Haemoglobin Ottawa, sickle cell trait and vaso-occlusive crises.

A girl with a sickle cell trait had severe VOCs (vaso-occlusive crises), her father also had a sickle cell trait but mild VOCs, and her mother had no symptoms. Electrophoresis on agarose gel under alkaline conditions showed haemoglobin AS (HbAS) in the girl and in her father, with an S band increased more than expected (46.2% and 41.2% respectively), and a band migrating at C (16.8% and 8.9% respectively) in both. There was a band at S (19.6 %) in her mother. The C band was attributed to a hybrid tetramer with haemoglobin S (HbS) and a Hb variant. A homozygous c.46G>C mutation (Hb Ottawa, the Hb variant) was detected by Sanger sequencing in the girl. Heterozygosity for Hb Ottawa by Sanger sequencing was shown in both the father and the mother. The father, with HbAS and heterozygous for Hb Ottawa, had mild VOCs. Heterozygosity only for Hb Ottawa did not produce any abnormality in the mother. A sister and two brothers of the index patient presented a Hb variant, probably Hb Ottawa, migrating to the S zone (all 20%) at electrophoresis, without HbS. These last three were asymptomatic. We conclude that Hb Ottawa, an α-globin variant, contributes along with haemoglobin S (HbS) to VOC symptoms.

CA125, Galectin-3 and FGF-23 are interrelated in heart failure with reduced ejection fraction.

Background: Carbohydrate Antigen 125 (CA125) is the most widely used biomarker in ovarian cancer screening. In patients with heart failure (HF), increased levels of CA125 have been observed and related to disease severity. Our objective was to determine the association of CA125 levels with two biomarkers of adverse remodeling in HF patients with reduced ejection fraction (HFrEF). Methods: CA125 circulating levels were determined with an electrochemiluminscent immunoassay. Concentrations of B-type natriuretic peptide (BNP), N-terminal proBNP (Nt-proBNP), Galectin-3 and Fibroblast Growth Factor 23 (FGF23) were also measured by immunoassays. Results: CA125 levels were increased in HFrEF, were associated to disease severity according NYHA classes. Median CA125 concentration was also significantly related to cardiovascular mortality. CA125 concentrations were positively and significantly associated to Galectin-3 and FGF23. Conclusions: Concentrations of CA125 are increased in patients with HFrEF, associated to disease severity and adverse cardiovascular outcomes. CA125 levels are also correlated to Galectin-3 and FGF-23, two biomarkers related to fibrosis and cardiovascular remodeling.

Biological variation of CA 15-3, CA 125 and HE 4 on lithium heparinate plasma in apparently healthy Caucasian volunteers.

OBJECTIVES: Tumor markers are well-known for being important tools in the support of diagnosis, monitoring of treatment efficacy and follow-up of cancers. CA 125, CA 15-3 and HE 4 have demonstrated potential efficacy in other clinical indications. The main objective was to evaluate the biological variation of these glycoproteins using two different immunoassays in an apparently healthy Caucasian population. METHODS: Nineteen healthy volunteers including 11 women and 8 men were sampled weekly for 5 consecutive weeks. Samples were analyzed in duplicate on Lumipulse® G600II (Fujirebio) and on the Cobas e602 (Roche Diagnostics) analyzers. After assessment of normality, exclusion of outliers and analysis of homogeneity of variance, analytical variation (CVA), within-subject biological variation (CVI) and between-subject biological variation (CVG) were determined using a nested ANOVA. RESULTS: CVA, CVI and CVG were determined on both analyzers and both genders. For CA 125, the CVA ranges from 1.0 to 3.4%, the CVI from 5.7 to 13.8% and the CVG from 32.2 to 42.9%. For CA 15-3, the CVA is between 1.1 and 3.4%, the CVI between 3.9 and 6.5% and the CVG between 43.7 and 196.9%. Lastly, HE 4 has CVA values between 1.4 and 2.4%, CVI between 5.1 and 10.5% and CVG between 7.1 and 12.6%. CONCLUSIONS: Our study provided updated data on the biological variation of CA 125, HE 4 and CA 15-3. These data allow to improve the clinical interpretation and thus the management of the patient.

Value of DNA testing in the diagnosis of sickle-cell anemia in childhood in an environment with a high prevalence of other causes of anemia.

BACKGROUND: Sickle-cell anemia (SCA) is the most common genetic disease worldwide caused by a single mutation in the gene HBB. DNA testing can help to clarify the diagnosis when Hb electrophoresis is inconclusive. We evaluated the usefulness and feasibility of DNA-based diagnosis of SCA in rural Central Africa. METHODS: This is a cross-sectional study conducted from November 2016 to end October 2017 in the Hôpital Saint Luc de Kisantu, located 120 km from Kinshasa. This hospital offers the management of SCA patients, mainly identified using the Sickling test (Emmel test) combined with clinical features. We included patients aged 6 months to 18 years locally diagnosed as SCA, and we collected clinical and hematological data. All patients were offered Hb electrophoresis and DNA testing at the Center for Human Genetics of the University of Kinshasa. RESULTS: This study included 160 patients. Hemoglobin capillary electrophoresis suggested that 136 (85%) were homozygote SS, 13 (8.1%) were heterozygote (AS), and 11 (6.9%) were homozygote normal (AA). DNA testing confirmed these electrophoresis findings, with the exception of four patients, two AS in electrophoresis were found SS due to recent transfusion, and two SS in electrophoresis were found AS because they have compound heterozygous form S/ß°-thalassemia. The diagnosis of SCA was therefore wrongly ascertained with Emmel test in 15% of patients. CONCLUSION: This study reveals a high proportion of false-positive SCA diagnoses in a rural environment in Central Africa. This underlines the importance of DNA testing in conjunction with Hb electrophoresis.

Biological variation and analytical goals of four thyroid function biomarkers in healthy European volunteers.

BACKGROUND: Interpretation of thyroid function tests by means of biological variation (BV) data is essential to identify significant changes between serial measurements at an individual level. Data on thyroid parameters in adults are limited. OBJECTIVES: We aimed at determining the BV of four thyroid function test (thyroid-stimulating hormone (TSH), free thyroxin (FT4), free triiodothyronine (FT3) and thyroglobulin (Tg)) by applying recent recommendations to acquire BV data on a latest generation of immunoassay. METHODS: Nineteen healthy volunteers (8 males and 11 females) were drawn every week during 5 consecutive weeks. Samples were analysed in duplicate on the Cobas 602 analyzer (Roche Diagnostics). After normality assessment, outlier exclusion and homogeneity of variance analysis, analytical variation (CVA ), within-subject biological variation (CVI ) and between-subject biological variation (CVG ) were determined using nested ANOVA. RESULTS: CVA , CVI and CVG were 0.9%, 19.7% and 37.6% for TSH; 3.6%, 4.6% and 10.8% for FT4; 2.2%, 6.0% and 8.6% for FT3; and 0.9%, 15.4% and 84.9% for Tg. Index of individuality (II) for all parameters was between 0.2 and 0.7. The percentage above which the change between two measures is truly significant (reference change value) was 54.7% for TSH, 16.2% for FT4, 17.7% for FT3 and 42.8% for Tg. CONCLUSION: Based on recent international recommendations, our study provides updated BV data for four thyroid function tests in European healthy volunteers. Reliable BV characteristics, and especially RCV, can facilitate the interpretation of consecutive thyroid function tests in an individual and therefore have the potential to efficiently support clinical decisions regarding thyroid diseases.

A rare case of tuberculosis-induced hypercalcemia.

Laboratory investigations of hypercalcemia involve testing of various biochemical parameters such as parathyroid hormone (PTH), 25-(OH) Vitamin D (25-(OH) VitD), 1,25-(OH)2 Vitamin D3 (calcitriol) and PTH related peptide (PTHrp). We herein present an atypical case of severe hypercalcemia in a patient with rheumatoid arthritis who has been treated for years by various biological disease-modifying antirheumatic drugs (DMARDs) and suddenly presented with general state alteration, oedema and ulceration of her right ankle. We illustrate how tuberculosis (TB) can cause high calcitriol concentration and subsequently lead to potentially severe hypercalcemia. Moreover, we highlight the importance of TB testing and follow-up in patients treated with biological DMARDs.

Could trisialotransferrin be used as an additional biomarker to CDT in order to improve detection of chronic excessive alcohol intake?

OBJECTIVES: Carbohydrate-deficient transferrin is a well-known biomarker widely used for detection of chronic excessive alcohol intake. However, under certain clinical conditions particularly frequently met amongst heavy drinkers (steatosis, fibrosis, cirrhosis…), it isn't a reliable biomarker. In this study, we tried to find additional biomarkers to CDT in order to improve detection of chronic excessive alcohol intake. DESIGN AND METHODS: We conducted a retrospective cohort study from December 2007 to December 2009. We focused mainly on three different groups: heavy drinking patients with active alcohol consumption (n=243), cirrhotic patients (abstinent patients and non alcoholic cirrhosis, n=44) and control group (n=85). RESULTS: In our study, CDT showed a poor sensitivity for diagnosis of heavy drinking patients (around 63%, and even lower) for cirrhotic patients and patients at advanced stage of fibrosis. Combination of CDT with trisialotransferrin enabled to improve significantly sensitivity and specificity (p-value AUC ROC<0.001). When adding mean corpuscular volume and gamma-glutamyltransferase to this first combination, performances were even better (p-value<0.001). This second cluster enabled to make a statistically significant difference between cirrhotic patients with active alcohol consumption compared to abstinent cirrhotic patients and to non alcoholic cirrhotic patients (p-value<0.001). CONCLUSION: From our study, trisialotransferrin seems to be a useful additional biomarker to CDT in order to improve detection of chronic excessive alcohol intake.

Significant, quantifiable differences exist between IgG subclass standards WHO67/97 and ERM-DA470k and can result in different interpretation of results.

OBJECTIVES: Accurate measurement of IgG subclass (IgGSc) levels are essential to aid in the diagnosis of disease states such as primary immunodeficiencies. However, there is no single standardisation of nephelometric and turbidimetric assays for these analytes and two reference materials have been utilised. We expand on previous reports and present data from a multi-site analysis that both identifies and quantitatively defines the differences in calibration resulting from the use of different reference materials. DESIGN AND METHODS: IgGSc antibodies in the serum specimens and reference materials were measured according to the manufacturers' instructions using commercially available IgGSc assays or components. RESULTS: Data from four independent sites showed that in spite of the different commercial suppliers of IgGSc assays calibrating to different reference materials, ERM-DA470k and WHO67 /97, the resulting calibrations were comparable for IgG1 and IgG2. However, for IgG3 and IgG4 the calibrations were significantly different. The use of assay specific normal ranges should compensate for these calibration differences, however, the two manufacturers' assays can give differing clinical classifications. The agreement between the different manufacturers' IgGSc assays was between 85.1% and 95.8% for all IgGSc assays, the discordance of sample classification for IgG1 and IgG2 assays was approximately 12% and 15% respectively, whilst that for IgG3 and IgG4 was 4% and 13% respectively. CONCLUSION: We discuss the similarities and differences between assays that utilise the different reference materials.

Quantification of serum free light chain kappa and lambda by the SPAPLUS analyser.

OBJECTIVE: Clinical assessment of the SPAPLUS® system for the determination of the serum free light chains kappa (κ FLC) and lambda (λ FLC) compared to the BNII®. DESIGN AND METHODS: 126 serum specimens from our routine activity were analysed on two different analysers: the BNII® (immunonephelometry, Siemens) and the SPAPLUS® (turbidimetry, Binding Site). We compared the absolute values of the serum κ FLC and λ FLC, as well as the FLC κ/λ ratio on both analysers. These results were further evaluated together with the clinical history of the patients. RESULTS: Regression analysis between the BNII® and the SPAPLUS® for κ FLC and λ FLC did not display any significant differences between both methods in the normal and pathological ranges. Nevertheless, some differences have been observed for some patients in the absolute value of the involved light chain, with potential clinical implications. CONCLUSION: The results show overall good concordance between both methods. However, it is recommended that the monitoring of patients affected by monoclonal gammapathies by measuring FLC, be performed in the same laboratory and by the same method. Moreover, the FLC results should always be interpreted together with other laboratory tests taking into account the patient's diagnosis.

Clauss assay and fibrinogen protein estimated by capillary zone electrophoresis.

BACKGROUND: The clottability and the amount of total protein in fibrinogen provide information about qualitative or quantitative alterations. We aimed to evaluate whether capillary zone electrophoresis (CZE) Capillarys II analyzer with the protein 6 buffer is able to estimate the amount of fibrinogen antigen. METHODS: Citrated plasmas were assayed for clottable fibrinogen, and any relationship with the ß(2)-globulin fraction (percentage of the area under the curve) was evaluated. The integration method used was "tangent skimming" in order to reduce the overestimation due to high protein background. Linearity was optimized according to the ICH Q2R1 recommendations and evaluated using polynomial regression. The precision was computed in accordance with the Clinical and Laboratory Standards Institute EP5-A2 protocol. In patients, clottable fibrinogen (Clauss method) was compared to its protein CZE amount by Passing and Bablok regression and the Bland-Altman plot. RESULTS: The correlation was linear y=0.0744+0.8991x (R(2)=0.9707) within the range of 2.26-17.26 µmol/L. The repeatability and the within-device precision were <15% for three levels of percentage of the ß(2)-globulin fraction (1.61%, 3.51%, and 9.24%). In patients, clottable fibrinogen and its protein amount were similar (-0.1779+0.9654x). The ratio activity/protein was 1.08 ± 0.32 (mean ± 2 SD). CONCLUSIONS: CZE with the Capillarys II and the buffer protein 6 is an easy method. It is a good candidate for estimation of the concentration of fibrinogen antigen, which may have diagnostic utility for the screening of quantitative or qualitative fibrinogen abnormalities.

Multicentre evaluation of the Tosoh HbA1c G8 analyser.

BACKGROUND: We report a Dutch-Belgian multicentre evaluation of the Tosoh HLC-723G8 glycohaemoglobin analyser, an ion-exchange HPLC instrument for the separation and quantification of haemoglobin A1c (HbA1c) in whole blood. METHODS: We evaluated the analytical performances of the Tosoh G8 analyser and compared the results for blood samples with its predecessor, the Tosoh G7, and with two other widely used analysers, the Bio-Rad Variant II and Adams Arkray HA-8160. RESULTS: Within- and between-batch imprecision [coefficient of variation (CVs)] was <0.5% and 2%, respectively, and compared favourably with the G7. The excellent performances in terms of speed (1.6 min/analysis) did not result in increased variability of the results or carry-over between samples. The method shows no interference from carbamylated haemoglobin, and recognises the presence of haemoglobinopathies, which triggers the correction of the HbA1c result. Comparison with established methods showed good correlation, not only with the G7 but also with the Variant II and HA-8160 systems. CONCLUSIONS: With respect to reproducibility, chromatographic resolution, speed of analysis and identification of Hb variants, the Tosoh G8 analyser can be considered to be state of the art.

Evaluation of a new Sebia kit for analysis of hemoglobin fractions and variants on the Capillarys system.

We evaluated the analytical performances of the new Sebia kit for quantification of hemoglobin fractions (HbA, HbF and HbA2) and structural hemoglobin variants on the Capillarys system. This automated capillary zone electrophoresis method uses an alkaline buffer with silica capillaries and spectrophotometric detection. Specimen stability was evaluated during 1 month. The reproducibility of migration and the imprecision of quantification were also investigated. Comparison with the Beckman P/ACE system was performed on 202 samples. A total of 131 subjects without any hematological abnormality were analyzed to establish the HbA2 reference ranges based on our local population. Quantification of the Hb fractions and variants exhibited excellent stability for 4 weeks of storage at 4 degrees C, with CVs < 0.3%. The imprecision of the migration normalized to that of HbA2 for all hemoglobins tested (fractions and variants) was low, with a CV of < 2.5%. At physiological and pathological levels, total imprecision ranged from 1.9% to 4.6% for HbA2, from 0.6% to 9.7% for HbF, and from 0.6% to 1% for HbS. Statistical analysis revealed a small proportional negative bias for HbA2 (-8.6%). Small systematic bias (-0.2%) and proportional bias (-28%) were observed for HbF. No statistically significant difference was found for HbS. The reference range for HbA2 was 2.1-3.2%. The Capillarys system is a fully automated and accurate system that gives high-resolution performance and displays appropriate characteristics for use as a routine method for the diagnosis of thalassemias and hemoglobinopathies.

Polyclonal versus monoclonal ELISA for the determination of fecal elastase 1: diagnostic value in cystic fibrosis and chronic pancreatic insufficiency.

BACKGROUND: Several clinical studies have evaluated the diagnostic efficiency of fecal elastase 1 (FE1) determination using monoclonal antibodies (ScheBo-Tech, Wettenberg, Germany). We report the results of the comparison of this method with a polyclonal based assay (BioServ AG, Rostock, Germany). METHODS: We collected single spot samples from two groups of patients. The group of adults included 13 healthy subjects (HS), 12 patients with non-pancreatic gastrointestinal disease (NPGD), 26 with chronic pancreatitis with presence of calcification (CCP) and 14 without calcification (NCP). The group of children included 17 cases of cystic fibrosis (CF) and 21 controls (CO). After a common extraction, both assays were performed as recommended by the manufacturers. RESULTS: Both tests showed a statistically significant difference between patients with normal pancreatic function and patients with pancreatic disorders. Neither showed a significant difference between HS and NPGD. CONCLUSIONS: Although a statistical difference was found between the two methods for the normal groups (HS and CO), both kits are suitable for the detection of severe pancreatic insufficiency either in adult patients or in children. However, caution should be taken in case of patients with liquid stool specimens.

Automated serum protein electrophoresis by Capillarys.

Capillary zone electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. In this report, we evaluate automated capillary zone electrophoresis by Capillarys (Sebia, France). Within-run and between-run imprecision for the five electrophoretic fractions was <2% and <6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel electrophoresis and Paragon CZE 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and alpha1-antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel electrophoresis, Capillarys, capillary electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology (e.g., spike) of the protein profile or hypogammaglobulinemia on agarose gel electrophoresis and/or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum electrophoresis.