HLA-DR typing for kidney transplants: advantage of polymerase chain reaction with sequence specific primers in a routine hospital laboratory.

AIMS: To determine whether the polymerase chain reaction with sequence specific primers (PCR-SSP) can assign HLA-DR type more accurately than serology in a routine hospital laboratory. METHODS: The 93 patients currently awaiting kidney transplants have been DR typed by serology over the past 14 years, 82% within the past five years. They have now been retyped using the PCR-SSP method described by Bein et al. Where the two results differed, PCR-SSP was repeated, once by the same method and once using the primer set of Olerup and Zetterquist. RESULTS: There were 13 (14%) discrepancies between the results. Of these, two were PCR-SSP failures, later overcome: three were failure to detect DRB1*0103 by serology; five assignment of other alleles by PCR-SSP to serological "blanks"; and three alleles were differently assigned by serology and PCR. The serological typing of the final patient when repeated for this study was at variance with the original findings (14 years ago), but in agreement with PCR. In the remaining patients, serology had not determined the split of 36 DR3 alleles (all DR17 by PCR-SSP) or 13 DR6 alleles (12 DR13 and one DR14 by PCR-SSP). One patient in each case had their antigen splits of DR2 and DR5 assigned by PCR-SSP (DR15 and DR11, respectively) but not by serology. CONCLUSIONS: PCR-SSP provides more reliable and detailed information on HLA-DR polymorphism than serology, and does so within a routine tissue typing laboratory.

Cytokine secretion in mixed lymphocyte culture: a prognostic indicator of renal allograft rejection in addition to HLA mismatching.

We have previously demonstrated significant inter-individual variations in cytokine protein secretion between normal individuals and patients prior to renal transplantation. In this study, pre-transplant patient vs. donor mixed lymphocyte cultures (MLC) were set up between 57 renal allograft patient/donor pairs, and secretion of cytokine protein (IL-2, IL-4, IL-6, IL-10 and IFN-gamma) into the culture supernatant measured by ELISA. Significant inter-individual variations in protein secretion in MLC were observed for all cytokines studied. Univariate analysis demonstrated that high levels of IFN-gamma and IL-10 in MLC and spontaneous IL-4, together with female donor sex and a high degree of HLA mismatching (especially HLA-DR) were significantly associated with rejection. However, multivariate analysis revealed the greatest risk of rejection (RR = 25.5, P = 0.003) was associated with a combination of high IL-10 secretion in MLC and mismatching for at least four HLA antigens (HLA-A, -B and -DR). It remains to be determined whether cytokine secretion in MLC is linked to cytokine gene polymorphisms. In future, assays for measuring either cytokine secretion or genetic polymorphisms may prove to be useful in aiding donor selection and tailoring immunosuppressive therapy.

A study of cytokine gene polymorphisms and protein secretion in renal transplantation.

Although there is evidence that cytokine gene polymorphisms are associated with varying quantities of cytokine protein production, the exact role of these polymorphisms in allograft rejection remains unclear. In a previous study, we demonstrated a significant association between high IL-10 secretion in mixed lymphocyte culture (MLC), together with HLA mismatching for at least 4-6 antigens, with the occurrence of acute rejection following renal transplantation. We, therefore, wished to ascertain whether cytokine gene polymorphisms are associated with varying levels of protein secretion and/or allograft rejection in the same group of patients. Cytokine protein secretion in MLC for IL-4, IL-6, IL-10 and IFN-gamma was measured by ELISA in 49 patient-donor pairs. Protein secretion for the above cytokines was also measured in phytohaemagglutinin (PHA) stimulated cultures in 30 normal controls. In both patient and control groups, single nucleotide polymorphism analysis for IL-4 G(-590)T, IL-6 G(-174)C, IL-10 G(-1082)A, IL-10 C(-819)T, IL-10 C(-592)A, TNF-alpha G(-308)A and microsatellite analysis for IFNG (CA repeat) was performed. No correlation was found between cytokine gene polymorphisms and cytokine protein secretion in either mitogen stimulated cultures (control group) or MLC (patient group). In addition, no correlation was demonstrated between cytokine gene polymorphisms and renal allograft rejection.

Effects of radiographic contrast media on leukocyte phagocytosis.

RATIONALE AND OBJECTIVES: Differences in leukocyte phagocytosis following exposure to different classes of radiographic contrast media (RCM) may help in the development of less toxic alternatives and be useful as a guide to contrast selection in certain patient groups. The effect of RCM on leukocyte phagocytosis of Escherichia coli was examined. METHODS: Cell population phagocytosis and individual cell phagocytic activity were assessed in a control group and in samples exposed both to the ionic RCM diatrizoate and ioxaglate and to the nonionic RCM iohexol and iotrolan by using a flow cytometer. RESULTS: The percentage of granulocytes undergoing phagocytosis was 88.4% in the control population. Following exposure to RCM, this value fell to 79.2% with iohexol, 77.6% with iotrolan, 70.5% with diatrizoate, and 68.7% with ioxaglate. The number of bacteria phagocytosed by active leukocytes was not affected by RCM. CONCLUSION: RCM adversely affect the percentage of granulocytes involved in phagocytosis but not the number of pathogenic bacteria that are phagocytosed by individual granulocytes.

The effects of radiographic contrast media on leucocyte orientation.

The effects of radiographic contrast media (RCM) on leucocyte orientation in vitro were studied using a Zigmond chamber. Leucocyte orientation was assessed following exposure of the leucocytes to iotrolan, iohexol, ioxaglate and diatrizoate. The RCM used were diluted to a concentration similar to that obtained in vivo during routine angiography. At this concentration there was a significant reduction in leucocyte orientation for all RCM investigated but the effect was more pronounced in the monomeric than the dimeric RCM. The results may have significance when deciding which radiographic contrast medium to use in selected patients, particularly those who are immunosuppressed or septicaemic.