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Paroxysmal nocturnal haemoglobinuria (PNH) is a rare stem cell disorder causing, in untreated patients, symptoms that include renal damage, thrombosis and increased mortality. When correctly diagnosed and treated, patients have reduced symptoms and normal life expectancies. Historically PNH testing resided within blood transfusion laboratories using techniques that were insensitive, for example, the Ham test. However, technology has evolved and flow cytometry is now regarded as the gold standard methodology. Given the clinical importance of diagnosing PNH correctly, we undertook a study to examine PNH testing procedures in blood transfusion laboratories within the UK and Ireland to determine implementation of best practices. An online survey was issued to 386 blood transfusion laboratories in the UK and Ireland requesting details of their current PNH testing practices and procedures. There were 143 responses, representing a 37% response rate. Of these, we identified seven laboratories undertaking PNH testing using obsolete methodologies. Furthermore, multiple centres did not refer samples for confirmatory testing by national PNH reference centres and inclusion on the national PNH disease registry. Staff handling requests for PNH testing should ensure that all samples are tested in accordance with current best practices using only flow cytometry.

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BACKGROUND: CD4(+) T-lymphocyte subset enumeration is routinely used for monitoring HIV disease progression, with approximately 300,000 tests performed annually in the UK alone. Technical variables can impact upon any laboratory test and therefore the final result obtained. Here, we report the findings of a survey questionnaire issued to 1,587 clinical flow cytometry laboratories to: (a) determine if the UK NEQAS for Leucocyte Immunophenotyping (UK NEQAS LI) lymphocyte subset external quality assessment (EQA) programme was suitable for current laboratory needs and practices; and (b) assess the impact of these responses on clinical practice where CD4(+) T-lymphocyte subsets analysis is undertaken. The survey covered areas not traditionally examined by EQA such as: staffing numbers, flow cytometer age and service intervals, plus six test specific sections covering: leukaemia immunophenotyping, CD4(+) T-lymphocyte subsets analysis (reported here), CD34(+) stem cell testing, low level leucocyte enumeration, minimal residual disease testing and PNH testing. RESULTS: The responses revealed major methodological variations between centres undertaking CD4(+) T-lymphocyte subset analysis. Significant differences existed in basic laboratory practices such as: normal range derivation; pipetting techniques; instrument maintenance and units of reporting, all of which results in non-adherence to international guidelines. DISCUSSION: Despite the availability of international guidelines our survey highlighted a lack of concordance amongst laboratory techniques. Such variation could adversely impact on patient care and clinical trial data. Therefore, it is recommended centres undertaking flow cytometric CD4(+) T-lymphocyte subsets analysis urgently review their methodologies and normal ranges to ensure they are fit for purpose and meet current international guidelines.

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This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.

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The diagnosis of plasma cell leukaemia, a rare disorder with an aggressive clinical course and poor prognosis, is not always straightforward and may be dependent on the results of immunophenotyping. Samples from two cases of plasma cell leukaemia have been issued by the UK NEQAS for Leucocyte Immunophenotyping Scheme during the last 5 years and on each occasion a significant number of laboratories failed to make the correct diagnosis. The details of the two samples issued and the results of both surveys are presented. The data highlights the need to adhere to guidelines for immunophenotyping, with respect to using the correct antibody panels, the importance of data interpretation in conjunction with morphological appearance as well as the need to participate in external quality assurance schemes.

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Leucocyte counts of < 5 x 106 per blood transfusion product are currently recommended in the UK in order to reduce transfusion-related infections and febrile reactions. Routine leucocyte depletion, however, requires the development of reliable internal and external quality assurance (EQA) programmes. We report preliminary findings from the UK NEQAS for Low-Level Leucocyte Counting from 18 UK Transfusion Centres over a four month period. Data analysis showed that the IMAGN 2000 had the lowest CVs (range 7.5-36%, mean 16.7) for samples with counts of 5-30 cells/microl when compared to the flow cytometric (range 13.8-88%, mean 29.5) and Nageotte methods (range 20.6-117%, mean 61.8). In addition, laboratories using commercial nuclear stains (LeucoCOUNTTM) had consistently lower CVs than those using 'in-house' propidium iodide staining methods. Important differences in flow cytometric gating strategies were also identified. This study highlights the current variability in low level leucocyte counting, especially within the critical range of 5-30 cells/microl (equating to < 5 x 106/l). The acceptance of consensus protocols, including gating strategies and nuclear staining techniques, is required to reduce the observed interlaboratory variation. Finally, we demonstrate that stabilized blood preparations can be successfully used to provide a national/international low-level leucocyte EQA scheme.

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The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single-platform flow cytometric protocol for the enumeration of CD34+ stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34+ stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34+ Stem Cell Quantification that analysed the same specimens using non-standardized methods. Two bead-counting systems, Flow-Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow-Count, the intralaboratory coefficient of variation (CV) was

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As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.

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To determine the potential advantage of single-platform technology in the enumeration of CD4+ T lymphocyte and CD34+ stem cells, data has been analysed from the UK NEQAS for Leucocyte Immunophenotyping schemes. The inter-laboratory CVs for CD4+ T lymphocyte counts were consistently lower for single-platform (mean 13.7%, range 10-18.3%) compared to dual-platform methodology (mean 23.4%, range 14.5-43.7%). Subgroup analysis of single-platform users demonstrated mean overall inter-laboratory CVs of 17.2%, 13% and 7.1% for the FlowCount, TruCount and volumetric approach respectively. The lowest inter-laboratory CVs obtained for a single sample by each single platform approach were 4% (TruCount), 4.4% (volumetric), 4.6% (FACSCount) and 12.7% (FlowCount). Similarly, the mean inter-laboratory CV for CD34+ stem cell enumeration using non-standardized single-platform approaches was 18.6% (range 3.1-36.9%) compared to 28.6% (range 19-44.2%) for the dual-platform technology. Our results suggest absolute cell subset enumeration should be performed by single-platform technology and that such an approach should improve the quality control of multi-centre clinical trial data for CD4+ T lymphocyte and CD34+ stem cells.

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One-year patient survival rates have improved remarkably, from 84% in 1968 to 97% in 1980 for parental donor grafts, and from 65% to 90% for cadaver donor grafts. In contrast, graft survival rates showed a steady decline from 1968 to 1975 but subsequently improved at a rate of 2.4% per year for parent donor transplants and 2.7% per year for cadaver donor transplants. During this period of improving survival rates, the pretransplant transfusion exposure rate increased from 52% in 1977 to 91% by 1981. We conclude that transplantation has now reached a new level of acceptability as a clinical treatment modality and that blood transfusion has produced its effect on graft survival when results are disseminated over a large number of transplant centers.

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The peak-monitor software of the Technicon SMAC system has been investigated by experiments designed to test its ability to detect abnormally shaped peaks, deliberately produced by presenting serum samples of inadequate volume for analysis. The software provided by the manufacturer is shown to detect faulty peaks inefficiently, and a set of modified parameters has been identified which greatly improves the performance of the peak-monitor software.

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A heat-inactivation method for determining absolute activities of liver and bone alkaline phosphatases in serum has been applied extensively in routine diagnosis. Values for each isoenzyme in healthy individuals of different ages are reported together with results obtained in various diseases. Data from normal subjects show that bone alkaline phosphatase contributes about half the total alkaline phosphatase activity in adults. Liver phosphatase shows a slight increase with age. The method is also able to detect reliably the presence of carcinoplacental isoenzymes.

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This article discusses the advantages and disadvantages of no penalty and penalty marking systems. It also reviews ways in which examiners have attempted to correct for guessing by candidates, and the use of 'don't know' options and confidence-weighting for attempting to assess the degree of certainty that candidates attach to their answers.

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A method is described for the determination of the percentage of liver alkaline phosphatase in serum samples in which measurements of residual activity are made after incubating the samples for 15 and 25 min at 56 degrees C. This method has the advantage of taking into account quantitative variations in the heat stability characteristics of liver alkaline phosphatase from one sample to another, in contrast to methods in which only a single period of exposure to 56 degrees C is employed.

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The course of the decline in alkaline phosphatase activity during exposureof serum samples to a temperature of 56 degree C can be resolved into two phases. These represent the exponential decay of an enzyme component with an average half-inactivation time of 112 seconds and of a second component with an average half-inactivation time of 456 seconds. The more rapid fall is due to inactivation of bone alkaline phosphatase and the slower to inactivation of liver and, when present, intestinal phosphatases. The half-inactivation times of the different enzyme species show considerable variation from one serum sample to another. The implications of this variation for methods of estimating the relative proportions of alkaline phosphatase isoenzymes by selective inactivation procedures are discussed.

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