Distribution of patients' test values and applicability of "average of normals" method to quality-control of radioimmunoassays

Applicability of the Hoffmann's average of normals (Mn) method was evaluated in quality control (QC) of radioimmunoassays (RIA) for thyroxine, 3,5,3'-tri-iodothyronine, thyrotropin, and insulin--assays that are performed routinely in the authors' laboratory. In the first three RIAs, the patterns of the distributions were almost constant and Mn showed significant correlations with values of QC sera and intercepts of the dose-response curve. In insulin RIA, the patterns varied appreciably and Mn showed correlations with parameters that reflect a disturbance in the distribution. Exclusion of assays with abnormal distributions, however, resulted in better correlations of Mn with other QC parameters. These results suggest that average of normals method can be a very useful adjunct to conventional QC methods for RIA. The possibility that the method may be affected by a disturbance in the distribution can be monitored by computation of parameters reflecting such disturbance.     

Evaluation and use of a synthetic quality control material, included in the European external quality assessment scheme for cystic fibrosis

Assuring high quality within the field of genetic testing is fundamental, as the results can have considerable impact on the patient and his or her family. The use of appropriate quality control (QC) samples is therefore essential. Diagnostic laboratories mainly use patient samples as QC material, which of course include a maximum of two mutations per sample. Bearing in mind that some assays (such as for cystic fibrosis [CF] testing) can test for more than 100 mutations, multiplex QC materials including more than two mutations could save valuable time and reagents. Based on this need, synthetic multiplex controls have been developed by Maine Molecular Quality Controls, Inc. (MMQCI) for CF. A synthetic control, containing six homozygous mutations and one polymorphism for CF transmembrane conductance regulator (CFTR), was evaluated by distributing it through the CF external quality assessment (EQA) scheme, along with the EQA samples in 2005. A total of 197 participants returned results of the yearly EQA scheme and 133 laboratories participated in the evaluation of the synthetic sample. Respectively, 76% and 73% of the participants were assigned as successful. This evaluation study revealed that the multiplex QC material performed well in the majority of assays and could be useful in method validation, as a tool to challenge interpretation skills, and as potential proficiency testing (PT) material.     

Serum prostatic acid phosphatase (PAP): monoclonal enzyme-linked immunoassay compared to polyclonal radioimmunoassay

A new enzyme linked immunosorbent assay (ELISA) kit which utilizes a monoclonal antibody against prostatic acid phosphatase (PAP) was compared with current radioimmunoassay (RIA) methodology which uses a polyclonal antibody. Both assays are double antibody immunoassays with the major difference being the method of antibody preparation. A study of intrarun precision using control material showed an average within run coefficient of variation (CV) percent as 7.0 percent and 6.9 percent for ELISA and RIA, respectively, while between run CV percent averaged 11.1 percent and 11.5 percent, respectively. Thus, precision results compare similarly between the two assays. The specificity showed significantly different results. Patterns of correlation between the two methods indicate differing specificities of the primary antibodies. The values for ELISA were greater than RIA for control sera and patient samples when values fell outside the reference range; however, RIA values exceeded ELISA values with patient samples which fell within the reference ranges as provided by each manufacturer. Therefore, there exists a question of specificity of antibody employed in each of the two assays. The PAP antigen is prepared from two different sources for each kit. The ELISA manufacturer prepares antigen from seminal fluid and RIA manufacturer prepares antigen from normal human prostate. The question of specificity may be influenced by: (1) source of antigen used in immunizing animals and (2) monoclonal versus polyclonal means of producing antibody.     

Performance evaluation and planning for patient-based quality control procedures

In a recent publication, the effect of the length of time between the routine testing of quality-control (QC) samples on QC performance was studied using a newly proposed performance measure: the average number of patient samples that contain unacceptable analytic error due to an out-of-control error condition (ANPTE). We show that ANPTE also perfectly suits the evaluation of patient-based QC procedures. We used ANPTE to study the effect of the number of patient results averaged and the width of the truncation limits on an average of normals QC procedure. Estimates of ANPTE were obtained by using computer simulations that use actual patient result distributions. Based on ANPTE, the conclusions about the effects of truncation limits and number of patient results averaged on the performance of an average of normals QC procedure are substantially different from those described by others using the probability of error detection to characterize QC performance.     

酶联免疫吸附试验检测HIV抗体的质量控制方法探讨

目的寻找一种适用于HIV抗体酶联免疫吸附实验室内质量控制（IQC）的方法。方法采用Levey-Jennings质控图法、即刻法和控制变异系数（CV）的方法（改良即刻法）同时统计规范操作前和规范后的两批各30个质控数据，制作质控图。结果规范前检测采用即刻法和Levey-Jennings质控图法考核显示在控．改良即刻法显示失控；规范后检测3种方法考核均为在控。结论采用即刻法考核，前3次结果对后续质控结果影响较大，前3个质控数据的cV值较大时，随后的结果会出现假在控。Levey-Jennings质控图法如果不对CV值进行考核，前3个质控数据的CV值较大时，随后的结果也会出现假在控。采用改良即刻法统计，只要设定好一个实验室或一个地区的允许CV值，就没有假失控和假在控的结果，因此改良即刻法较为适用于HIV抗体ELISA检测的室内质量控制。ELISA检测手工操作步骤较多，操作细节都对实验结果有很大影响，因此需要规范实验操作，并严格按照要求执行。     

Levels of Cytokines and Immune Activation Markers in Plasma in Human Immunodeficiency Virus Infection: Quality Control Procedures

Procedures for quality control (QC) in a laboratory that concentrates on cytokine and soluble marker measurements in biological fluids are outlined. Intra-assay, interassay, and interlaboratory experiences are presented. Plasma and serum β2-microglobulin (β2M) and neopterin test data are presented in greatest detail, along with substantial tumor necrosis factor alpha (TNF-α), gamma interferon, soluble interleukin-2 receptor-α (sIL-2Rα), sTNF-RII, IL-4, and IL-6 data. Recommended QC procedures for cytokine and soluble-marker testing include replicate testing of two or more reference samples provided by the kit manufacturer, replicate testing of in-house frozen reference QC samples that represent normal and abnormal analyte contents, retesting 15 to 20% of randomly selected samples, and comparing normal reference ranges each year. Also, eight cytokines and soluble markers were evaluated in human immunodeficiency virus (HIV)-seronegative and HIV-seropositive individuals stratified on the basis of CD4 T-cell numbers. Levels of some but not all cytokines in serum increased in HIV infection. There was a tendency for cytokines to increase with more advanced disease, defined by reduced CD4 T-cell numbers. Cytokine changes did not relate closely to CD4 level, indicating that separate information was provided by the measurements of TNF-α, sTNF-RII, sIL-2Rα, β2M, and neopterin. Serum IL-4 and TNF-α levels were not increased. The quality of laboratory data can impact on clinical relevance. Interlaboratory comparisons revealed substantial differences at some sites and documented the need for external proficiency-testing quality assurance programs.     

Evaluation of Clearview and Magic Lite tests, polymerase chain reaction, and cell culture for detection of Chlamydia trachomatis in urogenital specimens.

The Clearview Chlamydia test (CV; Unipath Ltd., Bedford, United Kingdom), the Magic Lite Chlamydia test (ML; CIBA Corning, Medfield, Mass.), a polymerase chain reaction (PCR), and cell culture (CC) were evaluated for detection of Chlamydia trachomatis in urogenital specimens. Specimens were collected from 283 men and 724 women visiting the outpatient clinic for Sexually Transmitted Diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands. ML, PCR, and CC were all performed on the same sample to prevent swab-to-swab variability. CV was performed on a separate sample. Analysis of discordant results was performed by application of the following confirmatory assays: first, PCR on the CC, second, ML was repeated, and third, PCR was repeated by using a different DNA extraction protocol. If more than one test was positive, the sample was considered true positive. If only one test was positive, which was confirmed by the confirmatory assay, the sample was also considered true positive. By using these interpretations, the following results were obtained. The sensitivity and specificity of CV for samples from men were 60.4 and 86.3%, respectively. For samples from women, these values were 62.3 and 99.7%, respectively. The low specificity for samples from men was caused by unidentified substances in the swab that was used. The use of CV on samples from men is not recommended by the manufacturer. For samples from women, the specificity of CV was high, but the low sensitivity of CV limits its use for diagnostic purposes. The sensitivities of ML were low for samples from both men and women (68.8% and 50.9% respectively), while specificities were excellent for samples from both groups (100 and 99.9%, respectively). The low sensitivity of ML limits its diagnostic value. The PCR technique was highly specific for samples from both men (99.6%) and women (99.9%). The sensitivity of PCR, however, was unexpectedly low for samples from both groups (men, 87.5%; women, 79.2%), most likely because of the sample treatment method used. The sensitivity and specificity values of CC for samples from men were 95.8 and 100%, respectively. For samples from women, these values were 100 and 99.9%, respectively. In the present study, CC was the most reliable technique for the detection of C. trachomatis.     

Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2.

Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase. Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells. Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate. In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5] were able to detect virus-infected cells. The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0]. Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells. The threshold of virus defection was between 10(5) and 10(6) PFU/ml. This test is sensitive and specific and therefore may be useful for diagnostic purposes.     

Validation of the Hemavet HV950FS multispecies haematology analyser for analysis of canine blood

The objective of this study was to evaluate the performance of the Hemavet HV950FS haematology analyser by comparison to a reference method. Over 5 days, canine blood samples submitted to the University of Cambridge’s Veterinary Clinical Pathology Service were analysed in triplicate on the Hemavet and also on the reference analyser (Celldyn 3500). One sample was run ten times. The results obtained from the two analysers were compared by linear regression, a concordance correlation and through difference plots. The coefficient of variance (CV) was calculated for each parameter measured. Twenty-five canine samples were analysed. The Hemavet compared favourably with the reference method for total white blood cell (WBC) count, neutrophil count, red blood cell (RBC) count, haematocrit, haemoglobin level (HB) and red cell distribution width. Although the Hemavet produced HB measurements closely correlated with the reference method, it was biased, routinely producing lower values. The manufacturer has produced CVs for five parameters, and with the exception of the platelet count CV, these results support their findings. The CVs were unacceptably high for monocyte, eosinophil and basophil counts. In conclusion, the Hemavet performed favourably for overall WBC and RBC count, but the WBC differential is less reliable. As far as can be assessed, the performance of the Hemavet was comparable to reports in the literature of other impedance analysers that have been validated.     

Reproducibility in quality control of protein (Western) immunoblot assay for antibodies to human immunodeficiency virus

The protein (Western) immunoblot assay (IB) for antibodies to human immunodeficiency virus, like other laboratory procedures, sometimes gives variable results. In the Transfusion Safety Study, indistinguishable aliquots of four quality control (QC) samples have been routinely submitted for IB with each group of patient specimens. A false negative IB result was obtained for 1.7% of 179 assays of two known positive (QC) standards and a false positive result for 2.0% of 101 assays of two known negative (QC) standards. In addition, a test panel of 24 samples was sent on a single occasion to three widely used laboratories. A false positive result was reported by one laboratory and a false negative by a second. Although generally reliable, IB results may occasionally be in error. There is much more technical variability in the relative frequencies of antigen-antibody bands than has been recognized. These interlaboratory and intralaboratory comparisons show quality control checks are essential for all laboratories, and more than one specimen should be tested if its applicability to a specific patient is questionable. Specific bands are sufficiently inconstant for the same specimen to make appearance or disappearance on successive specimens prognostically unreliable.     

Determination of histamine in human plasma: the European external quality control study 1988

There is an increasing interest in measuring human plasma histamine levels in various clinical conditions. A variety of 'old' and newly developed techniques are applied to meet this demand. However, the discrepancy between reported reference values for histamine in human plasma measured using this variety of techniques, suggests the existence of a certain degree of inaccuracy and imprecision. We therefore organized an external quality control study on the reliability of current histamine determinations in European laboratories. Three lyophilized plasma quality control samples, in duplicate, covering the normal and pathological range of histamine concentrations (0-45 nmol/l), two different aqueous histamine standard samples and one solvent sample were sent to 10 laboratories for the analysis of their histamine content. The following methods were used: gas chromatography-mass spectrometry (n = 2), enzymatic single isotopic assay (n = 1), fluorometric-fluoroenzymatic assay (n = 3), radioimmunoassay (n = 3) and high performance liquid chromatography (n = 2). The study was performed and evaluated according to the approved recommendations (1983) of the International Federation of Clinical Chemistry (IFCC). The target values +/- s.d. of the three plasma samples were: 39.5 +/- 4.6 nmol/l (CV = 11.6%), 2.3 +/- 2.2 nmol/l (CV = 96%) and 8.9 +/- 1.5 nmol/l (CV = 17%), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sérums de contrôle de qualité pour les immunodosages de thyroxine libre (T4 L) : congelés plutôt que lyophilisés ?

Important biases are reported between free thyroxine (FT4) determined by equilibrium dialysis (ED) and by most immunoassays when T4-binding capacity is decreased. Ten lyophilized and 32 frozen quality control (QC) sera were analyzed in the italian and french (lyophilized sera) and british (frozen sera) external quality assessment programs for FT4 using five immunoassays (Elecsys, ADVIA Centaur, Vitros ECi, Immulite and AxSYM). We determined the bias between the FT4 results reported with each immunoassay and FT4 measured in our laboratory with ED. We measured in each serum the concentration of T4-binding proteins and the T4-binding capacity estimated by the Elecsys T4-Uptake test (TU). Mean biases were less marked with frozen samples (from -9% to -23%) than with lyophilized samples (from -4% to -44%). Biases were correlated with TU decreasing (p < 0.0001). The biases appeared to be related to the processing of the QC sera causing a reduction in their T4 affinities. Frozen QC sera involving less processing seem to be more suitable for FT4 external quality assessment.     

Redesigned proficiency testing materials improve survey outcomes for prostate-specific antigen. A College of American Pathologists Ligand Assay Survey tool

CONTEXT: Large disparities in prostate-specific antigen (PSA) results from different assays have been observed in the College of American Pathologists (CAP) Ligand Assay Survey, with interassay results varying severalfold. Survey specimens are predominately composed of free PSA and do not reflect the composition of typical patient specimens. OBJECTIVES: To characterize a pilot material developed for CAP in which pooled sera samples were spiked with purified PSA and alpha(1)-antichymotrypsin-bound PSA at targeted concentrations and to compare it to CAP survey and reference materials. DESIGN: CAP survey, reference, and pilot materials were analyzed using 10 total PSA and 7 free PSA assays. These assays included Food and Drug Administration-approved assays and assays for research use only. RESULTS: Variability among the 10 total PSA methods was greatest for the 1997 ligand survey material (CV range, 56%-65%) followed by the pilot material (CV range, 10%-29%) and the reference material (CV range, 6%-13%). In contrast, interassay variability for the 7 free PSA methods was similar for the 3 preparations, with the exception of one specimen close to the limit of detection of the assays. As determined with the Hybritech Tandem-R method, the ligand survey specimens were essentially composed of all free PSA, whereas the reference and pilot materials were composed of approximately 10% and 35% free PSA, respectively. CONCLUSIONS: The newly formulated pilot material prepared using a human base that contained defined concentrations of free PSA and alpha(1)-antichymotrypsin-bound PSA more closely resembled patient specimens and minimized differences among methods compared with the semen-supplemented original survey material.     

Evaluating an internal quality control procedure: application to multidimensional control

Internal quality control keeps in constant evolution in the industrial world. Introducing in clinical chemistry new QC methods derived from the industrial practice raises the point of the means for their evaluation. The main evaluation criteria are discussed in this paper. The importance of ARL (average run length) as a key-criterion of the efficiency of a quality control procedure is stressed. These principles were applied to the multivariate approach of multilevel control with the Hotelling's T2. This method led to a better detection of random errors than the independently managed conventional Shewhart (Levey- Jennings) charts. Applied to systematic errors, both methods gave similar results with a certain lack of sensitivity. However the multivariate method can be sensitised using EWMA (exponentially weighted moving average), a method specific for improved bias detection. EWMA efficiency outperforms that of the older systems of rules. Moreover, in any kind of error, multivariate approach secures a well-defined false rejection rate, whereas this rate is dependant on unknown inter-level correlation coefficients in conventional QC.     

Proficiency testing of enzymes. Charting the way toward standardization

Laboratories participating in the College of American Pathologists Enzyme Survey (ES) and Comprehensive Chemistry Survey used diverse methods for the same analyte, resulting in a considerable range of values for the commonly performed enzyme measurements. Nevertheless, with the techniques developed for the ES, both the short-term (within-mailing) and long-term (between-mailings) coefficients of variation (CVs) can be determined. The ten-year experience of the ES has shown improvement in the short-term CV; however, long-term stability of enzyme testing requires more effort on the part of the instrument and reagent suppliers and participating laboratories. A reference material with an International Federation of Clinical Chemistry-established aspartate aminotransferase value, National Bureau of Standards RM 8430, is now available and was sent to three large peer groups as part of a special study. Correction of the results to the RM 8430 aspartate aminotransferase value resulted in reducing the range of data from peers using the duPont aca but not from those using the American Monitor KDA or Technicon SMAC. Based on our experience with the ES, goals of 5% for the short-term CV and 10% for the long-term CV are proposed; they are achievable by most laboratories and meet medical needs for biochemical screening. Fixed criteria for the evaluation of enzyme results appear to be appropriate given the way most enzyme data are used clinically.     

Sperm motility index: a quick screening parameter from sperm quality analyser-IIB to rule out oligo- and asthenozoospermia in male fertility study

The aim of this study was to evaluate the sperm quality analyser (SQA)-IIB, a new automated sperm analyser, and to compare its results with those obtained with a method based on the World Health Organization recommendations. Eighty-nine unprocessed semen samples and 53 selected sperm suspensions were analysed. Concentration, motility and morphology were evaluated using the routine laboratory method. The SQA-IIB measured the sperm motility index (SMI) and estimated the previously mentioned parameters. In the imprecision assay a maximal coefficient of variation (CV) of 18.8% was found. A semen sample with immunological factor showed a CV of 75.75%, which invalidates its use for these types of samples. A good correlation was obtained between SMI and concentration of progressively motile spermatozoa (CPMS) (r = 0.87), and a fair correlation with the other parameters. There was no statistically significant correlation between both methods for normal sperm morphology. The sensitivity and specificity of the SMI test in relation to CPMS were 96 and 84% respectively, for an SMI threshold value of 160. The results obtained make the SQA-IIB a good screening test to rule out oligozoospermia and asthenozoospermia when studying the male factor in the sterility outpatient clinics. However, the results suggested that it is not a valid method to evaluate morphology.     

纤维蛋白(原)降解产物定量检测方法的建立与评价

目的:建立定量检测血浆纤维蛋白(原)降解产物(FDP)含量的实验方法,评价该方法实验性能与临床应用价值。方法:采用ACLTOP全自动血凝仪,通过免疫比浊法定量检测血浆FDP含量。分析其重复性、稳定性、线性、抗生物性干扰等,评价和比较实验方法对弥散性血管内凝血(DIC)的诊断效能,并初步建立本地区FDP定量检测结果的参考区间。结果:高、低值FDP批内变异系数均小于3.2%,日间变异系数均小于10.0%;轻至中度黄疸、脂血、溶血对FDP检测的影响度均低于5%;FDP在0～120μg/ml内线性相关系数为0.9974;实验方法对DIC诊断的敏感度为96.6%,特异度为52.1%,优于对照方法;本地区FDP成人参考区间为0.67～3.18μg/ml。结论:FDP免疫比浊定量检测方法具有良好的实验性能,可用于临床纤溶状态的判断和监测。     

A time-resolved immunofluorometric assay for quantification of the bovine collectin conglutinin

A high capacity time-resolved immunofluorometric assay (TRIFMA) for the bovine collectin conglutinin was developed. The TRIFMA was constructed as a non-competitive sandwich assay based on polyclonal antibodies as the capture reagent and a novel monoclonal antibody raised against conglutinin as the detection reagent and was set up to run on an automatic analyzer designed for the TRIFMA detection system. Polyclonal antibodies immobilized on microtiter plate wells were incubated overnight at 4 degrees C with diluted plasma samples, including quality controls (QC) and dilutions of a plasma with known conglutinin concentration. Conglutinin was sandwiched between the capture antibodies and the monoclonal antibody and the detection optimised with biotin-labelled secondary antibodies and streptavidin-Eu(3+). Plates were washed four times between each step and finally incubated with enhancement solution before measuring the fluorescence. The assay detection limit was 0.34 ng/ml and the working range 0.80 ng/ml-0.20 microg/ml. Intra-plate and inter-plate coefficients of variation (CV) were in the range of 5.0-8.3% and 6.2-7.2%, respectively, at concentrations of 3.4 and 150 ng/ml. Recovery was 90.9+/-2.4% and 98.8+/-2.5% when samples were spiked with 20 ng/ml and 100 ng/ml purified bovine conglutinin (BK). No circadian rhythm (24-h variation) in conglutinin plasma levels was observed across animals, indicating that the plasma levels were not influenced by, e.g. feeding. Samples could be stored at -20 degrees Celsius and were not sensitive to repeated freezing and thawing. In conclusion, the developed TRIFMA for bovine conglutinin is specific and reliable over a measurement range covering most situations.     

A practical approach to glomerular filtration rate measurements: creatinine clearance estimation using cimetidine

Determination of creatinine clearance (Ccr) is not a reliable indicator of glomerular filtration rate (GFR), owing to tubular secretion of creatinine. It has been reported that Ccr measurements can approximate true GFR after cimetidine (Ci) administration. In this study, GFR was estimated by Cockcroft and Gault's equation (C(C-G)) based on measurement of plasma creatinine, and Ccr was determined by the standard clearance equation using 4- and 24-hr urine samples (Ccr4 and Ccr24, respectively) in 17 patients and 10 healthy controls. After cimetidine administration (800 mg, 3 times daily), GFR values were recalculated at the same time periods (C(CiC-G), CcrCi4 and CcrCi24, respectively). The results were all compared to those obtained by the 99mTc-DTPA protein-free double-sample method (C(DTPA)), which is a reference method for GFR determination. The coefficient of variation (CV%) for Ccr24/C(DTPA) was high before cimetidine administration; Ccr24 and CcrCi24 values were significantly different from C(DTPA) (CV 23.1%, Ccr24/C(DTPA) = 1.17, p 0.005; and CV 14.1%, CcrCi24/C(DTPA) = 0.92, p 0.006, respectively). Ccr4 values obtained before cimetidine ingestion showed large variation and were significantly different from C(DTPA) (CV 15.5%, Ccr4/C(DTPA) = 1.11, p 0.001). CcrCi4 values after cimetidine were similar to CDTPA (CV 6.9%, CcrCi4/C(DTPA) = 1.01, p 0.28). C(C-G) estimates were higher before cimetidine intake (CV 12.4%, C(C-G)/C(DTPA) = 1.21, p <0.001), whereas C(CiC-G) values were not significantly different from C(DTPA) values (CV 7.0%, C(CiC-G)/C(DTPA) = 1.01, p 0.67). This study shows that GFR estimations by C(C-G), Ccr4, Ccr24, or CcrCi24 are insufficiently reliable. On the other hand, C(CiC-G) and CcrCi4 results are acceptable for true GFR estimations.