Volume 47, Issue 11

Much emphasis continues to be placed on the Journal Impact Factor (IF), a measure of journal article citation rates, and typically used as a surrogate marker of quality of both the article and journal. The IF is both revered and reviled, and is neither a perfect nor comprehensive measure, having several limitations and being subject to easy manipulation. The IF holds ‘power’ for journals because it can influence their future success. Furthermore, the perceived utility of the IF has grown way beyond that of its original and still popular use as a surrogate marker of publication ‘quality’. The IF is increasingly being used (i) to objectively evaluate the scientific and academic value of scientists across a wide variety of disciplines, (ii) to short-list research projects for future financial support, (iii) to short-list or select applicants for academic promotion, and (iv) by researchers to measure the success of research institutes, research funding, or even entire countries. Accordingly, despite our love-hate relationship with the IF, don't expect its demise any time soon. Clin Chem Lab Med 2009;47:1319–24.

Prostate-specific antigen (PSA) assay-dependent variations could result in misinterpretation of individual PSA values. Therefore, the situation for clinical interpretation of PSA or percent free PSA (%fPSA) results is complicated. This review summarizes the differences in various total PSA (tPSA) and free PSA (fPSA) assays, and results obtained using the new World Health Organization (WHO) calibrated Access assays from various studies. Method comparisons between the traditionally calibrated Hybritech PSA and fPSA assays and the new “standardized” WHO calibrated assays yield results that are ∼25% lower for PSA and fPSA. A PSA cut-off of 3 or 3.1 μg/L should be considered for WHO calibrated assays in order to achieve the same sensitivity/specificity profile as with a cut-off of 4 μg/L in traditionally calibrated assays. The %fPSA cut-offs could be retained. Clin Chem Lab Med 2009;47:1325–31.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) both represent highly heterogeneous entities on the basis of diverse cyto- and molecular genetic alterations with considerable influence on prognosis and therapeutic decisions. In recent years, insights into the complex network of molecular markers underlying this diversity have shown marked progress due to the detection of novel mutations, such as nucleophosmin gene ( NPM1 ) in AML, and due to the description of cooperation pathways in leukemogenesis. Also, targeted therapeutic strategies are continuously expanding as illustrated by the tyrosine kinase inhibitor (TKI) imatinib for BCR-ABL positive ALL. Thus, molecular analysis based on various techniques, such as polymerase chain reaction (PCR) has become an essential part of the diagnostic panel for acute leukemia. In addition, cytomorphology, cytogenetics, fluorescence in situ hybridization (FISH), and immunophenotyping with multiparameter flow cytometry (MFC) need to be applied for diagnosis. During the course of disease, the residual leukemic cell load can be monitored by highly sensitive quantitative PCR techniques (“real-time PCR”). At present, new techniques, such as high throughput sequencing (next generation sequencing, NGS) or gene expression profiling with microarrays are being explored for use in hematological malignancies, and are being evaluated in preclinical studies. This demonstrates that molecular diagnostics for acute leukemias are in continuous development. This review summarizes the most important recurrent molecular markers seen in acute leukemias, their role in prognosis and therapy and provides an overview on the relevant PCR techniques. Clin Chem Lab Med 2009;47:1333–41.

Following the completion of sequencing of the human genome, there has been a very rapid increase in the development of new molecular diagnostic tests. However, the numerous genetic tests and genetic testing technologies offered do not always satisfy essential quality criteria required to ensure confidence in the results that are produced. This is of particular importance for genetic tests since many patients may be tested for a particular genetic defect only once in their lifetime. Thus, there is a pressing need for comprehensive guidelines for the validation of molecular diagnostic tests and procedures, including DNA sequencing, the latter being a fundamental aspect of the development and validation of most genetic tests. To that end, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee for Molecular Diagnostics has prepared the following paper that describes a possible approach to the development of a reference method for sequencing of haploid DNA. We discuss various aspects which should be considered before, during and after applying the sequencing procedure, in order to achieve results with a known level of confidence, including robustness and assessments of quality. Clin Chem Lab Med 2009;47:1343–50.

Background: Diagnostic reviews often include the sensitivity/specificity results of individual studies. A problem occurs when these data are pooled because the correlation between sensitivity and specificity is generally strongly negative, causing overestimation of the pooled results. The diagnostic odds ratio (DOR), defined as the odds of true positives vs. that of false positives, may avoid this problem. The aim of the study was to review the advantages and limitations of the DORs. Methods: A systematic review of 44 previously published diagnostic studies was used as an example. Results: DORs can be readily implemented in diagnostic research. Advantages include: (1) they adjust for the negative and curvilinear correlations between sensitivities and specificities, (2) they take account of the heterogeneity between studies with respect to the different thresholds chosen by the investigators in the original studies, and (3) it is easy to extend the model with covariates representing between-study differences in design. Limitations include: 1) the outcome parameter is a summary estimate of both sensitivity and specificity, and 2) the magnitude of the studies included is not taken into account. Conclusions: Reported sensitivities and specificities of different studies assessing similar diagnostic tests are not only negatively correlated, but also negatively correlated in a curvilinear manner. It is appropriate to take this negative curvilinear correlation into account in the data pooling of such meta-analyses. The DORs can be applied for that purpose. Clin Chem Lab Med 2009;47:1351–4.

Background: The verification/validation of laboratory test results is one of the most critical aspects of the total testing process, which may produce conflicts between competencies and duties at the point of professional crossroads. This process has centered for decades on the human component, with positive effects as well as potential adverse consequences (postanalytical errors). Manual validation of data is a time-consuming activity, is inherently subjective and arbitrary, and requires the constant presence of postgraduate physicians or biologists within the laboratory with adverse economical and organizational impacts. To overcome these inherent limitations, we have developed and implemented in our stat department an automatic system for verification, validation and delivery of laboratory results. Methods: The procedure is based on automatic validation of test results by an expert system, coupled with remote wireless connection, which allows the laboratory professional “on call” to access, visualize, analyze, validate and deliver alert values (suspect, erroneous or critical) using a small laptop. This system also provides five phases where preanalytical and analytical errors can be identified and handled. Results and conclusions: Six months following implementation of this innovative system, which can be customized to facilitate a wide variety of laboratory workflow models, the reporting efficiency of our stat laboratory has greatly improved, reducing manual data entry, and increasing the timeliness and utility of test results. Clin Chem Lab Med 2009;47:1355–60.

Background: Results from hemolyzed, icteric, and lipemic samples may be inaccurate and can lead to medical errors. These preanalytical interferences may be detected using visual or automated assessment. Visual inspection is time consuming, highly subjective and not standardized. Our aim was to assess the comparability of automated spectrophotometric detection and visual inspection of lipemic, icteric and hemolyzed samples. Methods: This study was performed on 1727 routine biochemistry serum samples. Automated detection was performed using the Olympus AU2700 analyzer. We assessed: 1) comparability of visual and automated detection of lipemic, icteric and hemolyzed samples, 2) precision of automated detection, and 3) inter-observer variability for visual inspection. Results: Weighted κ coefficients for comparability of visual and automated detection were: 0.555, 0.529 and 0.638, for lipemic, icteric and hemolyzed samples, respectively. The precision for automated detection was high for all interferences, with the exception of samples being only slightly lipemic. The best overall agreement between observers was present in assessing lipemia (mean weighted κ=0.698), whereas the lowest degree of agreement was observed in assessing icterus (mean weighted κ=0.476). Conclusions: Visual inspection of lipemic, icteric and hemolyzed samples is highly unreliable and should be replaced by automated systems that report serum indices. Clin Chem Lab Med 2009;47:1361–5.

Background : The prevalence of thyroid autoimmunity and subclinical primary hypothyroidism in persons with chronic kidney disease (CKD) not requiring chronic dialysis is not well defined. Methods : We studied 1000 consecutive adult outpatients who were referred by their general practitioner for blood testing over the last 2 years. We excluded those with abnormal serum free thyroxine (FT4) levels (n=85). No participants required chronic renal replacement therapy. Thyroid autoimmunity was defined as increased concentrations of serum anti-thyroid antibodies. Subclinical primary hypothyroidism was defined as a serum thyrotropin (TSH) concentration >4 mIU/L. CKD was defined as an estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m 2 . Results : Overall, 53 (5.8%) subjects had eGFR <60 mL/min/1.73 m 2 . Of these, 98 (10.7%) had subclinical hypothyroidism, and 213 (23.3%) subjects had increased anti-thyroid antibodies. Approximately 26% and 34% of those with eGFR <60 mL/min/1.73 m 2 had laboratory evidence of subclinical hypothyroidism or thyroid autoimmunity, respectively. In subgroup analysis stratified by TSH and thyroid autoimmunity, decreasing eGFR values appeared to be more strongly related with the increase in TSH rather than antithyroid antibodies. Conclusions : Thyroid autoimmunity and subclinical primary hypothyroidism are highly prevalent in persons with CKD not requiring chronic dialysis. Clin Chem Lab Med 2009;47:1367–71.

Background: The goal of this study was to detect modification in the expression of plasma proteins and/or post-translational modifications of their structure in patients with end stage renal disease. Methods: Serum samples from 19 adult patients treated by maintenance hemodialysis (MHD) were analyzed in comparison to sera from six healthy controls using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Spots of interest were identified by mass spectrometry analysis. In addition, the 2DE maps were incubated with a human anti-albumin polyclonal antibody. Results: SDS-PAGE gels, 2DE maps and matrix-assisted laser desorption/ionization time of flight analysis indicated over-expression of low-molecular weight proteins (LMWP) in sera from patients. Unexpectedly, another 15 spots with estimated M r of 12.5–29 kDa from the 2DE maps of six patients were identified as fragments of albumin. 2D immunoblotting of sera from 12 other patients detected numerous albumin fragments. Conclusions: These results indicate that in addition to increased expression of LMWP, a relevant amount of albumin fragments are detectable in the serum of patients undergoing MHD. Uremia appears to facilitate the fragmentation of albumin and/or the retention of albumin fragments in blood. Clin Chem Lab Med 2009;47:1373–9.

Background: Vitamin B12 deficiency and bone fractures are common in vegetarians. However, a direct relationship between vitamin B12 status and bone metabolism in vegetarians has not been tested sufficiently. Methods: Our study included 96 vegetarians (23 German vegans, and 54 German and 19 Indian lacto-, lacto-ovo-vegetarians) and 89 omnivores (Germans and Asian-Indian immigrants in Oman). Blood concentrations of total vitamin B12, holotranscobalamin (holoTC), 25OH-vitamin D (25(OH)D), total homocysteine (tHcy), methylmalonic acid (MMA), and the bone turnover markers (BTMs) bone alkaline phosphatase (BAP), osteocalcin (OC), pro-collagen type I N-terminal peptide (PINP) and C-terminal telopeptides of collagen I (CTx) were measured. Results: Vegetarians from both population groups exhibited significantly higher concentrations of tHcy, MMA, folate, and BAP, but lower concentrations of holoTC and cobalamin compared with omnivores from the same population. Additionally, German vegetarians had higher circulating activities of BAP as well as higher CTx, OC, and PINP compared with their omnivorous controls. HoloTC and MMA were correlated with OC, CTx and BAP. Subjects with low vitamin B12 status (holoTC ≤35 pmol/L and MMA >271 nmol/L) had significantly lower serum concentrations of 25(OH)D, but higher tHcy and the BTMs P1NP, BAP, OC, and CTx, compared with subjects with normal vitamin B12 status. Multiple regression analysis showed that the association between BTMs and markers of vitamin B12 status was independent from the association with 25(OH)D. Approximately 12%–14% of the variation in the concentration of BTMs was explained by a regression model including holoTC, MMA and 25(OH)D. The strictness of the diet was not related to the magnitude of change in BTMs. Conclusions: Low vitamin B12 status is related to increased bone turnover in vegetarians which is independent from vitamin D status. Clin Chem Lab Med 2009;47:1381–7.

Background: We evaluated prospectively the predictive value of serum apolipoprotein (apo) A-II, the second major apolipoprotein of high-density lipoprotein (HDL), for cardiometabolic risk in Turkish adults showing abnormalities in other proteins that normally confer protection. Methods: Determinants of apoA-II and its associations with coronary heart disease (CHD), metabolic syndrome (MetS) and diabetes were investigated at 4 years follow-up in 193 elderly men and women. Results: ApoA-II concentrations at baseline, in addition to being significantly related to HDL-cholesterol, were directly associated with complement C3 in multivariate linear regression analyses comprising nine variables. Following adjustment for gender, age and HDL-cholesterol (>30/>33 g/L, in men and women, respectively), low serum apoA-II concentrations predicted incident MetS [relative risk (RR) 3.5 (95% CI 1.4; 8.6)] and type 2 diabetes [RR 4.5 (95% CI 1.3; 15.6)] in both genders at an increment of 1 SD. Increased apoA-II values were not associated with prevalent or incident CHD, and tended to be marginally atheroprotective only in males. Conclusions: Serum apoA-II concentrations confer risk for MetS and diabetes and exhibit evidence of anti-inflammatory properties among Turks. These findings support the effects seen for several other HDL protein constituents. This finding may explain the increased cardiometabolic risk among Turks. Clin Chem Lab Med 2009;47:1389–94.

Background : Multiple sclerosis (MS) is a frequent and often severe autoimmune disease of the central nervous system. We describe a newly developed enzyme-linked immunosorbent assay (ELISA)-based test system for the assessment of neuronal autoantibodies in serum and cerebrospinal fluid (CSF). This tool could help define autoimmune status and thus be a potential means of therapeutic surveillance. Methods : We used an assay system (ELISA, E100, Mediagnost) based on purified bovine antigens [myelin-oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipid protein (PLP) and alpha-B-crystalline (CRY)] antibodies for the measurement of specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies. Assay characteristics and preliminary validation were conducted by measurement of serum and CSF samples from 41 MS patients and 128 patients with other neurological diseases (OND). Results : We measured the inter- (17.8/10.1%) and intra-assay variability (5.5/6.7%); linearity (1:250– 1:16,000), and specificity of IgG and IgM. We demonstrate that by the results of this test system MS patients can be differentiated from patients with OND. Conclusions : The ELISA kit we evaluated is suitable for the measurement of neuronal autoantibodies. The initial validation demonstrates its potential use in the differential diagnosis of central neuronal system diseases. Clin Chem Lab Med 2009;47:1395–400.

Background : The International Consensus Group for Hematology Review recommends manual blood smears for new findings from automated complete blood cell (CBC) tests. However, in outpatients, the rate of peripheral manual blood smear reviews is uncertain, as is the effect of historical results on this rate. Methods : In a prospective cohort study of 687,955 members of a health maintenance organization, we extracted 39,759 consecutive automated CBC test results and determined the rate of manual peripheral smear reviews that were indicated using different triggering criteria, and historical results. Results : Individual triggering values increased from 10.7% to 19.5% when comparing our criteria to those of the consensus criteria. The proportion of CBC test results with at least one triggering value was 7.2% using our criteria, compared to 13.9% with the inclusion of a mean corpuscular volume <75 and a plus one immature granulocyte flag according to the consensus committee. Availability of historical data decreased our peripheral smear review rate to 2.6%. Conclusions : The peripheral smear review rate in outpatients using the consensus criteria was higher than with our less stringent criteria. If historical data are available and no clinical utility is demonstrated for the additional consensus criteria, an acceptable out-patient review rate might be as low as 2.6%. Clin Chem Lab Med 2009;47:1401–5.

Background : Query-fever (Q-fever) is a zoonotic infection caused by the intracellular Gram-negative coccobacillus Coxiella burnetii . A large ongoing outbreak of Q-fever has been reported in the Netherlands. We studied various markers of infection in inpatients (hospitalised) and outpatients (treated by a general physician) with acute Q-fever in relation to disease severity. Methods : Leukocyte counts, C-reactive protein (CRP) and procalcitonin (PCT) concentrations were measured in 25 inpatients and 40 outpatients upon presentation with acute Q-fever. Chest X-rays, if available, were analysed and confusion, urea, respiratory rate, blood pressure-age 65 (CURB-65) scores, indicating severity of pneumonia, were calculated. Results : CRP was the only marker that significantly differentiated between inpatients and outpatients. It was increased in all patients from both groups. Leukocyte counts and PCT concentrations did not differ between inpatients and outpatients. Overall, only 13/65 patients had an increased leukocyte count and only 11/65 patients presented with PCT concentrations indicative of possible bacterial respiratory tract infection. Infiltrative changes on the chest X-ray were observed in the majority of patients. CURB-65 score was 0±1 (mean±SD). Conclusions : Acute Q-fever, a relatively mild pneumonia with low CURB-65 scores, specifically induces a response in CRP, while PCT concentrations and leukocytes are within the normal range or increased only marginally. Clin Chem Lab Med 2009;47:1407–9.

Background: New erythrocyte parameters are reported by the Sysmex XE 5000 analyzer. This instrument measures the hemoglobin (Hb) content of individual red cells, calculates the percentage of hypochromic red cells (%Hypo He) and the percentage of hyperchromic red cells (%Hyper He) and quantifies the proportion of marginally sized erythrocytes (%Micro R and %Macro R). The goals of the study were to establish the reference range for erythrocyte extended parameters, their value in different types of anemia and to investigate their reliability in the study of disorders of iron metabolism. Materials: Three hundred and ninety samples were analyzed. The Kolmogorov-Smirnoff test, independent samples t-test and Pearson correlation were calculated; receiver operating characteristic (ROC) curve analysis was used to determine their diagnostic performance. Results: The values of the four parameters studied were normally distributed and statistically different (p<0.0001) in the different groups of patients; the only exception was %Hypo He in cases of iron deficiency and thalassemia (p=0.3758). Results of ROC curve analysis for %Hypo He in the diagnosis of restricted erythropoiesis reticulocyte Hb content (reticulocyte hemoglobin content, Ret He <29 pg) were: area under the curve 0.963; cut-off 1.8%; sensitivity 98.3%; specificity 91.1%. Conclusions: The new parameters appear to be sensitive for detecting small changes in the number of red cells with inadequate hemoglobinization and volume. Clin Chem Lab Med 2009;47:1411–6.

Background: C-reactive protein (CRP) is able to bind phospholipids (mainly phosphocholine) in the presence of calcium ions. We investigated the use of this property for developing an affordable turbidimetrical CRP assay based on diluted soy oil. Methods : Serum (or heparinized plasma) was mixed with Intralipid ® 20% in Tris-calcium buffer (pH 7.5). After 30 min of incubation at 37°C, the CRP-phospholipids complexes were measured by turbidimetry (660 nm/700 nm) with a Cobas 6000 analyzer (Roche). Results were compared with those obtained using a typical immunoturbidimetric method (Roche). Results: Good correlation (r 2 =0.931) was obtained between the functional and the immunoturbidimetric CRP assay. Within-run and between-run %CV values for the functional assay were 2.4% (100 mg/L); 6.0% (50 mg/L); 10% (20 mg/L), and 3.6% (100 mg/L); 8.0% (50 mg/L); 11% (20 mg/L), respectively. The limit of detection was 7 mg/L. Results were not affected by serum calcium, triglyceride, or phospholipid concentrations. Conclusions: The functional CRP assay allowed measurement of CRP in serum and plasma in the range of 7 mg/L–400 mg/L. The assay is particularly suited in conditions where resources are restricted. Since the assay is species independent, the described functional CRP assay could be used for veterinary purposes as well. Clin Chem Lab Med 2009;47:1417–22.

Background : Most screening programs for sickle cell disease (SCD) utilize isoelectric focusing (IEF) or high performance liquid chromatography (HPLC) to detect haemoglobin (Hb) variants. The first method is not automated and becomes too tedious when many samples have to be investigated. The aim of this work is to explore the capacity of an automated capillary electrophoresis (CE) system, with full traceability, as a tool for newborn screening of SCD. Methods : The Capillarys ® neonat fast automated system has been developed by Sebia for newborn screening. We performed separate studies using different types of samples to evaluate the utility of the Capillarys ® for (i) separating Hb S and other variants, and (ii) for performing the routine activity of our laboratory for 20 working days. Results : A throughput of 48 samples per hour with a loading capacity of 192 samples was achieved. Migration times of the major Hb variants were distinct. There were few variants showing similar migration times to Hb S and Hb C and thalassaemia could be detected. In addition, late screening, screening of premature or transfused babies and screening performed using poor quality Guthrie's cards did not interfere with reporting of accurate phenotypes. Conclusions : Sebia Capillarys ® neonat fast automated system is a reliable tool for haemoglobinopathy neonatal screening. Clin Chem Lab Med 2009;47:1423–32.

Background : Many factors have been implicated in the pathogenesis of unexplained recurrent spontaneous miscarriage (URSM). Methods : The objective of this preliminary clinical study was to verify whether intravaginal sildenafil citrate tablets (25 mg intravaginally, 4 times/day for 24 days) are effective as a novel treatment of threatened miscarriage in patients with a history of URSM (n=4). We also evaluated changes in the concentrations of some antioxidants, the percent expression of cells positive for tumor necrosis factor-α (TNF-α), CD56+T-cells [CD3+CD56+ natural killer cells (NKT cells) (cluster differentiation 3, 56 NKT cells)], which express one of the NK cell markers, and uterine artery pulsatility index (PI). We compared these results with healthy women in their first trimester of pregnancy (FTP n=4). Results : Sildenafil improved the measured antioxidants concentrations [total antioxidant capacity (TAC), glutathione S-transferase (GSH-S-T) and catalase (CAT), superoxide dismutase (SOD)], and improved oxidative stress by reducing malondialdehyde (MDA) and nitric oxide (NO) in the four patients under investigation. The improvement in antioxidants was close to concentrations seen in women in their FTP. The four patients had progressed to their third trimester of pregnancy using this novel application. The percentage of [CD3+CD56+ NKT cells] and TNF-α in peripheral blood was higher in URSM patients compared to women in their FTP. However, concentrations were greatly reduced and improved after sildenafil administration. We demonstrated that intravaginal sildenafil citrate tablets can reduce vasoconstriction by increasing blood flow through relaxation of uterine arteries as indicated from measured PI in URSM patients. Conclusions : We conclude that intravaginal sildenafil citrate tablets used as suppositories might be a novel, interesting, safe antiabortive option in the treatment of threatened miscarriage in patients with a history of URSM. We are the first to report this novel clinical application in four case reports in Egypt. Clin Chem Lab Med 2009;47:1433–8.

Background: Insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) belong to a group of hypoxia related proteins. IGF-I induces expression of VEGF and decomposes wild type p53 in cancer cell lines. The goal of our study was to evaluate serum IGF-I, VEGF and p53 with respect to overall and disease free survival of patients with colorectal cancer (CRC) patients compared with healthy volunteers. Methods: Preoperative blood samples from 125 patients with CRC and 16 healthy volunteers were examined using ELISA for serum IGF-I, p53 and VEGF concentrations. Results: Concentrations of p53 and VEGF were significantly higher in CRC patients than in controls (p<0.0006 and p<0.0001, respectively). IGF-I was not statistically different between both groups. Serum IGF-I showed negative correlation with p53 in CRC patients (p<0.04, r=−0.193). IGF-I and VEGF showed negative correlation in poorly differentiated cancers (G3) (p<0.03, r=−0.339). Patients with VEGF concentrations that were above average for the cancer population survived for a shorter period of time (p=0.065 in evaluation of overall survival and 0.071 in estimation of disease-free survival during a 3-year follow-up) compared with patients with serum VEGF lower than the highest values seen in controls. Conclusions: Comparisons between serum IGF-I and p53 appear to confirm the metabolism of p53 by IGF-I. Serum VEGF showed prognostic significance in our study. Serum concentrations of IGF-I and VEGF did not show positive correlation, as expected due to IGF-I induction of VEGF in malignant colon cell lines. Clin Chem Lab Med 2009;47:1439–45.