Defects in the determination of left-right asymmetry

Approximately 70% to 80% of Rh-positive adults and children with acute or chronic immune thrombocytopenic purpura or HIV-related thrombocytopenia respond to infusions of anti-D immunoglobulin. The speed of onset of response is slower than that seen with intravenous immunoglobulin. Anti-D immunoglobulin is well tolerated, with occasional adverse reactions similar to those seen in treatment with polyclonal intravenous immunoglobulin, but anemia requiring blood transfusion can occur. Response is generally better in younger patients and those who have responded to other forms of treatment. Inhibition of Fc receptor-mediated platelet destruction by anti-D immunoglobulin-opsonized erythrocytes is the most likely mechanism of action, although the relative ineffectiveness of a monoclonal anti-D immunoglobulin preparation in treatment of immune thrombocytopenic purpura suggests that other mechanisms may exist. Hepatitis C has been transmitted by intravenous anti-D immunoglobulin preparations when used in the prevention of Rh immunization, prior to the introduction of screening donor plasma for hepatitis C virus antibodies. However, an intravenous solvent-detergent-treated preparation is now available.     

Asymmetric extractions used in the treatment of patients with asymmetries

Properties of a new anti-D immunoglobulin were assessed in Rh(D) negative healthy male adults. Six volunteers received intravenous, and five volunteers intramuscular injections of 200 micrograms anti-D, 48 hours after pre-treatment with 5 mL of Rh(D) positive erythrocytes. Immediately after intravenous administration of anti-D, a rapid decrease of the Rh(D) positive erythroyctes was noted. After intramuscular injection of anti-D, there was a lag phase of 6 hours until the erythrocytes decreased, and the elimination rate was slower. Twenty-four hours after injection of anti-D, the Rh(D) positive erythrocytes were at the detection limit or no longer detectable in all volunteers. After intravenous administration, anti-D serum levels decreased from 45 ng/mL at 2 hours to 29 ng/mL at 24 hours, whereas after intramuscular administration, anti-D became detectable at 4 hours and increased to 11 ng/mL at 24 hours. During subsequent months, anti-D serum levels decreased at similar rates in both groups. After six months, anti-D was not detectable in any of the volunteers. Thus, the new anti-D immunoglobulin induced elimination of the Rh(D) positive erythrocytes and suggested that Rh(D) immunization of the volunteers was prevented.     

Color Doppler ultrasound used for screening varicocele: the clinical significance as compared to physical examination

Here we present an enzyme-linked immunosorbent assay (ELISA) on microtiter plates for the quantitative determination of anti-D. This method is based on the solubilization of red blood cells sensitized with anti-D and the subsequent measurement of immunoglobulin G by ELISA. The measuring range of the assay is 40-5,000 IU/ml and the lowest quantifiable concentration in plasma is 0.5 IU/ml. The interassay relative standard deviation for concentrations above 130 IU/ml ranges from 3.2 to 8.1% and below 50 IU/ml from 10.5 to 19.7%. Comparison of ELISA with automated hemagglutination shows that the results of the two assays correlate well: r = 0.992, n = 26. The assay was validated for donor plasma samples and anti-D immunoglobulin preparations and it can also be used in assessing the severity of Rh (D) hemolytic disease during pregnancy.     

Treatment of chronic autoimmune thrombocytopenic purpura with monoclonal anti-D

BACKGROUND: The platelet count increases transiently after treatment with polyclonal anti-D in about 50 percent of D+ patients with autoimmune thrombocytopenic purpura (AITP). The effect is usually attributed to macrophage Fc-receptor blockade by antibody-coated red cells. As polyclonal anti-D is in limited supply, prospective testing was performed on a monoclonal anti-D (MoAb D) in such patients. STUDY DESIGN AND METHODS: Seven D+ patients with chronic AITP received MoAb D intravenously at doses of 47 to 95 microg per kg of body weight. Response was assessed by studying platelet count increment. Hemolysis and red cell-bound MoAb D were measured before and after MoAb D administration. RESULTS: MoAb D red cell binding was demonstrated in all patients at a ratio higher than that observed in AITP patients successfully treated with polyclonal anti-D. However, little or no platelet count increment was observed in six patients, while a transient response was observed in only one (platelet count 97 x 10(9)/L before MoAb D infusion and 163 x 10(9)/L 4 days later). Furthermore, because five patients showed signs of hemolysis and two became anemic, higher doses of MoAb D should be used only with caution in patients with AITP. CONCLUSION: The MoAb D used in this study cannot be proposed as an alternative treatment for patients with AITP.     

Purification of human albumin by the combination of the method of Cohn with liquid chromatography

Large volumes of plasma can be fractionated by the method of Cohn at low cost. However, liquid chromatography is superior in terms of the quality of the product obtained. In order to combine the advantages of each method, we developed an integrated method for the production of human albumin and immunoglobulin G (IgG). The cryoprecipitate was first removed from plasma for the production of factor VIII and the supernatant of the cryoprecipitate was fractionated by the method of Cohn. The first precipitate, containing fractions (F)-I + II + III, was used for the production of IgG by the chromatographic method (see Tanaka K et al. (1998) Brazilian Journal of Medical and Biological Research, 31: 1375-1381). The supernatant of F-I + II + III was submitted to a second precipitation and F-IV was obtained and discarded. Albumin was obtained from the supernatant of the precipitate F-IV by liquid chromatography, ion-exchange on DEAE-Sepharose FF, filtration through Sephacryl S-200 HR and introduction of heat treatment for fatty acid precipitation. Viral inactivation was performed by pasteurization at 60 degrees C for 10 h. The albumin product obtained by the proposed procedure was more than 99% pure for the 15 lots of albumin produced, with a mean yield of 25.0 +/- 0.5 g/l plasma, containing 99.0 to 99.3% monomer, 0.7 to 1.0% dimers, and no polymers. Prekallikrein activator levels were < or = 5 IU/ml. This product satisfies the requirements of the 1997 Pharmacopée Européenne.     

Extraesophageal varices

BACKGROUND AND OBJECTIVES: Alpha-proteinase inhibitor (PI) protects the lungs from proteolytic damage caused by elastase and can be used to treat congenital emphysema. We describe an improved method of purification of alpha 1 PI from redissolved fraction IV-1 paste. MATERIALS AND METHODS: The process used dimethylaminoethyl anion exchange chromatography, sulfopropyl cation exchange chromatography, virus inactivation by dry heat, and tri-n-butyl-phosphate/cholate treatment, followed by a second strong cation exchange chromatography. Optimizations of loading conditions for ion exchange chromatography at small scale (20-60 ml of suspension) are described. Virus inactivation was adjusted to provide the best yield of alpha 1 PI consistent with effective inactivation. The process has been effectively scaled up. RESULTS: The final product was approximately 90% pure by SDS-PAGE, with a 60-70% yield from starting fraction IV-1 paste. The process has been characterized by methods including nonreduced SDS-PAGE, alpha 1 PI inhibition assay, and biuret protein assay. CONCLUSION: The method described is an effective way of preparing large quantities of alpha 1 PI from fractionated plasma.     

The effect of Rh-D immunoprophylaxis with hyperimmune anti-D-immunoglobulin on the occurrence of RhD immunization in pregnancy

During prenatal immunohaematological examination in the period from January 1, 1991 to December 31, 1995, in the Croatian Institute of Transfusion Medicine we tested sera of 5107 RhD negative women. All of them had pregnancies in their medical history. The frequency of Rh immunization was 4.6% in 1991; 4.1% in 1992; 2.5% in 1993; 2.5% in 1994 and 2.4% in 1995. Rh immunization during the first pregnancy was observed in 0.46% of women, in 1.8% during the second, in 9.4% during the third, in 22.4% during the fourth pregnancy, and 33.8% in women with more than five pregnancies. In women that have no abortions in their medical history, anti-D alloantibodies were found with the frequency of 0.46% at the end of the first pregnancy, 1.2% at the end of the second pregnancy, 5.9% at the end of the third pregnancy, 14.3% at the end of the fourth pregnancy, and 15.3% in women with more than five pregnancies. The frequency of anti-D alloantibodies in women who in their medical history have only abortions is 3.4% after the first abortion, 10.5% after the second, 17.8% after the third and 20.8% after the fourth or more abortions. The frequency of antibodies of anti-D specificities in women who had abortions and births is 17.1% at the end of the third pregnancy, 26.2% at the end of the fourth pregnancy, and 42.7% after more than five pregnancies. The frequency of anti-D alloantibodies in women who were protected from Rh immunization by hyperimmune anti-D globulin is 1%. The obtained results demonstrate that prevention of Rh immunization by hyperimmune anti-D globulin does not comprise all the Rh negative women, and is especially inadequate after abortions and multiple pregnancies.     

Effects of thiols on prostaglandin synthesis in bovine bladder epithelium

Immunoaffinity chromatography was shown to be the method achieving the most complete elimination of antigenic admixtures from leukocyte interferon preparations without the loss of the preparation activity. An affinity sorbent has been developed on the basis of covalently linked polyvinyl alcohol (PVA). The immobilization of the antigen-specific rabbit globulin in the preparation of the sorbent is achieved by reaction of protein amino groups with the activated matrix. The proposed sorbent achieved the elimination of the antigenic admixtures from interferon preparations as effectively as those prepared on the basis of sepharose 4B, the productivity of the purification process being at least 5 times higher. The proposed sorbent is stable at the limit values of rH, is not destroyed by detergents, is sterilized in the process of preparation. Owing to the strong linkage of the immobilized immunoglobulin with the PVA-carrier, immunoaffinity chromatography on this sorbent does not involve contamination of the preparations with rabbit globulin allergenic for man. The combination of a large pore structure, wetting ability, stiffness, mechanical and chemical stability allows the proposed sorbent to be recommended for use in modern large-scale biotechnological production.     

Long-term evolution of the hypervariable region of hepatitis C virus in a common-source-infected cohort

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.     

High density lipoprotein conversion mediated by human plasma phospholipid transfer protein

Phospholipid transfer protein (PLTP) was purified from lipoprotein-free human plasma, obtained upon treatment of plasma with dextran sulfate and Ca2+, by employing a series of column chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified PLTP showed a single main band, corresponding to the molecular mass of 78 kDa. However, isoelectric focusing of the purified preparation gave multiple bands with pI ranging from 4.3 to 5.1, indicative of microheterogeneity. Purified PLTP was shown to possess not only phospholipid transfer activity, but also high density lipoprotein (HDL) conversion activity (Tu, A.-Y., Nishida, H. I., and Nishida, T. (1990), FASEB J. 4, A2148; Jauhiainen, M., Metso, J., Pahlman, R., Blomqvist, S., van Tol, A., and Ehnholm, C. (1993) J. Biol. Chem. 268, 4032-4036). Isolated HDL3 was enlarged to the size of HDL2b upon incubation with purified PLTP for 6 h at 37 degrees C at the PLTP/HDL3 molar ratio of approximately 1:45. Both the HDL conversion and the phosphatidylcholine transfer activities of purified PLTP were effectively inhibited by rabbit anti-PLTP immunoglobulin G. The primary importance of PLTP in the HDL enlargement that occurs in human plasma upon incubation at 37 degrees C was shown by the strong inhibitory effect of the anti-PLTP immunoglobulin G. The process of PLTP-mediated HDL enlargement was accompanied by the release of apoproteins, primarily apoA-I. HDL3 enlargement mediated by PLTP was effectively inhibited by the addition of free fatty acids.     

Colorectal stapled anastomoses. Experiences and results

The mechanism whereby passive Rh (D) immunoglobulins suppress the fetomaternal alloimmunization is still unclear. New in vitro tests are needed to better characterize the functional properties of polyclonal anti-Ds. The DAF assay was developed to monitor the antibody-dependent cell-mediated cytotoxicity (ADCC) and the phagocytosis of anti-Rh (D)-sensitized RBCs by effector cells. The principle of this test is based on the oxydization of the 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of free hemoglobin. The reaction is proportional to the hemoglobin concentration. This test was performed to determine and emphasize the efficacy of different polyclonal anti-D immunoglobulin preparations to mediate lysis and phagocytosis of sensitized RBCs by human peripheral mononuclear cells. The functional properties of different human RhD monoclonal antibodies were also analyzed and compared. The test was found to be convenient to perform and allowed the avoidance of radioactive labelling of RBCs for ADCC studies. It is mainly useful for the direct quantitation of phagocytosis.     

Rapid one-step purification of goat immunoglobulins by immobilized metal ion affinity chromatography

A rapid, single step purification of immunoglobulins from goat serum was achieved using immobilized metal ion affinity chromatography (IMAC) on a new high capacity gel, Novarose, coupled to tris(2-aminoethyl)amine (TREN) chelated with copper. When goat serum was adsorbed to this gel in buffer pH 7 at 11 cm/h (8.6 ml/h), the immunoglobulin fraction was recovered in a decreasing linear pH gradient at about pH 5.5. When the adsorption buffer was adjusted to pH 6.0 and the linear velocity increased to 110 cm/h (221 ml/h), an immunoglobulin fraction of greater than 95% homogeneity was obtained. Protein purity was assessed by silver-stained native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The capacity of the gel for immunoglobulins was 17 mg immunoglobulin/ml at the low flow rate with adsorption at pH 7 and 15 mg immunoglobulin/ml at the high flow rate with adsorption at pH 6. No problems of back pressure or gel compression were observed at the higher linear velocity. The mild elution pH, high flow rate, and synthetic nature of the ligand support make this new metal-chelating gel a powerful alternative to the use of other currently available commercial gels commonly used for immunoglobulin purification.     

Molecular insights into the development of the pancreas

Del (D-elute) in the Rh blood group system is a variant with very weak D antigen and no agglutination is found by the indirect antiglobulin test. This variant is characterized by the presence of anti-D eluate obtained after an adsorption-elution test using anti-D antibodies. We studied here the molecular genetic status of Del by using polymerase chain reaction with sequence-specific primers (PCR-SSP). We screened 306 serologically apparent D-negative Japanese donors comprising 102 Del types for exons 7, 4 and 10 of the RHD gene. No PCR product was found in all 204 non-Del samples from the D-seronegative donors. However, PCR products were found in all 102 Del samples and all 70 D-seropositive samples tested by the three PCR methods for exons 7, 4 and 10 analysis. Del was found with CCee, CcEe and Ccee, but not with CCEe, CcEE, ccEE, ccEe or ccee phenotype. The incidences of Del in the samples with the serological phenotypes CCee, CcEe and Ccee were 80.0% (4/5), 68.2% (45/66) and 61.6% (53/86), respectively. The results provide evidence that Del samples have exons 4, 7 and 10 of an RHD gene present in some form. This is consistent with the evidence that D antigen is present on the cells although only detected by antibody adsorption and elution. The PCR-SSP method in the present study is useful to confirm Del among serologically apparent D-negative samples.     

Rhesus incompatibility and anti-D prevention in the Düsseldorf region (a ten-year survey) (author's transl)

Among 116 120 children born between 1964 and 1973, 512 (0.44%) required treatment for haemolytic disease of the newborn caused by the presence of irregular antibodies. While the incidence between 1964 to 1970 ranged between 0.42 to 0.56%, it fell from 1971 to 1973 to 0.28%, evidence for effective anti-D prophylaxis since 1971. Those cases still occurring after 1971 were largely due to pregnancies which had started before 1971. In addition there were abortions, sensitizations during the first pregnancy, but also blood transfusions as cause for new sensitizations. Sensitization after pregnancy despite anti-D prophylaxis was observed twice. In the last few years there has been a relative increase of rare antibodies, increasing the complexities of serological diagnosis. Safety and speed of treatment can be further improved by more frequent identification of irregular antibodies in the mother and regular reports to the paediatrician.     

Aeromyocological survey in the Santiago Western district (author''s transl)

It has not been determined whether Rho (D)-negative infants born of Rho (D)-positive mothers are sensitized during gestation or during parturition. Sensitization before use precludes the efficacious use of human Rho immune globulin as a prophylactic. The purpose of the present study is to identify the time of sensitization. Cord blood was collected from the placentas of 68 Rho (D)-negative infants whose mothers were Rho (D)-positive. Sixty-three of the 68 infants had one blood sample obtained between 1 and 9 months later. The paired samples were analyzed for anti-D by standard Coombs test and by automated antibody detection techniques. With the technique of automated antibody detection, we have been unable to demonstrate antibody in cord blood of the Rho (D)-negative infants of whom at least 7 of 63 (11%) had detectable anti-D between 1 and 9 months of age. These data show that Rho (D)-negaitve infants do not have detectable antibody at birth but may develop detectable anti-D in the first months of life. This observation suggests that the sensitizing dose of Rho (D) antigen occurs at parturition rather than during gestation.     

Intravenous immunoglobulin: implications for use in the neurological patient

The efficacy of intravenous immunoglobulin (IVIG) in the treatment of the autoimmune disease, idiopathic thrombocytopenic purpura (ITP), has been well documented in the literature. This has encouraged researchers to examine possible therapeutic uses of IVIG in other immune-mediated disorders. Recent clinical reports have suggested that IVIG may have a role in the treatment of neurological disorders with a possible immunopathogenic etiology. Intravenous immunoglobulin, a blood product which contains immunoglobulin G and a trace amount of immunoglobulin A, is believed to work as an immunomodulating agent. However, its mechanism of action is not well understood. Nurses involved with the administration of IVIG must be well informed about the manufacturing and regulation, proper dose and administration, adverse effects, appropriate assessments and related patient education.     

Myocardial revascularization with one and two mammary arteries: clinical results and long-term follow-up

The manufacturing process for albumin in Australia is based primarily on ion-exchange chromatography. The capacity of ion-exchange matrices to remove non-enveloped viruses (canine parvovirus and poliovirus type 1) was assessed using a scaled-down chromatographic process which was shown to yield product meeting purity criteria set for the manufacturing process. Poliovirus type 1 and canine parvovirus were added at one tenth the volume of desalted and delipidated Supernatant II + III produced by traditional Cohn Fractionation from human plasma before the material was applied to DEAE and CM ion-exchangers connected in series. Samples were taken at equilibration, wash, elution and regeneration steps and the log clearance and reduction of the viruses calculated. The mean clearance and reduction factors for viral load of poliovirus type 1 were 5.3 logs and 3.2 logs, respectively and 1.8 logs and 1.8 logs for canine parvovirus.     

Effects of a colostrum replacement product derived from serum on immunoglobulin G absorption by calves

Calves are born hypogammaglobulinemic and rely on immunoglobulin (Ig) from colostrum to obtain passive immunity. Previous research has indicated that colostrum supplements derived from milk are less effective than is maternal colostrum in providing adequate IgG to neonatal calves. Our objective was to determine the absorption of IgG by newborn calves fed a USDA food-grade colostrum supplement derived from bovine serum or fed pooled maternal colostrum. Holstein calves (n = 20; 10 bulls) were removed from the dam within 1 h of birth and were housed in individual stalls for the 24-h study. Calves were fed 2 L of colostrum or colostrum replacer at 1.5 and 13.5 h (+/- 0.1 h). Calves were blocked by colostrum pool, and replacer was fed to provide equal intakes of IgG within blocks. Jugular blood was collected at 1 and 24 h (+/- 0.1 h) for analysis of IgG by radial immunodiffusion. At 24 h, calves were injected with 1.5 ml of Evans blue dye to estimate plasma volume. Mean plasma IgG at 24 h of age was 7.3 +/- 0.4 g/L and was affected by an interaction of block and treatment. Apparent efficiency of IgG absorption of 24 h was reduced when 750 g of the colostrum replacement product were fed but was increased when 266 g of colostrum replacement product were fed. Mean plasma volume was unaffected by treatment and was 3.5 +/- 0.2 L or 9.1% of BW. These data indicate that efficiency of IgG absorption from the colostrum replacement product may be affected by amount of material fed. Proteins other than IgG in the colostrum replacement product might have reduced the efficiency of IgG absorption.     

The use of anti-D to improve post-transfusion platelet response: a randomized trial

Patients undergoing induction chemotherapy for acute leukaemia often become refractory to platelet transfusions. Increased clearance of transfused platelets due to alloimmune destruction has been identified as one of the primary mechanisms contributing to this refractory state. We performed a double-blind randomized trial to determine whether the administration of anti-D to Rh-positive individuals could prevent the refractory state and improve post-transfusion platelet response. Rh-positive patients with acute leukaemia undergoing induction chemotherapy and requiring platelet transfusions were allocated to weekly intravenous anti-D (20 micrograms/kg) or placebo. Platelets and red cell concentrates were administered according to standardized transfusion guidelines. Outcome measures included platelet transfusion utilization, red cell utilization, platelet recovery 18-24 h post-infusion, and the percentage of patients refractory to platelet transfusion. There were 43 patients studied: 21 received anti-D and 22 saline placebo. The mean number of platelet concentrates required per day of observation was 0.59 (SD 0.22) in the anti-D group and 0.61 (SD 0.22) in the placebo group, P = 0.86. No difference was detected between groups in terms of platelet recovery post-infusion, refractoriness to platelet transfusion or frequency of infection (P = 0.97). Red cell concentrate utilization was significantly increased in the anti-D group compared to the placebo group, 0.58 units per day versus 0.37 units per day respectively, P = 0.005. We conclude that the use of anti-D did not improve post-transfusion platelet response in Rh positive patients with acute leukaemia, but did result in an increased need for red cell transfusion.