Volume 47, Issue 8

Neutrophilic granulocytes express Fcγ receptor (FcγR) [cluster of differentiation 64 (CD64) antigen] only when they are activated. Neutrophil CD64, demonstrated using flow cytometry, can be used as a diagnostic marker of infection and sepsis. Neutrophil CD64 is superior to C-reactive protein and hematological determinations for detecting systemic infection or sepsis, since it combines high sensitivity (90% or more) with high specificity (90%–100%) in both adults and children. In addition, the test performs well in distinguishing infection from flares in autoimmune inflammatory diseases and has somewhat more limited utility for differentiating bacterial from viral infection. This review summarizes the available literature regarding CD64 as a marker of infection, and outlines future clinical studies for confirming the diagnostic performance of this promising marker. Clin Chem Lab Med 2009;47:903–16.

Background: rs1333049 polymorphism on chromosome 9p21 has been shown to affect susceptibility to coronary artery disease (CAD) in Caucasians. This study examined the association of rs1333049 with myocardial infarction (MI), angiographic severity of CAD and clinical outcome after a first acute MI in Han Chinese. Methods: rs1333049 polymorphism was genotyped in 520 patients with a first acute MI and in 560 controls. The number of angiographically documented diseased coronary arteries (luminal diameter stenosis ≥50%), echocardiographic left ventricular ejection fraction (LVEF), and major adverse cardiac events (MACE) during follow-up (mean, 29±15 months) were recorded. Results: Patients with MI had higher frequencies of the CC genotype (30.0% vs. 20.7%) or C allele (55.5% vs. 46.2%) compared with controls (all p<0.01). rs1333049 polymorphism was strongly associated with MI [odds ratio (OR) 1.48, 95% confidence interval (CI) 1.22–1.79] after adjusting for traditional risk factors. Although longer hospitalization stay was observed in patients with the rs1333049- C allele, this polymorphism was not related to angiographic severity of CAD, LVEF, and occurrence of MACE after MI. Conclusions: This study demonstrates an association of rs1333049 polymorphism locus on chromosome 9p21 with risk for MI, but not with post-MI prognosis in Han Chinese. Clin Chem Lab Med 2009;47:917–22.

Background: The pathogenesis of pre-eclampsia (PE) is incompletely understood. The placenta is considered to play a key role in this disease. Recent research showed that many microRNAs (miRNAs) are expressed in human placenta. Our aim in this study was to determine differential expression of miRNAs in placenta with severe PE, and normal placenta. Methods: Differential expression of miRNAs in placenta (four severe PE and a control group of four normal pregnant women) was first screened using microarray analysis. Following this, some differential miRNAs were selected and validated using real-time quantitative reverse transcription-polymerase chain reaction in placenta from women with severe PE (n=24), and a healthy control group (n=26). Results: We found the following miRNAs were significantly increased in placenta from women with severe PE: miR-16, miR-29b, miR-195, miR-26b, miR-181a, miR-335 and miR-222. Gene ontology analysis of the target genes revealed enrichment for specific biological process categories, i.e., regulation of cellular physiological process including miR-16, miR-29b, miR-195, miR-26b and miR-335, and signal transduction including miR-181a and miR-222. Conclusions: These different miRNAs may play an important role in pathogenesis of PE and may become diagnostic markers for PE. Clin Chem Lab Med 2009;47:923–9.

Background: Hereditary tyrosinemia type 1 (HT1; MIM 276700) is caused by mutations in the fumarylaceto-acetate hydrolase ( FAH ) gene, and is the most severe disorder associated with the tyrosine catabolic pathway. HT1 is a very rare disorder and no genetically confirmed case of HT1 in Korea has yet been reported. In this study, we present a Korean neonate with clinical and biochemical features of HT1. Methods: A female neonate was admitted to our hospital for further work-up of an abnormal newborn screening test. We analyzed amino acids and organic acids in the patient's blood and urine. To confirm the presence of the genetic abnormality, all the coding exons of the FAH gene and the flanking introns were amplified by polymerase chain reaction (PCR). Results: The patient's newborn screening test revealed increased concentrations of methionine and tyrosine. Subsequent urine organic acid analysis showed increased urinary excretion of 4-hydroxyphenyllactate, 4-hydroxyphenylpyruvate, succinate, and succinylacetone. Gap-PCR and sequence analysis of the FAH gene revealed a homozygous large deletion mutation encompassing exons 12–14. The patient's parents were not consanguineous but were heterozygous carriers of the same mutation. Conclusions: The patient had a novel, large deletion mutation of FAH and is the first report of genetically confirmed HT1 in Korea. Clin Chem Lab Med 2009;47:930–3.

Background: In vitro hemolysis, the prevailing cause of preanalytical error in routine laboratory diagnostics, might influence the reliability of several tests, affect the quality of the total testing process and jeopardize patient safety. Although laboratory instrumentation is now routinely equipped with systems capable of automatically testing and eventually correcting for hemolysis interference, to our knowledge there are no reports that have compared the efficiency of different analytical platforms for identifying and classifying specimens with hemolysis. Methods: Serum from a healthy volunteer was spiked with varying amounts of hemolyzed blood from the same volunteer, providing a serum free hemoglobin concentration ranging from 0.0 g/L to 2.0 g/L as measured by the reference cyanmethemoglobin assay. The spiked serum samples were shipped to seven separate laboratories and the hemolysis index (HI) was tested in triplicate on the following analytical platforms: Roche Modular System P (n=4) and Integra 400 Plus (n=1), Siemens Dimension RxL (n=3), ADVIA 2400 (n=1) and ADVIA 1800 (n=1), Olympus AU 680 (n=1) and Coulter DXC 800 (n=1). Results: Satisfactory agreement of HI results was observed among the various analytical platforms, despite a trend toward overestimation by the ADVIA 2400 and 1800. After normalizing results according to the instrument-specific alert value, discrepancies were considerably reduced. All instruments except for the Dimension RxL gave values normalized to the instrument-specific alert value, <1.0 for the sample with 0.048 g/L free hemoglobin, and >1.0 for the sample with 0.075 g/L free hemoglobin. The results of the four Modular System P tests were also highly reproducible among the different facilities. When evaluating instruments that provided quantitative HI results, the mean intra-assay coefficient of variation (CV) calculated for the triplicate determinations was always between 0.1% and 2.7%. Conclusions: The results of this multicenter evaluation confirm that efficiency of different analytical platforms to correctly identify and classify unsuitable samples is satisfactory. However, more effort should be placed on the standardization of reporting HI. All the instruments that we tested provide either quantitative or qualitative results that are essentially comparable, but which should always be compared with the instrument-specific alert values to harmonize their efficiency. Clin Chem Lab Med 2009;47:934–9.

Background: Haemolysis is usually caused by inadequate specimen collection or preanalytical handling, and is suggested to be a suitable indicator of pre-analytical quality. We investigated the prevalence of detectable haemolysis in all routine venous blood samples to identify differences in preanalytical quality. Methods: Haemolysis index (HI) values were obtained from a Vitros 5,1 in the routine clinical chemistry laboratory for samples collected in primary health care centres (PHCs), nursing homes, and a hospital emergency department (ED). Haemolysis was defined as a HI ≥15 (detection limit). Results: Samples from the PHC with the highest prevalence of haemolysis were 6.1 times (95% confidence interval (CI) 4.0–9.2) more often haemolysed compared to the centre with the lowest prevalence. Of the samples collected in primary health care, 10.4% were haemolysed compared to 31.1% in the ED (p<0.001). A notable difference in haemolysed samples was found between the ED section staffed by emergency medicine physicians and the section staffed by primary health care physicians (34.8% vs. 11.3%, p<0.001). Conclusions: The significant variation in haemolysis indices among the investigated units is likely to reflect varying preanalytical conditions. The HI is a valuable tool for estimation and follow-up of preanalytical quality in primary health care. Clin Chem Lab Med 2009;47:940–4.

Background: Widespread use of D-dimer in recent years has led to the development of a number of new fully automated quantitative D-dimer assays. Methods: We evaluated the analytical performance of the particle-enhanced immunoturbidimetric assay Innovance D-DIMER (Siemens Medical Solutions) on the Behring Coagulation System (BCS) analyzer. Results: Within-run coefficients of variation (CVs) for samples with low, borderline, slightly, and extremely increased D-dimer concentrations were 2.1%–5.5%, whereas between-run CVs for control samples with low and extremely increased D-dimer were 5.5%–8.4%. The assay exhibited good linearity in the working range between 0.17 mg/L and 5.45 mg/L fibrinogen equivalent units (FEU), with the lower limit of detection of 0.099 mg/L FEU. The upper reference value determined in 40 plasma samples from healthy volunteers was 0.495 mg/L FEU. The results obtained in 457 fresh plasma samples were compared with results obtained with VIDAS D-Dimer Exclusion. Passing and Bablok regression analysis demonstrated highly significant correlation (y=1.370x–0.108, r=0.952, p<0.001). Bland and Altman difference plots demonstrated slightly higher results obtained with Innovance D-DIMER that was more pronounced with increasing values. Very good agreement between both assays was observed (κ=0.860; 95% confidence interval (CI), 0.811–0.908). Conclusions: This study demonstrates that Innovance D-DIMER fulfills all analytical requirements for daily routine use. Clin Chem Lab Med 2009;47:945–51.

Background: To examine the prevalence of significant interference from heterophile antibodies (HAb) in the measurement of serum thyroglobulin (Tg), we evaluated a large cohort of samples from patients with differentiated thyroid carcinoma (DTC). Methods: Serum Tg measurements were performed in 406 serum samples before and after incubation of each serum sample in heterophile-blocking tubes (HBT) at room temperature for 1 h. We calculated the difference between the original Tg value and the value obtained after HBT treatment. We considered any sample showing an absolute percent difference >3 SD from the mean percent difference as being affected by HAb interference. Results: We identified five patients (1%) as showing interference from HAb. Of these, three (60%) showed a false positive or falsely increased Tg concentration without any recurrence following clinical work-up; two (40%) showed a false negative or falsely reduced Tg levels, and metastases were detected in both cases by imaging procedures. Conclusions: HAb may increase as well as reduce the measured Tg in a significant number of patients. A positive HAb interference should be suspected if Tg elevation does not fit the clinical pictures. A negative interference is a more challenging problem because increases in Tg generally occur as the first sign of recurrence of DTC. Therefore, treatment using HBT tubes of all sera referred for Tg measurement should be considered in order to prevent both unwarranted investigations or therapy, and delayed diagnosis of recurrence in patients affected by DTC. Clin Chem Lab Med 2009;47:952–4.

Background: The aim of the present study was to analyze variations in permeability of albumin and immunoglobulin G (IgG) influx into cerebrospinal fluid (CSF) in a clinical setting. Methods: In a retrospective intra-individual comparison of CSF samples, we used the IgGIndex and its constituents to indicate alterations in IgG/albumin permeability. Results: We found altered IgGIndices in 25/64 patients (range –25% to +44%), with differently altered QAlb and QIgG values (–69% to +549%), unaltered IgG-Indices in a further 25/64 patients with equally altered QAlb and QIgG values (–46% to +107%), and no parameter alteration in 14/64 patients. Parameter alterations in 25/64 patients indicated that permeability of albumin was changed to different extents than for IgG. It changed in the same direction in 20/25 patients, and the opposite in five patients. In further 25/64 patients, equal QAlb and QIgG alterations indicated equally altered permeabilities and/or altered efflux of the proteins. In 14/64 patients, no alteration in permeability or efflux was seen. Conclusions: Results revealed surprisingly variable intra-individual changes in permeabilities for albumin and IgG in pathologic as well as normal CSF. Differing changes in permeability indicate that the diffusion paths of the two proteins may react to disturbances independently of each other. The details of the influx permeability for albumin and IgG into CSF illustrate the prospect of a more comprehensive insight into the protein exchange between blood and CSF. Clin Chem Lab Med 2009;47:955–8.

Background: Patients with end-stage renal disease (ESRD) and on hemodialysis (HD) are at increased risk for developing rheumatoid arthritis (RA), as a result of defective immunity. Our aim was to examine if ESRD and the length of HD treatment impact the clinical utility of antibodies to cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) as diagnostic tools for RA. Methods: We included 94 subjects in our study: 37 healthy volunteers and 57 patients with ESRD who had been undergoing HD for 1–12 years, and without confirmed RA. In order to test our hypothesis, we measured and correlated anti-CCP and RF as laboratory markers of RA. Results: Our study showed that there is no significant difference between values for anti-CCP (p=0.11) and RF (p=0.98) in control subjects as well as in patients undergoing HD, regardless of the length of time that patients had been undergoing HD treatment. Conclusions: Our study indicates that HD does not impair the specificity of anti-CCP and RF for RA in patients where the disease has not yet developed. Future prospective studies may show whether there is any use in determinating RF, and especially anti-CCP, as early predictors of RA in patients with ESRD who are at greater risk of developing this condition. Clin Chem Lab Med 2009;47:959–62.

Background: Interpretative commenting is an important activity of the clinical diagnostic laboratory. We describe a study of interpretative commenting abilities among senior laboratory professionals in the Asia-Pacific region and Africa. Methods: Five sets of laboratory results reflecting common and important problems encountered in clinical chemistry were distributed at 4-weekly intervals to 31 registered participants from countries in the Asia-Pacific region and Africa. Participants were asked to attach an interpretative comment to the results assuming that the requesting doctor had asked for an interpretation of the result. Results: Twelve pathologists and 19 scientists from seven countries registered to participate and the overall reply rate was ∼50% for the five cases. The quality of the comments returned by participants was diverse and some reflected incorrect or misleading interpretation and advice. Conclusions: While interpretative commenting is an important laboratory activity, the results of this study suggest that there is room for improvement in the quality of interpretative comments offered by senior laboratory professionals, even for commonly reported results relating to most prevalent and important public health conditions. Interpretative commenting should be formally taught during training of pathologists and scientists, and continuing professional development in this area is required for the provision of a quality interpretative service. Clin Chem Lab Med 2009;47:963–70.

Background: Recent guidelines recommend monitoring for opioid and drug use in patients with chronic pain to detect use of undisclosed or illicit substances, and to determine compliance with prescribed medications. We examined the technical and clinical impact of drug testing for patients on chronic pain therapy in our clinical laboratory. Methods: Testing volumes were obtained for 4 years. Volumes from three recent months were also examined in detail to determine average turnaround time, assay performance (i.e., number of positive screening and confirmatory tests and testing discrepancies) and consistency of patient results with medication history. Results: Our testing volume continues to grow significantly, especially in the primary care setting, with an average yearly increase of 34%. Approximately 70% of patients confirmed positive for an opioid. Other drugs were positive in <30% of patients. Twenty-nine percent of patients tested positive for a medication without a prescription. Overall, the compliance rate was 85% indicating that 15% of patients had negative test results despite being prescribed a chronic pain medication. Conclusions: Clinical laboratories should consider the impact of these guidelines and examine options to optimize testing such as limiting or modifying the test panel. Clin Chem Lab Med 2009;47:971–6.

Background: The epidermal growth factor receptor HER2 is overexpressed or amplified in 25%–30% of patients with breast cancer. The mechanism behind HER2 amplification is unknown, but may be a patho-physiological phenomenon caused by continuous stimulation and activation of the HER1-4 system. We have mapped the protein concentrations of HER1-4 in breast cancer tissue, autologous reference tissue, normal breast tissue and serum samples, to see whether non-cancer cells from these patients express a protein profile indicating general activation. Methods: Tissue samples from malignant and adjacent normal breast tissue (autologous reference tissue) were collected from 118 women consecutively admitted for surgical treatment of breast cancer. In addition, 26 samples of normal breast tissue were collected from healthy women having breast reduction surgery. The tissue samples were homogenized and the proteins extracted. The tissue and serum concentrations of HER1-4 were determined quantitatively using a commercially available enzyme linked immunosorbent assay (ELISA) method. Results: HER1 was down regulated in cancer tissue when compared to autologous reference tissue (p=8×10 −6 ), while HER2 (p<10 −7 ) and HER3 (p=3×10 −5 ) were up regulated. Comparing autologous reference tissue with normal tissue showed down regulation of HER1 (p=0.122) and up regulation of HER2 (p=10 −6 ), HER3 (p<10 −7 ) and HER4 (p<10 −7 ). Furthermore, we observed that correlations between the receptor combinations HER1-2, HER1-3 and HER1-4 were maintained from normal breast tissue to autologous reference breast tissue, but were lost in cancer tissue. Conclusions: We suggest that these findings indicate that breast cancer is a systemic disease where the HER1-4 system in autologous reference tissue is continuously activated, thus favoring the subsequent development of cancer. Clin Chem Lab Med 2009;47:977–84.

Background : Quality control results for serum MUC-1/CA 15-3 assays have always shown large discrepancies. Methods : This multicentre study of 15 methods (labelled M1-M15) measured coded sera from 35 patients with breast cancer without recurrence (group 1), 46 patients at 1st metastasis (group 2), and 39 patients with advanced metastases (group 3). Results were compared using parametric statistics, ANOVA, principal component analysis, and receiver operating characteristic (ROC) curves. Results : Mean MUC-1/CA 15-3 concentrations varied widely (75.1–303.0 U/mL, 24.8%) among methods. The false positive (FP) rate for group 1 was 8/521 (1.5%); for group 2 and group 3 false negative (FN) results were 21/680 (3.1%) and 11/583 (1.9%), respectively. Using the ROC cut-offs, we found no FPs for group 1 and no FNs for group 3. However, group 2 showed 16 FNs. All p-values for Pearson's correlation were <0.0001 between methods, except for M11. When comparing methods using different antibodies, discordance rates reached a maximum of 15.2%. Principal component analysis revealed a grouping of methods using: CanAg monoclonal antibodies (mAbs) (M2, M7 and M12); Centocor/Fujirebio mAbs (M3-M6, M8-M10, M14-M15) and Biomira mAbs (M1 and M13); and Centocor/Fujirebio mAbs (M11). Conclusions : Results were more consistent among methods using the same antibody type. Principal component analysis showed that antibody type was the strongest determinant of immunoassay results. Clin Chem Lab Med 2009;47:985–92.

Background: Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play an important role in tumor invasion and metastasis. This study aimed to determine the serum levels of MMP-2, MMP-9, 130- and 225-kDa gelatinolytic bands and conventional tumor markers, carcinoembryonic antigen (CEA) and cancer antigen (CA) 19-9, in patients with gastrointestinal cancers. The relationship between these parameters and clinicopathological factors was also studied. Methods: Sera from controls (n=19), and patients with colorectal (n=47) and gastric (n=34) cancer were collected prospectively. The gelatinolytic activities of MMP-2, MMP-9, 130- and 225-kDa bands were determined using gelatin zymography. CEA and CA 19-9 were determined using immunoradiometric assay (IRMA). Results: Serum levels of MMP-9, 130- and 225-kDa gelatinolytic bands, CEA, and CA 19-9, but not MMP-2, in colorectal and gastric cancer were significantly higher than that of controls. No significant correlation was found between histological grade or clinical stage and levels of MMP-9, 130- and 225-kDa gelatinolytic bands, which were correlated (r=0.61–0.89, p<0.005). Conclusions: Our findings suggest that zymographic determination of MMP-9, 130- and 225-kDa gelatinolytic bands in colorectal and gastric cancer may be useful in studying these types of cancer in parallel with conventional tumor markers. Clin Chem Lab Med 2009;47:993–1000.

Background: Thyroglobulin (Tg) measurement following thyrotropin (TSH) stimulation is used in the follow-up of patients with differentiated thyroid carcinoma (DTC). However, high-sensitive assays allow accurate measurement of serum Tg even without TSH stimulation. Here, we prospectively evaluated the impact of unstimulated high-sensitive Tg measurement in early and long-term outcome of patients with DTC. Methods: One hundred and ninety five patients affected with DTC were evaluated. Six months after thyroid ablation (i.e., thyroidectomy plus radioiodine) serum Tg was measured during TSH-suppressive thyroxine (T4) treatment (onT4-Tg). Patients with undetectable onT4-Tg and negative neck ultrasound (US) were considered disease free and onT4-Tg was measured every 12 months for a mean follow-up of 6.8 (4.7–8.9) years. Patients with an increase in onT4-Tg underwent specific diagnostic work-up and appropriate treatment if necessary. Results: Four patients showed recurrence at first follow-up visit with a corresponding increase in onT4-Tg concentrations (sensitivity 100%). Three patients had false positive onT4-Tg measurement (specificity 98%) with a spontaneous decrease within 3–6 months in all cases (specificity 100%). Three of 188 patients with undetectable serum onT4-Tg at first follow-up showed recurrence later with an increase in onT4-Tg as the first (n=2) or unique (n=1) sign of relapse (sensitivity 100%). Among 185 disease-free patients in a prolonged follow-up, 12 had a transient increase in onT4-Tg (specificity 91.6%). However, a spontaneous reduction within 3–6 months occurred in all cases (specificity 100%). Conclusions: Undetectable serum onT4-Tg using a high-sensitivity immunoradiometric assay 6 months after thyroid ablation predicts low-risk of DTC recurrence. When onT4-Tg became detectable during follow-up, the evaluation of Tg slope in a 3–6 months period accurately discriminated patients with DTC recurrence from those without recurrence. This helped avoid unnecessary diagnostic or therapeutic procedures. Clin Chem Lab Med 2009;47:1001–4.

Background: Although there have been some epidemiological studies on the effects of diet and nutritional status on cervical carcinogenesis, evidence for a protective effect of antioxidant micronutrients against cervical neoplasia is insufficient. The relationship between serum antioxidant micronutrients and sociodemographic factors and the risk of cervical neoplasia was investigated in this multi-center, case-control study. Methods: The study population included women with histopathological diagnosis of cervical intraepithelial neoplasia (CIN) 1 (n=147), CIN 2/3 (n=177), cervical cancer (n=160), and a control group (n=378). Epidemiological data were collected and the serum concentrations of β-carotene, lycopene, zeaxanthin plus lutein, retinol, α-tocopherol, and γ-tocopherol were measured using reverse-phase, gradient high-pressure liquid chromatography. Results: Cervical cancer was found to be associated with older age, increased body mass index, and lower socioeconomic status as measured by education level and income. The mean serum concentrations of β-carotene, lycopene, zeaxanthin plus lutein, retinol, α-tocopherol, and γ-tocopherol of cervical cancer patients were significantly lower than those of control subjects. Odds ratio adjusted for age, smoking status, alcohol consumption, and human papillomavirus infection status revealed a significant gradient of decreasing risk of CIN 1, CIN 2/3, and cervical cancer with increasing serum concentrations of most antioxidant micronutrients. Conclusions: The results of this study show an inverse association between serum antioxidant micronutrient concentrations and the risk of cervical neoplasia. These results suggest that antioxidant micronutrients play a role in the prevention of cervical carcinogenesis. Clin Chem Lab Med 2009;47:1005–12.