Volume 51, Issue 11

Subarachnoid haemorrhage (SAH) has a high mortality and morbidity rate. Early SAH diagnosis allows the early treatment of a ruptured cerebral aneurysm, which improves the prognosis. Diagnostic cerebrospinal fluid (CSF) analyses may be performed after a negative computed tomography scan, but the precise analytical methods to be used have been debated. Here, we summarize the scientific evidence for different CSF methods for SAH diagnosis and describe their implementation in different countries. The principle literature search was conducted using PubMed and Scopus with the search items “cerebrospinal fluid”, “subarachnoid haemorrhage”, and “diagnosis”. CSF analyses for SAH include visual examination, red blood cell counts, spectrophotometry for oxyhaemoglobin or bilirubin determination, CSF cytology, and ferritin measurement. The methods vary in availability and performance. There is a consensus that spectrophotometry has the highest diagnostic performance, but both oxyhaemoglobin and bilirubin determinations are susceptible to important confounding factors. Visual inspection of CSF for xanthochromia is still frequently used for diagnosis of SAH, but it is advised against because spectrophotometry has a superior diagnostic accuracy. A positive finding of CSF bilirubin is a strong indicator of an intracranial bleeding, whereas a positive finding of CSF oxyhaemoglobin may indicate an intracranial bleeding or a traumatic tap. Where spectrophotometry is not available, the combination of CSF cytology for erythrophages or siderophages and ferritin is a promising alternative.

Remarkable advances in understanding human biology in health and disease, propelled by technological innovations, have contributed to an increase in the number and quality of diagnostic tests. This evolving scenario has been accompanied by the proliferation of false myths and legends in laboratory diagnostics, consuming valuable human and economic resources and jeopardizing the clinical reasoning. The aim of this article is to provide a synthetic overview about some paradigmatic examples of false beliefs in laboratory diagnostics involving activated partial thromboplastin time (APTT), cardiospecific troponins, ischemia modified albumin (IMA), D-dimer, prostate specific antigen (PSA), dibucaine number, Bence Jones protein (BJP), lipoprotein(a), neutrophil gelatinase-associated lipocalin (NGAL), potassium and reference ranges. Although the suggestive cases described in this article are not intended to be comprehensive, we hope that their description may help remove some mysticisms in laboratory diagnostics.

Background: A national cross-sectional survey on the reporting of critical values was conducted to provide suggestions on monitoring of the quality indicator and obtain the goal of quality improvement. Methods: All clinical laboratories participating in the external quality assessment (EQA) programs of chemistry, blood gas, and hematology tests organized by the National Center for Clinical Laboratories (NCCL) were enrolled in our study. General information on the policy of critical values reporting and the particular problems about critical limits and practice of critical values reporting in the last 2 months were asked in the questionnaire. All collected data were analyzed by the special statistical software designed by the NCCL. Results: The return rate of surveys for critical values on clinical biochemistry, blood gas, and hematology testing were 46.11%, 41.14%, and 39.42%, respectively. Most laboratories in the surveys suggested they set different critical limits for children and adults. The analytes of critical values chosen most frequently were potassium, glucose, sodium, calcium, pH, pCO 2 , pO 2 , white blood cell (WBC) count, hemoglobin (Hb), prothrombin time (PT), activated partial thromboplastin time, and fibrinogen. The analytes with most critical values were potassium, blood gas, and creatinine for clinical biochemistry, pCO 2 for blood gas, and WBC, Hb, and PT for hematology testing. The medians of critical limits for potassium, sodium, calcium, and glucose were not significantly different from the results of the national survey conducted by Kost in 1990. Conclusions: Remarkable inter-laboratory difference in the development and implementation of critical values reporting procedure were found. Laboratories should communicate with clinicians to obtain consensus on the analytes and limits of critical values.

Background: Despite striking similarities among colorimetric lipase assay recipes, marked intervendor differences are noted in the reported lipase values. In the present study, the effect of physical properties of the cuvette surface on measurement of serum lipase was investigated. Methods: Lipase activity was measured concomitantly in cuvettes from three different analyzers: Vista (Siemens), Modular (Roche), and Synchron (Beckman Coulter). The surface/volume ratio of the cuvettes and the contact angle of the cuvette polymers were determined. The effects of various characteristics of serum (biochemical parameters, surface tension) were also examined. Results: Serum lipase activities based on the colorimetric methylresorufin assay differed markedly according to the cuvettes used. More specifically, in the lower activity rate, marked differences were reported. The physical properties of the various cuvettes showed remarkable differences, especially in the contact angles. Other biochemical parameters (bilirubin, alkaline phosphatase, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides) and serum surface tension did not affect the results. Conclusions: Serum lipase activity is affected by the physical properties of the cuvette surface.

Background: In Tunisia, thalassemia and sickle cell disease represent the most prevalent monogenic hemoglobin disorders with 2.21% and 1.89% of carriers, respectively. This study aims to evaluate the diagnosis reliability of a series of red blood cell indices and parameters in differentiation of beta-thalassemia trait (β-TT) from iron deficiency anemia (IDA) and between homozygous sickle cell disease (SS) and sickle cell-thalassemia (ST). Methods: The study covered 384 patients divided into three groups. The first one is composed of 145 control group, the second consists of 57 β-TT and 52 IDA subjects and the last one with 88 SS and 42 ST patients. We calculated sensitivity, specificity, positive-predictive values, negative-predictive values, percentage of correctly identified patients and Youden’s index for each indice. We also established new cut-off values by receiver operating characteristic curves for each indice. An evaluation study was performed on another population composed of 106 β-TT, 125 IDA, 31 SS and 17 ST patients. Results: Srivastava Index, mean corpuscular hemoglobin, red blood cell, Mentzer Index (MI) and mean corpuscular hemoglobin concentration show the highest reliability in discriminating β-TT from IDA with new cut-offs slightly different from those described in literature. Ehsani Index, mean corpuscular volume, MI, Shine and Lal Index and Sirdah Index are the most powerful in the differentiation between SS and ST. Conclusions: The effectiveness and the simplicity of calculation of these indices make them acceptable and easy to use for differential diagnosis.

Background: Measurement of immature platelets was introduced into routine diagnostics by Sysmex as immature platelet fraction (IPF) some years ago and recently by Abbott as reticulated platelet fraction (rPT). Here, we compare both methods. Methods: We evaluated the precision and agreement of these parameters between Sysmex XE-5000 and Abbott CD-Sapphire in three distinct thrombocytopaenic cohorts: 30 patients with beginning thrombocytopaenia and 64 patients with recovering platelets (PLT) after chemotherapy, 16 patients with immune thrombocytopaenia (ITP) or heparin-induced thrombocytopaenia type 2 (HIT) and 110 additional normal controls. Furthermore, we analysed, how IPF/rPT differed between these thrombocytopaenic cohorts and controls. Results: Both analysers demonstrated acceptable overall precision (repeatability) of IPF/rPT with lower precision at low PLT counts. IPF/rPT artificially increased during storage of blood samples overnight. Inter-instrument comparison showed a moderate correlation (Pearson r²=0.38) and a systematic bias of 1.04 towards higher IPF-values with the XE-5000. IPF/rPT was highest in recovering thrombopoesis after chemotherapy and moderately increased in ITP/HIT. The normal range deduced from control samples was much narrower with CD-Sapphire (1.0%–3.8%, established here for the first time) in comparison to XE-5000 (0.8%–7.9%) leading to a smaller overlap of samples with increased PLT turnover and normal controls. Conclusions: IPF and rPT both give useful information on PLT turnover, although the two analysers only show a moderate inter-instrument correlation and have different reference ranges. A better separation of patient groups with high PLT turnover like ITP/HIT from normal controls is obtained by CD-Sapphire.

Background: Evaluation of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM) is not standardized and is associated with consistent inter-laboratory variability. Methods: In order to rule out the influence of particular approach in generating final results, we analyzed the performance characteristics of individual consensus strategies for small to intermediate (1%–20%) and minor (<1%) PNH clones within the white blood cell (WBC) and red blood cell (RBC) compartments with sensitivity up to 0.1%. Results: Coefficient of variation (CV) for precision/reproducibility analysis ranged from 0.67%/1.49% to 2.56%/3.09% for granulocytes, from 0.93%/3.09% to 7.76%/12.06% for monocytes and from 0.41%/4.73% to 6.53%/5.1% for RBCs. Coefficient of determination (r 2 ) for linear regression analysis ranged from 0.95 to 0.99, Wilcoxon ranks test showed no statistically significant differences (p>0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from −0.18 to 1.24. Conclusions: Our results confirmed very good performance characteristics for precision and reproducibility analysis, excellent correlation and favorable agreement between strategies, suggesting that reported inter-laboratory variability is related mainly to incorrect performance and/or insufficient experience with PNH testing by flow cytometry, rather than to relevant limitations of any particular approach.

Background: PR3-ANCA, the serological marker of granulomatosis with polyangiitis (GPA), is usually detected by immunometric assays, with purified PR3 directly coated onto the solid-phase. Novel methods for PR3-ANCA detection have been developed to improve the performance of traditional PR3-ANCA specific assays, but little is known about their diagnostic performance in real-life clinical settings. This study aimed to compare the performance of nine different commercial PR3-ANCA specific assays, including traditional and newer ones, for the diagnosis of GPA. Methods: The evaluated assays for PR3-ANCA detection were representative of the first, second, and third generation tests ( direct, capture and anchor assays, respectively). A third-generation assay employing both human and recombinant PR3 was also evaluated. The study population consisted of 55 GPA patients, 175 disease controls (representing most diseases in differential diagnosis with primary small-vessel vasculitis) including 52 with microscopic polyangiitis, and 20 healthy subjects. We performed the primary evaluation of test sensitivity using cut-off points which provided adequate and identical specificity for each test. Results: Although specificity and area under the ROC curve did not differ significantly between the different assays, substantial differences in sensitivity at 98%-specificity were found in some instances (p<0.001). Compared to first generation direct PR3-ANCA specific assays, some of the second and third generation tests increased the positive predictive value (PPV) for GPA diagnosis. Conclusions: Some of the newer PR3-ANCA specific assays have better PPV than traditional ones.

Background: Detection of systemic sclerosis-associated autoantibodies (SSc-Ab) is mostly restricted to anti-centromere and anti-topoisomerase-I. However, anti-RNA-polymerase-III and anti-PM/Scl are also important diagnostic markers for the disease supporting their incorporation in the laboratory repertoire. The aim of this study was to compare different testing strategies integrating the identification of these extra SSc-Ab in a routine testing algorithm. Methods: Sera from 144 consecutive SSc-patients and 265 controls were screened for antinuclear antibodies (ANA) by indirect immunofluorescence (ANA IIF) and tested for anti-extractable nuclear antigen (ENA) using five different assays that differ in their ability to detect SSc-Ab [two screening enzyme immunossays (EIA) with antigen mixtures, one multi-parameter line-immunoassay and two EIA with individual antigens]. Results: The application of SSc-Ab testing in cascade with the routine ANA/anti-ENA tests improved diagnostic performance characteristics. Besides the type of algorithm, also the number of antigens included in the screening EIA as well as the expected patient/control ratio, influenced the average expected costs and the number of additional SSc-Ab tests to be performed. In laboratories with an expected patient/control ratio of 0.002, cascade testing was most exploited by the use of a screening EIA that included all SSc-Ab as a secondary test after ANA IIF. Conclusions: Restriction of the performance of additional SSc-Ab assays based on the results of prior ANA/anti-ENA tests is a cost-effective strategy allowing optimized use of laboratory resources with minimal loss in diagnostic capacity.

Background: Little attention has been paid to the maximum number of specimens for reference interval calculation, i.e., the number of specimens beyond which there is no further benefit in reference interval calculation. We present a model for the estimation of the maximum number of specimens for reference interval studies based on setting the 90% confidence interval of the reference limits to be equal to the analyte reporting interval. Methods: Equations describing the bounds on the upper and lower 90% confidence intervals for logarithmically transformed and untransformed data were derived and applied to determine the maximum number of specimens required to calculate a reference interval for 12 common chemistry and hematology analytes. Results: Maximum sample sizes ranged from 126 to 18,171 and depended on the standard deviation of the population, any transformation involved and on the chosen reporting interval. Conclusions: This paper demonstrates the importance of the influence of reporting interval on reference intervals. Using this technique can reduce the cost of determining a reference interval by identifying the maximum number of specimens required.

Background: A number of methods have been developed to measure small dense low-density lipoprotein cholesterol (sd-LDL-C) to evaluate atherogenic risk in the population. However, to our knowledge there are no reports about the biologic variability of these lipoproteins. Therefore, the aim of this work was to estimate sd-LDL-C biological variability, and with this information establish quality specifications, index of individuality (II) and reference change values (RCV). Methods: To estimate within- and between-subject biological variability, sd-LDL-C in serum was measured in 24 individuals (11 female and 13 male) for 5 consecutive days and then, at 2 and 3 weeks. Quality specifications, II, and RCVs were estimated according to procedures described. Results: Total within- and between-subject biological variability, expressed as coefficient of variation, was 9.1% and 20%. Meanwhile, within- and between-biological variability in female and men was 10.9% and 6.7%, and 22% and 17%, respectively. Desirable quality specification to the sd-LDL-C method was 4.6% for analytical imprecision, bias 5.5% and total allowable error of 11.4%; the II was 0.46 and the RCV (calculated at 95% and 99% of significance) was 27.1% and 35.7%, for the total data. Conclusions: Short-term biological variability components were determined, and then used to estimate quality specifications, II and RCV for sd-LDL-C precipitation assay. To our knowledge, this is one of the first reports about sd-LDL-C biological variability, so that this information can be used as a starting point to develop long-term studies of biological variability for sd-LDL-C.

Background: There is currently very little data available on the consistency of quantitative and qualitative faecal immunochemical test (FIT) for colorectal cancer screening. Methods: A representative random population (n=1889, 40–74 year olds) in Jiashan, China was invited for FIT screening in 2012. Faecal samples were collected by a single specimen collection device and simultaneously tested by a quantitative FIT (OC-SENSOR, OC) and two qualitative FITs (FIT A and FIT B with intrinsic positive haemoglobin cut-off concentrations of 20 μg Hb/g faeces and 40 μg Hb/g faeces, respectively). The observational criteria for a positive result of the qualitative FIT were set according to the density of the colour appearing in the test strip. The results produced by the quantitative and qualitative FIT for each sample were compared. κ coefficient was used to measure consistency. Results: A total of 1368 (72.4%) individuals returned faecal samples. Both FIT A and FIT B precisely identified all faecal samples with haemoglobin concentration above 100 μg Hb/g faeces, but the overall consistency was poor for OC & FIT A (κ=0.32, 95% CI 0.20–0.44) and was moderate for OC & FIT B (κ=0.74, 95% CI 0.64–0.85). A more favourable consistency (κ=0.64, 95% CI 0.57–0.72) was achieved when a different positive criterion was employed for FIT A. Conclusions: The diagnostic inconsistency between quantitative and qualitative FITs mainly exists in the faecal samples with low haemoglobin concentrations. Refining the criterion for a positive result may be a feasible way to improve the accuracy of qualitative FIT.

Background: The IFCC Task Force on Clinical Applications of Cardiac Biomarkers suggests comparing several contemporary sensitive troponin assays in the same, sufficiently large reference population. Methods: Three contemporary sensitive assays were used to measure troponin I concentration in samples from a uniquely large healthy population (2404 individuals) and in a sub-group with tighter inclusion criteria of 908 individuals. The 99th percentiles were calculated using quantile regression which takes the entire population into account. Results: The 99th percentile for the ARCHITECT STAT Troponin I assay was 21 ng/L, 31 ng/L for the ADVIA Centaur Troponin I-Ultra assay and 28 ng/L for the Dimension Vista cTnI assay. Significantly higher values were found in males than in women only in the Dimension Vista cTnI assay and in the subgroup for the ARCHITECT STAT Troponin I assay. Conclusions: Quantile regression provides a tool to accurately estimate the 99th percentile and establish a continuous function of the relation between the 99th percentile and the age and gender. There was no age dependency demonstrated. A gender difference was found in one assay but inconclusive in another and not demonstrated in a third.

Background: The aminothiols homocysteine and, to a lesser extent, cysteine have been associated with adverse cardiovascular outcome, whereas glutathione, as an antioxidant, may protect against atherosclerosis and thrombosis. Potentially, the combined assessment of these aminothiols may provide a more accurate association with future cardiovascular outcome. We evaluated the association between recurrent atherothrombotic events and the concentration of total plasma cysteine, homocysteine, and glutathione and their combination. Methods: Respective aminothiols were measured by high-performance liquid chromatography in blood plasma of consecutive first-day survivors admitted for an acute coronary syndrome between April 2002 and January 2004. The combined score was calculated using the combination of the individual aminothiols. The end point was the composite of cardiovascular death, myocardial infarction, and/or stroke. Results: A cohort of 375 consecutive patients (median age 66 years, 66% male) were followed for a median duration of 2.7 years. The end point occurred in 82 patients (22%). In univariate analyses, all aminothiols were significantly associated with the composite end point. After correction for possible confounders, only cysteine and glutathione remained significantly associated. The strongest association with the end point was observed for the combined score (adjusted hazard ratio, 1.40 per standard deviation increase; p=0.005). Conclusions: Although homocysteine is generally considered the aminothiol of interest with respect to cardiovascular disease, in our prospective study, only cysteine and glutathione appeared independently associated with recurrent atherothrombotic events. Moreover, we showed that an imbalance in the combination of aminothiols could be of more importance than investigating the individual metabolites.

Background: Estimated average glucose (eAG) is a value calculated from hemoglobin A 1c (HbA 1c ) that reflects average glycemic status over the preceding few months. A linear relationship between HbA 1c and eAG was demonstrated by the International HbA 1c -Derived Average Glucose (ADAG) Trial in 2008. We investigated the relationship between fasting plasma glucose (FPG) and eAG. Methods: This retrospective study was conducted by reviewing the medical records of 6443 subjects, including 5567 diabetic patients and 876 non-diabetic subjects. The levels of HbA 1c and FPG were reviewed and eAG was calculated using the regression equation published by the ADAG trial: eAG mmol/L =1.59×HbA 1c (NGSP, %)–2.59[eAG mg/dL = 28.7×HbA 1c (NGSP, %)−46.7]. Results: In all subjects, FPG showed a moderate correlation with eAG (r=0.672, p<0.001). When diabetic and non-diabetic subjects were divided into subgroups according to FPG level, the correlation between eAG and FPG decreased in both diabetic [FPG ≥10.0 mmol/L (180 mg/dL), r=0.425; FPG 7.2–9.9 mmol/L (130–179 mg/dL), r=0.373; FPG <7.2 mmol/L (130 mg/dL), r=0.202] and non-diabetic [FPG 5.6–6.9 mmol/L (100–125 mg/dL), r=0.363; FPG <5.6 mmol/L (100 mg/dL), r=0.186] subgroups as the FPG level decreased. The differences between eAG and FPG were statistically significant (p<0.001). Only 81% (4487/5567) of diabetic patients had a lower FPG level than eAG level. Conclusions: Our results suggest that the relationship between eAG and FPG may depend on glycemic control, thereby enhancing our understanding of eAG.

Background : A novel enzymatic HbA 1c assay was introduced for use in an automated chemistry analyzer. With this unique method, HbA 1c and plasma glucose can be measured from the same EDTA tube. We evaluated the analytical performance of this enzymatic HbA 1c assay in a JCA-BM6010/C analyzer and compared the HbA 1c values with the results from other widely used methodological instruments. Methods : The imprecision, linearity, carry-over and concordance rate of the enzymatic HbA 1c test (BM Test HbA 1c ) using the JCA-BM6010/C analyzer were evaluated. Three hundred and seventy-seven specimens with HbA 1c concentrations from 16 to 133 mmol/mol were used for a comparison study with two high performance liquid chromatography methods: Variant II Turbo and Tosoh HLC 723 G8 and the AutoLab Hemoglobin A 1c immunoturbidimetry reagent using a Hitachi 7600-110. Forty specimens were used for the glucose method comparison. Results : The HbA 1c coefficients of variation of the within-run imprecision for low and high levels were 0.6% and 0.4%, respectively. The linearity of the BM Test HbA 1c using the JCA-BM6010/C analyzer was excellent in the range between 31 mmol/mol and 143 mmol/mol. The carry-over rate was 0.2%. The relationships between the BM test and the other three methods were 0.916×Tosoh G8+3.644, r=0.986; 0.887×Bio-Rad Variant II+1.896, r=0.972; and 0.941×AutoLab+4.532, r=0.977. The concordance rates using a cut-off of 48 mmol/mol were 91.5% with Tosoh G8, 82.8% with Bio-Rad Variant II, and 91.0% with AutoLab. The simultaneously assayed plasma glucose with HbA 1c was 1.002×Routine plasma glucose+0.625, r=1.000 Conclusions : The enzymatic BM Test HbA 1c in the JCA-BM6010/C analyzer showed excellent precision and linearity, and a minimal carry-over rate. The simultaneously assayed plasma glucose analysis showed good performance.