The role of standardization in quality assurance.

The main organizations involved in standardization of laboratory medicine are described. At the international level these are mainly: ISO, IEC, WHO, ICSH, IFCC and COWS/WASP, while at the European level CEN, CENELEC and BCR are involved in standardization activities in Europe. At the present the European Commission is preparing a directive on in vitro diagnostic. This document will rely, for its implementation, on standards prepared by CEN/CENELEC.

Monocyte counting: discrepancies in results obtained with different automated instruments.

To determine the accuracy of several methods for measuring the monocyte count, the results obtained by a number of different automated cell counters were analysed. Considerable discrepancies occurred for monocyte counts obtained in normal blood among the counters. The results of a visual monocyte count on a total of 800 leucocytes were used as the reference method. The technique of measuring the monocyte count by using dual staining with monoclonal antibodies CD45 and CD14 provided the closest agreement with the reference method. Six other automated counting systems were assessed. Two of these systems (Coulter VCS and Technicon H1) gave results, which, although under-estimating monocytosis, correlated well with the results obtained by the reference technique. A third system (Toa Sysmex NE-8000) gave unreliable results. Three of the automated systems evaluated measured a "third population"--that is, monocytes together with other leucocytes. One of these systems (Ortho ELT 1500), overestimated the count, as expected, but correlated well with the reference method. The second of these "third population counters" (Coulter S Plus IV) correlated moderately well with the reference monocytosis, while the Toa Sysmex E-5000 correlated poorly. It is clear that problems exist in the evaluation of different instruments for counting monocytes. An accurate and reliable reference method is a pre-requisite to evaluate this aspect of cell counters. As the visual method is too cumbersome a different reference method would be useful. Based on the results of this study, it is suggested that the technique using fluorescence labelled monoclonal antibodies should be regarded as an acceptable alternative.

K2- or K3-EDTA: the anticoagulant of choice in routine haematology?

The choice of K2- or K3-EDTA as the preferred anticoagulant for blood count remains controversial. We compared the effect of different concentrations of both anticoagulants on normal blood. In optimal conditions (appropriate anticoagulant concentration and measurements done between 1 and 4 h after phlebotomy), no marked differences are seen between either EDTA salt. Important discrepancies appear, however, in less optimal conditions, as often happens in day to day practice. The packed cell volume, when measured on centrifuged blood, decreases with increasing anticoagulant concentrations and this is most pronounced with the K3 salt. This phenomenon has been reported by different authors and is ascribed to shrinking of erythrocytes in an hypertonic medium. Automated instruments react in a different way, their MCV is not influenced by K3-EDTA concentrations up to ten times normal, while K2-EDTA, at high concentrations, results in a slight increase in MCV, as measured with three of the instruments. With most instruments, the accuracy of the white cell count is not markedly influenced. However, when measured with the Unipath CD 3000 all tested blood samples, taken in high K3 (but not in K2) concentrations, showed an appreciable decrease in the leucocyte count (to less than 50% of the original value, at a concentration of 15 g/l, 24 h after blood collection). Measurement of RDW and automated differentials is also influenced by the choice of anticoagulant when determinations are done in less than optimal conditions. We conclude that the choice of anticoagulant, its use at an appropriate concentration and the age of the blood sample are important matters and should be given due consideration.(ABSTRACT TRUNCATED AT 250 WORDS)

Reticulocyte count using thiazole orange. A flow cytometry method.

Recently flow cytometry techniques have been developed to replace the microscope reticulocyte count. We used thiazole orange, a RNA binding fluorochrome, to discriminate reticulocytes from mature erythrocytes. Thiazole orange and the Retic-COUNT software package were evaluated for performance of routine analysis on different flow instruments. The applied methodology analysed 10(4) cells semi-automatically in an easily performed manner. Consistent results were obtained with dipotassium EDTA anticoagulated blood (stable for 30 h after venesection), with incubation times in thiazole orange solution ranging from 2 to 7 h at 25 degrees C. This allowed flexibility in specimen collection and storage and assay performance with no change in results. Changes of incubation temperature up to 30 degrees C had no measurable effect. The values obtained showed good linearity, precision and accuracy for normal, low and high reticulocyte counts. However interferences were observed: RBC autofluorescence, nucleated RBC, Howell-Jolly bodies, high leucocyte count, high platelet count and giant platelets, all falsely increased the number of reticulocytes. These artifacts were eliminated by software gate corrections, thus leaving less than 5% of the specimen to be reanalysed by the microscopic method. The thiazole orange flow cytometric method was determined to be a fast, reliable method for the routine clinical quantitation of reticulocytes.

Congenital macrothrombocytopenia, leucocyte inclusions, deafness and proteinuria: functional and electron microscopic observations on platelets and megakaryocytes.

We report a patient with a variant of Alport's syndrome: macrothrombocytopenia, leucocyte inclusions, deafness and proteinuria. Ultrastructural studies revealed giant spheroid platelets with a high density of organelles and a disorganized microtubular system. In addition, Fechtner inclusions were observed in neutrophils of the patient and her mother. In platelet rich plasma platelets aggregated normally for the low platelet number, although no shape change was visible. Platelet studies in whole blood using impedance aggregometry gave supernormal aggregation curves; this is not in agreement with the abnormally long bleeding time, showing the limited usefulness of this technique in patients with such large platelets. The megakaryocytes (MK) showed two different distribution patterns of the demarcation membrane system (DMS), which may explain the production of few large platelets. The formation of platelets occurred by fragmentation of the granular zone of the MKs, which seemed to be followed by expulsion of platelets through openings of the peripheral zone. The involvement of cytoskeletal structures in the organization of the DMS and the expulsion of platelets is discussed.

The role of intensive remission induction and consolidation therapy in patients with acute myeloid leukaemia.

Sixty-one patients with AML, 59 adults and two children, were treated with intensive remission induction and consolidation therapy. The median age was 36 years. Forty-four (72%) patients entered complete remission (CR); 11 patients received a bone marrow transplantation. The median survival of complete remitters was 26.5 months; the probability of remaining in CR at respectively 1 and 2 years was 75% and 62%. The only factor significantly correlated with the outcome of remission induction, survival and duration of CR was age. Patients less than 30 years fared significantly better than those 30 years or older; no difference in outcome was observed between patients aged 30-50 and those over 50 years. In patients less than 30 years the CR rate was 95%; 75% of them were still alive at 2 years and only one (5%) has relapsed. In contrast, in patients 30 years or older the CR rate was 60% and the median survival only 11.5 months, 50% of the complete remitters in this age group have relapsed. Morbidity from intensive consolidation therapy was considerable; more than 50% of consolidation courses were complicated by high fever, needing urgent admission; only four (3%) courses had a fatal event. It is concluded that intensive consolidation therapy may be considered as a major advance in the treatment of younger patients with AML, while its role in older individuals remains questionable. A possible explanation for the completely different outcome in younger and older patients with AML is discussed.

The myelodysplastic syndromes.

The myelodysplastic syndromes constitute a fascinating model for monoclonal premalignant disorders. Haemopoiesis is 'dysplastic' with inefficient maturation of a slowly expanding or sometimes of a stable population, of blood cell precursors. About one third of the patients evolve into acute leukaemia, the result of either a progressive expansion of the original clone or a new mutation producing a more malignant subclone. The majority of patients suffer from the results of bone-marrow insufficiency, with pancytopenia and possibly immune deficiency. Characteristic karyotype anomalies involving mainly chromosomes 5, 7 and 8 are seen in half the patients. These same chromosomes are known to carry different oncogenes. The myelodysplastic syndrome occurs mainly in the aged and there is a moderate male preponderance. The incidence is still unknown but is probably similar to that of acute leukaemia. The etiology is also unknown; however, a secondary myelodysplastic syndrome precedes acute myeloid leukaemia, as a late consequence of chemo- and radio-therapy in treated Hodgkin's disease. This suggests that environmental mutagens might also be involved in primary myelodysplastic syndromes. Treatment remains highly unsatisfactory but a few recent developments improve prognosis, at least in the younger patient.

Effect of acute lead intoxication on the ultrastructure of rat erythroblasts and reticulocytes. Morphometric analysis and röntgen micro-analysis.

Effect of 17 hr intoxication of lead on the different maturation stages of erythroid cells were studied in rat. Morphometric methods were used to analyse the lead-induced ultrastructural changes in the early, intermediate, late erythroblasts and reticulocytes. It was found that the toxic effect of lead increases with maturation. Energy-dispersive Röntgen micro-analysis showed that fibrillar structures within vesicles and endoplasmic reticulum contained lead.

The spleen and haemolysis: evaluation of the intrasplenic transit time.

The mean intrasplenic red cell transit time (STT) and the slow mixing splenic red cell volume (SSV) have been measured in patients with hereditary spherocytosis (HS), autoimmune haemolytic anaemia (AIHA) and lymphoproliferative disease (LD). There was an inverse relationship between the mean red cell life span (MRCLS) and the STT in HS (r = -0.96, P less than 0.001) and in AIHA (r = -0.90, P less than 0.001). No such relationship existed in LD. The size of the spleen and the SSV were not related to the severity of haemolysis. Our data offer strong evidence for the conditioning effect of the spleen on HS- and AIHA red cells and suggest that the STT is an index of the adverse effect of the spleen on red cells in patients with HS or AIHA.

Platelets augment granulocyte aggregation and cytotoxicity: uncovering of their effects by improved cell separation techniques using Percoll gradients.

The 'standard' technique of granulocyte preparation for in vitro studies uses dextran removal of erythrocytes and Ficoll-Hypaque gradient centrifugation to increase granulocyte purity. The procedure is lengthy, approximately 150 min in our hands, and provides granulocytes significantly contaminated with platelets (approx. 5 platelets/PMN). We report a technique that replaces dextran with hydroxy-ethylstarch and Ficoll-Hypaque with Percoll. Preparation time is reduced by approximately 40% and platelet contamination by more than 80%. Granulocytes, so prepared, function metabolically (O2-generation, chemiluminescence, HMP-shunt maxima) and, in motility/phagocytosis assays, identically to 'standard' preparations. However, an augmentatory effect of platelets in granulocyte aggregation responses and their mediation of cytotoxicity is uncovered. Ficoll-Hypaque purified cells (platelet-rich) aggregate to a significantly greater degree with FMLP or activated complement lectins and excessively kill 51Cr-labelled target cells when compared to Percoll-preparations (platelet-poor). Re-addition of purified platelets or of platelet release supernatants to the latter reproduces results using the 'standard' preparations.

The histological characterization of ALIP in the myelodysplastic syndromes.

Bone marrow biopsies of patients with a myelodysplastic syndrome (MDS) may, in the absence of an increased number of blasts in the bone marrow smears, contain small clusters of immature precursors. The presence of these cell nests, previously described as "abnormal localized immature precursors" or ALIP, bears a strong prognostic value predisposing patients to early death with an increased risk to develop myeloid leukaemia. In order to describe and delineate this histological characteristic more precisely, we compared bone biopsies of patients with MDS, used in these previous studies, with bone marrow biopsies performed for staging procedures in patients with Hodgkin's disease, non-Hodgkin's lymphoma and carcinoma. From this comparison we conclude that immature precursors are readily differentiated from proerythroblasts, myeloblasts and small-sized megakaryocytes, and that they most probably represent precursors of the myelo-monocytic-erythroid series. A clearcut increase in their number, mostly resulting in the formation of small clusters, is only found in biopsies from patients with MDS.