Evaluation of schistocyte analysis by a novel automated digital cell morphology application.

INTRODUCTION: The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBC, which automatically performs a preliminary characterization and grouping of RBC into 21 morphological categories, including schistocytes. Upon automated classification, the software offers the possibility of reclassification of RBC by the operator. The aim of this study was to evaluate the schistocyte analysis by the CellaVision Advanced RBC Application. METHODS: Schistocyte counts were evaluated comparing the automated count on a CellaVision DM96, both before and after reclassification, with the reference manual microscopic method according to the ICSH criteria. Thirty-six samples of hospitalized patients and 40 samples of controls were analyzed. RESULTS: Within-run, between-run and between-observer coefficients of variation were lower when counted with the CellaVision compared to the manual microscopic count. The very high sensitivity but rather poor specificity implicates the need for reclassification by the operator, following automated analysis. After reclassification, method comparison studies revealed good agreement with the manual microscopic method, with however slightly higher values of schistocytes for the automated analysis. CONCLUSION: The CellaVision Advanced RBC Software Application provides a sensitive and reproducible measurement of schistocytes in peripheral blood, but still requires manual revision. Furthermore, it is an easy-to-use software and an excellent teaching tool that might contribute to standardization in the investigation of schistocyte-related conditions.

Autologous transplantation of bone marrow mononuclear cells for limb ischemia in a caucasian population with atherosclerosis obliterans.

BACKGROUND: Autologous transplantation of bone marrow mononuclear cells (ATBMMNC) has been used successfully in critical limb ischemia. All reported patients were of Asian descent, however, and several studies included only young patients with thromboangiitis obliterans. Whether the beneficial results can be extrapolated to older Caucasian patients with atherosclerosis obliterans and a heavy burden of cardiovascular risk factors remains unclear. METHODS: We enrolled 16 patients (age 78 +/- 2 year) with critical limb ischemia and a high prevalence of hypertension, smoking, diabetes, hypercholesterolemia and uremia. Mononuclear cells were isolated from the bone marrow and injected in the gastrocnemius muscle of the affected limb. RESULTS: Four patients died because of progressive gangrene (two) or unrelated causes (two). Three patients required an amputation and one patient a femorocrural bypass within 12 weeks. The remaining eight patients had a modest improvement of resting pain and/or trophic lesions. Transcutaneous oxygen pressure (ratio lesion/reference) improved from 0.51 +/- 0.11 before to 0.86 +/- 0.03 (P < 0.001) after 12 weeks, whereas ankle-brachial index did not change significantly (0.42 +/- 0.15 vs. 0.59 +/- 0.1; P = 0.23). The number of visible collateral vessels on digital subtraction angiography changed with 0.89 +/- 0.86 on a scale of 1-4 (P = 0.33). Capillary surface area in a biopsy of gastrocnemius, evaluated by immunostaining for endothelial nitric oxide synthase, increased from 0.61 +/- 0.07% to 2.38 +/- 0.73% (P < 0.05). CONCLUSIONS: Although ATBMMNC was associated with objective signs of neovascularization, symptomatic improvement was only modest and restricted to the least affected patients. The discrepancy with previous findings may be related to the high prevalence of cardiovascular risk factors which causes endothelial progenitor cell dysfunction.

A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias.

Chromosomal translocations with breakpoints in T-cell receptor (TCR) genes are recurrent in T-cell malignancies. These translocations involve the TCRalphadelta gene (14q11), the TCRbeta gene (7q34) and to a lesser extent the TCRgamma gene at chromosomal band 7p14 and juxtapose T-cell oncogenes next to TCR regulatory sequences leading to deregulated expression of those oncogenes. Here, we describe a new recurrent chromosomal inversion of chromosome 7, inv(7)(p15q34), in a subset of patients with T-cell acute lymphoblastic leukemia characterized by CD2 negative and CD4 positive, CD8 negative blasts. This rearrangement juxtaposes the distal part of the HOXA gene cluster on 7p15 to the TCRbeta locus on 7q34. Real time quantitative PCR analysis for all HOXA genes revealed high levels of HOXA10 and HOXA11 expression in all inv(7) positive cases. This is the first report of a recurrent chromosome rearrangement targeting the HOXA gene cluster in T-cell malignancies resulting in deregulated HOXA gene expression (particularly HOXA10 and HOXA11) and is in keeping with a previous report suggesting HOXA deregulation in MLL-rearranged T- and B cell lymphoblastic leukemia as the key factor in leukaemic transformation. Finally, our observation also supports the previous suggested role of HOXA10 and HOXA11 in normal thymocyte development.

Simultaneous occurrence of myelodysplastic syndrome and monoclonal B lymphocytes with a different clonal origin.

Bone marrow and peripheral blood from a myelodysplastic syndrome patient with trisomy 13 and monoclonal B lymphocytes (without evidence of systemic lymphoma) were investigated for clonal lymphoid lineage involvement using interphase fluorescence in situ hybridization (FISH) and X-chromosome inactivation assay (HUMARA) on CD19+ and CD34+ sorted cells. Trisomy 13 was detected in 55% of CD34+ cells and in 5.5% of CD19+ cells, the latter being comparable to the negative control specimen. X-chromosome inactivation showed both CD34+ and CD19+ cells to be monoclonal, though their inactivated X-chromosome was different. The results strongly suggested that both populations of CD34+ and CD19+ cells have originated from a different progenitor stem cell.

Haptoglobin phenotype 2-2 overrepresentation in Cys282Tyr hemochromatotic patients.

BACKGROUND/AIMS: Patients with genotypic Cys282Tyr homozygous hemochromatosis differ largely in phenotypic presentation. The HFE mutation on itself does not explain the different manifestations of hemochromatosis. We hypothesized that the genetic haptoglobin (Hp) polymorphism, because of its effect on iron metabolism, could be a modifying factor that influences the clinical presentation of hereditary hemochromatosis. METHODS: In 167 Cys282Tyr homozygous hemochromatotic patients, the frequencies of Hp types (1-1, 2-1 and 2-2) and alleles (Hp1, Hp2) were compared with those in 918 healthy subjects. Clinical and laboratory indices of iron overload were incorporated in the analysis. RESULTS: The Hp 2-2 type was overrepresented in the patient group (P<0.01). Male patients carrying Hp 2-2 had higher serum iron (P=0.003) and ferritin levels (P=0.03) than those with a Hp 1-1 or 2-1 type. The amount of iron removed with phlebotomy was also higher in Hp 2-2 patients (P=0.03). CONCLUSIONS: The Hp 2-2 type is overrepresented among Cys282Tyr homozygous hemochromatotic patients. At diagnosis, iron overload was more pronounced in male patients carrying Hp 2-2. Our data suggest that Hp polymorphism affects iron metabolism in hereditary hemochromatosis.

Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing.

To evaluate the origin of cells after allogeneic haematopoietic stem cell transplantation we optimised and evaluated two commercially available systems (AmpFlSTR Profiler Plus and GenePrint Powerplex-16) which are based on multiplex fluorescent short tandem repeat (STR) analysis. A standard procedure for interpretation of electropherographs was found essential to obtain reproducible results. On the basis of the relative length of donor and recipient alleles, TYPE-I (no shared alleles are used to calculate chimerism), TYPE-II (one shared and one unshared allele is used to calculate chimerism) or TYPE-III (not informative) allelic distribution types were distinguished. Also, stutter peaks were recognised as an important criterion to exclude a marker for analysis. Intralaboratory and multicentre evaluation of the AmpFlSTR Profiler Plus system showed that mixed blood samples could be determined with an absolute deviation of less than 2%. A sensitivity threshold was set at 5% for TYPE-I and 10% for TYPE-II markers since relative imprecision increases at low chimerism values. No significant difference of calculated chimerism values was observed between STR markers shared between both systems. By monitoring 26 allogeneic peripheral blood stem cell transplants, the applicability of the proposed method was demonstrated.

Further characterization of morphologically defined typical and atypical CLL: a clinical, immunophenotypic, cytogenetic and prognostic study on 390 cases.

We analysed a group of 390 patients, diagnosed with chronic lymphocytic leukaemia (CLL). Cases were subclassified as morphologically typical and atypical CLL according to the criteria of the FAB proposal. Typical CLL cases were mostly diagnosed at a low-risk stage (Binet A/Rai 0), required no immediate treatment and expected a long survival; atypical CLL cases mostly presented at a more advanced risk stage (Binet B/Rai I-II), usually required immediate treatment and their survival was shorter. Moreover, clinical staging was of prognostic significance in typical but not in atypical cases. In typical CLL, del(11q) was the most common chromosomal abnormality (21%) whereas in atypical CLL trisomy 12 was found in about 65% of the cases documented with an abnormal karyotype. Although chromosomal abnormalities were associated with a poor survival in typical CLL, they are of no prognostic significance in atypical CLL. Based on these data, we conclude that subtyping CLL by morphology enables the identification of two groups of cases, each characterized by a specific clinical presentation, different cytogenetic abnormalities and prognostic parameters. We speculate that these two groups may represent two related, but different, diseases with different prognostic parameters and a different survival.

Relationship between nuclear morphology and DNA content in large cell lymphomas.

The relationship between DNA content and nuclear morphology in large cell lymphomas (LCLs) was investigated on lymph node imprints. Mean maximum nuclear diameter (mean MND), nuclear shape and chromatin pattern were evaluated microscopically on May-Grünwald-Giemsa-stained slides. DNA content and nuclear area were measured on Feulgen-stained slides by image analysis. Twelve of the 24 cases were DNA diploid and 12 tetraploid. The DNA diploid cases were characterized by medium large (mean MND 11.2-13.7 microns), round nuclei with a fine chromatin pattern. The DNA tetraploid cases had significantly (P < .01) larger (mean MND 13.0-19.1 microns) nuclei and a higher frequency of coarse chromatin pattern, nuclear irregularities and multilobation. One of the researchers, unaware of the DNA index, could predict the ploidy level in 80% of cases on morphology. The linear coefficient of correlation between mean MND and mean nuclear area was 0.84. We also found that nuclear area increased as the cell moved through the cell cycle. DNA content, related to ploidy and position in the cell cycle, is an important explanation for the variable nuclear morphology in LCLs.

Prevalence and induction of circulating antibodies against recombinant staphylokinase.

Streptokinase (SK) is a routinely used thrombolytic agent but it is immunogenic and allergenic; staphylokinase (STA) is a potential alternative agent which is under early clinical evaluation. The comparative prevalence of antibodies against recombinant STA (STAR) and against SK was studied in healthy subjects and their induction with intravenous administration in small groups of patients. Enzyme-linked immunosorbent assays, using microtiter plates coated with STAR or SK and calibration with affinospecific human antibodies, revealed 2.1 to 65 micrograms/ml (median 11 micrograms/ml) anti-STAR antibodies and 0.9 to 370 micrograms/ml (median 18 micrograms/ml) anti-SK antibodies (p < 0.001 vs anti-STAR antibodies) in plasma from 100 blood donors, with corresponding values of 0.6 to 100 micrograms/ml (median 7.1 micrograms/ml) and 0.4 to 120 micrograms/ml (median 7.3 micrograms/ml), respectively, in 104 patients with angina pectoris. Three out of 17 patients with Staphylococcus aureus bacteremia had significantly increased anti-STAR antibody levels (150, 75 and 75 micrograms/ml), and STAR neutralizing activities (2.2, 3.6 and 4.1 micrograms STAR neutralized per ml plasma, respectively). In 6 patients with acute myocardial infarction, given 10 mg STAR intravenously over 30 min, median anti-STAR antibody levels were 3.5 micrograms/ml at baseline, 2.9 micrograms/ml at 6 to 8 days and 1.2 mg/ml at 2 to 9 weeks, with median corresponding titers of STAR neutralizing activity at 2 to 9 weeks of 42 micrograms/ml plasma. Conversely, in 5 patients treated with 1,500,000 units SK over 60 min, median anti-SK antibodies increased from 2.9 micrograms/ml at baseline to 360 micrograms/ml at 5 to 10 days, with corresponding median SK neutralizing activities of 13 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukaemic intermediate lymphocytic lymphomas: analysis of twelve cases diagnosed by morphology.

Twelve cases of leukaemic intermediate diffuse lymphocytic lymphoma (ILL), diagnosed by morphology, were analysed. The morphology of the ILL cells was so typical that it allowed ready distinction from chronic lymphocytic leukaemia (CLL) and other related B cell disorders. All cases were of B derivation, had strong mu and chi or lambda immunoglobulin (Ig) staining, were CD5 and FMC7 positive and CD10 negative. Cytogenetic abnormalities were found in 8 patients all having t(11;14)(q13;q32). DNA analysis revealed a relatively high incidence of hypoploidy. At diagnosis all the patients (9 males, 5 females; median age 68) had a low degree of absolute lymphocytosis but the disease was advanced and mostly widespread. The course of the disease appears to be aggressive and incurable with conventional combination chemotherapy.

Plesiomonas shigelloides meningitis and septicaemia in a neonate: report of a case and review of the literature.

A 2-day-old neonate was admitted to hospital with septicaemia and meningitis found to be caused by Plesiomonas shigelloides. In spite of a course of intravenous ampicillin and cefotaxime he died 2 days later. We could not isolate the bacterium from the faeces of the mother and her dog, nor from the aquarium-water of her parents-in-law nor from the aquarium-water and fish of the doctor who delivered the baby. We briefly review the literature on P. shigelloides meningitis and its epidemiology.

Survey of total and respirable suspended particulate matter in an iron foundry. Comparison of stationary sampling and personal monitoring.

In the workshop of an iron foundry total and respirable suspended particulate matter was collected. The performances of (1) filtration systems with 47 mm membrane filters, (2) Andersen cascade impactors, and (3) personal total or respirable monitoring, were compared at a position away from intense sources of particulate debris. Using 15 stationary samplers a survey was made of the particulate levels in the workshop, over a period of 2 weeks. Very large concentration gradients and concentration variations as a function of time were measured for total suspended particulate matter. In the three major source areas, i.e. the pouring department, the core-making department and the shake-out department, special studies were performed to compare stationary and personal monitoring. In the immediate vicinity of intense point sources of coarse particles, such as core-making or shake-out, stationary sampling cannot be used to estimate the personal exposure to total suspended particulates. For respirable particles, however, one or two well-situated stationary size-selective samplers can provide a good estimate of the personal exposure as measured with a personal respirable monitor. The differences found are in the order of 10-20%.