Diverse and novel RHD variants in Australian blood donors with a weak D phenotype: implication for transfusion management.

BACKGROUND AND OBJECTIVES: Variant RHD genes associated with the weak D phenotype can result in complete or partial D-epitope expression on the red cell. This study examines the genetic classification in Australian blood donors with a weak D phenotype and correlates RHD variants associated with the weak D phenotype against D-epitope profile. MATERIALS AND METHODS: Following automated and manual serology, blood samples from donors reported as 'weak D' (n = 100) were RHD genotyped by a commercial SNP-typing platform and Sanger sequencing. Two commercial anti-D antibody kits were used for extended serological testing for D-epitope profiles. RESULTS: Three samples had wild-type RHD exonic sequences, and 97 samples had RHD variants. RHD*weak D type 1, RHD*weak D type 2 or RHD*weak D type 3 was detected in 75 donors. The remaining 22 samples exhibited 17 different RHD variants. One donor exhibited a novel RHD*c.939+3A>C lacking one D-epitope. Weak D types 1·1, 5, 15, 17 and 90 showed a partial D-epitope profile. CONCLUSION: The array of RHD variants detected in this study indicated diversity in the Australian donor population that needs to be accommodated for in future genotyping strategies.

Development of human anti-murine antibody (HAMA) response in patients.

Human anti-mouse antibody (HAMA) response was determined in the serum of 67 patients who received subcutaneously administered radiolabelled murine monoclonal antibodies (MoAb) (50 micrograms-3 mg) for immunolymphoscintigraphy and of 10 patients with advanced colorectal cancer who received murine MoAb-N-acetyl melphalan (MoAb-N-AcMEL) conjugates (amount of MoAb ranged from 120 mg/m2 body surface area to 1000 mg/m2 body surface area) as therapy. A pre-existing low level of apparent human anti-mouse antibody reactivity could be detected in the serum of normal subjects and patients prior to administration of murine MoAb. Subcutaneous administration of low doses of murine MoAb, as used in immunolymphoscintigraphy, was associated with a low incidence (4/67 or 6%) of elevated HAMA response; the use of F(ab')2 fragments was associated with the development of elevated HAMA response in one of three patients. By contrast, therapy with hepatic artery infusion of murine MoAb-N-AcMEL conjugates in three repetitive daily doses (each infusion lasting 2 h) elicited elevated HAMA responses in 10/10 (100%) patients, usually 1-3 weeks after the start of therapy. The HAMA response of patients in the therapy group was higher than those in the immunolymphoscintigraphy study and the use of steroids did not prevent the development of the HAMA response. Further administration of MoAb-N-AcMEL conjugates to a patient, who had already developed HAMA, led to 'serum sickness'-type symptoms and a transient reduction in the HAMA titres. The elevated HAMA response was polyclonal, containing increased levels of both immunoglobulin M and G (IgM and IgG) and was directed against mouse-specific determinant, the isotype (presumed to be the Fc portion), the F(ab')2 and the 'idiotype' of mouse immunoglobulins.

Improvement in sensitivity of enzyme-linked immunosorbent assay for tumour necrosis factor.

Various protocols were used in the development of enzyme-linked immunosorbent assays (ELISA) to improve the sensitivity and range of detection of human tumour necrosis factor-alpha (TNF-alpha). ELISA can provide a specific, sensitive and rapid method for detection of TNF-alpha in patient's sera, and it is important that the assay used should be sufficiently sensitive to detect low levels of TNF-alpha. The double sandwich ELISA proved to be the most sensitive, detecting less than 0.080 ng/mL TNF. Of eight different protocols, one assay using a purified monoclonal antibody to human TNF-alpha and rabbit polyclonal anti-TNF-alpha antibody had the greatest sensitivity and range of detection. The study illustrates methods for the development of sensitive immunoassays which may have applications in many assay systems.