Validation of the Sysmex CS5100 coagulation analyzer and comparison to the Stago STA-R analyzer for routine coagulation parameters.

INTRODUCTION: The CS5100 analyzer (Sysmex) was validated for the determination of routine coagulation parameters. This fully automated coagulation analyzer uses multiple wavelength technology to perform coagulation (e.g., activated partial thromboplastin time - APTT, prothrombin time - PT, fibrinogen - FBG), chromogenic (e.g., antithrombin - AT) and immunological (e.g., D-dimers - DDi) assays. METHODS: A comparison with the currently used STA-R Evolution (Stago) was performed. Validation and verification of reference values of the CS5100 was performed in accordance to CLSI guidelines (H57-A, H47-A2, and C28-A3). RESULTS: As a different detection system and reagents were used, significant differences were observed (e.g. APTT). The within-day and between-day imprecision, accuracy and total error were all acceptable. The reference values defined by the manufacturer could be used except for APTT. In our settings, the therapeutic anti-Xa range of 0.3-0.7 IU/mL corresponded to an APTT range of 60-100 s (Dade actin FS reagent). The APTT reagent showed factor sensitivities between 46 and 72% for FVIII, IX, XI and XII while the PT reagent showed sensitivities between 34 and 52% for FII, FV, FXII, and FX. CONCLUSION: In conclusion, the CS5100 instrument is suitable for the determination of the APTT, PT, FBG, DDi and AT in routine analysis.

Creating a robust framework for the analysis of cryopreserved samples in quantitative immunological experiments.

Longitudinal clinical or experimental immunological studies warrant the use of cryopreserved samples for flow cytometric phenotyping. Most notably CD62L+ and CD25+Foxp3+ counts were shown to be reduced by past studies. Here we are the first to compare the effects of cryopreservation on cell type calculations performed on a longitudinal dataset. We first compared lymphocyte subpopulation counts from fresh samples with those from samples frozen for either 5 or 6 months coming from 9 individuals. This way, we found that the cell counts obtained after basic lymphocyte differentiation in CD3+CD4+, CD3+CD8+, CD3-CD19+ and CD3-CD56+ were relatively robust for cryopreservation. However, when further subtyping CD4+ and CD8+ cells, we only found CCR7 and CD45RA to have a relation between fresh and cryopreserved counts, but we could not conclude the same for CD62L. Also, CD4+CD25+Foxp3+ were shown to be approximately 0.5 times less counted after cryopreservation. Next, we performed basic longitudinal calculations for which we either subtracted the cell counts at time 1 from the cell counts at time 2 or calculated the ratio between the cell counts at time 2 and time 1, for both fresh and cryopreserved samples. This way, we found that the use of absolute cell counts supported a good one-to-one relation between fresh and cryopreserved counts for all markers except CD62L and CD4+CD25+Foxp3+. In conclusion, we found no support for the use of CD62L and CD4+CD25+Foxp3+ as markers for calculations on flow cytometric counts from cryopreserved longitudinal datasets. However, all other basic lymphocyte markers proved to be relatively robust if absolute counts were used.

Evaluation of the Sysmex pocH-1001 haematology analyser in an outdoor oncology service.

Since rapid blood count analysis as near patient testing is expanding, we evaluated the use of a Sysmex pocH-100i compact haematology analyser in an outdoor oncology setting according to the recently published International Council for Standardization in Haematology (ICSH) guidelines. In total, 838 blood samples from oncology patients were analysed by pocH-100i and re-analysed by a high-throughput haematology analyser for comparison (Abbott CD-4000 or Sysmex XE-2100) to evaluate in use imprecision, comparability and vote-outs. Imprecision was less than 5%, except for platelet enumeration in the low range (within-run imprecision 7%). Good comparability was found even for platelet enumeration in the low range (r2 = 0.82). Vote-outs were found in 10.6% of examined samples. In conclusion, the Sysmex pocH-100i demonstrates good imprecision conform with former publications, produces reliable results in normal and in lower ranges comparable to the results of high throughput haematology analysers. In a well controlled management plan the Sysmex pocH-100i is suitable for near patient testing in oncology.

Performance evaluation of the Sysmex XS-1000i automated haematology analyser.

The Sysmex XS-1000i is a compact new, fully automated haematology analyser, designed to generate complete blood counts with five-part leucocyte differential. In our study, a Sysmex XS-1000i instrument was evaluated according to Clinical Laboratory Standards Institute (CLSI) and International Council for Standardization in Haematology (ICSH) guidelines. Precision, carry-over and linearity were determined. Using a total of 700 patient samples, results from the Sysmex XS-1000i were compared with those from a Sysmex XE-2100, an Abbott Cell Dyn 4000 and the manual reference leucocyte differential. Using quality control material, total and within-run imprecision was less than 3% except for platelets. The system demonstrated good linearity over the entire reporting range and no carry-over (<0.5%). The Sysmex XS-1000i showed good correlation with XE-2100, CD-4000 and the manual reference leucocyte differential. Overall flagging sensitivity and specificity were 91% and 48%, respectively. In conclusion, the Sysmex XS-1000i demonstrated good analytical performance, is able to generate a complete blood count with five-part differential on low blood volumes and has considerable back-up capacity.

Comparison of the reticulocyte mode of the Abx Pentra 120 Retic, Coulter General-S, Sysmex SE 9500, Abbott CD 4000 and Bayer Advia 120 haematology analysers in a simultaneous evaluation.

The Abx Pentra 120 Retic, Coulter General-S, Sysmex SE 9500, Abbott Cell Dyn 4000 and Bayer Advia 120 were evaluated simultaneously in a general hospital laboratory. Linearity, precision, sample stability, carry-over and comparability of the reticulocyte mode were determined following International Council for Standardization in Haematology guidelines for the evaluation of blood cell analysers. All analysers showed good results for dilution, stability and carry-over testing. The between-batch coefficient of variation of the General-S was high compared to the other analysers evaluated. Multiple correlation studies showed good agreement for all analysers in the normal and high reticulocyte range, with correlation coefficients above 0.7. Multiple correlation studies for reticulocytopenic samples (< 15.109/l) were less satisfactory, with a wider range of correlation coefficients (r-values 0.0-0.9). Overall, the General-S, SE 9500 and Advia 120 gave lower reticulocyte counts than the Pentra 120 Retic and CD 4000. Reagent costs were also evaluated. Reagent consumption was close to the manufacturers' specifications for the SE 9500 (Search reagent), CD 4000 (CD Retic) and Advia 120 (Retics) but was higher than stated for the Pentra 120 Retic (Retix), General-S (Retic kit) and SE 9500 (Sheath reagent). Our results show that these new generation haematology analysers will meet the needs of hospital laboratories for reliable and cost-effective reticulocyte counting.

Immunoglobulins D and M multiple myeloma variants are heavily mutated.

Multiple myeloma (MM) is a B-cell malignancy characterized by the expansion of malignant plasma cells within the bone marrow. Previous studies that have examined the Ig VH genes of IgG and IgA MMs have shown the presence of somatic mutations, suggesting that in these cases, the myeloma precursor cell passed through the phase of antigenic selection within the germinal center but is no longer exposed to the somatic mutation process. However, no information about this matter is available in the rare IgD and IgM MM variants. Therefore, we have analyzed the Ig VH genes of three IgD, one IgM, and one biclonal (IgG and IgM) MM for the presence of somatic mutations. Our study demonstrates that all of these myeloma clones have accumulated a high number of somatic mutations within their Ig VH genes but show no intraclonal variation. Moreover, proof that the clone sustained a strong antigenic selection pressure could be provided in three cases (one IgD and two IgMs). Therefore, this study strongly implies that IgD and IgM MMs emerge from a postgerminal center preswitched B cell that is no longer exposed to the somatic mutation process or able to undergo further isotype switching in vivo.

Oligoclonal bands in cerebrospinal fluid detected by PhastSystem isoelectric focusing.

Pharmacia's "PhastSystem" for semi-automated isoelectric focusing (IEF) in thin precast polyacrylamide gels (PAGE) was found to be as sensitive as high-resolution protein electrophoresis (HRPE) in agarose gels and conventional PAGE-IEF for detection of oligoclonal banding (OB) in concentrated cerebrospinal fluid (CSF) samples. Both PhastSystem IEF and HRPE revealed OB in CSF from eight of nine multiple sclerosis patients and four of 10 patients with various types of infection of the central nervous system as opposed to only two of 70 patients with miscellaneous neuropsychiatric disorders. The PhastSystem also frequently detected OB in silver-stained, unconcentrated CSF from patients with multiple sclerosis.

Identification and quantification of N-methylepinephrine in human urine.

N-Methylepinephrine was analysed in human urine using a high-performance liquid chromatographic catecholamine assay. The identity of the compound was confirmed by gas chromatography-mass spectrometry. The excretion of N-methylepinephrine is slightly less than that of epinephrine: excretion values were in the range 2-65 nmol per 24 h (0.4-13 micrograms per 24 h) in 150 hypertensive patients. In addition to increased epinephrine excretion, increased urinary excretion of N-methylepinephrine was found in three out of five patients with histologically proven pheochromocytoma.

Rhabdomyolysis in hypokalaemic periodic paralysis: a clue to the mechanism that terminates the paralytic attack?

The changes in serum levels of myoglobin (Mb) and creatine kinase (CK) during a spontaneous attack of hypokalaemic periodic paralysis were studied. During paralysis, serum Mb and CK were normal. A rise in plasma potassium, resulting in clinical recovery, was associated with a simultaneous rise in serum Mb, and followed by a rise in serum CK. It is postulated that hypokalaemia might cause muscle ischaemia, which would result in an accumulation of free fatty acids (FFA) within the muscle cells. High concentrations of FFA may induce molecular changes and increase the permeability of the sarcolemma. This might be the mechanism by which potassium is released from muscle cells into the circulation and muscle membrane excitability is restored.

Hormonal changes during a spontaneous attack of hypokalemic periodic paralysis.

We studied the hormonal changes during a spontaneous attack of hypokalemic periodic paralysis in a 20-year-old man, before and after treatment with potassium chloride. During paralysis, we observed high circulating levels of insulin, epinephrine, norepinephrine, growth hormone, ACTH and cortisol, most likely reflecting a condition of stress. Normalization of all these hormones occurred with recovery. Plasma aldosterone concentrations were normal. The increased plasma levels of insulin, but also of catecholamines and growth hormone, created a condition promoting potassium uptake in muscle cells. We suggest that stress may play a role in the pathophysiology of the paralytic attacks in this disorder.

High-performance liquid chromatographic assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid.

A procedure is described for the concurrent assay of free norepinephrine, epinephrine, dopamine, vanillylmandelic acid and homovanillic acid in physiological fluids using high-performance liquid chromatography with electrochemical detection. The column packing is an octadecyl-bonded silica. A single mobile phase containing 1-octanesulphonate is used for the assay of catecholamines and for the assay of the acidic metabolites. An efficient sample preparation scheme is presented for the isolation of the catecholamines and their acidic metabolites from the same sample aliquot. Catecholamines are extracted by ion exchange on small columns and adsorption on alumina, using dihydroxybenzylamine as an internal standard. Vanillylmandelic acid and homovanillic acid are recovered from the combined loading and washing effluents of the ion-exchange column by a solvent extraction procedure. Recovery of catecholamines averages 67%. The limit of detection for individual catecholamines is ca. 30 pg. Recoveries of vanillylmandelic acid and homovanillic acid average 77% and 87%, respectively. The use of the same mobile phase for the concurrent assay of catecholamines and their acidic metabolites considerably increases the throughput of samples in the chromatographic system by eliminating the time-consuming column-equilibration periods.

Electrophoresis of serum protein to detect alpha 1-antitrypsin deficiency: five illustrative cases.

We describe five cases of severe alpha 1-antitrypsin (AAT) deficiency to illustrate the importance of visual inspection of electrophoretic patterns of serum proteins. In four patients the diagnosis of AAT deficiency was clinically unsuspected; in the other patient, the electrophoretic pattern was the first clue to confirm the diagnosis. Densitometric scanning of these patterns invariably overestimated the concentration of alpha 1-globulin. By visually inspecting electrophoretic strips instead of relying on densitometry, clinical chemists can help detect AAT deficiency earlier.

Bromism after prolonged use of carbromal.

Most of the brominated monoureide and bromide salt containing drugs are obtainable in Belgium without prescription. Apart from the regularly encountered suicidal attempts, these drugs can also, without the intention of the patient, cause bromism . We report two cases of patients recently admitted because of bromism after prolonged use of Carbromal . Bromism now has become a rather unfamiliar condition. Therefore diagnostic and therapeutic aspects are briefly discussed. Because we dispose now, for the same indications, of more efficient and much less toxic drugs, we suggest that drugs containing a significant amount of brominated monoureides or bromide salts should be removed from the market.