Background:A simple point-of-care method for measuring leukocyte counts in a doctor's office or emergency room could be of great importance. We developed a protocol for measuring cell count by disrupting the cell membrane and analyzing specific proteins within the cells and used it to analyze proteins from eosinophils and neutrophils. Methods: Lateral immunochromatographic (ICR) assays have been developed for eosinophil protein X (EPX) and human neutrophil lipocalin (HNL) as measures of the concentration of eosinophils and neutrophils. The correlation between the lateral ICR assays and cell counting of eosinophils and neutrophils was performed manually and with an automated cell counter. RIA assays measuring the same analytes were also compared with the results from cell counting and lateral ICR assays. Results: The optimized assays showed analytical detection limits below the clinical ranges of 3.36 g/L and 2.05 g/L for EPX and HNL, respectively. The recovery was 114.8%-122.8% for EPX and 94.5%-96.9% for HNL. The imprecision was 3%-17% CV for EPX over the whole range and 5%-16% CV for HNL. The correlation coefficients between manually counted cells and lateral ICR assays were 0.9 and 0.83 for EPX and HNL, respectively.

Transferrin (Tf) has different isoforms based on the degree of sialylation of its two N-linked oligosaccharide chains. The least sialylated isoforms of Tf; with 0 (asialo Tf), 1 (monosialo Tf), and 2 (disialo Tf) sialic acids are referred to as carbohydrate-deficient transferrin (CDT). CDT has been reported to be a specific and sensitive marker for the detection and monitoring of alcohol abuse. However, the possible differences between the three CDT isoforms in males and females relative to alcohol consumption has not been known. The present study included 82 males (M) and 43 females (F) with well documented drinking habits. The Tf isoforms were separated by FPLC and measured by RIA in the collected fractions, as well as by a commercially available method (CDTect RIA). The results were expressed as relative values and absolute values. Female low consumers compared to male low consumers had higher levels of asialo Tf (p < 0.01) and monosialo Tf (p < 0.01), but not of disialo Tf or sum of asialo, monosialo, and disialo Tf. Male high consumers and chronic consumers compared to male low consumers had 53% and 219% higher levels of asialo Tf, 4% and 28% higher monosialo Tf, 57% and 148% higher disialo Tf, and 48% and 134% higher sum of CDT isoforms, respectively. The corresponding increases in females were for asialo Tf 68% and 249%, for monosialo Tf 36% and 58%, for disialo Tf 54% and 225%, and for sum of CDT isoforms 52% and 192%, respectively. For both genders, total Tf, trisialo Tf, and the levels of more sialylated transferrin isoforms were constant when comparing the consumption groups. Results expressed as relative values and absolute values were in good agreement. In conclusion, the present study indicates that alcohol consumption strongly increases the levels of asialo Tf and disialo Tf and slightly increases the level of monosialo Tf. However, women had higher asialo Tf and monosialo Tf levels than men. Alcohol consumption does not increase trisialo or more sialyated Tf subfractions. Expressing the CDT results as absolute or relative values made no obvious difference in diagnostic efficiency.

Different methods for detecting carbohydrate-deficient transferrin (CDT) were compared. In addition, their efficiency for detecting alcohol abuse among men not having clinical evidence of liver disease was studied in controls (n = 26), weekend (n = 16) and daily (n = 12) heavy drinkers, and alcoholics (n = 28). Comparisons were made between anion-exchange separation of iron-saturated transferrin (Tf) by microcolumns (CDTect) and by the Fast Protein Liquid Chromatography (FPLC% and FPLC-MG), followed by double-antibody radioimmunoassay of collected fractions. Tf fractions with pl > or = 5.7 were also measured by two different isoelectric focusing (IEF) methods, followed by immunofixation (SA-IEF-CDT and IEF-CDT-TOT), the latter method being used also for detection of asialotransferrin (IEF-CDT-AS). The cut-off was 20 units/liter for CDTect, 4.4% of total Tf for SA-IEF-CDT, and the mean +2 sd of the control group for FPLC-MG (as mg/liter of Tf), FPLC-%, IEF-CDT-TOT, and IEF-CDT-AS (all as percentage of Tf). The overall accuracies (combining sensitivity and specificity) for detecting heavy drinkers of CDTect, FPLO (mg/liter), FPLC (%), SA-IEF-CDT, IEF-CDT-TOT, and IEF-CDT-AS were 63%, 59%, 61%, 74%, 57%, and 63%, respectively; for detecting alcoholics, 87%, 83%, 81%, 89%, 37%, and 76%, respectively. In conclusion, the methods were in rather good agreement with each other. Diagnostic characteristics among heavy drinkers and correlations between methods differed slightly, probably depending on the ability of different methods to separate and detect asialo-, monosialo-, and disialotransferrin.(ABSTRACT TRUNCATED AT 250 WORDS)

CDTect-RIA and CDTect-EIA for determination of serum carbohydrate-deficient transferrin (CDT) by radioimmunoassay and enzyme immunoassay respectively were tested for equality and precision in four European laboratories. For correlational studies, serum samples with CDT concentrations up to 130 U/l were analysed in accordance with a uniform trial schedule. The regression of CDT values obtained by the two procedures was computed for each laboratory using the method of Passing and Bablok. Slopes and intercepts of the regression functions did not differ significantly from the values 1 or 0, as proved by the corresponding 95% confidence intervals. Precision studies were computed using analysis of variance. For CDT concentrations at the upper reference limit for men, the within-day coefficients of variation (CVs) ranged between 0.7 and 6.4% (median 5.2%) for CDTect-RIA and from 4.3 to 9.2% (median 6.2%) for CDTect-EIA. The corresponding pure between-day CVs were 5.0-18.5% (median 9.8%) and 3.5-14.5% (median 10.9%). The study demonstrates the equality of CDT values obtained by CDTect-RIA and CDTect-EIA. According to this study, the two methods can be used interchangeably without getting fluctuating CDT values, e.g. in longitudinal studies.

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