Examination of peripheral blood films using automated microscopy; evaluation of Diffmaster Octavia and Cellavision DM96.

BACKGROUND: Differential counting of peripheral blood cells is an important diagnostic tool. Yet, this technique requires highly trained staff, is labour intensive and has limited statistical reliability. A recent development in this field was the introduction of automated peripheral blood differential counting systems. These computerised systems provide an automated morphological analysis of peripheral blood films, including a preclassification of both red and white cells (RBCs and WBCs, respectively). AIMS: To investigate the ability of two automated microscopy systems to examine peripheral blood smears. METHODS: Two automated microscopy systems, the Cellavision Diffmaster Octavia (Octavia) and Cellavision DM96 (DM96), were evaluated. RESULTS: The overall preclassification accuracy values for the Octavia and the DM96 systems were 87% and 92%, respectively. Evaluation of accuracy (WBC analysis) showed good correlation for both automated systems when compared with manual differentiation. Total analysis time (including post classification) was 5.4 min/slide for the Octavia and 3.2 min/slide for the DM96 (100 WBC/slide) system. The DM96 required even less time than manual differentiation by an experienced biomedical scientist. CONCLUSIONS: The Octavia and the DM96 are automated cell analysis systems capable of morphological classification of RBCs and WBCs in peripheral blood smears. Classification accuracy depends on the type of pathological changes in the blood sample. Both systems operate most effectively in the analysis of non-pathological blood samples.

[Reports of transfusion incidents: experiences from the first year of hemovigilance in the region of the former ZWN (South West Netherlands) blood bank in Rotterdam]. / Meldingen over transfusie-incidenten: ervaringen in het eerste jaar van hemovigilantie in de regio van de voormalige Bloedbank ZWN Rotterdam.

OBJECTIVE: Itemize blood transfusion incidents in the South-West Netherlands region (about 3.5 million inhabitants), where a regional reporting system for transfusion incidents was introduced in January 2001. DESIGN: Prospective, descriptive. METHOD: In the period 1 January 2001-31 December 2001, 22 hospitals voluntarily reported transfusion incidents in patients to the blood bank. All incidents were anonymously recorded in a standardised report and registered in 14 categories. RESULTS: A total of 119 transfusion incidents were reported and categorised as: incorrect blood component transfused (n = 8), mild fever 1-2 degrees C (n = 14), non-haemolytic fever > 2 degrees C (n = 36), acute haemolytic transfusion reactions (n = 3). delayed haemolytic transfusion reactions (n = 18), allergic reactions (n = 11), bacterial contamination (n = 3), transfusion-related acute lung injury (n = 1), near accidents (n = 6) and product recalls (n = 19). There were no reports in the categories anaphylactic shock, post-transfusion purpura, transfusion-acquired viral infection, and transfusion-related graft versus host disease. In the same year of haemovigilance, the blood bank issued a total of 158,000 blood products. A complication rate of 1:700 blood products was calculated. It is estimated that 53% of all incidents were reported. CONCLUSION: Despite all of the safety measures taken, severe adverse events still occurred. A well-run system for haemovigilance can contribute to the knowledge of transfusion incidents. The safety and quality of blood transfusions can be improved if this knowledge is incorporated into ongoing education about blood transfusions and in the prevention and treatment of transfusion reactions.

Different strategies in the laboratory diagnosis of autoimmune disease: immunofluorescence, enzyme-linked immunosorbent assay or both?

We investigated the clinical utility of different strategies for antinuclear antibodies (ANA) and antibodies to extractable nuclear antigens (ENA) testing. All requests for ANA and ENA (n = 485) in a 20-week period were tested by immunofluorescence (FANA) and immunodiffusion (strategy 1), enzyme-linked immunosorbent assay (ELISA) techniques (strategy 2) or a combination of FANA and ELISA (strategy 3). Results of strategy 1 were positive by FANA in 8% (by immunodiffusion in 2%). By ELISA, 11% of the samples tested positive. In 12% (n = 60) of the cases the two strategies did not agree. The positive predictive value (PPV) for autoimmune disease of strategy 1 was significantly higher than that for strategy 2, but after exclusion of rheumatoid arthritis this difference was abolished. In strategy 2 reagent costs were high but working time comparably shorter. With strategy 3 PPV results were not better, whereas costs and working time were higher. The most frequently occurring reasons for ANA/ENA test requests were: joint symptoms (37%), follow up (30%) or abnormal laboratory result (7%). In a survey of the clinicians 66% replied that the test result did not have any consequences, irrespective of the result or the strategy used. We conclude that FANA and immunodiffusion are superior to ELISA techniques. However, the clinical value of ANA/ENA testing is low and more selective test ordering is strongly recommended.

[Survey on blood transfusion policy within Dutch hospitals: wide discrepancies. Central Guidance Organization for Peer Review]. / Enquête bloedtransfusiebeleid binnen de Nederlandse ziekenhuizen: grote variaties.Centraal Begeleidingsorgaan vor de Intercollegiale Toetsing.

OBJECTIVE: To describe the transfusion practices in Dutch hospitals. DESIGN: Descriptive. METHOD: Late in 1995 a questionnaire was sent to the heads of the transfusion laboratories of all Dutch hospitals. RESULTS: The response was 91% (117/128). A large variation was found not only in the ways transfusion medicine was organised but also in laboratory methods used. CONCLUSION: The implementation of the consensus agreements on blood transfusion in the Netherlands (1983, revised in 1989) is poor. Continuous education of the participants should be encouraged. Implementation of these agreements could be facilitated by active participation of the blood bank in the transfusion committees of each hospital.

[2nd revision consensus on blood transfusion. Central Guidance Organization for Peer Review]. / Tweede herziening consensus bloedtransfusie. Centraal Begeleidingsorgaan voor de Intercollegiale Toetsing.

Since 1982 several consensus conferences regarding the transfusion practice in hospitals have been organized in the Netherlands. Repeated updating of the consensus text such as described in this article is required to keep abreast of new developments and changes in clinical practice. Guidelines concerning compatibility testing (the result of compatibility testing is valid for three days at most), the organization of responsibilities and the clinical use of red cell concentrates, including a protocol for transfusion in patients with massive bleeding, were changed. No consensus was reached concerning the need to use only red cells that are antigen c, E and K compatible in women younger than 45 years of age and in patients with abnormal erythropoiesis. Although the need of systematic reviews to support guidelines was recognized, literature reviews revealed that only few studies were designed well enough to allow definitive conclusions regarding the benefits and risks of red cell transfusion.

Application of a computer program to exclude additional unexpected antibodies.

BACKGROUND: When a patient has produced red cell (RBC) antibodies in the past, he or she is at risk of producing additional antibodies after antigen challenge. The presence of these antibodies should be excluded before each transfusion. The following criteria are applied when using commercial test RBCs: RBCs should not express the antigen against which the previously documented antibody is directed. For other clinically significant antigens, at least one RBC sample should be from a donor who is homozygous for the encoding gene. The manual selection of such RBCs is tedious and requires experience. STUDY DESIGN AND METHODS: A computer program has been developed that generates exclusion panels (EPs) by selecting a minimum number of RBCs from commercial test panel complying with current criteria. When RBCs from a donor who is homozygous for the encoding gene are absent, the program selects, as a second-best option, RBCs from a donor who is heterozygous for that gene. The computer program developed for this study investigated the usefulness of commercially available panels from separate manufactures in excluding the presence of additional antibodies. A list of 488 antibodies detected by a regional blood bank in 1994 was used as cases of antibodies documented in the past. RESULTS: In 61 percent of the cases, successful EPs (i.e., those complying with the criteria), consisting of three to four different phenotypes, were selected. In the remaining 39 percent of cases, it was impossible to generate successful EPs: 1 to 2 additional antibodies could not be excluded or could be excluded only by using RBCs from heterozygotes. Commercial panels differed only slightly in their efficiency in providing suitable RBCs. None of the commercial panels could provide suitable RBCs to exclude all additional antibodies in the presence of anti-c, anti-e, or anti-M. Increasing the number of RBCs from which to select EPs only slightly increased the percentage of success. CONCLUSION: Computer-aided construction of EPs quickly shows whether strict criteria can be met or whether alternative techniques should be used. It leads to a significant reduction in the number of RBC suspensions necessary to exclude additional antibodies. Results with various commercial panels differed only slightly.

Four aged siblings with B cell chronic lymphocytic leukemia.

All four aged siblings (>80 years) of one family presented with B cell chronic lymphocytic leukemia (B-CLL). In an attempt to find common characteristics in the four patients, we performed detailed immunological marker analysis, Southern blot analysis of immunoglobulin (Ig) genes, and cytogenetic studies. In three patients clonality of the B-cells could be proven by single Ig light chain expression, but in the fourth patient no Ig light chain expression was detected and clonality of the B cells could only be demonstrated by Southern blot analysis of the Ig genes. Interestingly, in two patients, the Ig gene rearrangement patterns were compatible with the presence of two independent B cell clones, whereas in the two other siblings a monoclonal rearrangement pattern was found. All four patients showed clonal chromosome aberrations, which were different in each patient. In the two patients with biclonal Ig gene rearrangement patterns, two unrelated clones could also be demonstrated by the cytogenetic studies. These combined Ig gene and cytogenetic data indicate the presence of two different B-CLL in two of the four patients. Remarkably, the B-CLL cells of the two oldest patients expressed the CD8 antigen, which is rarely observed. Our finding of six different B-CLL in the four living siblings indicates that the members of this family are highly susceptible to the development of B-CLL. However we could not identify a common factor to explain this susceptibility further. In contrast to the literature, the occurrence of two B-CLL in one patient and the expression of CD8 were not associated with clinically aggressive disease in this family.

A patient with a variant form of hairy cell leukaemia.

A 57-year-old woman was admitted because of weakness, fatigue, abdominal discomfort, easy bruising and splenomegaly. A highly elevated leukocyte count with hairy-cell-like cells was found, the cells being positive for the monoclonal antibodies CD19, FMC7, CD11c and B-ly-7 and negative for CD24 and CD25. Blood and bone marrow were investigated not only in our own laboratory but also in several other laboratories resulting in a variety of possible diagnoses. Only after combining all data could a definitive diagnosis of variant hairy cell leukaemia be made. The patient was treated initially with a splenectomy and later on with interferon-alpha-2b, resulting in a steady decrease in the leukocyte count. After a follow-up of 2 years a nearly complete remission was obtained with a good quality of life. The differential diagnosis of this rare disorder is discussed with emphasis on the relative contribution of different diagnostic procedures.

Multivariate statistical modeling: alternative approach to test evaluation, applied to counting reticulocytes by flow cytometry.

We present a statistical path analysis model for the evaluation of two tests in the absence of a "gold standard" method. This model is applied to the evaluation of flow-cytometric and visual reticulocyte counting by using as the comparison method a combination of three hematological measurements: hemoglobin concentration (HGB), mean cellular volume (MCV), and erythrocyte density width (EDW). We assumed that, in general, a higher reticulocyte count is associated with a lower HGB value and with greater values for MCV and EDW. Applying this assumption and the statistical model, we demonstrated that flow cytometry was superior to visual reticulocyte counting in the low-value range studied. The path analysis model is potentially applicable in other cases where two tests are to be compared, and when no gold standard is available.

Bhattacharya: a new application for quality control.

The Bhattacharya method and the 'average of normals' method for internal quality control were compared. Both are based on unselected patient test results. The Bhattacharya method is mostly used in clinical chemistry for calculation of reference intervals but could be modified for quality control procedures. Using the data of a coagulation test and stimulating systematic errors, it was concluded that the Bhattacharya method is more sensitive to expected shifts and is more flexible than the 'average of normals' method.

Metabolic studies of erythropoietin (Ep): I. A reliable micro assay method for Ep in rat plasma using fetal mouse liver cells (FMLC).

It is a well recognized problem that sample derived transferrin-bound iron (Tf-Fe) interferes with radio-iron incorporation into heme in the in vitro assay of erythropoietin (Ep) using fetal mouse liver cells (FMLC). This paper describes a mathematical procedure to correct for the unknown quantity of "cold" Tf-Fe in a plasma sample, being assayed for Ep in the FMLC. Excellent recovery of Ep activity was obtained with this method of correction, when testing solutions containing Step III sheep Ep with and without human Tf-Fe. The distorted dose response curves, obtained, when testing specimens of rat plasma, showed, after correction, linearity and parallelism to a Step II Ep dose response curve, permitting valid estimation of the potency ratio. Reproducibility of this method was found to be excellent. Accuracy was acceptable with plasma samples containing more than 50 mU Ep/ml. The FMLC and the exhypoxic polycythemic mouse assay were compared using several batches of rat plasma. Good agreement was found between the results in both assays. As only a small amount of sample is needed (0.2 ml plasma) this method enabled us to estimate EP activity in single rats serially without disturbing their plasma volume significantly.

Metabolic studies on erythropoietin (EP): II. The role of liver and kidney in the metabolism of Ep.

Half plasma disappearance time (HDT) of endogenous erythropoietin (Ep) was measured in single rats, using an in vitro assay system for EP. In rats treated with the hepatotoxic agent d-Galactosamine-HCl (GalN), a small but significant elevation of HDT was found as compared with control rats (164 and 105 min, respectively). In bilaterally nephrectomized rats mean HDT was significantly elevated: 266 min. Combination of nephrectomy and GalN treatment did not result in a significant further elongation of HDT (mean = 301 min). In experiments using isolated liver perfusion, rat livers (with and without GalN treatment) were shown to be unable to change perfusate Ep titre during 4 h of perfusion. It is concluded that hepatic degradation of Ep in rats is only minimal. The kidney however is important in the catabolism of Ep.