Measurement of feto-maternal haemorrhage: a comparative study of three Kleihauer techniques and tow flow cytometry methods.

In the UK a Kleihauer test is routinely performed on all RhD-negative women after the birth of an RhD-positive child to ensure that an adequate dose of anti-D immunoglobulin is given. The results of Kleihauer testing are interpreted to assess the volume of any feto-maternal haemorrhage and additional anti-D immunoglobulin is administered if necessary. A similar procedure is followed ante-natally when incidents occur known to be associated with alloimmunization. The performance of Kleihauer tests is difficult to standardize and there can be problems in interpreting the volume of feto-maternal haemorrhage. The use of flow cytometry to measure feto-maternal haemorrhage is reported to give more accurate and reliable results. This study compared three different Kleihauer methods and two different flow cytometry techniques all performed using the same maternal sample and within a single laboratory. The results demonstrated variability between the Kleihauer tests used and also in the flow cytometry measurements. A well-performed Kleihauer test would still appear to be useful as a screening technique for detection of feto-maternal haemorrhage. However, accurate quantitation of size of feto-maternal haemorrhage is more reliably determined by flow cytometry.

The use of pooled red cells and column techniques for routine red cell antibody detection.

The object of antibody screening is to detect all clinically relevant antibodies. In order to do this effectively red cells are selected with an appropriate antigen profile. The introduction of column techniques for antibody screening by indirect antiglobulin testing (IAT) and two-stage enzyme testing (ETC) is perceived to lead to an increased sensitivity and an ability to detect red cell antibodies more easily than by traditional tube techniques because reactions in columns are more easily read and are stable. We evaluated the use of a column technology with pooled red cells for routine antenatal screening. The pooled cells used contained at least one cell with homozygous antigen expression for the majority of clinically significant antibodies known to be present, except for Kell. Pooled cell results were not as easy to read in gel columns when compared with single cell results due to weaker reactions which were often diffused throughout the gel in the column. We concluded that the use of pooled cells led to a decreased sensitivity which proved problematic for the interpretation of results. We used a two-cell and a three-cell pool and found that detection of known antibodies was reduced in IAT and ETC methods.

A retrospective study of red cell maternal antibodies by chemiluminescence.

Plasma samples from 109 patients with maternal IgG alloantibodies were investigated using a chemiluminescence (CL) assay, a functional test, to predict which antibodies were clinically significant. The CL assay was able to distinguish between those patients who were unaffected or mildly affected requiring only phototherapy, and those patients with moderate or severe haemolytic disease of the fetus or newborn (HDN) requiring transfusion therapy. The CL result was compared with the anti-D quantification result, the number of IgG molecules bound per red cell and, in 80% of the cases, the monocyte monolayer assay. If mothers carrying Rh-negative fetuses were ignored, then the CL assay correctly predicted the outcome for 93.4% of all cases (including those other than D), while the AutoAnalyzer and monocyte assay predicted correctly 92.7% (of the anti-D patients) and 81.5%, respectively. Greater than 80% of patients with severe or moderate HDN had both IgG1 + IgG3 subclasses in the maternal plasma, while those infants who were unaffected or only mildly affected had a greater chance of having IgG1 only (44%) in the maternal plasma, IgG3 only (27%) or both subclasses (29%).

Assessment of severity of haemolytic disease of newborn by monocyte monolayer assay.

The present study was undertaken to estimate the predictive value of antibody titration, antibody quantitation and monocyte monolayer assay (MMA) in assessment of severity of haemolytic disease of the newborn (HDN). Serum samples from 45 alloimmunized mothers, with anti-D(23), anti-c(10), anti-K(6), anti-E(5) and anti-e(1) were taken for the study. The results obtained were compared and the efficiency of each technique in predicting the severity of HDN was assessed. Antibody quantitation and MMA (phagocytic index) correlated well with severity of HDN in mothers with anti-D antibodies. Antibody quantitation (anti-D) had a positive predictive value of 54.5 per cent and negative predictive value of 85.7 per cent while MMA had a positive predictive value of 75 per cent and a negative predictive value of 100 per cent. These findings suggest MMA to be a good negative predictor of HDN but not a good positive predictor of haemolytic disease of the newborn.

A case of unexplained mild Rh (D) haemolytic disease in utero.

This report describes the case of a patient with a history of HDN complicated by fetal losses, in which the alloantibody in this particular pregnancy did not appear to cause HDN in utero. No protective HLA-DR antibodies could be demonstrated, and transport of IgG across the placenta appeared to be normal. The infant's red cells possessed a normal D antigen and his mononuclear phagocyte system appeared unimpaired. However, the number of molecules of IgG bound in vivo per fetal red cell was below the level usually associated with significant haemolysis and HDN.

Haemolytic disease of the newborn due to anti-M.

A patient with haemolytic disease of the newborn (HDN) due to anti-M which required exchange transfusion is described. Anti-M antibodies are usually assumed to be naturally occurring and to consist of immunoglobulin M (IgM); many however have an immunoglobin G (IgG) component. In view of this and the described occurrence of HDN, recommendations are made regarding the management of a pregnancy in which anti-M antibodies are detected.

Red cell antibody screening and identification: a comparison of two column technology methods.

Two commercial column techniques for use in antibody screening and identification procedures were tested in parallel with 1000 random samples sent for ante-natal serological investigation. The DiaMed ID microtyping system uses a sephadex gel contained in microtubes, either neutral or impregnated with anti-human globulin (AHG), for use in two-stage enzyme methods and LISS indirect antiglobulin testing (IAT) respectively. The Ortho Biovue technique consists of a slurry of micro glass spheres which act as the filter to retain haemagglutination reactions within the matrix. Columns containing AHG also possess a macromolecular density barrier to prevent test serum from passing into the column and neutralising the AHG. Both systems offer the advantage of 'no-wash' IAT, which minimises the potential for problems and errors associated with conventional spin-tube techniques. In this comparison of the two column methods, antibody detection rates were found to be similar and the sensitivity of both methods was comparable, although the Biovue technique was prone to exhibit equivocal results, particularly in the IAT.

Molecular genetic analysis of the ABO blood group system: 4. Another type of O allele.

We have encountered an allele which seems to be another type of O allele at the human histo-blood group ABO locus. We have determined the nucleotide sequence of this allele over the coding region in the last two coding exons. This allele does not possess the single-nucleotide deletion found common among all the O alleles previously analyzed. Compared with A1 allele, this allele has three nucleotide substitutions resulting in two amino acid substitutions. The introduction of these amino acid substitutions into the A1 transferase expression construct apparently abolished the enzymatic activity of A1 transferase.

Gel techniques in blood group serology.

Serological investigation involves the interpretation of haemagglutination reactions, conventionally obtained in liquid phase systems. Gel centrifugation techniques utilise buffered dextran gels which separate red cells from their suspension media during a centrifugation phase, negative reactions resulting in the formation of a pellet of cells whereas agglutinations are trapped on top of, or throughout, the gel. The gels may be neutral or impregnated with specific reagents such as antihuman globulin, or antisera for phenotyping red cells. Gel systems have been found to be more sensitive than conventional serological techniques, particularly for the detection and identification of clinically significant antibodies. The elimination of the wash phase in the antiglobulin test, and the stability of the completed tests, provide greater reliability of results and enhance standardisation and control of laboratory procedures.

Evaluation of the ID-gel test for antibody screening and identification.

A gel technique for the detection of red blood cell (rbc) antigen-antibody reactions was evaluated for use in antenatal antibody screening and identification procedures. The evaluation was undertaken on 3,900 random antenatal samples. Results obtained in the gel test system were compared with those obtained from parallel testing using conventional serological methods. The ID gel system detected 148 (3.7%) red cell antibodies, compared with 95 (2.4%) using traditional techniques. The number of non-specific antibodies and false-positive screens were reduced using the gel test system. Antibody titres performed using the gel system were more sensitive than with our tube IAT method. The gel system was easy to use and gave reliable, reproducible results. Antibody detection rates were enhanced compared with our existing routine techniques.

Blood group chimaerism: a possible further example.

A blood group chimaera is described whose red blood cells exhibit a dual population of group A1 and O, in the proportions 20% and 80% respectively. These results could also indicate a blood group mosaic of the Amos type, but family studies, secretor and transferase investigations on this patient suggest that blood group chimaerism of unestablished type is more likely. There was no further evidence of chimaerism found during cytogenetic investigations, immunoglobulin allotyping or studies of red cell enzyme systems. Blood group chimaeras were previously thought to be rare, as dual red cell populations and the resulting mixed-field agglutination patterns are not always easy to recognise. This case is the second example of chimaerism found within two years in a single routine laboratory, appearing to confirm the view that chimaerism is not as uncommon as previously thought.

The Liverpool chimaera.

A dispermic human chimaera is described who is a fertile female XX/XY chimaera. The chimaera was discovered due to anomalies found during routine antenatal testing. The patient had two red cell populations differing in three blood group systems; ABO, Rh and MN. Analysis of cultured lymphocytes showed 70% of cells to have the 46, XY male karyotype and analysis of cultured skin cells showed 30% of cells to have the 46, XY karyotype.