Klas Böer

Evaluation of the XE-5000 for the automated analysis of blood cells in cerebrospinal fluid

OBJECTIVES Counting of cells in cerebrospinal fluid is an important clinical laboratory test and elevated white blood cell counts in cerebrospinal fluid are frequently seen in CNS disorders. Quantification of red blood cell concentrations in CSF may help to interpret certain diagnostic constellations and may result from subarachnoid haemorrhage, surgical procedures or contamination due to traumatic puncture. Table top analyser XE-5000 (Sysmex, Norderstedt, Germany) offers, beside its use as a haematology analyser, a protocol for the quantification of red and white blood cells in body fluids such as CSF including the differentiation between polymorphonuclear and mononuclear cells. A detection limit of 1 cell/mm(3) would render this device suitable for automated CSF analysis. DESIGN AND METHODS White blood cell counting was compared between Fuchs-Rosenthal counting chamber and XE-5000 in 273 routinely collected lumbar and ventricular CSF samples. Red blood cell counting was compared between UF-100 and XE-5000. Differentiation was performed on a slide stained after Pappenheim and compared to the differential count of the XE-5000. RESULTS Linearity was established between 1 and 10,000 cells/mm(3) for white blood cells and between 1000 and 110(3) particles/mm(3) for red blood cells. Functional sensitivity was established at 20 cells/mm(3) for white blood cell counting and at 1000 particles/mm(3) (lowest reported concentration) for red blood cell counting. When comparing between microscopic and automatic white blood cell counts no statistically significant slope and offset were detected in lumbar CSF samples while a significant slope and offset were detected when comparing ventricular CSF samples. Most patients were classified correctly according to their WBC count (non-pathologic, mildly, moderately, and highly elevated) by both methods although more patients had pathologic white blood cell counts on XE-5000. A significant slope and offset were detected when comparing red blood cell counts between UF-100 and XE-5000. CONCLUSIONS In summary despite its high imprecision at low white blood cell counts (<20 particles/mm(3)) most patients were classified correctly and therefore XE-5000 is suitable for automated quantification of white blood cells in cerebrospinal fluid in a defined diagnostic setting. This could significantly improve automation in the relatively time- and manual work-intensive field of cerebrospinal fluid diagnostics. However, careful review of plausibility of the results continues to be compulsory.

Comparison of the ecarin chromogenic assay and different aPTT assays for the measurement of argatroban concentrations in plasma from healthy individuals and from coagulation factor deficient patients.

INTRODUCTION The aim of this study was to assess the usefulness of different aPTT assays and the ecarin chromogenic assay reaction time (ECA) for measurement of argatroban concentration in plasma from healthy persons as well as in different patient subgroups. METHODS We spiked plasma samples from healthy individuals, patients under oral anticoagulation (OAT) or with liver dysfunction (LD) with increasing argatroban concentrations (0-2000 ng/ml) and performed 4 different aPTTs assays and the ECA. RESULTS Depending on argatroban concentrations aPTTs increased in a curvilinear fashion; in plasma from healthy individuals means of calculated argatroban concentration at 2-fold aPTT differed extensively depending on the aPTT reagent used (725 ng/ml to 1136 ng/ml) and were even more pronounced in plasma from coagulation factor deficient patients (460 ng/ml in patients with LD vs. 1172 ng/ml in patients with OAT), whereas ECA showed linear argatroban influence and reliable results in all subgroups. CONCLUSIONS Because of wide differences in aPTT measurements depending on the aPTT reagent used, interindividual variations and different clinical conditions the aPTT is not the method of choice for monitoring argatroban and the ECA should be preferred.

Influence of direct thrombin inhibitor argatroban on coagulation assays in healthy individuals, patients under oral anticoagulation therapy and patients with liver dysfunction.

We assumed that argatroban, a direct thrombin inhibitor, has a strong influence on different coagulation tests which is even more pronounced in patients with an established reduced factor activity like those under oral anticoagulation therapy or with liver dysfunction. To validate this influence we spiked plasma samples from healthy individuals, patients under oral anticoagulation therapy or with liver dysfunction with increasing argatroban concentrations (0–2000 ng/ml) and performed routine laboratory coagulation tests. Consequently, prothrombin time, activated partial thromboplastin time, thrombin time, batroxobin time, coagulation factor activity (FII-FXIII), protein S (activity), protein C (chromogen) and fibrinogen (derived and Clauss fibrinogen method) were measured. Furthermore, the influence of argatroban on the induced platelet aggregation was evaluated. Argatroban interference on standard coagulation assays differed markedly depending on the different subgroups of patients investigated. Prolongation of prothrombin time by argatroban (at 2000 ng/ml 2.7-fold in healthy persons) was significantly higher in oral anticoagulation therapy (3.9-fold) and even more pronounced in liver dysfunction (6.0-fold). The fibrinogen concentration was determined falsely even at low-argatroban concentrations using functional methods in healthy persons and all patient subgroups. The influence of argatroban on standard laboratory coagulation tests is significantly increased by a preexisting factor deficiency. Functional fibrinogen measurement may be helpful to assess in-vivo fibrinogen function but should be avoided to evaluate fibrinogen concentration in argatroban treated patients. Argatroban had no influence on chromogenic protein C measurement, batroxobin time and induced platelet aggregation. Knowledge of argatroban interference is a prerequisite for the reliable interpretation of coagulation assays.

Quantitation of serum free light chains does not compensate for serum immunofixation only when screening for monoclonal gammopathies

Abstract Background: Detection of plasma cell dyscrasias (PCD) requires screening of serum and urine for monoclonal proteins. Several studies have demonstrated increased sensitivity and specificity when measurement of serum free light chain (SFLC) is part of the screening protocol. In addition, omission of immunofixation (IFE) in the standard work-up that includes SFLC assay has been proposed. This study attempts to define the role of the SFLC assay in a screening strategy limited to serum only. It compares outcomes to a serum-only screening strategy that omits serum IFE. Methods: Serum from 691 patients was analysed for the presence of monoclonal protein using standard serum IFE, serum protein electrophoresis (SPE) and measurement of SFLC. Data were analysed retrospectively. Results: Specificity and sensitivity of abnormal SFLC-ratios for the detection of monoclonal protein using IFE were 96% and 41%, respectively. Eighteen patients with negative monospecific and Bence Jones IFE, but abnormal SFLC-ratios were identified. In most cases, this could be attributed to kidney and inflammatory disease or haematological disorders. In four cases, this resulted in further diagnostic investigation and light chain disease was later detected in two cases. Light chain disease was confirmed in one case but not confirmed in the other patient. In 14 patients, Bence Jones IFE was negative, although the concentrations of SFLC suggested the presence of monoclonal Bence Jones protein at concentrations detectable by IFE. Thus, either the anti-serum failed at detection, there was polymerisation of the free light chains or the SFLC assay overestimated protein concentrations. Simulating a work-up that included IFE only if abnormalities were detected by SPE or the SFLC assay would have resulted in 26% fewer IFEs being performed, but three patients with monoclonal proteins by IFE would have been missed. Conclusions: Abnormal SFLC concentrations are neither sensitive nor specific for the detection of monoclonal proteins by IFE. Not all PCD are accompanied by excessive production of SFLC, and several other conditions, such as renal disease are associated with increased SFLC concentrations. An abnormal SFLC-ratio is a specific marker for PCD, and occurs primarily in patients with haematological disease. If renal and inflammatory diseases are excluded, this should prompt further diagnostic investigation. Screening of serum without performing an IFE as a standard procedure leads to a reduction of sensitivity when compared to screening of serum that includes IFE. Clin Chem Lab Med 2009;47:1109–15.

Comparison of HbA1c Measurements using 3 Methods in 75 Patients Referred to One Outpatient Department

OBJECTIVE HbA1c is the most important surrogate parameter to assess the quality of diabetes care and is also used for the diagnosis of diabetes mellitus (DM) since 2010. We investigated the comparability of 3 HbA1c methods in the city of Jena (Germany). METHODS The HbA1c determination was carried out in 50 healthy subjects and 24 people with DM (age 51.2±16.3 years, HbA1c 6.8±2.2%) with 3 different hemoglobin A1c testing methods at 4 locations in one city. Our laboratory (HPLC method) served as a reference for comparing the results. All methods are IFCC standardized and all devices are certified by the interlaboratory test. RESULTS The mean HbA1c of people without diabetes was: laboratory A (TOSOH G8, HPLC) 5.7±0.3%; laboratory B (TOSOH G8, HPLC) 5.5±0.3%, laboratory C (VARIANT II) 5.2±0.3%; laboratory D (COBAS INT.) 5.6±0.3%. All differences are significant (p=0.001).The mean HbA1c of patients with mild to moderate elevated HbA1c was: Laboratory A 7.5±0.9%; B 7.3±1.0%; C 7.0±0.9%; D 7.5±1.1%. Differences are significant (p=0.001) except between laboratory A and D (p=0.8).The mean HbA1c of patients with massively increased HbA1c was: laboratory A 11.5±1.8%; laboratory B 11.4±1.8%; laboratory C 10.8±1.6%; laboratory D 11.5±1.5%. Differences between laboratory A and C, as well as between C and D were significant (p=0.001). CONCLUSION The mean IFCC standardized HbA1c from 75 people differs by up to 0.5% absolute between 4 laboratories. This difference is clinically significant and may lead to misdiagnosis and wrong treatment decisions, while HbA1c value from one patient were analyzed in different laboratories within a short time.

Workflow restrictions on hematology analyzers XE-2100 and XS-800 when assaying pediatric samples with limited volume.

Abstract Background: Samples with limited volume is a common problem in laboratories receiving samples from pediatric patients. Also, pediatric samples may contain nucleated red blood cells (NRBC) which distorts the white blood cell (WBC) count and which can only be measured by some automated cell counting systems. Differential counts are sometimes required, posing the question of validity of flagging depending on age of the patients and on predilutions. Methods: We evaluated the hematology analyzers XE-2100 and XS-800 for their suitability in measuring hematological parameters in such samples. Results: With the exception of the MCHC and partly the MCH, we observed very good agreement between complete blood counts (CBC) in diluted and undiluted samples. Dilution did not impair sensitivity in the clinically relevant range nor, accuracy of the NRBC count on XE-2100. Flagging was ineffective in undiluted samples from children <1 year of age and in all diluted samples when measuring differential counts. Conclusions: In summary, while automated measurement of CBC and NRBC is possible in diluted samples, measurement of differential counts is restricted by loss of flagging efficiency. In addition, flagging is also ineffective in children <1 year of age using the analyzers evaluated and should, for diagnostic purposes, be performed manually. Clin Chem Lab Med 2009;47:607–11.

Strong interference of hemoglobin concentration on CSF total protein measurement using the trichloroacetic acid precipitation method

Abstract Background: Among other methods, trichloroacetic acid precipitation is used to quantify total protein in cerebrospinal fluid (CSF). Methods: We analyzed the influence of hemoglobin on total protein concentration assayed by the trichloroacetic acid method and compared the results to the benzethonium chloride method. Results: Four CSF samples were spiked with different amounts of hemoglobin, leading to overestimation of protein concentration when assayed by the trichloroacetic acid method. Using the benzethonium chloride method, measurement of protein concentration was minimally disturbed. In addition, albumin and total protein concentrations were measured in 135 clinical samples. The total protein/albumin ratio remained constant when protein was measured with the benzethonium chloride method, while ratios increased when protein was assayed by the trichloroacetic acid method. Conclusions: Strong interference by hemoglobin leads to overestimation of the total protein concentration in CSF when assayed by the trichloroacetic acid method and may lead to false conclusions when evaluating the blood-brain barrier. Clin Chem Lab Med 2007;45:112–3.

CD62L on neutrophil granulocytes, a useful, complementary marker for the prediction of ventriculitis in blood-containing CSF

OBJECTIVES Presence of residual blood is a common problem in cerebrospinal fluid (CSF) diagnostics of ventriculitis. We hypothesised that neutrophil granulocytes in infected, blood-containing CSF lose CD62L expression. Therefore CD62L expression on neutrophils may present a complementary marker to distinguish between patients with residual blood and infection. DESIGNS AND METHODS Evaluation was performed in 64 ventricular CSF samples sent to the laboratory for diagnostic investigation. Cell count, microbiological culture, total protein and flow cytometric analysis of CSF were performed. RESULTS Cell counts and CD62L expression were significantly different between the culture positive and negative group. ROC-analysis revealed a significant predictive value for cell count and CD62L expression. Optimal cut-offs were calculated and a decision tree was established to predict a positive culture. CONCLUSIONS Cell count and CD62L expression were predictive for a positive culture and the combination helped to increase specificity and sensitivity for the detection of ventriculitis in blood-containing CSF.

Lactic acid is of low predictive value for the diagnosis of bacterial infection in ventricular cerebrospinal fluid samples containing residual blood

Abstract Background: Lactic acid concentrations (LA) are an established marker of bacterial infection in cerebrospinal fluid (CSF). However, use of LA for the detection of infection in CSF with residual blood has not been fully evaluated. Methods: Analysis of LA and total protein, cell count and bacterial culture were performed in 90 lumbar and ventricular CSF samples contaminated with blood. Results: Bacterial culture was positive in six CSF samples. The diagnostic value of the cell count was significantly higher than that of LA for the prediction of a positive culture, even if all culture positive and all likely infected samples were included in the analysis. There was no significant difference in LA concentrations between positive or likely positive ventricular CSF samples and all negative, ventricular samples. Conclusions: Although LA concentrations in CSF are evidently a predictor of bacterial infection, its diagnostic value for the detection of bacterial infection in ventricular CSF with residual blood is limited. Clin Chem Lab Med 2010;48:1777–80.

Implausible elevation of peripheral thyroid hormones during therapy with a protein supplement

To evaluate thyroid function, the measurement of free triiodothyronine (fT3), free thyroxin (fT4) and thyrotropin (TSH) is part of the diagnostic routine. As elderly patients with hyperthyroidism as well as with hypothyroidism are frequently asymptomatic or oligosymptomatic [1], a careful clinical examination belongs to the routine, too. However, taking the clinical presentation into account is also necessary in younger patients with discrepancies between the laboratory findings and the clinical presentation. Our patient was a 55-year-old who suffers from multiple food allergies (confirmed elevated IgE against pork, beef, rye, wheat, nut, celery). He underwent subtotal thyroidectomy (autonomic adenoma) in 2010, peripheral thyroid hormones and TSH were measured 6-monthly and were within the normal range. Thyroid hormone replacement therapy was recommended in 6/15 by his general practitioner, when TSH was measured 41.7 mU/L; the reference range was 0.4–2.7 mU/L. One hundred micrograms L-thyroxine were not tolerated and reduce soon after being proscribed it to 25 or 12.5 μg by the patient and stopped in 7/15. At the same time, due to assumed malnutrition, a nutritionist recommended Master Amino Acid Pattern (MAP) tablets, a dietary protein substitute, providing eight essential amino acids for “building protein”. The patient had no further medication. Whereas TSH levels initially decreased, fT3 levels were measured about three-fold and fT4 about four-fold elevated from 8/15 on several occasions by an electrochemiluminescenct one-step competitive binding immunoassay (Elecsys® fT3 III and fT4 II tests performed on a Cobas e601 platform, Roche Diagnostics) although thyroid hormone replacement had been stopped (hormone levels see Table 1). The patient had no clinical signs of hyperthyroidism at all. After communication with the chemical pathologist and obtaining the patient’s consent, fT3 and fT4 concentrations were measured in the archived samples with another two-step competitive binding immunoassay on the ARCHITECT® i8200system platform (Abbott Laboratories) and provided hormonal results within the reference ranges. Subsequently an aliquot of the patient’s serum was sent to the Research and Development Department at Roche Laboratories (Penzberg, Germany) for further investigation. The analysis by the provider revealed an interference factor against streptavidin. In competitive immunoassays such anti-streptavidin antibodies bind to the solid phase and prevent binding of biotinylated immune complexes, decreasing the chemiluminescent signal detected and falsely increasing concentration. We assume an association with the intake of MAP with the formation of an endogenous antibody in the patient, which is cross reacting with streptavidin. It remains speculative, in how far this very patient with multiple allergies was predisposed to antibody formation against the exogenous amino acids or other contents of the tablets. MAP intake had been stopped in 10/15. This may explain a continuous decrease of these antibodies with a reversal towards normal peripheral thyroid hormones and TSH between 3 and 8/16 and their good match with the measurements on the ARCHITECT® i8200system platform. Streptavidin antibodies seem to be a rare occurrence. The ARCHITECT® assay is a two-step assay which does not involve streptavidin-biotin binding. *Corresponding author: Prof. Dr. Igor A. Harsch, MD, Thuringia Clinic Saalfeld “Georgius Agricola”, Department of Internal Medicine II, Division of Endocrinology and Metabolism, Rainweg 68, 07318 Saalfeld/Saale, Germany, Phone: +49(0)3671/541569, Fax: +49(0)3671/541403, E-mail: iharsch@thueringen-kliniken.de Peter C. Konturek: Department of Internal Medicine II, Thuringia Clinik “Georgius Agricola” Saalfeld, Saalfeld, Germany Klas Böer and Mike Reinhöfer: Institute of Laboratory Medicine, Greiz, Germany