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Laboratory detection of Rh immunization2. Laboratory detection of Rh immunizationAntenatal testing of maternal bloodSerological investigations on maternal blood samples are a crucial in management of HDN. The investigations at this stage have two principal aims: * To detect irregular antibodies, identify the antibody and assess the likelihood and severity of HDN. * To ensure availability of compatible blood to the mother and the baby. If unusqal antibodies are detected, other family members may be grouped or autologous maternal blood may Ie collected. Routine ABO and Rh(D) grouping All worsen should have their ABO and Rh(D) grouping determined during their first visit to the antenatal cljnic. If possible, tests on two separate occassions with two different anti-D reagents or techniques may be done. Antibody Screening All material blood samples must be tested for irregular antibodies. A combination of techniques i.e. saline RT, saline 37°C, albumin, enzymes and indirect antiglobulin test (both normal saline and LISS) shoulà be employed to detect all clinically significant antibodies in the maternal serum. If an antibody is detected, it should be identified using a panel of red cells. Frequently of testing (See Table below) The frequency at which the samples should be examined varies according to the * Rh (D) group * Antibody status * Previous obstetric history Frequency of antinatal testing
Antenatal tests to assess and predict the severity of Rh-HDN The tests done during this period are directed towards the prevention of stillbirth and morbidity associated with HDN. These include : * Complete obstretric history * Serological testing of father * Maternal anti-D levels * Amniocentesis * Cellular assays * Ultrasound foetal assessment * Foetal blood sampling 1. Complete obstetric history The severity of Rh-HDN is somewhat consistent in successive pregnancies; mild cases are followed by severe ones and a woman with a previous severe affected infant is at high risk of having a stillbirth. However, history alone is of limited preditive value as stillbirths have been reported in the first affected pregnancy also. 2. Serological testing of father The father’s Rh phenotyping shoud be borne by testing the cells with anti-D, -C, -E, -e, and -c. The probable genotype can then be determined. HDN cannot occur unless the foetal red cells carry the antigen against which the maternal alloantibody is directed. As the antigens are paternal in origin, typing of paternal blood should be done to determine the degree of risk to which the infant is exposed e.g. there will be no risk to foetus if mother has anti-K antibodies and father is K-negative. In such case the antibody may have been produced in response to previous transfusion rather than pregnancy. Paternal samples should be examined in all cases where the antibody is clinically significant (all antiglobulin reactive antibodies). If the father is heterozygous for the particular antigen, there is a 50% chance of the child being unaffected. 3. Maternal anti-D levels All Rh negative women should be tested for anti-D at about 12 weeks of gestation. In primigravida, anti-D will only be found at 12 weeks in a few subjects who have had a previous transfusion. In mulgravida if antibody is detected at 12 weeks, it indicates failure to administer anti-D immunoglobulin following an earlier pregnancy or miscarriage. The patient should be followed up closely to detect a rise in titre. The levels of antibody in the maternal serum may be assessed by: a. Antibody titre b. Antibody quantitation Antibody titration It is useful to perform antibody titration in two situations : 1. To distinguish between miidiyaffected infants and others. A titre of 1/16 or less by IAT is seldom associated with severe disese. 2. An increase in antibody level may be detected on serial testing. When a change in antibody level is reported, it is necessary to compare the present sample with the earlier sample by testing the two samples together. the serum can be stored in a deep freeze for a long time without change in titre. When the test is to be set up, the previous serum sample should be thawed and throughly mixed before titration. A fourfold increase in titre is significant and implies that the foetus is Rh(D) positive and is potentially affected. The titration technique should inlcude use of albumin, indirect antiglobulin tests or enzyme-treated red cells Limitation of antibody titration 1. The expression of titres in terms of dilution does not give an idea about the avidity of reaction. Different sera with the same titre differ in their avidity. 2. Some immunized women maintain a fixed antibody titre even when the foetus is Rh-positive. Quantitation of maternal antibody Quantitative assays are available which measure the level of anti-D in maternal serum. These include a. Serological assays The maternal antibody level is measured using an autoanalyzer. It is observed that when the level of anti-D is less than 4 lU/mI, the foetus has only mild disease. b. Quantitative assays (Radiometric antiglobulin test & ELISA) Results from these assays correlate better with disease severity than the antibody titres.
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