Harmonisation of seven common enzyme results through EQA.

BACKGROUND: Equivalent results between different laboratories enable optimal patient care and can be achieved with harmonisation. We report on EQA-initiated national harmonisation of seven enzymes using commutable samples. METHODS: EQA samples were prepared from human serum spiked with human recombinant enzymes. Target values were assigned with the IFCC Reference Measurement Procedures. The same samples were included at four occasions in the EQA programmes of 2012 and 2013. Laboratories were encouraged to report IFCC traceable results. A parallel study was done to confirm commutability of the samples. RESULTS: Of the 223 participating laboratories, 95% reported IFCC traceable results, ranging from 98% (ASAT) to 87% (amylase). Users of Roche and Siemens (97%) more frequently reported in IFCC traceable results than users of Abbott (91%), Beckman (90%), and Olympus (87%). The success of harmonisation, expressed as the recovery of assigned values and the inter-laboratory CV was: ALAT (recovery 100%; inter-lab CV 4%), ASAT (102%; 4%), LD (98%; 3%), CK (101%; 5%), GGT (98%; 4%), AP (96%; 6%), amylase (99%; 4%). There were no significant differences between the manufacturers. Commutability was demonstrated in the parallel study. Equal results in the same sample in the 2012 and 2013 EQA programmes demonstrated stability of the samples. CONCLUSIONS: The EQA-initiated national harmonisation of seven enzymes, using stable, commutable human serum samples, spiked with human recombinant enzymes, and targeted with the IFCC Reference Measurement Procedures, was successful in terms of implementation of IFCC traceable results (95%), recovery of the target (99%), and inter-laboratory CV (4%).

Evaluation of point-of-care analyzers' ability to reduce bias in conductivity-based hematocrit measurement during cardiopulmonary bypass.

Most point-of-care testing analyzers use the conductivity method to measure hematocrit (hct). During open-heart surgery, blood-conductivity is influenced by shifts in electrolyte and colloid concentrations caused by infusion media used, and this may lead to considerable bias in the hct measurement. We evaluated to what extent different analyzers correcting for 0, 1, 2, or 3 factors, respectively, compensated for this electrolyte/colloid interference: (1) the conductivity method with no correction (IRMA), (2) with a [Na(+)]-correction (GEM Premier 3000), (3) with a [Na(+)]/[K(+)]-correction (i-STAT), and (4) with a [Na(+)]/[K(+)]-correction in combination with an algorithm that estimates the protein dilution [i-STAT in cardiopulmonary bypass (CPB)-mode]. Bias in hct was measured during three consecutive stages of a CPB procedure: (I) before CPB, (II) start of CPB and (III) after cardioplegia. In order of high to low electrolyte/colloid interference: the analyzer with no correction, [Na(+)]-correction, [Na(+)/]/[K(+)]-correction, and [Na(+)/]/[K(+)]/estimated protein-correction showed a change of bias from stage I to stage III of -3.9 ± 0.5, -3.4 ± 0.4, -2.1 ± 0.5, -0.3 ± 0.5%. We conclude that correcting for more parameters (Na(+), K(+), estimated protein) gives less bias, but residual bias remains even after [Na(+)/]/[K(+)]/estimated protein-correction. This suggests that a satisfactory algorithm should also correct for other colloidal factors than protein.

Introduction of a new cobalamin (vitamin B12) assay: lessons from a flawed validation study.

BACKGROUND: Plasma vitamin B12 [cobalamin (Cbl)] concentrations are usually measured as a screening marker for vitamin B12 deficiency. Siemens Healthcare Diagnostics has introduced Cbl assays for various platforms, i.e., the immulite (IML) 2000 and 2500. In our laboratories, regular validation studies for the IML 2500 were conducted and showed acceptable quality specifications. After the introduction of the IML 2500 Cbl assay, clinicians in the department of internal medicine reported an increased frequency of patients with Cbl-concentrations less than 148 pmol/L. METHODS: In order to investigate this claim from the clinicians, we retrospectively analyzed the internal and external quality control (QC) of the Cbl assay. In addition, the monthly patient means for the Cbl assay were analyzed both before and after the introduction of the new Cbl assay. RESULTS: No abnormalities were found in the internal and external QCs. However, the monthly patient means for the Cbl assay showed a statistically significant decrease in cobalamin concentrations. Siemens acknowledged the problems and formulated a new Cbl assay, which was subsequently validated in our laboratories and showed equivocal Cbl results when compared to the IML 2000 Cbl assay. CONCLUSIONS: We report a flawed validation study conducted by the manufacturer that resulted in an undetected analytical problem in the IML 2500 Cbl assay, its subsequent introduction on the market, the final recognition of the poor performance of the assay by our clinicians, and the eventual resolution by the manufacturer. Hence, it emphasizes the utmost importance for thorough comparison between assays over the entire measurement range, even when both assays are produced by the same manufacturer.

Hematological indices, inflammatory markers and neutrophil CD64 expression: comparative trends during experimental human endotoxemia.

CD64 is a high-affinity Fc(gamma)RI receptor expressed by activated neutrophils that has been recently evaluated as a potential sepsis parameter. In the present study, the kinetics of neutrophil membrane CD64 expression were examined during a standardized inflammatory response, using a human endotoxemia model, and compared with hematological indices, CRP, cytokines and interleukins. Ten healthy subjects received 2 ng/kg intravenous Escherichia coli lipopolysaccharide (LPS). After administration of LPS, neutrophil CD64 showed a biphasic response, characterized by a first increase from 108.5 +/- 7.5 to 133 +/- 6 AFU after 1 h (P = 0.047) and a second increase that started at 6 h and reached its maximum of 167 +/- 13 AFU at 22 h (P < 0.0001). CRP concentrations increased to 40 +/- 5 mg/dl 22 h after the administration of LPS. The cytokines and interleukins reached their maximum response within 1-2 h. The maximum values of pro-inflammatory cytokines (TNF-alpha, IFN-gamma and IL-6) correlated with the CD64 expression at 22 h after LPS administration (r(2) = 0.76, r(2) = 0.78, r(2) = 0.81, respectively, all P < 0.05), whereas this correlation was not found for the anti-inflammatory IL-10 (r(2) = 0.058, P = 0.54), suggesting that neutrophil CD64 expression might be a quantitative marker for innate immunity that could easily be used in the clinical setting.

Simultaneous determination of membrane CD64 and HLA-DR expression by blood neutrophils and monocytes using the monoclonal antibody fluorescence capability of a routine haematology analyser.

This study reports the design of an immunofluorescent method for the co-determination of neutrophil CD64 (PMN-CD64), monocyte CD64 (MON-CD64) and monocyte HLA-DR (MON-Ia) expression with the Cell-Dyn CD4,000 haematology analyser. Normal PMN-CD64, MON-CD64 and MON-Ia expression, defined as the mean+/-2SD of 25 healthy adults after correction for isotype control staining, corresponded to 17-67, 515-1045 and 170-670 AFU respectively. Analytical reproducibility determined by duplicate analysis of 12 random samples revealed good assay consistency for all three analysed antigens, with day to day variation in normal subjects being relatively minor in significance. CD4,000 PMN-CD64 and HLA-DR values showed good inter-method correlation with flow cytometry although short term (12 h) stability studies suggested an in vitro trend for increasing PMN-CD64 and variable HLA-DR antigen expression with progressive storage. Observed ranges of PMN-CD64, MON-CD64 and MON-Ia for 109 randomly-selected clinical samples were 31-1058, 307-2843 and 10-876 AFU. Abnormal PMN-CD64 and MON-CD64 shared the same trend (upregulation) while abnormal monocyte MON-Ia was characterised by declining expression. Normal PMN-CD64 was only seen with normal (45/52) or intermediate (7/52) MON-CD64, while high PMN-CD64 was mostly associated with intermediate (18/22) or high (3/22) MON-CD64. MON-Ia expression was largely independent (p=0.04) of PMN-CD64 although marked decreases in MON-Ia were invariably associated with intermediate or high PMN-CD64. MON-Ia expression was inversely related (p<0.0001) to absolute granulocyte counts, and patients with high PMN-CD64 were more likely (8/25) to have in excess of 10% Band Cells compared to samples with normal/intermediate PMN-CD64 (0/84). When compared to C-reactive protein (CRP), high PMN-CD64 and MON-CD64 were always associated with an increased CRP concentration, but minor proportions of samples with normal PMN-CD64 (11/52) or normal MON-CD64 (11/65) could also have an increased CRP. The procedures described in this communication overcome a number of limitations associated with flow cytometry, and co-determination of CD64 and HLA-DR antigen expression may provide complimentary insights into patient heterogeneity in the assessment of suspected sepsis compared to CD64 analysis alone.

Recommended intakes of vitamin D to optimise health, associated circulating 25-hydroxyvitamin D concentrations, and dosing regimens to treat deficiency: workshop report and overview of current literature.

Vitamin D is a fat-soluble hormone that traditionally has been linked to bone health. Recently, its involvement has been extended to other (extra-skeletal) disease areas, such as cancer, CVD, energy metabolism and autoimmune diseases. Vitamin D deficiency is a worldwide problem, and several recommendation-setting bodies have published guidelines for adequate vitamin D intake and status. However, recommendations from, for example, the Health Council of the Netherlands do not provide advice on how to treat vitamin D deficiency, a condition that is often encountered in the clinic. In addition, these recommendations provide guidelines for the maintenance of 'minimum levels', and do not advise on 'optimum levels' of vitamin D intake/status to further improve health. The NutriProfiel project, a collaboration between the Gelderse Vallei Hospital (Ede, the Netherlands) and the Division of Human Nutrition of Wageningen University (Wageningen, the Netherlands), was initiated to formulate a protocol for the treatment of vitamin deficiency and for the maintenance of optimal vitamin D status. To discuss the controversies around treatment of deficiency and optimal vitamin D status and intakes, a workshop meeting was organised with clinicians, scientists and dietitians. In addition, a literature review was conducted to collect recent information on optimal intake of vitamins, their optimal circulating concentrations, and effective dosing regimens to treat deficiency. This information has been translated into the NutriProfiel advice, which is outlined in this article.

Measurement of neutrophil membrane CD64 and HLA-Dr in a patient with abdominal sepsis.

A patient with abdominal sepsis, had both intra and extracellular bacteria in a blood smear, and high levels of neutrophil membrane CD64 and HLA-Dr. Intracellular bacteria are only observed in the terminal phase of a sepsis. Our patient recovered, suggesting that a high expression of neutrophil CD64 is indicative for a good prognosis.