Volume 41, Issue 5

Quality control plays a vital role helping to ensure the reliability of laboratory test results. The application of statistical quality control has been a component of laboratory medicine for approximately 50 years. Many of the control rules based on the early applications of statistical quality control have remained essentially unchanged since their initial introduction. Optimization of quality control rules can vary depending on the application for which a test is to be used. This review explores the various applications of laboratory quality control procedures and their role in identifying laboratory error. The ubiquitous use of computers in today's laboratories has enabled the development of more sophisticated means of assessing laboratory quality. The use of the Six Sigma technique and its adoption by the laboratory community is one example. Other examples include the use of patient-derived quality control procedures as a means of assessing laboratory performance. Early examples of these types of applications include use of Bull's algorithm, anion gap measurements, and delta checking. More recent applications include the correlation of laboratory test results, the average of normals procedure, and the Bhattacharya method.

Apolipoprotein E (apoE) has important functions in systemic and local lipid transport, but also has other functions. The gene ( APOE ) shows a common polymorphism with three alleles – APOE*2 , APOE*3 , and APOE*4 . Their frequencies vary substantially around the world, but APOE*3 is the most common almost everywhere and is often considered to be the ancestral or “wild-type” allele for that reason. However, there are several arguments for APOE*4 being the ancestral allele. But then, why has APOE*3 become so frequent? And why has APOE*4 not become extinct? The proportion of APOE*4 carriers increases from only 10–15% in southern Europe to 40–50% in the north. The gradient may be a trace of the demic expansion of agriculture that began about 10,000 years ago, but it may also reflect the possibility that APOE*4 carriers are less likely to develop vitamin D deficiency. The common APOE polymorphism is associated with varying risk of cardiovascular disease and Alzheimer's disease, but other interesting aspects may emerge in the future.

It is known that pentanucleotide repeat polymorphism, consisting of varying number of (TTTTA) repeats in a promoter, is associated with plasma lipoprotein(a) levels. G/A −914 base substitution has also been correlated with pentanucleotide repeat polymorphism in individuals with high and low lipoprotein(a) levels. To facilitate future analysis of these polymorphisms we have developed a quick and reliable assay for the detection of both polymorphisms in a single polymerase chain reaction.

Cardiovascular risk is associated with high lipoprotein( a) (Lp(a)) concentrations and low molecular weight apolipoprotein(a) (apo(a)) isoforms. We studied the relationship between these two biological parameters, particularly in subjects expressing two apo(a) isoforms. Plasma Lp(a) was measured by immunonephelometry in 530 unrelated Caucasian patients at high cardiovascular risk, and apo(a) size determined by immunoblotting using a recombinant standard. Two, one, or no apo(a) isoforms were detected in 258, 270, and 2 subjects, respectively. Lp(a) concentrations showed a non-Gaussian distribution, being higher in the ‘double band’ than in the ‘single band’ group (median 0.42 vs. 0.11 g/l, p < 0.0005). Apo(a) size distribution was bimodal, with two frequency peaks at 18 kringles (K) and 27 K. Small size apo(a) isoforms were more frequently found in the ‘double band’ group, where major isoforms were of lower size than minor isoforms (median 20 vs. 27 K). Regression analysis showed that apo(a) gene length accounted for 33% of Lp(a) variation, with a threshold effect at 20 K, no correlation being found over this value. The minor apo(a) isoform did not significantly influence Lp(a) concentration. These data confirm the relationship between apo(a) size and Lp(a) concentration and suggest that the assessment of cardiovascular risk should take into account the threshold effect at 20 K and the absence of influence of the minor apo(a) isoform.

Continuous ambulatory peritoneal dialysis (CAPD) carries a risk of peritonitis which is accompanied by mild symptomatology. Culture of effluent has yielded organism in 50% of cases. Peritoneal phagocytes produce tumor necrosis factor-α and interleukin (IL)-1 in response to contact with bacteria, initiating an inflammatory cascade which leads to IL-6 and IL-8 secretion. Additonally, neutrophils undergo an increase in oxidative metabolism. We have evaluated the diagnostic accuracy of effluent measurements of TNF-α, IL-6, IL-8, and oxidative metabolism markers in these patients. Dialysate fluids (n = 65) were collected from non-infected patients and those presenting with acute peritonitis. Positive culture proved the diagnosis. Oxidative markers and nitric oxide were measured by chemiluminescence. Cytokines were measured by solid phase chemiluminescent immunometric assay (Immulite, DPC, USA). Receiver operating characteristic (ROC) curves were used to assess the diagnostic accuracy and the areas under curves were calculated for comparison. All effluent cytokines and oxidative markers were significantly higher in patients with peritonitis when compared to those without (p < 0.05). Significant correlations were evident between IL-6 and IL-8, lucigenin chemiluminescence and luminol chemiluminescence, lucigenin chemiluminescence and IL-6 or IL-8, and luminol chemiluminescence and IL-6 or IL-8. ROC curves showed that the ability of IL-6, IL-8, lucigenin chemiluminescence, and luminol chemiluminescence to differentiate CAPD patients with peritonitis from non-infected cases exceeds that of polymorphonuclear leukocyte count.

Hematopoietic growth factors (HGFs) are involved in the regulation of growth and spread of cancer. Therefore, in the present study, we have investigated in colorectal cancer patients the serum levels of selected HGFs, such as stem cell factor (SCF), interleukin 3 (IL-3), granulocyte-macrophage-colony stimulating factor (GM-CSF), and M-CSF in relation to controls and to the classical tumor markers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) in colorectal cancer. Additionally, we have compared the serum levels of cytokines with tumor site and stage and other clinical characteristics such as age and sex. We also defined the receiver-operating characteristics (ROC) curve for HGFs and classical tumor markers. The tested cytokines were measured in 70 patients with colorectal cancer and in 40 healthy subjects. HGFs were determined using enzyme-linked immunosorbent assay (ELISA). CEA and CA 19-9 were measured by microparticle enzyme immunoassay. There were significant differences in the levels of circulating SCF, IL-3, M-CSF, GM-CSF, and CEA and CA 19-9 in the colorectal cancer patients compared to the control group. The levels of M-CSF and CEA were significantly higher in patients with a more advanced tumor stage. The significant positive correlation was observed between the CEA and CA 19-9 concentrations. The M-CSF serum levels correlated positively with the tested tumor markers. The M-CSF area under the ROC curve was the largest. These results suggest that M-CSF is, among the tested HGFs, the best candidate for a colorectal cancer tumor marker.

Persisting chylomicron remnants have been linked to premature atherosclerosis. The analysis of chylomicron remnant concentrations by an oral triglyceride tolerance test, however, is time-consuming for the study subjects and requires large resources in the laboratory. Therefore, only small numbers of subjects have been studied in the past. The aim of this study was to elucidate the prevalence of elevated chylomicron remnants, to identify effectors of chylomicron remnant clearance and to compare chylomicron remnants in the prediction of coronary artery disease with other risk factors. We applied a novel oral triglyceride tolerance test to 423 patients (368 males, 55 females) with a confirmed diagnosis of coronary artery disease (CAD) and to 390 control subjects (295 males, 95 females) in a case-control setting. This study revealed that elevated chylomicron remnant concentrations (retinyl esters >1.5 μmol/l) are present in 20% of all subjects. Male gender, the apolipoprotein E2 isoform, and higher body mass index were associated with increased chylomicron remnant concentrations. However, chylomicron remnants were lower and plasma triglycerides higher in patients with CAD. We conclude that screening for a delayed clearance of chylomicron remnants is of little clinical value in CAD.

The initial hypothesis was that the follicular fluid prorenin concentration was higher in women with polycystic ovaries (PCO) compared to normally cycling women stimulated by gonadotrophins. In a controlled prospective study, 47 women undergoing in vitro fertilisation (IVF) were enrolled: 20 women with PCO represented the study group and 27 normally cycling women the control group. Plasma prorenin, active renin, serum oestradiol, and androstenedione concentrations were measured on the day of human chorionic gonadotrophin (HCG) administration and 36 hours after HCG administration. Follicular fluid prorenin, active renin, oestradiol, and androstenedione concentrations from the dominant follicle and pooled follicles were measured 36 hours after HCG administration. Plasma prorenin concentration 36 hours after HCG administration was significantly higher in the PCO group (1867 μU/l; range 1137–3162 μU/l) than in controls (860 μU/l; range 433–1763 μU/l) (p = 0.007). The follicular fluid prorenin concentration in the dominant follicle and in the pooled follicles was lower in the PCO group (20190 μU/l; range 11130–25955 μU/l) than in controls (46930 μU/l; range 20671–66171 μU/l) (p = 0.003). We conclude that plasma prorenin concentrations in gonadotrophin-stimulated PCO cycles are augmented due to numerous follicles. Lower follicular fluid prorenin concentrations in women with PCO show that the synthesis of prorenin in theca cells starts later after HCG administration than in normally cycling women.

Few studies have demonstrated the optimal usage of common inflammatory markers, alone or in combination, based on the cost-effectiveness. We analyzed the yield and cost of C-reactive protein (CRP), white blood cell count (WBC), erythrocyte sedimentation rate (ESR), sialic acid, and protein fractionation in 177 new primary care outpatients with inflammation-related symptoms. A useful result (UR) was assigned if tests contributed to a change in physician's diagnosis or decision-making. Costs of testing were calculated based on either single or simultaneous measurement. Five inflammatory markers generated 147 URs in 123 patients. CRP showed the best contribution to generation of UR, followed by sialic acid, protein fractionation, WBC, and ESR. CRP demonstrated poor correlation with WBC (r = 0.458), while sialic acid strongly correlated with total absolute amount of α 1 and α 2 fractions in protein fractionation (r = 0.855) and moderately with ESR (r = 0.651). The combination of CRP and WBC produced the best cost-effectiveness at a cost of ￥ 1169 (US$ 9.6 or Euro 9.7)/additional UR against CRP testing alone. Sialic acid, an automated multichannel analyzer-based test, demonstrated the favorable cost-effectiveness over ESR or protein fractionation when combined with CRP (and WBC). Our results indicate that the optimal usage of these inflammatory markers should involve careful cost-effectiveness considerations.

The study reports on plasma total homocysteine (tHcy) levels in Tunisian patients with chronic renal failure (CRF) and those treated with hemodialysis (HD) and renal transplant (RT). The aims of the study were to identify the determinants of tHcy concentration and to test the association between hyperhomocysteinemia and atherothrombotic disease in end-stage renal disease (ESRD). A total of 35 CRF patients on conservative treatment, 50 HD patients, and 30 RT recipients, and 31 age-and sex-matched healthy subjects were included. Plasma tHcy was assessed by a fluorescent-polarizing immunoassay method. Multivariate analysis was applied to identify the main determinants of tHcy concentration and to assess the relationship between hyperhomocysteinemia and cardiovascular disease. Plasma mean tHcy concentration was significantly increased (p < 0.001) in CRF patients (mean ± SD) (28.9±9.8 μmol/l), in HD patients (29.4±11.1 μmol/l), and in RT (19.3±6.3 μmol/l) patients compared to controls (11.9±4.1 μmol/l). Multivariate analysis using GLM ANOVA modeling demonstrated that tHcy was significantly higher in males (p = 0.02), and was related to age (p = 0.008), albumin (p = 0.005), vitamin B 12 (p = 0.002), folate (p = 0.00001), and creatinine clearance (p = 0.0008). However, tHcy was not associated with C-reactive protein and did not significantly differ between CRF, HD, or RT patients. The upper quartile of tHcy concentration was significantly associated with atherothrombotic cardiovascular disease (unadjusted odds ratio (OR) = 3.09; 95% CI, 1.11–8.61; p = 0.01). This association remained significant after adjusting for sex, age, hypertension, and smoking (multi-adjusted OR = 4.78; 95% CI, 1.92–11.9; p = 0.0008). The mean tHcy concentration was 2 to 3 times higher in ESRD patients than in subjects with normal renal function. This increase could be related to glomerular filtration rate reduction and functional B vitamins deficiency, but was not associated with inflammation. The upper quartile of tHcy concentrations confers 4.78-fold increased independent risk for atherothrombotic events in ESRD patients.

Homocysteine (Hcy) is the demethylated derivate of methionine and can be metabolized by two pathways. It is either catabolized by the transulfuration pathway to cysteine or remethylated to methionine mainly by the folate- and cobalamin-dependent enzyme methionine synthase. Previous findings suggest that folate is the most important vitamin determinant of plasma total homocysteine (tHcy) concentration but also that the relation between plasma tHcy levels and levels of cobalamin and folate in the circulation might differ in different populations. In the present study, we have analyzed these parameters in different age groups of healthy subjects and psychogeriatric patients, who are known to have increased plasma tHcy. The present study shows that serum cobalamin concentration is a more important determinant of plasma tHcy concentration than blood folate concentration in the age groups <65 years in both psychogeriatric patients and control subjects, whereas with increasing age blood folate concentration becomes the most important vitamin determinant. The findings of increased plasma tHcy with folate being the main vitamin determinant in the oldest age groups of patients and controls, suggest that tissue levels of folate in elderly subjects are too low and that vitamin supplementation should be given.

In order to study the influence of gender on circulating levels of cardiac natriuretic hormones (CNHs) in heart failure, we measured the plasma levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) by means of highly sensitive and specific IRMA methods in 239 consecutive patients (age 64.7 ± 11.6 years, range 21–89 years; 170 men and 69 women) with cardiomyopathy. There was different response of CNH according to gender in patients with heart failure, as indicated by the ratio between the individual CNH values of patients and the gender-specific cut-off values. Indeed, the mean ratio for ANP found in men (3.6±3.6) was significantly higher (p = 0.0075) than that found in women (2.4±2.1). The mean ratio for BNP was on average 2.3 fold higher (15.9±27.1 in men and 6.9±6.8 in women, p = 0.0084). Moreover, age, ejection fraction, and disease severity independently and significantly contributed to regression with both ANP (R = 0.612, F = 39.969, p < 0.0001) and BNP (R = 0.656, F = 49.957, p < 0.0001) values, while gender did not. In conclusion, our study suggests a different, gender-specific activation of the CNH system in this clinical condition, although age, ejection fraction and disease severity seem to be more powerful predictors than gender of circulating levels of ANP and BNP in patients with heart failure.

Among the various models proposed to meet requirements for metrological performance in clinical laboratory measurements, that most widely used is intraand inter-biological variation. However, this model has weaknesses: a) in some cases, intra-individual biological variation is so small that requirements based on such data may not be met by routine methods; b) there is great variation among the intra- and inter-individual biological variations themselves. We therefore propose integration between “state-of-the-art” methods to minimize imprecision, and also suggest that requirements calculated on the basis of biological variations should be used in setting requirements for minimizing imprecision. Furthermore, we describe a new scale for setting requirements from biological variation data and suggest that systematic error should be corrected whenever it is statistically significant.

Measurement of S-100B protein in serum or cerebrospinal fluid is increasingly used as a molecular marker of brain damage. However, there is no information about the pre-analytical in vitro stability of S-100B. We have investigated whether storing blood samples at different time periods and conditions affected the measured levels of S-100B protein. Blood samples were taken from 29 neurosurgical patients with normal serum S-100B values and from seven patients with increased serum S-100B values. Blood samples were immediately divided into nine aliquots for measuring S-100B immediately and after 4, 8, 12, and 24 hours, stored at room temperature or at 4 °C. Measurement of S-100B was performed using the LIAISON ® assay (Byk-Sangtec Diagnostica, Dietzenbach, Germany). Moreover, in 10 additional patients the effect of freezing the serum and thawing the sample after 24 hours was investigated. There were no differences between the results of S-100B measurements after storing the sample at different temperatures and time periods. There was no trend towards higher or lower values in all three groups. Therefore, blood samples may be collected as part of the daily clinical routine without time constraint and even stored overnight without affecting S-100B serum levels when measured with the LIAISON ® Sangtec ® 100 assay.

Capillary zone electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. In this report, we evaluate automated capillary zone electrophoresis by Capillarys ® (Sebia, France). Withinrun and between-run imprecision for the five electrophoretic fractions was <2% and <6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel electrophoresis and Paragon CZE ® 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and α 1 -antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel electrophoresis, Capillarys, capillary electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology ( e.g. , spike) of the protein profile or hypogammaglobulinemia on agarose gel electrophoresis and/or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum electrophoresis.

This study explores the feasibility of using computer tutorials to train laboratory personnel in Nepal. Training incorporated three software programs that teach microscope-based laboratory tests (peripheral blood smears, urinalysis, Gram stains). Forty-seven participants attended training sessions and completed a questionnaire. The participants' overall perception was: 1) the software was superior to formal lectures for learning image-based laboratory tests (43 participants, 92%); 2) the software would enhance job performance (43 participants, 92%); 3) more subjects should be taught using software (40 participants, 85%); and 4) the software helped participants learn new materials (38 participants, 81%). Considering that 79% of the participants were novice computer users, it is noteworthy that 38 (81%) participants thought the method of instruction was easy to understand. Factors contributing to learning included: 1) the resemblance of the computer images to actual microscope images derived from patient samples (37 participants, 68%); 2) the use of multiple examples of cells and other microscopic structures (28 participants, 60%); 3) the ability to interact with images and animations (23 participants, 49%); 4) the step-by-step explanation of laboratory techniques (21 participants, 45%); and 5) the self-pacing of the tutorial (12 participants, 26%). Overall, the pilot study suggests that educational software could help train clinical laboratory personnel in developing countries.