A “miniaturized” method for the karyotypic analysis of bone marrow or blood samples in hematological malignancies

Standard techniques of karyotypic analysis of bone marrow or peripheral blood cells generally use large numbers of cells. Thus, the quantity of cells harvested from one bone marrow or blood puncture frequently represents a limiting factor for other assays such as molecular biology or flow cytometry, which are often essential for the diagnosis of hematological diseases. To resolve this problem, we developed a miniaturized technique of cytogenetic analysis that we tested on bone marrow (BM) cells from 20 patients with multiple myeloma (MM), 5 patients with acute leukemia (AL), as well as on CD34+ cells purified from the blood of 8 patients with agnogenic myeloid metaplasia (AMM). We used 3 × 10⁶ BM cells from MM patients (for testing 6 different culture conditions), 10⁶ BM cells from AL (2 culture conditions) and 4 × 10³ CD34+ cells from AMM patients. In most patients, 20 good quality metaphases per slide were easily analyzed, showing that a 10–40 times reduction of the number of cells used for cytogenetics allows a reliable karyotypic analysis in hematological malignancies.     

A method for the automated processing and analysis of images of ULVWF–platelet strings

We present a method for identifying and analysing unusually large von Willebrand factor (ULVWF)–platelet strings in noisy low-quality images. The method requires relatively inexpensive, non-specialist equipment and allows multiple users to be employed in the capture of images. Images are subsequently enhanced and analysed, using custom-written software to perform the processing tasks. The formation and properties of ULVWF–platelet strings released in in vitro flow-based assays have recently become a popular research area. Endothelial cells are incorporated into a flow chamber, chemically stimulated to induce ULVWF release and perfused with isolated platelets which are able to bind to the ULVWF to form strings. The numbers and lengths of the strings released are related to characteristics of the flow. ULVWF–platelet strings are routinely identified by eye from video recordings captured during experiments and analysed manually using basic NIH image software to determine the number of strings and their lengths. This is a laborious, time-consuming task and a single experiment, often consisting of data from four to six dishes of endothelial cells, can take 2 or more days to analyse. The method described here allows analysis of the strings to provide data such as the number and length of strings, number of platelets per string and the distance between each platelet to be found. The software reduces analysis time, and more importantly removes user subjectivity, producing highly reproducible results with an error of less than 2% when compared with detailed manual analysis.     

High dose chemotherapy followed by autologous peripheral blood stem cell transplantation or conventional pharmacological treatment for refractory rheumatoid arthritis? A Markov decision analysis

OBJECTIVE: To evaluate the effect of high dose chemotherapy (HDC) followed by autologous hematopoietic stem cell transplantation (ASCT) in comparison to conventional pharmacological therapy in the treatment of patients with refractory, progressively erosive rheumatoid arthritis (RA). METHODS: Decision analysis using a Markov model with a 5.5 year time horizon. Probabilities of transition towards 5 different health states, ranging from 70% improvement to death, were derived from published case reports, patient series, and expert panels. Quality of life (QOL) estimates were obtained from 2 RA clinical trials. Patients were hypothetical cohorts of 50-year-old female patients with progressively erosive, active RA, who failed treatment with methotrexate, combination therapy, and tumor necrosis factor blocking agents. Interventions were HDC + ASCT versus conventional pharmacological treatment with a (combination) therapy of disease modifying antirheumatic drugs. As main outcome measures, we included the number of quality adjusted life years (QALY) after HDC + ASCT compared to conventional therapy. Sensitivity analysis was performed to investigate the influence of treatment related mortality (TRM) and the influence of QOL during HDC + ASCT, and to assess the minimal desired effectiveness of HDC + ASCT for a given TRM of 1% and 10%. RESULTS: HDC + ASCT and conventional pharmacological treatment were equally effective in the base-case analysis (3.48 vs 3.46 QALY). A TRM of less than 3.3% favored HDC + ASCT as the preferred treatment. The analysis showed that when TRM was set at 1%, a relatively short period of efficacy was sufficient to remain the preferred strategy, whereas a TRM of 10% would require a sustained response for several years. CONCLUSION: This model predicted equally favorable effects of HDC + ASCT and conventional therapy in the treatment of refractory RA in the base-case. The minor differences in terms of QALY seem to indicate that clinical decision making should be guided by patient preferences. However, better clinical efficacy might be achieved by adaptation of the treatment regimen of HDC + ASCT and patient selection. The model supports the need for randomized clinical trials and may contribute to an optimal study design.     

Is the poly A (T>C) mutation a causative factor for misdiagnosis in second trimester prenatal diagnosis of β-thalassemia by fetal blood analysis on high performance liquid chromatography?

We report the problems in diagnosis faced by two families referred for prenatal diagnosis of thalassemia where cordocentesis and fetal blood analysis by high performance liquid chromatography (HPLC) had to be done. The Hb A levels of the fetal blood measured by HPLC on the VARIANT? Hemoglobin Testing System were 1.2 and 6.7%, respectively, suggestive of a heterozygous β-thalassemia (β-thal) fetus in the first case and a normal fetus in the second case. In one family, one of the parents had a borderline Hb A(2) level and in the other, one parent had normal RBC indices. However, DNA sequencing, done later, showed that in the first case the fetus was a compound heterozygote for the IVS-I-5 (G>C) and the polyadenylation signal site [poly A (T>C)] mutation, while in the second case, the fetus was homozygous for the poly A mutation. This emphasizes that characterization of β-thal mutations must be done whenever one of the parents has a borderline Hb A(2) level or normal RBC indices, and one should not rely on fetal blood analysis by HPLC for prenatal diagnosis of β-thal so as to avoid misdiagnosis.