Detecting human CD34+ and CD34- hematopoietic stem and progenitor cells using a Sysmex automated hematology analyzer.

In clinical medicine, particularly in the newly developing stem cell therapies required to support the practice of regenerative medicine, the measurement of both CD34+ and CD34- hematopoietic stem cells (HSC)/hematopoietic progenitor cells (HPC) is important in obtaining more accurate information about the total HSC/HPC content in various stem/progenitor cell sources. We report the results of an investigation into methods of detecting CD34+ and CD34- HSC/HPC using the immature information (IMI) channel incorporated into the Sysmex XE-2100 and SE-9000 automated hematology analyzers. In this study, CD34+ and CD34- HSC/HPC were separated by immunologic methods and quantified by flow cytometry (FACScan) and IMI channel analysis. In addition, CD34-/CD133+ HSC were prepared by a sequential antibody-based positive selection strategy. These cells appeared in the same area as CD34+ cells in the IMI channel of the automated hematology analyzer. These findings confirmed that an automated hematology analyzer can be used to measure both CD34+ and CD34- HSC. These results may explain the difference in HSC/HPC counts sometimes observed between the automated hematology analyzer and flow cytometric methods for CD34+ measurement. The results of this study demonstrated the potential of automated cell counting methods for measuring HSC content in cellular products for both research and clinical applications.

International Council for Standardization in Haematology (ICSH) recommendations for "surrogate reference" method for the packed cell volume.

The spun packed cell volume (PCV, hematocrit) is a key measurement on which are based hematology instrument calibration, reference range determination, and assignment of values to calibrators/controls. In 2001, the International Council for Standardization in Haematology (ICSH) recommended a Reference PCV method, which is fully traceable to the ICSH reference hemoglobin method. Because of its complexity, however, this method is impractical for occasional use in routine laboratories and is therefore intended primarily for use by manufacturers of capillary microhematocrit tubes, liquid calibrators, and multichannel analyzers. In response to the need for a simpler method--accessible to all routine laboratories--the ICSH offers this "Surrogate Reference" PCV procedure. It is traceable to the original ICSH Reference PCV method and is based on spun PCVs obtained using borosilicate capillary tubes with an already-known relationship to this reference procedure. This ICSH "Surrogate Reference" PCV method is substantially simpler, thus putting it within the reach of most routine hematology laboratories.

Evaluation of Sysmex UF-100 urine flow cytometer vs chamber counting of supravitally stained specimens and conventional bacterial cultures.

We evaluated the Sysmex UF-100 urine flow cytometer (TOA Medical Electronics, Kobe, Japan) with 269 uncentrifuged urine specimens by comparing it with Sternheimer staining and particle counting in 1-microL disposable chambers with both brightfield and phase-contrast microscopy (the reference method). Results of routine test strip analysis, sediment microscopy (182 specimens), and bacterial culture (204 specimens) were also available. Detection of urinary WBCs and RBCs was highly reliable with the UF-100 compared with manual chamber counting (r = .98 and .88, respectively). Identification of bacteria was equal to that with visual microscopy of uncentrifuged specimens; sensitivity was 55%, and specificity 90%, compared with bacterial cultures at a cutoff of > 10(3) colony-forming units per milliliter. Renal damage was difficult to evaluate even with manual methods because of the low counts of renal tubular cells and casts; with standard manual Sternheimer-stained sediment analysis, sensitivity was 65% to 69% and specificity 66% to 91%, compared with the uncentrifuged chamber method at a cutoff of 3 and 10 particles per microliter, respectively. Renal damage was demonstrated with the UF-100 with a sensitivity of 26% to 69% and specificity 92% to 94%, compared with chamber counts. Automated urinalysis with the UF-100 urine flow cytometer offers considerable savings in time and labor. When high sensitivity is needed, visual microscopic review should be performed to detect renal disease.

Icsh activities and Asia.

The International Council for Standardization in Haematology (ICSH), an international organization promoting international agreement on hematological testing, is now restructuring to strengthen its activities. In Asia, a diversity of testing methods exists and the resulting testing levels make it difficult to compare test results internationally among Asian countries. Fortunately, the ICSH is considering regionalizing its organization to 5 sub-societies to increase its activity, and we have been able to establish a new society, ICSH-Asia, under the ICSH umbrella.

Guidelines for organisation and management of external quality assessment using proficiency testing. Expert Panel on Cytometry of the International Council for Standardization in Haematology.

This document is intended to assist towards the WHO objective that external quality assessment (EQA) schemes be established at national and/or regional levels world-wide. Quality assurance is defined as all steps taken by the director of a laboratory to ensure reliability of laboratory results and to increase accuracy, reproducibility and between-laboratory comparability. This includes the use of internal quality control procedures and participation in external quality assessment. Internal quality control provides the means for evaluation of analytic test results at the time of testing in order to decide whether they are reliable enough to be released to the requesting clinicians. EQA, on the other hand, refers to a system of retrospective and objective comparison of results from different laboratories by means of proficiency testing (PT) organised by an external agency. The main purpose is to establish between-laboratory and between-method (including between-instrument) comparability, and agreement with a reference standard where one exists. Internal quality control and EQA complement each other and must never be considered as alternatives.

In vitro stability of the reticulocyte count.

A collaborative study was undertaken to investigate the effect of storage conditions on the measured reticulocyte count in venous blood samples. It was designed to cover variation in the three main determinants, namely time, temperature and anticoagulant. The aim was to determine the time for which samples could be stored for subsequent analysis for clinical purposes.

The campus at the University of Maryland at Baltimore.

The University of Maryland at Baltimore (UMAB) is an urban professional school campus. Its 36 acres include 39 buildings, both historic and new. The campus fabric is marked by diversity. The new facilities master plan was developed to reflect the strategic planning process for the campus. Design guidelines are intended to create a common theme while still encouraging each building to have a unique identity. The new Health Sciences Library/Information Services building will sit diagonally from Davidge Hall, built in 1812 and the symbolic center of the campus.

Discriminant analysis of macrocytic red cells.

Laboratory classification of red cell disorders uses the red cell indices (MCV, MCH, MCHC, RDW) and information gleaned from microscopic evaluation of a blood film. Additional red cell information is now available using the H series of automated blood cell analysers (Ames Technicon Division of Bayer Diagnostics). This study involved the development of a discriminant rule which would differentiate between three causes of macrocytosis (vitamin B12/folate deficiency, alcohol excess/liver disease and a reticulocytosis) using the information available on Research Screen 1 and Report Screen 3 of the H*1 instrument (Report Screen 3 is a graphical display of the trimmed scattergram of red cell volume and red cell haemoglobin concentration and Research Screen 1 displays the associated numerical data). Three methods of analysis were assessed to define a suitable discriminant rule. The percentages of patients correctly classified by the three methods were: 92.1%, 82.0% and 89.2% for Methods 1, 2 and 3 respectively. Method 1 involved the application of quadratic discrimination to transformed variables and produced the best results. Although complex, it could easily be applied using the microprocessor capability of the average multiparameter haematology analyser.

Evaluation of a prototype for a reference platelet counter.

A semiautomated single channel aperture-impedance particle counter was developed as a prototype for a reference platelet count for assigning values to reference preparations used in automated blood cell counts. The instrument is equipped with sheath flow and an aperture orifice of 50 microns in diameter and 60 microns in length to eliminate non-axial flow and minimise coincidence errors. Use of fixed volume and red blood cell:platelet ratios obviate dilution errors. The counter was assessed in accordance with the International Committee for Standardization in Haematology protocol for evaluation of automated blood cell counters. The counter provided a high level of linearity and precision, accurate coincidence correction, controlled volume, stability and negligible carryover.

Surrogate materials for calibration and control: the use of latex particles as calibrants for red cell volume measurements.

Latex particles, including BCR Certified Reference Material CRM 166a, have important applications for checking linearity and for calibrating aperture-impedance instruments used to determine red-cell volumes. Studies performed on Coulter Counters, e.g. the ZBi/C1000, ZM/C256 and Model S Plus IV, using a series of three different mono-sized latex particles have shown linearity but non-zero intercepts. This emphasizes the importance of calibrating instruments at more than one point. Latex particles also have an important role in calibration of red-cell volume measurements provided that a relative latex: red-cell shape factor has been assigned when the instrument is calibrated with fresh blood. Such relative shape factors are independent of latex particle size but need to be specific for instrument type and probably also for the orifice and diluent to be used. Nevertheless latex calibrants can serve an important role in standardizing red-cell volume measurements.

T4: T8-lymphocyte ratios in human bone marrow: the effect of sampling technique.

Marrow aspirate and trephine biopsy T4 : T8-lymphocyte ratios were compared with peripheral blood values. To do this, a new technique for harvesting marrow cells from trephine biopsies is described. Results show that blood and trephine ratios did not overlap. Marrow aspirate ratios were intermediate suggesting that peripheral blood contamination was causing aspirate T4 : T8 ratios to be falsely elevated.

Cyclical neutropenia and T8 lymphocyte mediated stimulation of granulopoiesis.

A 33-year-old female with cyclical neutropenia and a reciprocally cycling T8 (suppressor/cytotoxic) lymphocytosis was investigated. T8 lymphocytes ranged between 1.4 and 5.6 X 10(9)/l and a significant proportion (50-75%) were preactivated (1a+). Fc gamma receptors were detected in only a minority (7-10%). Functional studies on the lymphocytes indicated that despite their phenotype, little natural killer and reduced suppressor activities were present. Anti-granulocyte antibodies were not detectable in the serum. Production of colony stimulating activity (CSA) was assessed in the patient and control subjects' lymphocytes. Using a methylcellulose marrow culture system, the CSA production by the patient's lymphocytes was markedly increased compared with the control. Monoclonal antibody cytotoxic experiments confirmed that the T8 lymphocytes were responsible. As peaks of circulating T8 lymphocytes were synchronous with granulopoietic activity in the marrow, the above findings may represent a homeostatic mechanism which is attempting to compensate for an underlying stem cell defect.