Semiautomated data analysis of flow cytometric estimation of fetomaternal hemorrhage in D- women.

BACKGROUND: Accurate and reliable measurement of the volume of fetal D+ cells in D- women is required for adequate anti-D prophylaxis. A semiautomated flow cytometry assay based on a standardized calibration curve that was created with simulated fetomatemal hemorrhage (FMH) mixtures was developed. STUDY DESIGN AND METHODS: A calibration range of 0.083- to 2-percent D+ cells in the D-RBC mixtures (2-44 mL calculated FMH) was analyzed by use of a flow cytometer (XL-MCL, Coulter Electronics Ltd). Linear regression analysis of the calibration curve data with computer software (Excel, Microsoft) allowed semiautomated determination of the FMH volume. To optimize the assay, fresh versus frozen and thawed RBCs, RBCs from adults who are heterozygous for D or cord RBCs, and indirect- or direct-labeling techniques were evaluated by use of MoAbs. RESULTS: Fresh RBCs from adults heterozygous for D were chosen for routine use, although equivalent calibration curves were obtained with all cells tested (n = 12 calibration assays; r2 = 0.999; mean SD, 14%). A monoclonal anti-D reagent (Therad 10, Diagnostics Scotland) worked well in both indirect-(anti-IgG F(ab)-FITC) and direct-(anti-D-FITC) labeling methods compared to the use of BRAD-3 FITC. In routine practice, the FMH volumes obtained were mainly lower than those obtained in the Kleihauer Betke test when there was less than 4 mL of FMH. CONCLUSION: Semiautomated data acquisition and calibration curve analysis represents a further step toward standardization of flow cytometry for accurate FMH quantification and facilitates evaluation and control of day-to-day variations between laboratories, flow cytometers, and operators.

Limited efficacy of universal leucodepletion in reducing the incidence of febrile nonhaemolytic reactions in red cell transfusions.

This article demonstrates a 62% reduction in the number of febrile nonhaemolytic transfusion reactions (FNHTRs) and 50% reduction in febrile reaction rate associated with red cell transfusions following graded introduction of universal leucodepletion. Though this is a statistically significant reduction (P = 0.009), it shows limited efficacy in abrogating this complication. We also found a reduction in the proportion of cases of FNHTRs with lymphocytotoxic antibodies over the period studied from 54% in 1998, 28% in 1999 to 23% in 2000. This corresponds to a relative increase in the number of febrile reactions without human leucocyte antigen (HLA) antibodies following full implementation of universal leucodepletion, as the total number of reported reactions actually fell considerably during the period. The increase in the number of cases without HLA antibodies was directly proportional to the increase in the number of leucodepleted units used.

RhD haemolytic disease of the fetus and the newborn.

When an RhD negative mother is exposed to the RhD positive red cells (usually as transplacental haemorrhage), she develops allo-anti-D which crosses the placenta and then results in the destruction of fetal red cells. Clinical manifestations of RhD haemolytic disease (HDN) range from asymptomatic mild anaemia to hydrops fetalis or stillbirth associated with severe anaemia and jaundice. HDN was a significant cause of fetal mortality and morbidity until the introduction of amniocentesis, intrauterine transfusion, controlled early delivery and exchange transfusion in the management of severely alloimmunised women and their fetuses. The objective of monitoring alloimmunised women is to identify fetal anaemia and prevent the development of life-threatening hydrops. Evaluation involves assessing the history of previous pregnancies; serial estimation of maternal anti-D levels; serial ultrasound measurements; serial amniocentesis; fetal blood sampling, and intrauterine transfusion when indicated. Diagnostic genotyping by DNA-based methods can identify at-risk RhD positive fetuses early in gestation. Identification of transplacental haemorrhage (TPH) as the stimulus for anti-D antibody production led to the development of anti-D immunoglobulin prophylaxis for at-risk RhD negative women who are not already alloimmunised. Prevention includes administration of anti-D immunoglobulin for any event associated with TPH during pregnancy, and at delivery of an RhD positive infant. Prophylactic routine administration of anti-D immunoglobulin at 28 (and 34) weeks gestation, in addition to the above, has reduced alloimmunisation to <1% of RhD negative women carrying an RhD positive fetus.

A prospective study of routine antenatal enzyme antibody screening demonstrates lack of clinical value in predicting haemolytic disease of the newborn.

A prospective study of 7065 consecutive new pregnancies identified 230 with a positive screen, of which 27% (62/230) were 'enzyme-only' antibodies. 32 of these (52%) were potentially clinically important and were all of Rh specificity: 22 anti-E, seven anti-Cw, two anti-D and one anti-c. However, only three of these enzyme-only antibodies (one anti-D, one anti-c and one anti-E) became reactive by the indirect antiglobulin test (IAT) during the course of pregnancy, and all were detected in the routine 34-36-week maternal sample. No babies were affected, and we reaffirm that routine antibody screening by enzyme techniques is unnecessary.

Functional assessment of therapeutic anti-D immunoglobulin using Fc-mediated assays.

Therapeutic anti-D immunoglobulin preparations issued by the Scottish National Blood Transfusion Service, between 1980 and 1986, were evaluated using in-vitro Fc-mediated functional tests that reflect potential in-vivo mechanisms of specific red cell destruction and clearance. All batches tested were found to: (a) contain anti-D of mainly IgG1 subclass and lesser amounts of IgG3; (b) mediate lymphocyte and monocyte rosetting; and (c) produce lytic activity in both K cell and monocyte ADCC. The functional activity of the therapeutic immunoglobulin preparations over this period of production had not altered despite increased plasma contributions latterly to the pool from deliberately immunized male donors. This is the first in-vitro study of the Fc-mediated function of therapeutic polyclonal anti-D preparations. As these preparations were clinically effective in the prophylactic anti-D programme, such bioassays of FcRI/II and FcRIII activity are justified for the future evaluation of immune plasma before blending for fractionation and production of therapeutic anti-D immunoglobulin.

ADCC lysis of human erythrocytes sensitized with rhesus alloantibodies. IV. Characterization of anti-D sera which are inactive in ADCC.

Certain anti-D sera, selected on the basis of their agglutination characteristics in vitro, fail to induce lysis of Rh(D) positive red cells by lymphocyte mediated antibody dependent cell mediated cytotoxicity (ADCC). Further investigation revealed that the non-lytic anti-D blocked in an antigen specific manner the effect of other anti-D sera which were normally lytic in ADCC. Absorption selection studies and fractionation of a non-lytic anti-D serum showed that the blocking effect was associated with IgG anti-D. Antigen binding and lymphocyte Fc-receptor binding studies indicated that the non-lytic anti-D was bound to Rh(D) positive red cells and enabled them to be bound by lymphocytes, but failed to mediate ADCC.

Prediction of the outcome of rhesus haemolytic disease of the newborn: additional information using an ADCC assay.

An in vitro test system is described which measures the ability of anti-D sera to lyse Rh(D)-positive red cells. This test was applied to anti-D sera from 11 cases of HDN selected in Glasgow and tested 'blind' in Edinburgh. Evidence is presented to support the view that the ADCC (antibody-dependent cell-mediated cytotoxicity ) assay can correctly identify those cases where there is a satisfactory clinical outcome despite a high level of anti-D suggesting otherwise. Amniocentesis might therefore be avoided in this group.

Rhesus immunization in male volunteers: changes in lymphocyte functions following secondary immunizations in anti-D responders and non-responders.

After secondary immunizations of rhesus(D)-negative male volunteers with Rh(D)-positive red cells, changes were found in the in vitro transformation and antibody-dependent cell-mediated cytotoxicity (ADCC) capacities of the volunteers' lymphocytes. Responders, who produced anti-D, showed marked depressions of ADCC which were not found in non-responders. Responders and non-responders in general showed similar changes in lymphocyte transformation. The relationships between altered lymphocyte functions following immunization, immunoregulatory activity and responsiveness to the Rh(D) antigen are discussed.

ADCC (K-cell) lysis of human erythrocytes sensitized with rhesus alloantibodies. III. Comparison of IgG anti-D agglutinating and lytic (ADCC) activity and the role of IgG subclasses.

Conventional manual (enzyme and antiglobulin titres) and AutoAnalyser quantitation of anti-D sera were compared with the ability of the same sera to mediate lysis of Rh(D) positive red cells in an ADCC assay. AutoAnalyser (AA) quantitation correlated significantly with manual titration methods, although there were wide discrepancies between individual sera. The ADCC activity of the anti-D sera did not correlate with any of the conventional assays (AA, enzyme, antiglobulin). There were significant differences between anti-D sera obtained from females immunized during pregnancy and male volunteers immunized by deliberate injection. The female sera contained significantly less anti-D when assessed by AA, yet were significantly more active in ADCC activity at equivalent concentrations. These functional differences in anti-D activity could not be attributed to the absence of IgG subclasses (IgG1 and IgG3) known to induce ADCC. However, there was a relationship between ADCC and IgG1/IgG3 titres in that high ADCC activity was seen where the IgG3 titre was higher than the IgG1 titre. In the male anti-D sera IgG1 titres were greater than or equal to IgG3 titres and ADCC activity was very low.

Adverse effect of plasma exchange on anti-D production in rhesus immunisation owing to removal of inhibitory factors.

Intensive plasma exchange was used to reduce the maternal anti-D concentration in case of severe rhesus haemolytic disease. Initially the concentration fell from 30 to 4 IU/ml, but after six exchanges it increased to 490 IU/ml despite continued exchanges, and intrauterine fetal death eventually ensued. The increase in the rate of maternal anti-D production coincided with, and may have resulted from, removal of plasma immuno-regulatory factors that inhibited in-vitro lymphocyte functions. These results that the role of plasma exchange in haemolytic disease of the newborn is more complex than simply removing the antibody and that further investigations are needed.