Effects of bacterial contamination of reagent water on selected laboratory tests.

The effects of three widely spaced levels of bacterial contamination of reagent water on several chemistry, radioimmunoassay, and coagulation procedures were studied. These included determinations of lactate dehydrogenase, creatine kinase, aspartate transaminase, alkaline phosphatase, blood urea nitrogen, total protein, thyroid-stimulating hormone, digoxin, thrombin time, activated partial thromboplastin time, and prothrombin time. Statistical analyses included calculations of means and coefficients of variation, and analysis of variance, as well as correlation coefficients for test results versus logarithm of bacterial contamination. Statistically and clinically significant differences occurred together only for an elevated level of creatine kinase.

Flow cytometric reticulocyte analysis. Multiinstitutional interlaboratory correlation study.

Reticulocyte analysis by flow cytometry offers precision and sensitivity greater than those of conventional morphologic methods and permits derivation of a reticulocyte maturity index. However, interlaboratory variability has not yet been reported. The authors analyzed 310 samples at eight sites using 11 instruments over a 4-month period to examine intermethod and interlaboratory variabilities. Stains included thiazole orange, ethidium bromide, and auramine O. Instruments included models by Coulter, Becton Dickinson, TOA Medical Electronics, and Ortho Diagnostics. The coefficient of variation (CV) among all sites and methods on these samples varied as a function of the reticulocyte percentage, ranging from a mean CV of 69% for samples with < .5% reticulocytes to 24.1% for those with > 2.5% reticulocytes. The best performance was observed with the TOA R-1000 dedicated reticulocyte analyzers, with a mean CV of 18.4% for samples with < .5% reticulocytes and 4.6% for samples with > 2.5% reticulocytes. The reticulocyte maturity index showed comparable intersite precision, with a mean CV of 16% for samples with > 2.5% reticulocytes with multipurpose flow cytometers and a mean CV of 7.3% with the TOA R-1000 instruments. Interclass correlations among all sites ranged from .79 to .99 for the reticulocyte counts and .41 to .88 for the reticulocyte maturity index. The authors conclude that flow cytometric reticulocyte analysis is more precise than manual reticulocyte analysis. With greater automation of this methodology, further interlaboratory standardization of reticulocyte counts and the reticulocyte maturity index can be achieved.

Evaluation of an automated method for leukocyte differential counts based on electronic volume analysis.

An electronic blood cell counter (Coulter S-plus IV) classifies leukocytes as lymphocytes, large mononuclear cells, or granulocytes based on volume after treatment with a reagent. Correlation coefficients as compared with stained film microscopy are .94 for lymphocytes, .91 for granulocytes, and .49 for mononuclear cells. As a screening tool, it could reduce the number of traditional manual differential cell counts. We devised criteria for identifying those specimens requiring additional visual examination, giving particular attention to "low-density" abnormalities. Over 2,200 specimens were studied. Among 791 specimens obtained primarily from inpatients, the screening procedure yielded 267 true-positive, 298 false-positive, 213 true-negative, and 13 false-negative specimens. The 1,216 specimens obtained primarily from outpatients included 54 true-positive, 234 false-positive, 912 true-negative, and 16 false-negative specimens. All false-negative specimens represented low-density abnormalities. The usefulness of this approach is partially dependent on patient population.

Variation of free and total prostate-specific antigen levels: the effect on the percent free/total prostate-specific antigen.

OBJECTIVE: Recent studies have shown that calculations of the percent free/total prostate-specific antigen (PSA) improves the specificity of PSA testing. Characterizing the variability of free PSA and total PSA is necessary to evaluate the utility of an isolated free/total PSA measurement. We investigated the total variation of free and total PSA levels to determine how the percent free/total PSA was affected. DESIGN: Serum was obtained from nine urological patients on 5 different days over a 2-week period. Free and total PSA levels were measured on the day of collection. The total variation expressed in terms of percent coefficient of variation (%CV) was calculated, and the biological variation was derived taking analytical variation into consideration. SETTING: Patients were from Seattle (Wash) Urological Associates, and samples were processed at the Dynacare Laboratory of Pathology, Seattle, Wash. PATIENTS: Nine men (aged 48 to 69 years) were evaluated; three had been diagnosed with prostate cancer, three with benign prostatic hyperplasia, one with chronic prostatitis, one with high-grade prostatic intraepithelial neoplasia, and one was clinically normal. MAIN OUTCOME MEASURES: Total variation for free, total, and percent free/total PSA. RESULTS: The average total variation was 13.9% CV, 7.5% CV, and 10.6% CV for free, total, and percent free/ total PSA, respectively. Biological variation was derived to be 13.0% CV, 5.6% CV, and 8.0% CV for free, total, and percent free/total PSA, respectively. CONCLUSIONS: When applied, these results suggest that there are significant random changes in the numerator and denominator of the free PSA-total PSA ratio that could result in clinical misinterpretation. Clinicians must be aware that free PSA and total PSA levels will fluctuate owing to nonpathologic variation.

The College of American Pathologists Laboratory Accreditation Programme.

The Laboratory Accreditation Programme (LAP) offered by the College of American Pathologists (CAP) was begun in 1961. It is a voluntary peer review programme with the goal of laboratory improvement to excellence. It presently accredits more than 4300 laboratories throughout the world, although the majority are in the United States and Canada. Accreditation is contingent upon continuing successful performance in the CAP proficiency testing programmes, as well as passing biennial on-site laboratory inspections. These on-site inspections are done by practising laboratorians who use checklists appropriate for the various laboratory disciplines. Several governmental regulatory agencies (e.g. the Health Care Financing Agency) as well as private agencies (e.g. the Joint Commission on Accreditation of Healthcare Organizations) accept the LAP in place of their own programmes for laboratory accreditation.

Measurement of prostate-specific antigen by radioimmunoassay.

We describe the performance of an RIA for the measurement of prostate-specific antigen (PA). Between-assay precision (CV) for control sera with various analyte concentrations was as follows: mean = 1.67 micrograms/L, CV = 7.1%; mean = 4.47 micrograms/L, CV = 5.6%; mean = 7.15 micrograms/L, CV = 5.5% (n = 19 each). Analytical recovery of PA (nine concentrations ranging from 2.3 to 21.1 micrograms/L) added to a serum pool averaged 101.8% (range 96.1 to 116.1%). Sensitivity (detection limit) of the RIA was 0.25 micrograms/L. Cross reactivity of prostatic acid phosphatase (PAP) in this assay was less than 0.022%. The mean percent B/B0 for 74 specimens from women was 98.9%, not statistically different from that for the zero standard. The normal reference interval for men was 0-2.7 micrograms/L ( 99th percentile), as established by assay of specimens from 276 apparently normal men. Measurement of PA and prostatic acid phosphatase in 205 consecutive serum specimens from patients with clinical evidence of prostate disease similarly placed patients into normal (98) or abnormal groups (54) in 152 cases. However, in 49 cases only the concentration of PA in serum was abnormal. Sequential measurement of both tissue markers in specimens from several patients who were undergoing therapy for prostate cancer appeared to provide supplemental information regarding treatment success.

Use of the Du Pont aca to measure cholesterol in high-density lipoprotein fractions prepared by the heparin/Mn2+ precipitation method.

The enzymatic cholesterol method used with the Du Pont aca has been modified to provide a reliable measurement of high-density lipoprotein cholesterol in serum after heparin/Mn2+ precipitation of the low- and very-low-density lipoproteins. Interference by Mn2+, equivalent to about 90 mg of cholesterol per liter, is decreased to less than 40 mg of cholesterol per liter by the presence of ethylenediaminetetraacetate (8 mmol/liter) in the diluent; the residual effect of Mn2+ is compensated by calibrating the aca with standards containing Mn2+ and heparin. With an 80-microliter sample, the sensitivity is 236 muA/mg per liter and linearity ranges from 50 to 1500 mg/liter. Average analytical recovery of cholesterol added to the high-density lipoprotein fraction was 103%. Diluted fractions give the expected results. Between-run reproducibility (CV) is 1.3 and 1.6% at 463 and 554 mg/liter. Correlation with the Lipid Research Clinics' procedure (25 samples) gave a regression line of y(aca) = 1.039x- 15, and a correlation coefficient of 0.997.

Modified assay of prostate-specific antigen with a detection limit < 0.01 microgram/L.

We modified the Hybritech Tandem-E prostate-specific antigen (PSA) assay by increasing the sample volume, increasing enzyme-substrate incubation time, and using diethanolamine buffer. Our modified method has a detection limit of 0.009 microgram/L (P < 0.01). The assay curve is linear from 0.01 to 1.0 micrograms/L and has an overall assay time of about 4 h. Linear plots are obtained when the 1.0 micrograms/L standard is diluted with either matrix buffer or serum from men containing PSA < 0.01 microgram/L. Recovery of PSA (0.10 microgram/L) added to serum from men averaged 94%. Interassay CVs were 13%, 7%, and 4% at PSA concentrations of 0.04, 0.07, and 0.30 micrograms/L, respectively (n = 33). This assay should be useful in the detection of early recurrence of prostate cancer after radical prostatectomy.

Centrifugal analysis with automated sequential reagent addition: measurement of serum calcium.

We describe an automated method for calcium assay, for use with the Cobas-Bio centrifugal analyzer. Calcium is reacted with cresolphthalein complexone and the absorbance of the calcium--dye complex at 575 nm is measured. EDTA is then added to break up the calcium--dye complex and the absorbance at 575 nm is re-measured, to correct for endogenous color and turbidity. Day-to-day precision data, determined over four months, were as follows: mean = 92.9 mg/L, CV = 1.47%; n = 216; mean = 128.7 mg/L, CV = 1.72%; n = 216. Comparison of the Cobas-Bio method (y) with an atomic absorption spectrometric method (x) gave the following results: y = 1.012x--2.05, r = 0.991, Sy/x = 1.2, mean x = 92.63 mg/L, mean y = 91.69 mg/L, n = 74. Hemoglobin, bilirubin, or turbidity does not interfere. At the medical decision value (110 mg/L), the overall analytical error is 4.6 mg/L, which is less than the 5 mg/L allowable (95% confidence limit) error.

Evaluation of a radioimmunoassay for estradiol in unextracted serum.

We describe the performance of a commercial (Steranti/EIR) RIA reagent kit for measuring 17 beta-estradiol directly in serum. Day-to-day precision data for control sera were as follows: mean = 102.8 ng/L, CV = 6.8%, n = 20; mean = 231.1 ng/L, CV = 5.3%, n = 21; mean = 747.7 ng/L, CV = 9.4%, n = 21. Analytical recovery of added estradiol from seven different serum pools from men, to which three different concentrations of estradiol had been added, was (mean +/- SD): 98.6 +/- 7.0% at 107.5 ng/L added; 98.8 +/- 4.7% at 322.5 ng/L added; 108.2 +/- 4.8% at 645 ng/L added. Overall recovery of estradiol in these experiments (mean +/- SD for 21 determinations) averaged 101.9 +/- 7.0%. Assay of 32 serum specimens from women by both the direct (y) and an extraction method (x) gave the following linear regression statistics: y = 1.12x - 1.3, r = 0.998, Sy/x = 30.2 ng/L, mean y = 438.2 ng/L, mean x = 391.4 ng/L. Hemoglobin, bilirubin, and moderate lipemia do not interfere. Sensitivity of the direct assay was 2.6 ng/L. Compared with the extraction assay, the direct estradiol assay has advantages of speed and simplicity.

125I-radioimmunoassay for unconjugated estriol in serum of pregnant women.

An 125I-radioimmunoassay is described which measures unconjugated estriol in the serum of pregnant women. Estriol is extracted into ethyl acetate/hexane, an aliquot is evaporated, and the residue is redissolved in phosphate buffer. The sample is incubated with tracer and antibody at 37 degrees C for 15 min and then at 4 degrees C for 1 h and 45 min. The antibody-bound fraction is then precipitated with polyethylene glycol and isolated by centrifugation. Because the antigen-antibody complex is stable in the presence of polyethylene glycol, the separation steps are not influenced by timing. Extraction recovery of 3H-labeled estriol added to a pool of sera from pregnant women averaged 96.7% (SD = 1.3, n = 10). Estriol-supplemented (5, 10, and 20 microgram/liter) serum from men, carried through the entire procedure, showed analytical recovery ranging from 94 to 106%. Structurally analogous steroids normally present in serum of pregnant women exhibit negligible cross reactivity. Day-to-day precision (CV) is 13.3% (3.1 microgram/liter), 6.4% (7.6 microgram/liter), and 5.6% (21.4 microgram/liter) for n = 21. The current reagent cost (about 17 cents per tube), and a total procedural time, including counting, of 5.5 h make this an acceptable assay for routine use.

Prostatic acid phosphatase by radioimmunoassay. Sensitivity compared with enzymatic assay.

Prostatic acid phosphatase values in 98 patients with prostatic carcinoma were measured by a commercial radioimmunoassay (RIA) and by enzymatic assay. Forty-three carcinomas were staged by rigorous pathological criteria. Patients (N = 129) with benign prostatic hyperplasia were the control group. At 94% specificity, sensitivities of the RIA vs the enzymatic assay for clinically staged patients were as follows: stage A, 22% vs 6%; B, 29% vs 10%; C, 52% vs 38%; and D, 87% vs 80%. However, none of the seven patients with pathological stage A and B disease had a positive test result, and we suggest that variability in staging criteria accounts for the discrepant sensitivity claims reported. Prostatic acid phosphatase RIA should not be used for screening but as an adjunct for staging known prostatic carcinoma.

Serum albumin concentration-related Health Care Financing Administration quality assurance criterion is method-dependent: revision is necessary.

The objective of this study was to examine quantitative differences between the two commonly used methods for determining serum albumin concentration, bromcresol green (BCG) and bromcresol purple (BCP), in normal subjects and in 235 unselected dialysis patients in view of recently established Health Care Financing Administration (HCFA) quality assurance review criteria. The mean of normal results by the BCG method was 4.4 g/dL, and 97.5% of values were 3.8 g/dL or higher. The mean of normal results by the BCP method was 3.9 g/dL, and 97.5% of values were 3.3 g/dL or higher. Serum albumin concentrations in samples from the dialysis patients had respectively lower mean values by both methods. For the BCG method, the mean was 3.8 g/dL, and 82% of values were 3.5 g/dL or higher; for the BCP method, the mean was 3.3 g/dL, and 82% of values were 3.0 g/dL or higher. Likewise, for the reference immunonephelometric procedure, the mean value for the dialysis patients was 3.3 g/dL, and 82% of values were 3.0 g/dL or higher. For the samples from the dialysis patients, in comparison with the immunonephelometric method, the BCG method exhibited both constant (intercept = 9.3 g/L) and proportional error (slope = 0.87). The mean albumin value for the BCG method was 3.8 g/dL, 15% higher. In contrast, the BCP method compared closely with the reference method: slope = 1.00, intercept = 0.8 g/L, mean x = 3.3 g/dL, mean y = 3.3 g/dL. The HCFA quality assurance criteria are valid only for the BCG method.(ABSTRACT TRUNCATED AT 250 WORDS)