Artificially Positive Crossmatches Not Leading to the Refusal of Kidney Donations due to the Usage of Adequate Diagnostic Tools.

Allografting patients with human leukocyte antigens (HLA) which are recognized by preformed antibodies constitutes the main cause for hyper-acute or acute rejections. In order to select recipients without these donor-specific antibodies, the complement-dependent cytotoxicity crossmatch (CDC-CM) assay was developed as a standard procedure about forty years ago. The negative outcome of pretransplant crossmatching represents the most important requirement for a successful kidney graft survival. The artificially positive outcomes of CDC-based crossmatches due to the underlying disease Systemic Lupus Erythematosus (SLE), however, may lead to the unjustified refusal of adequate kidney grafts. Two prospective female recipients destined for a living as well as for a cadaver kidney donation, respectively, exhibited positive CDC-based crossmatch outcomes although for both patients no historical immunizing events were known. Furthermore, solid phase-based screening or antibody differentiation analyses never led to positive results. Immediate reruns of the CDC-based crossmatch assays using the alternative antibody monitoring system (AMS-)crossmatch ELISA resulted in unequivocally negative outcomes. Consequently both transplantations were performed without any immunological complications for the hitherto follow-up time of 25 and 28 months, respectively. We here show two case reports demonstrating an alternative methodical approach to circumvent CDC-based artefacts and point to the urgent need to substitute the CDC-based crossmatch procedure at least for special groups of patients.

Rat complement factor H: molecular cloning, sequencing and quantification with a newly established ELISA.

Factor H (FH) is the predominant soluble regulatory protein of the complement system. With a concentration of 300-600 microg/ml in human plasma it acts as a cofactor for the FI-mediated cleavage of the component C3b to iC3b. Furthermore, it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex (i.e. it has decay accelerating activity). FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of nearly 60 amino acid residues. For the screening of a rat liver cDNA library, we used two hybridization probes which had been produced by polymerase chain reaction (PCR). The probes were generated using degenerated primers which corresponded to conserved parts of the human and the murine factor H nucleotide sequences. The entire rat sequence spanned 4240 nucleotides with an open reading frame of 3708 nucleotides. These were preceded by 23 nucleotides of the 5' untranslated region, followed by a stop codon and a 3' untranslated region of 478 nucleotides including the polyadenylation-signal up to the beginning of the poly A tail. Comparison of the rat cDNA-derived coding sequence revealed identities of 74% to the human and 87% to the mouse FH nucleotide sequence. The translation product of rat FH mRNA was 1236 aa in length (leader sequence included) with an identity of 63% to the human and 81.5% to the murine protein. The degree of glycosylation of rat FH-Mr is about 9.5%. To quantitate FH in rat serum and supernatants of primary cultures of rat hepatocytes (HC), a reliable and sensitive sandwich-enzyme-linked immunosorbent assay (ELISA) was established. The concentration of FH in rat serum was calculated to be 238 microg +/- 21 microg/ml (mean +/- SD). Its concentration in the culture supernatants of HC was upregulated about three-fold by interferon (IFN)-gamma (100 U/ml).

Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease of the intestine that is characterized by mononuclear cell infiltration and a predominant Th1 lymphocyte response. We tested the hypothesis that CC chemokine receptors CCR2 and CCR5 might be important in the regulation of the intestinal immune response in this disease, and we speculated that carriers of a defective 32 base pair deletion mutant of CCR5, CCR5Delta32, which results in a non-functional receptor, might be protected from CD. Using polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) gene frequencies of CCR5Delta32 and of CCR2-641 (replacement of valine-64 by isoleucine in the CCR2 gene) in healthy controls (n=346) and in CD patients (n=235) were determined. In CD patients, subgroup phenotypic analyses were performed according to the Vienna classification. The overall gene frequency of CCR5Delta32 (9.8%) and CCR2-641 (7.6%) in CD patients did not deviate significantly from healthy controls (9.2 and 8.2%, respectively), nor did we observe a significant deviation from the predicted Hardy-Weinberg distribution. No significant differences in the CD phenotype classification for the different CCR5 and CCR2 alleles were observed, except for a trend to disease sparing of the upper gastrointestinal tract (carrier frequency 0 versus 19.6%, Delta=1 9.6%, P=0.079) as well as a more stricturing disease behaviour (23.5 versus 16.2%, Delta=7.3%, P=0.136) in carriers of the mutant CCR5Delta32 allele. These results indicate that the different CCR5 but not CCR2 alleles may influence disease behaviour and thereby contribute to the observed heterogeneity of CD. However, the associations observed are limited and await replication in other datasets. CCR2 and CCR5 polymorphisms are unlikely to be important determinants of overall disease susceptibility.

[The use of a C1-inhibior concentrate for short-term preoperative prophylaxis in two patients with hereditary angioedema]. / Die Anwendung eines C1-Inhibitorkonzentrats zur präoperativen Kurzzeit-prophylaxe bei zwei Patienten mit hereditärem Angioödem.

A commercially available C1 inhibitor (C1-INH) concentrate was used for short-term prophylaxis before surgery in two patients with hereditary angioneurotic oedema. The patients suffered from recurrent subcutaneous and submucosal oedema of the face, extremities, and gastrointestinal tract as the result of a hereditary C1-INH deficiency. Both patients were receiving tranexamic acid or danazol therapy as oral long-term prophylaxis. Over the years the patients underwent several operations in regional and general anaesthesia, with mask ventilation or intubation. The C1-INH plasma concentrations measured preoperatively were always very low (0.02-0.06 g/l, normal range 0.15-0.35 g/l), despite the oral long-term prophylaxis. Substitution treatment with 500-1000 U C1-INH was performed 1 h before surgery. No side effects were seen following the concentrate infusions. With this substitution treatment no specific symptoms of hereditary angioneurotic oedema were recognized in either case. The measurement of C1-INH plasma concentration 2 h or 4 h after C1-INH substitution showed a marked rise in both patients, though normal values were not reached in either. We suggest that infusion of C1 concentrate is an appropriate form of preoperative substitution treatment in patients with hereditary angioneurotic oedema, in view of the lower risk of infection than with infusion of fresh-frozen plasma and the observed effectiveness.