The gel agglutination test: a new technique for semiquantitative determination of fetomaternal hemorrhage in Rhesus-incompatibility

Background: The postpartum administration of an adequate amount of anti-D i mmunoglobulin to the mother in cases of Rhesus incompatibility requires the exact quantification of the amount of Rh-positive fetal cells that may be present in the Rh-negative maternal circulation. The classical methods to d etect an intrapartum fetomaternal hemorrhage are either time-intensive (suc h as the Kleihauer-Betke test), of low specificity (such as the indirect Co ombs test), or technically cumbersome (such as flow cytometry). The goals o f our study were to develop a simple screening test that may be used routin ely to quantify fetomaternal hemorrhage in cases of Rhesus incompatability and to evaluate this test in clinical practice. Study design and methods: In cases of Rhesus-negative mothers of Rhesus-pos itive neonates, 2.5 mi of maternal Rhesus negative blood was sampled in an EDTA tubes immediately postpartum and was incubated with anti-D antibodies. Thereafter, a semiquantitative determination was made of the amount of ant ibody that remained unbound in the serum via a gel agglutination test (GAT) (DiaMed., Switzerland) after mixing with test red blood cells. The amount of anti-D consumed (bound to fetal cells in the first phase) is the semi-qu antitatively indicated by the degree of positivity in the second phase the weaker reaction - the more anti-D absorbed in the first phase - the more Rh esus-positive fetal cells present in the maternal sample - the larger the f etomaternal hemorrhage. Following the development of a discrimination zone using this GAT which could ascertain an Rhesus-positive erythrocyte concent ration of over 0.2%,the test was applied in a clinical setting. Between Sep tember 1995 and April 1998 in unselected postpartum blood samples from 603 Rhesus negative parturients, the GAT was used to test the same blood sample s as those requiring evaluation for HbF concentration using the traditional Kleihauer-Betke test. Results: In 585 of the 603 cases (97%) there was no evidence of a fetomater nal transfusion following testing using both methods. Furthermore, both tes ts showed significant evidence for a fetomaternal transfusion in five cases . The Kleihauer-Betke test was false-positive in three cases of mothers who had a hereditary elevation of the HbF concentration. The GAT showed three false-positive reaction due to a D-weak maternal varient. In two cases, the disparity between the GAT and the Kleihauer-Betke test could be attributed to an antecedant dose of anti-D anti-body. In the two cases, the Kleihauer -Betke test results were 0.3% while the GAT was only 0.2%. Conclusion: The GAT may be used as a screening method in routine clinical p ractice. This is a quick test that allows for the specific determination an d semiquantitative evaluation of the Rh-positive erythrocyte concentration in clinically relevant concentrations. Thus, following a positive GAT scree ning test, a further specific test such as the Kleihauer-Betke test may be utilized to absolutely quantify the amount of blood transfused from fetus t o mother. It is also possible to perform such a quantification test with th e GAT by eventually using a diluted maternal blood sample.