The influence of curve-fitting procedures on radioimmunoassay results

142 .Ti% INFLUENCEOF CURVE-FITTING PROCEDURESON RADIOIM?lUNOASSAY RESULTS;Groom G; Wilson D; ~~~ c:$ Nix B. NEQAS Laboratory, Tenovus Institute for Cancer Research, Heath Park, Cardiff, WALESU.K. National External Quality Assessment Schemes(NEQAS)attempt to determine the disparity between methods (usually radioimmunoassay for hormones) used in different laboratories to measure a particular analyte. Such exercises are limited by many factors including the nature and stability of the samples, relative infrequency of testing assay performance, as well as the inherent differences in the kits or methods employed in each participating laboratory. In the I the coefficient of variation (CV) between laboratories is about 10-20’6 for oestradiol, cortisol progesterone and testosterone, though up to 50% differences in bias can be seen with certain methods. As part of a series of experiments to investigate these factors in more detail, a set of calibration data, ie counts and concentrations, from a typical assay were distributed to the 260 laboratories. Each analysed the data by whatever curve-fitting procedures were available and determined concentrations of QC samples presented to them as count-rates. This exercise theIS by-passed the problems associated with the sample and the assay itself. The results indicated that not only were the values different if different curve-fit algorithms were used, but also using a particular curve-fit in programs from various sources, i r different machines from a particular manufacturer, and even in different models of the same machine from a manufacturer. The magnitude of these errors could cause an artificial bias in the results of 5-10X. There was greater variation between results obtained using linear interpolation and logit-log methods than from h-parameter logistic, ‘Amersham’ or spline type could account for a major part of betweenmethods. The results suggest that curve-fitting laboratory errors seen by NEQAS. Kit manufacturers should specify the method to be used.

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_! 143 ?%lIOIMMUNOASSAY OF 1%HYDROXY-1-PREGNENE-3,2U-DIONE(Zl-DEDXYCORTICOSTERONE) IN HIIMAN PLASMA. Gueux, B; Fiet, J; Galons, H; Bonete, R; Villette, J-M; Vexiau, P; Brerault, J-L; Pham-HuuTrung, M-T; Raux-Eurin, M-C; Gourmalen, M; Julien, R; Dreux, C. iaboratoire de Biochimie et de Neuroendocrinologie, HBpital Saint-Louis, 75010 PARIS, FRANCE. high levels of plasma 21-deoxycortisol were In CAHpatients with 21-hydroxylase deficiency, found (Gueux 8. et al). In these patients 21-deoxycortisol is likely synthesized by direct llBhydroxylation of 17-OH progesterone in the glucocorticoid pathway. We postulated that a direct 11B-hydroxylation of proqesterone could also occurinthe minerals icorticoid pathway with formation of 21-deoxycorticosterone (115 OH progesterone). To check this ‘hypothesis a radioimmunoassay of plasma 21-deoxycorticosterone was developed by using an antiserum raised in the rabbit with 22-deoxycorticosterone-3-(D-carboxymethyl) oxime/BSA. 21-deoxycorticosterone extracted from plasma by cyclohexane/ethylacetate, was separated from the steroids which cross-reacted with the sntiserum (progesterone 10 %, corticosterone 1 %, pregnenolone 1.3 %, 11 cetoprogesterone 0.5 76) by celite chromatography. The intra and interassa variations were B % and 11 X respectively. Mean plasma 21-deoxycorticosterone levels (pg/ml + SD) was 5.9 2 4.90 in healthy subjects an There were no difference it increased up to 28 2 8.71 after an ACTHstimulati& (Synacthen). Bycontrastvery highlevels of plasma 21-deoxycorticosterone betueen males and females subjects. (16+13000 pg/ml) were measured in the treated patients suffering from21-hydroxylase deficiency. The strongest values were found in the salt-loser patients which exhibited high plasma renin activity. From these results, 21-deoxycorticosterone may he considered as a new marker for adrenal 21-hydroxylase deficiency. Gueux B. et al, Acta Endocrinologica, 1985, 108 : 537-544. _

144 - A NOVEL DERIVA'ITVE FOR DIRECT IODINATION IN STEROID RADIOIMMUNOASSAYS; E. Dept. of Endocrinology, Institute of Stupnicki, R; +Przewloka, T; %la, and +kstitute of Animal Physiology and Nutrition; Sports; 01-809 Warsaw, 05-110 Jablonna; POLAND

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Steroid derivatives oontaining histidine methyl ester /IiME/, instead of were prepared by mixed anhydride ooupling. The derivatives were crystalline, and when labelled in miorogram quantities by using Iodogen /erposure time one hr./ the yield of the immunoreotive fraotion was 40 - 50%. The roduots were similar in ismunoreaotivity and stability to the tiown hietamine erivatives. Assay parameters obtained with EDB-derivatives were oompared with those obtained with tritiated steroid6 and with analogous TME-derivatives. A heterologic assay of progesterone /3 antisera against ia-suooinyl-BSA+ and methylsuocinyl derivatives for lobelling aubatituted at lla-position/, and a homolof oortieol /4 antisera against 210suocinyl-BSA, and Xl-oarbonylerivatives for labellfns/ were studied. The HK&derivatives produoed lo&t-log ourves with slopes oomparable to as expressed by ED50 values, thoee in trltium-based a6naysb The eensitivity, by 66s higher than in tritium assays, and 50% higher than for the kyramassay for oortin a similar heterologvus assay. The -based is01 was by 26s less sensitive oompared to tritium, but several times more sensitive than in oaee of analogous WE-based assay.

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