First clinical evaluation of the CELL-DYN® 3200 haematology analyser

A prototype of the CELL-DYN 3200® haematology analyser was evaluated in a tertiary care hospital laboratory. Precision, effects of sample ageing, linearity, carry-over, and com-parability of cellular blood counts and five-part leucocyte differentiation were determined in accordance with the ICSH guidelines for the evaluation of blood cell analysers; the results were satisfactory for all parameters tested: haemoglobin concentration, RBC, MCV, WBC, platelet count, and counts of neutrophils, lymphocytes, monocytes, and eosinophils. Two-hundred and forty-seven routine blood samples were used for the comparability studies. The cellular blood count results from the CELL-DYN 3200® and the Bayer Diagnostic H-1 systems corresponded closely (correlation coefficient r > 0.96 for all parameters). For 201 samples without an instrument-generated suspect flag the same was true with regard to the dif-ferential parameters, although somewhat lower correlation was observed for monocyte counts (r = 0.88). Comparisons to 400-cell microscopic differentials gave similar results (r > 0.93 for neutrophil, lymphocyte and eosinophil counts). Our results suggest that the CELL-DYN 3200® analyser will serve the needs for automated blood cell counting and differential leucocyte counting in a tertiary care hospital laboratory.     

Interleukin-10 promoter polymorphisms in rheumatoid arthritis and Felty's syndrome

OBJECTIVE: To examine whether promoter polymorphisms associated with variation in interleukin-10 (IL-10) production are relevant to the development of rheumatoid arthritis (RA) or Felty's syndrome (FS). METHODS: DNA was obtained from 44 FS patients, 117 RA patients and 295 controls. The promoter region between -533 and - 1120 was amplified by polymerase chain reaction, and polymorphisms detected by restriction enzyme digest or sequence-specific oligonucleotide probing. RESULTS: We found no significant difference in allele or haplotype frequencies between the groups. CONCLUSION: There is no association between FS or RA and these recently identified IL-10 promoter polymorphisms. Other genetic or environmental factors could explain the alterations in IL-10 levels seen in these conditions.      

Hyperlipidämie und Fibrate unter besonderer Berücksichtigung des Diabetes

The lipid triad: hypertriglyceridemia, low HDL, small dense LDL represents a high risk group for excessive cardiovascular morbidity and mortality in type 2 diabetes. In poorly controlled diabetes LDL are increasingly oxydized and glycosylated whereas HDL degradation is accelerated. The high lipid risk in coincidence with the diseases of the metabolic syndrome leads to the conclusion that diabetes today is a cardiovascular disease demanding an aggressive correction of the lipid triad. The benefit of lipid lowering treatment has been proven not only for statins but there is more and more evidence also in favour of fibrates. Fibrates are particularly useful in the treatment of hypertriglyceridemia/low HDL. This also leads to a reduction in small dense LDL. In the case of insufficient correction of LDL a combination with a low dose of a statin is recommended. A definite answer with respect to the benefit/risk ratio of fibrates should be provided by large ongoing studies with these drugs in representative groups of diabetes patients (LDS, TrUMPET, FIELD).     

Biodegradation of Titanium Implants After Long-Time Insertion Used for the Treatment of Fractured Upper and Lower Jaws Through Osteosynthesis: Element Analysis by Electron Microscopy and EDX or EELS

Twelve patients underwent an osteosynthesis with titanium to treat upper and lower jaw fractures. Six to 12 months later, the miniplates were removed. Tissue samples were analyzed by light and electron microscopy for detection of a metallosis. The analysis showed new bone formation like callus tissue around the miniplates. In some cases small, rounded deposits and accumulation of colloid-like particles located next to bigger titanium artifacts were detected in the cytoplasm of histiocytes and in the matrix of connective tissue. The titanium was identified by elemental analysis using EDX in SEM as well as by EELS and electron diffraction in TEM. Both kinds of particles contain titanium, but they seem to be different in composition and derivation. The bigger particles seem to consist of metallic titanium and sourced by working on the metallic implants during the implantation itself. On the other hand, the colloidal-like, small, rounded particles in tissue macrophages and outside the cells in the matrix of connective tissue are presumably of other origin; for example, they could be derived from biodegradation and chemical conversion of the metallic implants. The titanium miniplates were examined before and after implantation by using ESCA technique and revealed metallic titanium and different compositions with other elements. The amount of titanium load of the tissue was very low in most cases and presumably not of biomedical relevance.     

Ultra‐deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas

The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α‐ and β‐chains (TCRb). Our aim was to assess whether ultra‐deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra‐deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR‐sequencing data. A polyclonal T cell repertoire with 367–16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, –0.218 to 0.465). 3–93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra‐deep TCR‐sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid- dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506- FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.