Rhmod phenotype: a parentage problem solved by denaturing gradient gel electorphoresis of genomic DNA.

Initial Rh phenotyping of a man with hemolytic anemia, his wife, and son appeared to exclude paternity. No exclusion was found in other blood groups or in the human leukocyte antigen (HLA) system; excluding Rh, the paternity index was 98.58 percent. Samples from these three family members, and two other family members, were tested with additional Rh antisera. The results indicated that the propositus has an Rhmod phenotype with expression of c, weak e, and very weak D, E, and G antigens. To support this hypothesis, DNA analysis of the RHD and RHCE genes was performed on the five family members. Polymerase chain reaction (PCR) products from exons 2 and 5 were analyzed by denaturing gradient gel electrophoresis (DGGE). The DNA results corroborated the serologic findings and refuted the exclusion of paternity.

Glycophorin A-deficient red cells may have a weak expression of C4-bound Ch and Rg antigens.

The blood group antigens Ch and Rg are polymorphisms of C4d. Antigen-positive red blood cells (RBCs) treated with proteases type as Ch-, Rg-. Although RBCs treated with sialidase may type Ch+ Rg+, they cannot be coated with C4 by the 10 percent sucrose method. Since studies of complement binding have shown that glycophorin A (GPA) is an important component for the uptake of C4 by RBCs, we tested all available GPA-deficient RBCs for their Ch and Rg status. Using eluates of human anti-Ch and anti-Rg, and monoclonal anti-Rg, we found that the Ch antigen was only weakly expressed on these RBCs, while Rg expression was variable. Our results imply that in the absence of GPA, C4 binds in vivo to a component or components other than GPA on RBCs.

The Rh antigen D: partial D antigens and associated low incidence antigens.

The expression of the Rh antigen D varies quantitatively and qualitatively (partial D); published information and 15 years' work studying D variants are discussed in this review. D epitopes correspond to the reaction patterns of monoclonal anti-D with partial D antigens. Partial D antigens can be reported in terms of their D epitopes but the epitope profile of cells with a quantitative variant of D (weak D) is difficult to determine reliably by haemagglutination tests. Nine partial D antigens, categories II-VII, DFR and two not previously reported, are identified by their epitope profiles and by association with low incidence antigens. Monoclonal anti-D recognize 16 D epitopes and more epitopes are anticipated. The specificities of polyclonal anti-D made by people with partial D antigens are considered in terms of possible D epitope specificities: recognized epitope specificities, or combination thereof, were not able to account for all observed reaction patterns of anti-D made by immunized individuals with partial D phenotypes. An attempt is made to understand partial D antigens and their associated low incidence antigens in terms of the molecular genetic information available.

The D antigen characteristic of RoHar is a partial D antigen.

The results of testing RoHarr cells with panels of monoclonal anti-D suggest that the D antigen (referred to as DHar) encoded by the haplotype (D)c(e) Rh33 is a partial D antigen. IgM monoclonal anti-D are more efficient than IgG monoclonal anti-D in detecting DHar.DHar expresses some but not all of both epD5 and epD6/7 and appears to lack epD1-epD4, epD8 and epD9.

Pulsatile GnRH stimulates normal cyclic ovarian function in amenorrheic lactating postpartum women.

The postpartum period is characterized hormonally by elevated levels of PRL and low levels of gonadotropins and sex steroids. In breast feeding, this state of postpartum amenorrhea can persist for an extended period, even though PRL levels decrease slowly. Although the action of PRL on multiple target sites has frequently been suggested as the cause of this ovarian quiescence, a suckling-induced alteration in hypothalamic gonadotropin-releasing hormone (GnRH) production has also been hypothesized. To test this latter hypothesis, we provided a uniform pulsatile GnRH stimulus to eight exclusively breast-feeding women for an 8-week duration beginning at 4 weeks postpartum. Five women with functional hypothalamic amenorrhea served as a comparison group. All women received GnRH administered at a dose of 200 ng/kg every 90 min sc via a portable infusion pump. Serial blood sampling for LH, FSH, and PRL was performed weekly for 5 h at 10-min intervals beginning immediately before initiation of GnRH, during the period of GnRH, and 1 week after the cessation of GnRH. The women collected daily urine aliquots for estrone-3-glucuronide, pregnanediol-3-glucuronide, and LH determinations. Serial transvaginal sonography was used to monitor follicular development. Before GnRH treatment the urinary steroid and serum gonadotropin levels of the two groups were low and similar. As expected, PRL levels were higher in the postpartum women (87 micrograms/mL vs. 4.25 micrograms/L, P < 0.05). After initiation of pulsatile GnRH, LH values increased and FSH values decreased in both groups. The LH increase with GnRH was significantly greater in the breast-feeding group than in the hypothalamic amenorrhea group (19.75 mIU/mL vs. 12.34 mIU/mL, P < 0.05). Analysis of pulse frequency and amplitude revealed a nearly complete 1:1 induction of LH pulses by the exogenous GnRH in both groups, with the breast-feeding group showing a greater amplitude (12.26 mIU/mL vs. 5.34 mIU/mL, P < 0.05). The cycle lengths, urinary steroids, and vaginal ultrasonography demonstrated a more rapid initial ovarian responsiveness in the breast-feeding group, as determined by the length of the first follicular phase. The breast-feeding group also showed a brisker ovarian response, as evidenced by a greater number of follicles that were 12 mm or greater (2.3 vs. 1.2, P < 0.05), and a greater luteal phase peak and integrated pregnanediol excretion, respectively (3.02 micrograms/L creatinine and 39.87 micrograms/L creatinine/cycle vs. 1.89 micrograms/L creatinine and 7.69 micrograms/L creatinine/cycle, P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

What is important on the red blood cell surface.

Blood group antigens have provided tools for investigation of the red cell surface and been very useful as genetic markers in family, population and forensic studies. Precise definition of phenotypes is very important. Application of MAIEA (monoclonal antibody-specific immobilisation of erythrocyte antigen), a recently reported technique, to identify antigens and to assign red cell antigens to a particular membrane component is described: location of Knops system antigens on CR1 is confirmed and provisional assignment of Cromer system antigens to the different short consensus regions of decay accelerating factor (DAF) is described. Variability of red cell antigen expression is considered. The possibility is discussed that factors other than alterations in Rh genes may be responsible for some Rh variant phenotypes. Some C variants, two of which are associated with low incidence antigens, are described. The relationship of Xga with the quantitative polymorphism of 12E7 antigen is reconsidered in light of some recent immunochemical studies.

Investigation of the biochemical relationship between the blood group antigens Xga and CD99 (12E7 antigen) on red cells.

The biochemical relationship between the red cell antigens Xga and the MIC2 gene product, CD99--previously designated the 12E7 antigen--has been examined by immunoblotting and immunoprecipitation analyses of the protein molecules bearing these antigens. Immunoblotting of membrane components and Xga-immunoprecipitates with anti-Xga has shown that Xga antigen is carried on a broad band of apparent molecular weight (Mr)) 24,500-29,500, which consists of a dark stained component at M(r)24,500 and a more diffusely stained component at approximately M(r) 26,500-29,500. Immunoblotting of membrane components and 12E7-immunoprecipitates with 12E7, and RFB-1 and NaM123 which also recognise CD99, distinguished two bands of M(r) 30,000 and 32,000. A non-radioactive immunoprecipitation technique was employed, which uses chemiluminescence detection of biotin-labelled red cell proteins. The protein of M(r) 32,000, which carries CD99, was identified by this method and the red cell quantitative polymorphism of CD99 was demonstrated. When the Xga protein was precipitated from biotin-labelled red cells, a protein of M(r) 32,000 was coprecipitated. This suggests that the proteins carrying the Xga antigen and CD99 are associated in the membrane.

PBDX is the XG blood group gene.

We have identified the Xga antigen, encoded by the XG blood group gene, by employing rabbit polyclonal and mouse monoclonal antibodies raised against a peptide derived from the N-terminal domain of a candidate gene, referred to earlier as PBDX. In indirect haemagglutination assays, these anti-peptide antibodies react with Xg(a+) but not Xg(a-) erythrocytes. In antibody-specific immobilization of antigen (ASIA) and immunoblot assays, the anti-peptide antibodies react with the same molecule as does human anti-Xga. Therefore, by its identity with PBDX, Xga is identified as a cell-surface protein that is 48% homologous to CD99 (previously designated the 12E7 antigen), the product of MIC2 which is tightly linked to XG. PBDX is renamed here XG.

FPTT is a low-incidence Rh antigen associated with a "new" partial Rh D phenotype, DFR.

BACKGROUND: Several Rh D phenotypes with partial D antigens are recognized. Some partial D antigens are associated with low-incidence Rh antigens. New partial D antigens are revealed by an atypical pattern of reactions with anti-D. STUDY DESIGN AND METHODS: The reactions of D variant cells with panels of monoclonal anti-D and with antibodies to low-incidence antigens were compared to those of known D categories to identify a new Rh D phenotype. The inheritance of partial D antigens was studied by Rh phenotyping of the families of the probands. Standard serologic methods were used and family data were analyzed. RESULTS: A new Rh D phenotype, to be called DFR, was identified in 17 probands, two of whom had made anti-D. The partial D antigen carries epD3, epD4, and epD9 and lacks epD8. The presence of other D epitopes is ambiguous; different answers were obtained for the same sample with different monoclonal anti-D of the same apparent epitope specificity. The immunoglobulin class of the anti-D was important: IgG were more successful than IgM monoclonal anti-D in detecting the partial D of DFR. Family studies showed that DFR traveled with Ce more frequently than with cE. The low-incidence antigen FPTT (International Society of Blood Transfusion number 700048) was found on all DFR samples. Family studies demonstrated that FPTT is, as suspected, part of the complex Rh system. CONCLUSION: The partial D of the Rh D phenotype, DFR, is recognized by its pattern of reactions with monoclonal anti-D and its association with the low-incidence antigen FPTT, FPTT has now been numbered Rh50.

Capillary gas chromatographic drug screen for use in forensic toxicology.

A screening method is presented involving the use of capillary gas chromatography using a BP-5 column and nitrogen-phosphorous detection. This method is a quick yet reliable procedure for a large range of neutral and basic drugs and uses 1 mL or less of blood. Running standards that contain a number of commonly observed drugs with each batch of cases allows for more accurate tentative identifications of likely drugs in unknown cases and also provides a measure of quality assurance. This method is suitable for postmortem and clinical blood samples as well as plasma and serum.

Production of a murine monoclonal antibody to the low-incidence red cell antigen Wra: characterisation and comparison with human anti-Wra.

The production of a monoclonal antibody which detects the low-incidence red cell antigen Wra is described. The antibody (BGU1-WR) was isolated following immunization of BALB/tk mice with Wr(a+) cells. BGU1-WR is an IgG1 antibody and reacted in a manner similar to human polyclonal anti-Wra with untreated, protease treated and chemically modified Wr(a+) cells.

Comparative immunochemical analysis of Wra and Wrb red cell antigens.

This paper describes immunoblotting and immunoprecipitation studies using monoclonal anti-Wra and anti-Wrb antibodies to investigate the nature of the low-incidence blood group antigen, Wra and its high-incidence allelic antigen Wrb. No membrane components were identified by the immunoblotting experiments. Immunoprecipitation studies confirmed that the Wrb antigen involves both glycophorin A and band 3. The monoclonal anti-Wra, BGU1-WR, failed to immunoprecipitate these or any othe red cell membrane component. The significance of these findings is discussed.

Mg+ MNS blood group phenotype: further observations.

Red cells carrying the low-frequency MNS antigen Mg reacted with the only example of anti-DANE, an antibody which had previously defined the GP. Dane (Mi.IX) phenotype. Furthermore, Mg+ cells reacted with the original anti-Mur (serum of Mrs. Murrell), but with none of 14 other anti-Mur. Therefore, Mg+ cells carry both DANE antigen and an atypical Mur antigen. Immunoblotting of membranes from Mg+ cells with anti-M, and with eluates prepared from anti-Mg and Mrs. Murrell's serum demonstrated a glycophorin A (GPA) molecule whose mobility was increased by an apparent M(r) of about 3,000 presumably due to the loss of the three O-glycans known to be absent from Mg-active GPA.

Application of the MAIEA assay to the Kell blood group system.

The monoclonal antibody-specific immobilisation of erythrocyte antigens (MAIEA) test has been employed to investigate the Kell system using five monoclonal antibodies which recognise high frequency epitopes on the 93,000-molecular weight Kell glycoprotein: BRIC 18, BRIC 68, BRIC 107, BRIC 203 and 6-22. BRIC 107, which has anti-k-like (KEL2) specificity, identifies a distinct epitope, whilst competitive binding assays suggested that BRIC 203 (anti-Kpbc), BRIC 18, BRIC 68 and 6-22 (anti K14) comprise an overlapping set of epitopes. The MAIEA assay has been very successful in confirming the assignment of most of the Kell and para-Kell antigens to the Kell protein. Due to the competitive nature of the assay and the fact that the monoclonal antibodies bind to different regions, the results also suggest the relative positions of some of the Kell antigens on the Kell protein; these appear to be located in at least five spatially distinct regions.