Summary Schistocytes are fragments of red blood cells (RBCs) produced by extrinsic mechanical damage within the circulation. The detection of schistocytes is an important morphological clue to the diagnosis of thrombotic microangiopathic anemia (TMA). Reporting criteria between different laboratories, however, are not uniform, owing to variability of shape and nature of fragments, as well as subjectivity and heterogeneity in their morphological assessment. Lack of standardization may lead to inconsistency or misdiagnosis, thereby affecting treatment and clinical outcome. The Schistocyte Working Group of the International Council for Standardization in Haematology (ICSH) has prepared specific recommendations to standardize schistocyte identification, enumeration, and reporting. They deal with the type of smear, method of counting, morphological description based on positive criteria (helmet cells, small, irregular triangular, or crescent‐shaped cells, pointed projections, and lack of central pallor). A schistocyte count has a definite clinical value for the diagnosis of TMA in the absence of additional severe red cell shape abnormalities, with a confident threshold value of 1%. Automated counting of RBC fragments is also recommended by the ICSH Working Group as a useful complement to the microscope, according to the high predictive value of negative results, but worthy of further research and with limits in quantitation.

The detection of circulating melanoma cells in peripheral blood by RT-PCR correlated with the clinical stage of patients with melanoma and was an independent prognostic factor for recurrence. Further studies are warranted to better assess the significance of this test in the evaluation of prognosis, early detection of relapse, and in monitoring the effectiveness of systemic therapy.

The actuarial probability of malignant transformation and the impact on expected survival were analysed in a series of 128 persons diagnosed with monoclonal gammopathy of undetermined significance (MGUS) over a 20-year period. At a median follow-up of 56 months the M-component remains stable in 101 patients (78.9%), 14 patients (10.9%) have died from non-related disorders and 13 (10.2%) have developed malignant transformation of MGUS (multiple myeloma, 10; primary amyloidosis, two; Waldenström's macroglobulinaemia, one). The actuarial probability of malignant transformation at 5 and 10 years was 8.5% and 19.2%, respectively. When different presenting features were analysed for predictive value of the malignant transformation, the IgA type of MGUS was the only variable associated with a higher probability of such an event (P less than 0.025). Although no significant difference was observed between the survival probability of persons with MGUS and that of the control population, the development of malignant transformation was associated with a shorter survival (P less than 0.001).

This paper, prepared by the EFLM Task and Finish Group on Allocation of laboratory tests to different models for performance specifications (TFG-DM), is dealing with criteria for allocating measurands to the different models for analytical performance specifications (APS) recognized in the 1st EFLM Strategic Conference Consensus Statement. Model 1, based on the effect of APS on clinical outcome, is the model of choice for measurands that have a central role in the decision-making of a specific disease or clinical situation and where cutoff/decision limits are established for either diagnosing, screening or monitoring. Total cholesterol, glucose, HbA 1c , serum albumin and cardiac troponins represent practical examples. Model 2 is based on components of biological variation and should be applied to measurands that do not have a central role in a specific disease or clinical situation, but where the concentration of the measurand is in a steady state. This is best achieved for measurands under strict homeostatic control in order to preserve their concentrations in the body fluid of interest, but it can also be applied to other measurands that are in a steady state in biological fluids. In this case, it is expected that the "noise" produced by the measurement procedure will not significantly alter the signal provided by the concentration of the measurand. This model especially applies to electrolytes and minerals in blood plasma (sodium, potassium, chloride, bicarbonate, calcium, magnesium, inorganic phosphate) and to creatinine, cystatin C, uric acid and total protein in plasma. Model 3, based on stateof-the-art of the measurement, should be used for all the measurands that cannot be included in models 1 or 2.

Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing, or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extent has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 h of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin, and citrate-based CPDA) for two temperatures (4°C and room temperature) were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination), red blood cell (RBC) volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations, and formation of micro vesicles), Ca2+ handling, RBC metabolism, activity of numerous enzymes, and O2 transport capacity. Our findings indicate that individual sets of parameters may require different shipment settings (anticoagulants, temperature). Most of the parameters except for ion (Na+, K+, Ca2+) handling and, possibly, reticulocytes counts, tend to favor transportation at 4°C. Whereas plasma and intraerythrocytic Ca2+ cannot be accurately measured in the presence of chelators such as citrate and EDTA, the majority of Ca2+-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using an optimized shipment protocol, the majority of parameters were stable within 24 h, a condition that may not hold for the samples of patients with rare anemias. This implies for as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the patients to the specialized laboratories may be the only option for some groups of patients with highly unstable RBCs.

Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.

Adenosine deaminase, which is essential for lymphoid differentianon and function, has previously been considered to be a cytosolic enzyme. In this report we demonstrate that it can be found associated with the plasma membrane of lymphocytes. By means ofi,mmunological techniques using both light and electron microscopy, adenosine deaminase was localized on the external side of the plasma membrane of normal lymphocytes and monocytes. Since the enzyme

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