Volume 46, Issue 3

Background : Single nucleotide polymorphisms (SNPs) of mitochondrial DNA (mtDNA) are involved in physiological and pathological conditions. We developed a rapid, accurate, highly sensitive and high-throughput approach with low cost to identify mtDNA SNPs. Methods : Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect 18 SNPs of mtDNA by uniplex and multiplex assays. The sensitivity and specificity of the MALDI-TOF MS were evaluated. The accuracy of the approach was validated by the comparison of using the robust sequencing analysis. Results : The detection limit achieved with the assays corresponded to the identification of five-genome equivalence of mtDNA per reaction after first round PCR amplification. The testing system enabled the discrimination of as little as 5% of mtDNA polymorphism in the predominating background of mtDNA not containing the SNP. No false positive and false negative results were obtained using the uniplex and multiplex MALDI-TOF MS assays for the analysis of the 18 SNPs compared with those obtained by sequencing analysis. Conclusions : Possible fields which could benefit from this powerful and sensitive tool include forensic medicine, tracing of matrilineage, transplantation immunology, transfusion medicine, the diagnosis of mtDNA mutation related disorders, and the research regarding aging, apoptosis and carcinogenesis based on physiologic and pathogenic alterations of mtDNA for the analysis of large-scale samples, multiple SNPs or rare mtDNA. Clin Chem Lab Med 2008;46:299–305.

Background : Previous genomic linkage studies have produced evidence linking sodium-lithium countertransport activity (Na/Li CT) with various chromosomal regions including loci harbouring glutathione S transferase ( GST ) genes. The aim of this study was to examine the putative association of erythrocyte Na/Li CT activity with GST T1 and M1 gene null polymorphisms. Methods : Na/Li CT activity was determined in erythrocytes isolated from 85 individuals, using a standard assay procedure employing atomic absorption spectroscopy. Genotyping of the GST T1 and GST M1 null polymorphisms was accomplished with a multiplex PCR method. A general linear model using age, sex, smoking, dyslipidaemia and hypertension as covariates was used to examine the association of Na/Li CT activity with the GST T1 and GST M1 genotypes. Results : Individuals with the GST T1 null genotype displayed marginally significantly (p=0.049) lower values of Na/Li CT activity compared to those harbouring at least one copy of the GST T1 gene. The significance of this association was eliminated following adjustment for covariates (p=0.150), but survived as a trend when the sample was limited to normotensive and normolipidaemic individuals (p=0.070). No association was detected between the GST M1 null polymorphism and Na/Li CT activity. Conclusions : The suggestive association of the GST T1 null polymorphism with erythrocyte Na/Li CT activity is in line with previously published data from genetic linkage and biochemical analyses and may be of potential prognostic value as regards the behaviour of the countertransport and the development of related pathologies under conditions of oxidative insult. Clin Chem Lab Med 2008;46:306–10.

Background : The integrity of circulating cell-free DNA (cf-DNA) in serum or plasma appears to be of diagnostic and prognostic value in cancer. Here, we investigated the dynamics of serum DNA levels and the size distribution of cf-DNA during adjuvant chemotherapy of patients with breast cancer (n=73). Methods : By evaluating sera taken at the beginning and the end of the adjuvant chemotherapy, variations of serum DNA levels and the size distribution were analyzed, based on quantification of shorter apoptotic and longer non-apoptotic fragments from abundant genomic ALU fragments amplified by quantitative real-time PCR. Results : The mean DNA level did not change significantly during chemotherapy. However, individual cases revealed considerable variation in the amount of serum DNA. It increased in 43.8% of the patients, whereas it decreased in the remaining majority (56.2%). By calculating a “coefficient of variation” (both decrease and increase) in the level of total DNA and non-apoptotic DNA fragments, we compared the values at the beginning and the end of the therapy. For total DNA, the range was between 1.02- and 26-fold (mean 3.76-fold), whereas for non-apoptotic fragments it ranged from 1.01- to 73-fold (mean 6.9-fold) (p=0.033). In accordance with these findings, the integrity of serum DNA was higher in patients with increasing DNA levels and vice versa. Conclusions : Our findings suggest that non-apoptotic fragments contribute to a higher degree to the change of the DNA level during adjuvant chemotherapy. Clin Chem Lab Med 2008;46:311–7.

Background : Optimum sample quality is a crucial requirement for molecular monitoring of patients with chronic myeloid leukemia (CML) on therapy. Bedside RNA stabilization systems (e.g., PAXgene™) have been developed to inhibit RNA degradation during shipment of samples from the clinical site to the specialized laboratory. In CML, blood but not bone marrow samples have been examined using RNA stabilization in previous studies. Therefore, we sought to investigate the applicability of the PAXgene™ system for bone marrow samples in CML. Methods : Simultaneously stabilized blood and bone marrow samples were obtained from 55 imatinib-resistant CML patients to compare RNA yield and purity, expression of two housekeeping genes (total ABL and β-glucuronidase; GUS) by quantitative reverse-transcriptase polymerase chain reaction, BCR-ABL expression (ratios BCR-ABL/ABL and BCR-ABL/GUS), and BCR-ABL kinase domain mutations analyzed by denaturing high-performance liquid chromatography and direct sequencing. Results : RNA extraction revealed high-quality RNA derived from both stabilized blood and bone marrow samples. RNA yield was significantly higher in bone marrow (median 9.9 μg RNA/mL bone marrow) than in blood (median 4.3 μg RNA/mL blood) (p=0.0005). The number of housekeeping gene transcripts was comparable in blood and bone marrow (median ABL copies/2 μL cDNA 13,260 vs. 25,590; median GUS copies/2 μL cDNA 35,490 vs. 60,200; n.s.). Further, ratios BCR-ABL/ABL (blood vs. bone marrow, median 47% vs. 57%) and ratios BCR-ABL/GUS (blood vs. bone marrow, median 26% vs. 21%) were not significantly different. Results of mutation analysis corresponded in 51 out of 55 patients (93%), whereas moderate differences were observed in four patients. Conclusions : We conclude that bone marrow can be effectively stabilized using the PAXgene™ system and shows concordance with blood in terms of BCR-ABL mRNA quantification and mutation analysis in imatinib-resistant CML patients. Clin Chem Lab Med 2008;46:318–22.

Background : Alcohol is a major risk factor for susceptibility to oesophageal cancer in the South African population. The role of polymorphisms in alcohol metabolising enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH2) in predisposition of this population to oesophageal cancer is unknown. Alcohol metabolising enzymes exhibit polymorphisms that result in variant alleles with either increased or decreased activity. Methods : The role of these polymorphisms in increased risk of oesophageal cancer was investigated in 238 patients and 268 controls from Black and Mixed Ancestry South Africans, using the PCR/RFLP technique. Results : The ADH3*2/*2 genotype was significantly associated with increased risk for oesophageal cancer amongst Black subjects (odds ratio, 2.19; p=0.004). The low activity ALDH2*2 allele was significantly associated with increased risk for oesophageal cancer amongst the Black subjects (odds ratio, 2.35; p=0.0084). Conclusions : It was observed that ADH variants, ADH2*1 and ADH3*2 , were associated with increased risk for oesophageal cancer, possibly due to the tolerance of the carriers of these alleles to alcohol consumption compared to those with high activity alleles ( ADH2*2 and ADH2*3 ) which are associated with higher production of the unpleasant acetaldehyde intermediate. Clin Chem Lab Med 2008;46:323–8.

Background : Studies that considered apolipoprotein B ( APOB ) gene polymorphisms as risk factors for coronary artery disease (CAD) have reported conflicting results. We sought to analyze the association between 5′ ins/del and 3′ VNTR polymorphisms of APOB , lipid parameters and CAD risk. Methods : We recruited 251 patients with CAD, documented by coronary angiography, and 94 controls. Genotyping was performed by PCR. Lipids and apolipoproteins were measured. Results : 5′ ins/del ( ins/ins , ins/del , del/del ) and 3′ VNTR ( LL , SS , LS ) polymorphism frequencies were significantly (p<0.05) different between controls and CAD patients. LL and del/del were significantly associated with higher levels of apolipoprotein B (apoB), total cholesterol/high-density lipoprotein cholesterol ratio and apoB/apoA-I ratio (p<0.05) and with increased risk of CAD. The odds ratio for significant coronary stenosis associated with del/del was 3.2 (95% CI 1.6–36.42) (p=0.032) and with LL was 2.2 (95% CI 1.1–5.1) (p=0.042). Conclusions : The two polymorphisms exert an impact on lipid levels and contribute to the susceptibility to the development of CAD. Clin Chem Lab Med 2008;46:329–34.

Background : The presence of monoclonal immunoglobulin free light chains (FLC) in the serum is commonly associated with the gammopathies, including multiple myeloma, systemic light chain amyloidosis and non-amyloid light chain deposition disease. Although a sensitive nephelometry-based assay is used for quantification of serum FLC and patient follow-up, this method does not provide information regarding the biochemical properties of these proteins. The present study focused on the development of the procedure for isolation and biochemical characterization of monoclonal FLC in small plasma specimens from patients with these disorders. Methods : Methods used in this study were ultrafiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein elution from gel and support membranes, dialysis, lyophilization, isoelectric focusing (IEF) and Western blotting. Results : The isolation, concentration and partial purification of FLC was based on micro-preparative SDS-PAGE employing analytical scale gels. For the determination of the isoelectric point of FLC, the developed protocol included consecutive IEF, electrotransfer of IEF-separated proteins onto and elution from support membranes, and their analysis by SDS-PAGE-based Western blotting. The procedures, which require only 20–50 μL of starting plasma, allow biochemical characterization of the monomeric, dimeric and truncated forms of FLC, including their charge properties. Conclusions : The developed procedure may be applied to reveal distinguishing chemical features of FLC in serum, which could be important in predicting the pathologic form of disease, and in yielding information to better understand the mechanism(s) involved in the deposition of light chains in tissues. Clin Chem Lab Med 2008;46:335–41.

Background : 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radical decomposition in alcohol solution is widely used, characterizing plant antioxidants that can rise in serum after fruit and vegetable intake. However, this test failed reproducible results with serum due to protein precipitation. We describe the application of serum deproteinization with acetonitrile relating to the DPPH test. Methods : Assay sensitivity, linearity, repeatability and storage effect were determined in serum samples deproteinized with an equal volume of acetonitrile. Associations between the DPPH test and the ferric reducing ability of serum (FRAP) method, measuring total antioxidant potential, were evaluated in sera from 78 healthy non-smoking men. The effect of a single ingestion of 1 L of cloudy apple juice on the serum DPPH radical scavenging activity in healthy volunteers was also investigated. Results : Assay linearity was within 5–25 μL (r=0.99, p<0.01). With 25 μL-deproteinized serum, coefficient of variation was 4.2% and detection limit was 0.5% of the initial amount of decomposed DPPH radical over 30 min incubation. There was no sera activity decrease over 14 days storage at –20°C. Mean values of DPPH radical scavenging activity and FRAP obtained in human serum were 11.2±3.3% and 382.0±88.1 μmol/L, respectively. A positive significant linear correlation was observed between these two methods (r=0.42, p<0.01). Serum supplementation with 50 μmol/L of catechin, gallic acid, ascorbic acid or uric acid enhanced DPPH test results. One brisk serving of 1 L of apple juice caused a significant increment of serum DPPH radical scavenging activity (1.9±1.9%, p<0.01) in 12 healthy subjects 1 h after juice ingestion. Conclusions : Applicability of the DPPH test to deproteinized serum with acetonitrile revealed numerous advantages, validating its practicability, simplicity and cost effectiveness as a tool in the estimation of antioxidant status in humans. Clin Chem Lab Med 2008;46:342–9.

Background : The aim of our study was to evaluate, retrospectively, the frequency of anti-thyroid antibodies (ATA) in coeliac disease (CD) patients. Methods : ELISA was used to determine the frequency of anti-thyroid stimulating hormone receptor antibodies, thyroperoxidase antibodies and thyroglobulin antibodies in sera of 104 adult patients with CD. Patients were divided into three groups: group I, 56 untreated patients; group II, 21 patients on a strict gluten-free diet (GFD); and group III, 27 patients who did not comply with a GFD. Sera of 189 healthy blood donors served as controls. Results : Out of 104 patients with CD, five (4.8%) had ATA. The frequency of ATA found in the control group (1.6%) was not significantly different from that found in all CD patients. However, the frequency of ATA in CD patients on a GFD was significantly higher than that found in the control group (8.3% vs. 1.6%, p=0.03). The frequency of ATA in groups I, II and III was 1.8%, 9.5% and 7.4%, respectively. Conclusions : ATA were found in CD patients even on a GFD. Clin Chem Lab Med 2008;46:350–3.

Background : A newly developed dried serum spot (DSS) vitamin B 12 assay compares well with a conventional reference serum vitamin B 12 microbiological assay (r=0.97, n=161) and demonstrates adequate within (CV% <6) and between assay (CV% <10) reproducibility. Methods : The consistency of long-term vitamin B 12 assay performance was supported and validated using a reconstituted International Reference Serum (IRR 81/563) stored at −70°C as both whole serum aliquots and as DSS. The inclusion of such reference sera also allows accurate comparisons to be made with data from other laboratories and studies. Results : The DSS matrix displays excellent characteristics of pre-analytical serum vitamin B 12 stability at ambient temperatures with less than 5% loss of activity occurring at 4°C, 20°C and 37°C after 7 days of storage in the dark. Conclusions : These qualities underline the suitability of the DSS matrix for epidemiological screens of serum vitamin B 12 levels by obviating the need for costly refrigeration and specialised handling of serum samples and allowing economic transportation using the basic postal service. Clin Chem Lab Med 2008;46:354–8.

Background : Accurate outcome prediction in gastroesophageal malignancies is challenging. Acute-phase response proteins (APRPs) have been claimed to be independent prognosticators, although the basis for their association with prognosis remains unexplained. We hypothesized that, similarly to pancreatic and lung cancers, changes in APRPs in gastroesophageal malignancies are associated with cachexia and accelerated angiogenesis. Methods : C-reactive protein (CRP), albumin and transferrin serum levels were evaluated and the Glasgow Prognostic Score (GPS) calculated. These data were compared with concentrations of circulating interleukin (IL)-1, IL-6 and IL-8, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF)-A, VEGF-C and midkine in 96 gastroesophageal cancer patients (49 with cachexia) and 42 healthy subjects. Results : Albumin and CRP levels were altered in the cancer patients, with further CRP elevation in those with cachexia. Transferrin was decreased only in the cachectic patients. The interrelationships between the APRPs were strengthened in cachexia and only then were APRPs correlated with the cytokines elevated in gastroesophageal cancer-related cachexia: IL-6, IL-8, VEGF-A and midkine. GPS corresponded well to transferrin, IL-1, IL-6, IL-8, TNF-α, VEGF-A and midkine concentrations. Conclusions : Cachexia in gastroesophageal cancers is associated with changes in APRP concentrations. This, together with a direct relationship of APRPs with accelerated angiogenesis, may constitute a foundation for the association of APRPs and GPS with outcome in these malignancies. Clin Chem Lab Med 2008;46:359–64.

Background : Vitamin D binding protein (DBP) is a multifunctional transport protein with a decreased serum concentration in cystic fibrosis (CF). The present study investigates the importance of DBP and its role as an alternative nutritional marker in CF. Methods : DBP phenotypes of 116 CF patients were determined electrophoretically. Serum DBP was measured by immunonephelometry. Parathormone was assessed by an immunoradiometric assay. Serum 25OH vitamin D 3 , 1,25(OH) 2 vitamin D 3 and leptin concentrations were determined by a radioimmunoassay. Serum α-tocopherol was measured by HPLC. Routine chemistry parameters were assessed using commercial methods. The Prognostic Inflammatory and Nutritional Index was calculated. Results : Decreased serum DBP concentrations were observed in the CF group. Total cholesterol, low-density lipoprotein (LDL)-cholesterol, triglyceride concentration, α-tocopherol and a low nutritional status correlated positively with DBP. Lipoprotein precipitation demonstrated a partial presence of DBP in very-low-density lipoprotein and LDL. Furthermore, DBP was positively correlated with serum leptin concentration. Conclusions : The present study demonstrates a positive correlation between DBP and serum lipids. Serum DBP concentration can be considered as a nutritional marker (especially for lipids). Clin Chem Lab Med 2008;46:365–70.

Background : We elucidate how the triglyceride (TG) and total cholesterol (TC) concentrations affect the percentage difference (%ΔLDL) between the low-density lipoprotein cholesterol (LDL-C) concentration evaluated by direct measurement (DLDL-C) and calculated using the Friedewald formula (FLDL-C), under conditions allowing the calculation. Methods : Serum concentrations of TC, TG, high-density lipoprotein cholesterol (HDL-C), and DLDL-C were measured and the FLDL-C and %ΔLDL were calculated for 38,243 Koreans who had TG values <4.52 mmol/L. The DLDL-C was measured using the homogeneous Kyowa Medex assay (Kyowa, Tokyo, Japan). The %ΔLDL was calculated using the equation: [(FLDL-C–DLDL-C)/DLDL-C]×100. Results : The mean %ΔLDL-C was –9.1±6.4%. The %ΔLDL differed by more than ±5% in 75.4% of the subjects, and the FLDL-C was lower than the DLDL-C in 96.3%. The mean %ΔLDL-C for the group with the highest TG and lowest TC was 11.8-fold that for the group with the lowest TG and highest TC. Conclusions : Under conditions satisfying the requirements of the Friedewald formula, the DLDL-C and FLDL-C differed significantly over the concentration ranges of both TC and TG. In an evaluation of patients with hyperlipidemia, the Friedewald calculation may underestimate the risk for coronary heart disease. Clin Chem Lab Med 2008;46:371–5.

Background : Gallstone analysis is important in determining the possible etiology of stone formation and the pathophysiology of cholelithiasis. Physical analysis using Fourier transform infrared spectroscopy (FT-IR), compared to chemical analysis, requires minimal sample volume, shows uniform sensitivity and specificity for all components and provides quantitative results with greater reproducibility. We studied the characteristics and distribution of gallstones using FT-IR in addition to the risk factors for gallstone formation in Korean patients. A better understanding of the mechanism underlying stone formation may help prevent gallstone development. Methods : Physical analysis of gallstones in 490 patients who underwent cholecystectomy was carried out using the FT-IR system 2000 (Perkin-Elmer Co.) and Spectrum™ software (Perkin-Elmer Co.). Visual inspection of the size, color, consistency and surface of the stones was compared with the physical characteristics. Clinical, demographic and laboratory findings were evaluated and compared with the gallstone components. Results : The FT-IR evaluation showed that most gallstones were composed of a single component (84.1%); cholesterol was the most commonly observed element among the major components (50%, 245/490). Morphological classification according to color, consistency and surface was different from the FT-IR composition analysis. There were significant differences in the components based on age, obesity, education level and the presence of diabetes mellitus. Conclusions : The results of this study show that physical analysis of gallstones with FT-IR provides important information on stone composition, distribution and risk factors. These study results will help improve our understanding of the pathophysiology of gallstone disease in the Korean population, where there is a high frequency of hepatobiliary disorders. Clin Chem Lab Med 2008;46:376–81.

Background : Cardiomyopathy (CMP) is a common debilitating illness, associated with a high mortality and poor quality of life. There is extensive evidence from in vitro and animal experiments that CMP is a state of increased oxidative stress. Coenzyme Q10 (CoQ10) and high-sensitivity C-reactive protein (hs-CRP) are important markers to evaluate the oxidative stress and inflammatory status of patients with CMP. Methods : A total of 28 patients with chronic stable heart failure (21 men and 7 women, ages 18–76 years) were included in the study. Causes of heart failure were ischemic CMP in 17 patients and idiopathic dilated CMP in 11 patients. A total of 28 patients (12 men and 16 women; ages 30–71 years) with normal coronary angiography were enrolled as a control group. Levels of CoQ10, albumin, total thiol groups (T-SH), bilirubin, uric acid as plasma antioxidants, hs-CRP as an inflammation marker and lipid profile were studied in patients and controls. Results : Plasma CoQ10, T-SH and albumin levels were significantly decreased in patients compared to controls. Uric acid, bilirubin and hs-CRP levels were found to be significantly increased compared to controls. Conclusions : In this study, evidence of decreased antioxidant status was determined in CMP patients together with vascular inflammation. CoQ10, other plasma antioxidants and hs-CRP measured routinely can reflect decreased antioxidant status and inflammatory process in patients with dilated CMP. These markers can be used to monitor the status of patients with CMP. Clin Chem Lab Med 2008;46:382–6.

Background : We investigated the levels of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA), as well as homocysteine and cysteine thiols, in a cohort of subjects affected by retinal vein occlusion (RVO) disease. Methods : Capillary electrophoresis analysis was performed in both RVO subjects (n=54) and in a control group (n=32). Results : No differences were found between controls and patients; however, after categorisation for RVO type, central RVO (CRVO) patients showed higher levels of ADMA (0.710±0.139 μmol/L) than controls (0.635±0.117 μmol/L) and branch RVO patients (0.642±0.096 μmol/L). Moreover, cysteine plasma levels were also significantly higher in CRVO patients than in controls (265.8±46.9 vs. 226.7±51.9 μmol/L, p<0.01), while homocysteine plasma concentration was more or less identical in all groups. Conclusions : We hypothesise that the elevated levels of cysteine in CRVO patients may post-translationally inhibit dimethylarginine dimethylaminohydrolase enzyme activity, as already described for homocysteine, thus contributing to the accumulation of ADMA in this patient group. Clin Chem Lab Med 2008;46:387–92.

Background : Endothelial dysfunction plays a significant role in the pathogenesis of essential hypertension (EH). This trial was undertaken to reveal the effects of extended-release felodipine on endothelial vasoactive substances in EH patients. Methods : A colorimetric chemical method was employed to measure the level of nitric oxide (NO) and nitric oxide synthase (NOS), while radioimmunoassay was employed to measure endothelin (ET), angiotensin-II (Ang-II), thromboxane A2 (TXA 2 ) and prostacyclin I 2 (PGI 2 ) in plasma of the subjects. Group 1 consisted of 120 patients who were treated with a 4-week mono-therapy of felodipine. Group 2 consisted of 70 patients who were participating in fitness programs during this period. Another group comprising 80 individuals was selected as controls. Data from both the starting point and the ending point were collected and analyzed. Results : After a 4-week mono-therapy of extended-release felodipine in Group 1, the levels of plasma ET, Ang-II and TXA 2 decreased significantly, while levels of NO, NOS and PGI 2 did not noticeably change. In Group 2, there were almost no changes in levels of ET, Ang-II, TXA 2 , NO, NOS and PGI 2 . Conclusions : From these results, we conclude that felodipine reduced blood pressure by decreasing the secretion of ET, Ang-II and TXA 2 . Consequently, felodipine can revitalize the endothelial function in EH patients. Clin Chem Lab Med 2008;46:393–5.

Background : The aim of this study was to investigate the cause of markedly low albumin values determined by a bromcresol green (BCG) method in patients on haemodialysis. Methods : Serum and heparinised plasma from haemodialysis patients and normal controls were collected. Albumin was measured using Beckman bromcresol purple (BCP) and Roche BCG methods on the Beckman Synchron LX20. Results : The albumin in heparinised plasma determined by a BCG method was 33.3% lower than that of the BCP method in a haemodialysis patient. The albumin values determined by the BCP method were comparable to those measured by immunonephelometric analysis for this patient. Significantly lower albumin levels were also observed in lithium heparin plasma by a BCG method compared to the BCP method in both non-renal patients (31.2±3.8 vs. 34.1±4.1 g/L, p<0.001, n=30) and haemodialysis patients (28.6±3.5 vs. 32.8±3.7 g/L, p<0.001, n=30). This negative bias was directly correlated with heparin concentrations in the plasma. The BCP method did not show this dose-dependent bias. Conclusions : Lithium heparin plasma can cause falsely low albumin values by an automated BCG method and the suitability of lithium heparin blood tubes should be carefully assessed for haemodialysis patients. The BCP method is free of this bias. Clin Chem Lab Med 2008;46:396–400.

Background : We investigated the prevalence of a low level of red blood cell (RBC) folate in individuals suspected to be deficient and further evaluated the clinical consequence of identifying an individual with a low folate level in a population where no fortification with folate has been introduced. Methods : We conducted a retrospective review of all RBC folate analyses requested in one of the university hospitals in Denmark and identified patients with abnormally low (<350 nmol/L) and high (>1700nmol/L) folate levels. For requests from the hospital, we evaluated the clinicians' response to a decreased level of folate. Results : A total of 12,932 RBC folates requested from the hospital (26%) or from general practitioners (GPs) (74%) were investigated. RBC folate levels were low in a comparable fraction of those requested from the hospital (1.7%) and from the GPs (1.3%). A high level of RBC folate was observed more often than a low RBC folate and occurred considerably more frequently (p<0.0001) in patients referred from the hospital (4.9%) than amongst those referred from GPs (1.2%). The clinicians' response rate to a low folate was around 60% and occurred more often for patients with a low level of cobalamins than in other patients. Conclusions : Low RBC folate values are rare in Danish patients despite no folate fortification program. Further, the clinical reaction to a low folate value is unexpectedly low. Clin Chem Lab Med 2008;46:401–4.

Background : Cystatin C (CysC) has been identified to be an alternative marker of glomerular filtration rate (GFR), but no CysC-based equation has been developed for patients receiving liver transplantation. Methods : Serum CysC and 99m Tc-DTPA clearance (true GFR) were measured simultaneously on post-transplant days 1, 4 and 7. A new equation was constructed based on an observation group of 30 patients and its predictive ability was compared with three other CysC-based equations (Hoek, Filler and Larsson) based on a validation group of 30 patients. Results : The new equation for calculating GFR was defined as 19.12+96.21×(1/CysC) and the derived GFR was estimated at 97.4±30.2 mL/min/1.73 m 2 and was close to the true GFR (96.8±32.8 mL/min/1.73 m 2 ). Estimates of GFR by Hoek, Filler and Larsson formulas (61.4±25.4, 73.8±31.9 and 61.3±29.6mL/min/1.73 m 2 , respectively) differed significantly from the true GFR. Correlation between the true GFR and all formulas showed no significant difference. Bias was neglectable for the new equation (mean difference: 0.6 mL/min/1.73 m 2 ) but remarkable for the other three equations (mean difference: −22.9 to −35.4 mL/min/1.73 m 2 ). Accuracy within 10%, 30% and 50% of the true GFR for the new equation (30.0%, 76.7% and 93.3%) was significantly higher than those of the other three equations (p<0.001 for all). Conclusions : A new serum CysC-based equation was established in this study and it was shown to be accurate in estimating GFR after liver transplantation, compared to the formulas of Hoek, Filler and Larsson. Clin Chem Lab Med 2008;46:405–10.

Background : Des-γ-carboxy prothrombin (DCP), Lens culinaris agglutinin-reactive α-fetoprotein ratio (AFP-L3) and total α-fetoprotein (AFP) are tumor markers useful for diagnosing and determining the prognosis of hepatocellular carcinoma (HCC). There is a real need for measurement of these three markers on a one-assay platform. Methods : A method of DCP measurement in human serum was developed using liquid binding assay (LBA), which enables rapid antigen-antibody reaction and bound/free separation on the LiBASys clinical analyzer. Results : The dilution curve for DCP was linear up to 500 ng/mL. The limit of detection of DCP concentration was 0.5 ng/mL. Intra- and inter-assay coefficients of variation of DCP were 0.7%–2.4% and 2.2%–6.5%, respectively. This method was free from interference by hemoglobin, bilirubin, ditaurobilirubin, intrafat, ascorbate, galactose, glucose and rheumatoid factor. The analytical recoveries of DCP added to serum were 91.7%–108.2%. DCP concentration measured with the LBA method was linear and was significantly correlated with that measured with the ELISA method. Conclusions : The LiBASys clinical analyzer made possible measurement of the complementary tumor markers, HCC, total AFP, AFP-L3 and DCP. Clin Chem Lab Med 2008;46:411–6.

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