The half-life of the RhIG in the absence of significant mixing of fetal with maternal circulation, called fetal-maternal hemorrhage, is approximately 21 days. Two tests are commonly used to detect and quantify FMH: Rosette test and Kleihauer-Betke test.
i. Rosette test
o Qualitative test.
o Used to detect FMH greater than 30mL.
o Procedure:
I. Incubate suspension of maternal blood with anti-D
II. Fetal Rh-positive RBCs in maternal blood react with anti-D.
III. Wash unbound antibody.
IV. Add suspension of group O, Rh-positive cells.
V. Reaction between anti-D with both O Rh-positive and fetal Rh-
positive RBCs, form agglutination in a rosette form.
VI. Examine the suspension microscopically.
o Presence of a certain number of clumps in a defined number of microscopic fields indicates positive result.
o Less than 30mL indicates negative result.
o Quantitative test must be performed to estimate the number of vials of RhIg needed.
ii. Kleihauer-Betke (KB) test.
o Quantitative test.
o Must be performed if positive rosette test.
o Done to estimate the needed number of RhIG vials.
o Principle is based on the resistance of fetal hemoglobin to acid elution.
o The percentage of fetal cells in maternal sample can be used to calculate the volume of FMH and the appropriate number of vials of RhIG.
o Indication:
- Determine possible FMH in the newborn.
- Aid in diagnosis of certain types of anemia in adults
- Assess the magnitude of FMH
o Calculate dosage of RhIg to be given through the percentage of fetal cells in maternal sample.
iii. Flow cytometry
o Similar purpose with KB test.
o Less available compare to KB test.
o Done by analyzing mixture of maternal sample, anti-D and fluorescein-labeled IgG by fluorescein-activated cell sorter.
CLINICAL FINDINGS
· Anemia
· Hyperbilirubinemia
· Reticulocytosis
· Thrombocytopenia
· Increase nucleated RBC count
· Leucopenia
· Hypoalbuminemia
· Smear: polychromasia, anisocytosis, no spherocytes.
· Rh negative blood type
· Positive DAT