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.

In recognition of the need for a standardization of the measurement of the erythrocyte sedimentation rate (ESR), the International Council for Standardization in Haematology makes the following recommendations: (i) The reference method for measurement of the ESR should be based on the Westergren method, which is a specific test for the ESR, with modifications, (ii) The reference method for measurement of the ESR should use either whole blood anticoagulated with EDTA and later diluted with sodium citrate or saline (4 : 1) or whole blood anticoagulated with sodium citrate (4 : 1) in Westergren pipettes, (iii) The ESR pipettes can be of glass or plastic (with specific characteristics). It must be colourless; a minimum sedimentation scale of 200 mm, a minimum bore of 2.55 mm, which should be constant within 5%. A protocol for the evaluation of alternative methodologies against the reference method is outlined: The new technologies must be tested over a range of ESR values of 2–120 mm. In this comparison, 95% of the differences should be 5 mm or less, with larger differences associated with higher ESR values. A minimum of 40 samples should be tested in 3 different groups of values: 1–20, 21–60 and more than 60 mm. The statistical methods recommended for ESR evaluations are the coefficient of correlation, the Passing‐Bablock regression and the Bland‐Altman statistical method. This reference method replaces all earlier standardized and reference methods.

Summary The Spanish haematology external quality assessment scheme (EQAS), established in 1984, is run by the Spanish Haematology and Haemotherapy Association (AEHH) [ Quality Assurance in Health Care 3 (1991) 75] and functions to evaluate the quality and reproducibility of the assessment of diagnostic samples by clinical laboratories. The Hospital Clinic of the University of Barcelona (HCB) serves as the EQAS Coordination Centre and follows the guidelines established by the International Committee for Standardization in Haematology [ Annali dell'Istituto superiore di Sanità 31 (1995) 95; International Journal of Hematology 68 (1998) 45]. During the period 2001–2006, replicates of 25 different blood films were sent to 604 EQAS participants for cell morphology evaluation. Some patient details corresponding to the samples were disclosed, such us age, sex, haemoglobin value and white blood cell count. The participants were asked to select up to four significant morphology features using a coding list, provided by the Coordination Centre, which included significant morphological alterations that appear in haematopoietic cells. For each survey, individual results were assessed against the morphological reference results (MRR) established by the Cytology Group of the AEHH (‘true’ answers). This paper describes the organization of the 6‐year‐long study and the evaluation of laboratory performance for blood smear interpretation by the Spanish haematology EQAS. Different performance levels were detected relative to the laboratory category. Laboratories providing services to hospitalized patients showed higher performances compared with laboratories providing services to nonhospitalized patients. Pathological lymphoid cells were the most difficult to identify by the participants. To improve the results in EQAS peripheral blood morphology, the development of specific cytology educational trainings is discussed.

Based on the presence of immature cells in fetal blood, and in an attempt to shorten the cytogenetic reporting time, three simultaneous one-day culture regimes were established in 23 fetal blood samples: (a) the standard phytohemagglutinin (PHA)-stimulated lymphocytes culture, (b) a culture using the granulocyte-macrophage colony-stimulating factor (GM-CSF) as an alternative mitogen, and (c) an unstimulated culture. Diagnostic success rates achieved by these three methods were as follows: 43 per cent (95 per cent CI: 23-64) (GM-CSF), 30 per cent (95 per cent CI: 12-49) (PHA) and 9 per cent (unstimulated). These three regimes were also assayed in three-day cultures giving 100 per cent diagnostic success rate for the PHA and GM-CSF, and 62 per cent (95 per cent CI: 41-83) for the unstimulated. A moderate correlation was found between the initial concentration of cultured erythroblasts and the metaphase count in one-day GM-CSF-stimulated (r=0.43, p=0.01) and unstimulated (r=0.35, p=0.05) cultures, suggesting that erythroblasts may be in part responsible for the mitotic index observed in these two regime cultures. In conclusion, our experience suggests that immature cells in fetal blood may be successfully cultured for diagnostic purposes.

Dengue infection is nowadays considered a re-emergent disease. It has a worldwide tropical and subtropical distribution. The dengue virus in a member of the flavivirus family composed by 4 different serotypes. The virus is transmitted by mosquitos of the Aedes genus. With the increment of travels to the endemic areas, dengue is now observed frequently in our country. We analyzed 57 patients, 30 with imported dengue (ID) and 27 with dengue fever suffered during the trip (DDT). This series is compared with other published ones and a review of the subject is presented. Patients with ID followed a protocol as a febril syndrome returning from the tropics. Dengue was diagnosed through a compatible clinico-epidemiological history, the absence of other ferbil illness and positivity of specific serology. All patients had travelled to endemic areas (Central America 28 cases, Indian subcontinent 15, South-East Asia 10, South America 2, West Africa one, and Pacific one). The following were the most important clinical characteristics: fever and asthenia (100%), headache (98%), mialgia (84%), arthralgia (72%), morbilliform rash (61%) and retroocular pain (65%). For ID cases, the most helpful analitical results were: leucopenia (70%), reactive lymphocytes in peripheral blood smear (70%), thrombocytopenia (70%), and increased hepatic enzymes ALAT (53%), ASAT (63%) and LDH (100% in the 7 patients tested for this enzyme). Dengue must be included in differential diagnosis of fever in patients coming back to travels to tropical areas.

BACKGROUND Platelet counts between 150 x 10(9)/l and 400 x 10(9)/l are considered as normal in the adult population. However in Spain it is not unusual to find lower counts in healthy people. SUBJECTS AND METHODS We have studied platelet counts in 1,430 prospective healthy platelet donors. In 93 we measured mean platelet volume (MPV) in a blood sample collected in citrate (1:4 v/v) in order to avoid the platelet swelling induced by EDTA. Complete blood counts were performed on a Bayer-Technicon H*1. A reference range of 95% was calculated for platelet count and MPV, and the relationship between platelet count and sex, age, hemoglobin and MPV was studied. RESULTS Mean (SD) platelet count (in x 10(9)/l) in men (195 [42]; n = 1,053) is lower (p < or = 0.0005) than in women (213 [47]; n = 377). The reference range in men is 123-295, in women 137-319 and in both 125 to 300. The mean (SD) for MPV (in fl) is 9.6 (0.7) (no significant differences between sexes) and the reference range is 7.8-11. There is an inverse linear relationship between the circulating platelets and their MPV measured in citrate at high concentration (r = -0.282, p = 0.006, n = 93) and in EDTA (r = -0.364, p < or = 0.0005, n = 1,430) and also between platelet count and Hb levels (r = -0.166, p < or = 0.0005). CONCLUSIONS The mean of platelet count in women is higher than in males in a sample of Spanish population. There is an inverse linear relationship between platelet count and their MPV measures measured in citrate at high concentration and in EDTA. The reference range for platelet count seems to be lower than in other populations of North European origin.

PURPOSE:To assess the reliability of the differential leucocyte count (DLC) and the left shift flagging (LSF) system provided by the Coulter MAXM (MAXM) haematology analyzer.MATERIAL AND METHODS:380 blood specimens (drawn with tri-K EDTA as anticoagulant) were studied.RESULTS:By using the reference method (NCCLS H20-A), 50 out of the 380 blood specimens presented abnormal DLC (bands > 6%). Of from these, in 39 (80%) the MAXM displayed LSF of "bands 1 or 2". In 118 left shift flagged specimens (MAXM) with normal manual DLC, 87 (74%) had the "bands 1" alarm and 31 (26%) the "bands 2" alarm. Accordingly if the LSF "bands 1" is overlooked, the percentage of FP decreases from 36% to 10% but the percentage of false negatives (FN) increases from 22% to 58%. In order to improve the appreciation of LSF by decreasing the need of manual revisions, the visual examination of the leucocyte distribution scattergram (LDS), also provided by the MAXM, was conveniently evaluated. This study was performed on 190 blood specimens from which the MAXM displayed a normal DLC in 122 (64%), the LSF of "bands" in 44 (23%) and the LSF of "bands 2" in 24 (12.6%). Of from the 122 specimens with normal DLC, four were FN, of from the 44 specimens with "bands 1" LSF, 37 were FP and of from the 24 specimens with "bands 2" LSF, 16 were FP. The visual appreciation of the LDS showed in the majority of samples with "bands 1" and "bands 2" a definitely different shape consisting in a sharper image up to the top of the picture when compared to samples with normal DLC (without flags). According to this criteria, all the 122 specimens with normal DLC displayed a normal LDS and all the 24 specimens with "bands 2" flag displayed abnormal LDS. Of from the 44 specimens with "bands 1" flag, 26 (59%) showed an abnormal LDS and 18 (41%) a normal LDS. It is noteworthy that of from the 26 specimens with abnormal LDS only 7 were true positive (TP), whereas the 18 specimens with normal LDS all showed a normal DLC according to the reference method. These data allow us to conclude that manual revision was required in 26 out of 68 specimens with "bands 1" and abnormal LDS (13% of the total) and in all the 24 specimens with "bands 2" flag. Therefore by using the information provided by the LDS the need of manual revision decreases to 73% of the total sample with LSF.CONCLUSION:Our results give further support to the idea that th VCS method used by the Coulter MAXM provides a high quality DLC with specific left shift detection.

The Abbott (R) Cell-Dyn 3500 (Abbott CD 3500, Abbott Diagnostics Division, Mountain View, CA) is a fully automated hematology analyzer capable of providing a complete blood count (CBC) profile, including a five-part differential leukocyte count (DLC) and flagging system in this study. The CBC profile and DLC flagging system of the Abbott CD 3500 were evaluated according to the HA-20 protocol of the National Committee for Clinical and Laboratory Standards (NCCLS) and compared to the Technicon H*2 blood analyzer currently used in the authors' laboratory. Linearity, carryover, precision, and stability were all within the limits established by the manufacturer. No significant break-downs were found during the evaluation period. Evaluation of DLC indicated an excellent correlation with the manual reference method for neutrophils, lymphocytes, and eosinophils (r = 0.916, 0.936, 0.967, respectively), a good correlation for monocytes (r = 0.811) and a poor correlation for basophils (r = 0.224). Overall flagging for morphologic abnormalities displayed higher sensitivity (85%) than specificity (67%), with a false-positive ratio of 33%. In general, these results are in accordance with those obtained by other authors in the same period of time.

The increased precision of flow cytometric techniques permits the recognition of small differences even in the low or normal range of the reticulocyte count. Moreover, measurement of the RNA content of reticulocytes makes possible the identification of the youngest highly fluorescent macroreticulocytes (HFR) prematurely delivered from bone marrow in conditions of increased erythropoietic stimulation. The aim of this study was the definition, using the dedicated flow cytometers Sysmex R-1000 or R-3000 (Toa Medical Electronics Ltd, Kobe, Japan), of reticulocyte absolute number and HFR percentage in patients with haematological disorders prior to any treatment. Analysis of 54 healthy subjects and 100 untreated patients with five types of haematological disease is presented. In haemolytic anaemias (15 cases) both the reticulocyte count and HFR were greatly increased and the reticulocyte count was inversely correlated with Hb level, as in the reference population. In polycythaemia vera (20 cases) reticulocytes were moderately increased and directly correlated with Hb. In dyserythropoietic syndromes (20 cases) reticulocytes were low and HFR moderately increased; HFR showed an inverse correlation with Hb. In acute myeloid leukaemia (30 cases) reticulocytes were low and HFR increased; reticulocytes correlated with both HFR and Hb. In acute lymphoid leukaemia (15 cases), while the reticulocyte count did not differ from the reference group, the HFR was increased. These results provide reference values for the evaluation of reticulocyte counts and HFR in haematological diseases. From a physiopathological standpoint, they suggest that in anaemic patients the reticulocyte count directly reflects effective bone marrow erythrocyte production, while the proportion of circulating HFR more closely reflects the intensity of erythropoietic stimulation.

Flow cytometric reticulocyte counting including their maturation fractions was performed with a Sysmex R-3000 automated analyser during follow-up after induction and/or consolidation with combination chemotherapy in patients with acute leukaemias (AL, n = 39; 58 courses) and malignant lymphomas (ML, n = 21; 29 courses). The ML patients received granulocyte colony stimulating factor (G-CSF) in addition after chemotherapy. During the leucopenic phase only reticulocytes of low fluorescence ratio (LFR) at extremely low concentration (< 10 x 10(9)/l) were found. After a median interval of 17 days (range 8-43), the middle fluorescence fraction (MFR) began to rise, preceding high fluorescence ratio (HFR) reticulocytes by a median of 1 day in AL patients with complete or partial remission. In ML patients, MFR and HFR reticulocytes appeared more often simultaneously after a median interval of only 11 days (range 8-15) and increased faster during the first week of marrow recovery showing a pattern different from AL. Granulocytes passed the critical limit of 0.5 x 10(9)/l at a median of 5 days after appearance of MFR reticulocytes in AL but in ML on the same day as MFR and HFR (day 0). The absolute reticulocyte concentration reached the lower limit of the reference range after about 10 days in AL. Thus, finding MFR and, to a lesser extent, HFR at very low cell concentrations, may serve as sensitive early indicators of marrow recovery after chemotherapy and are much more sensitive parameters than the absolute reticulocyte concentration. The higher median values for reticulocytes (total, HFR and MFR) after G-CSF therapy suggests that G-CSF is not lineage specific and may also stimulate erythroid precursor cells.