The role of a new noninvasive imaging technology in the diagnosis of anemia.

Traditionally, numerical data regarding the status of a patient are a combination of measurements made at the point of care (POC) and those made in the laboratory on specimens withdrawn from the patient. We report here on our experiences with a new method for a noninvasive determination of anemia, as defined by blood hemoglobin (Hb) concentration. This method is based on a novel technology, orthogonal polarization spectral imaging, which provides high quality digitized images of the microcirculation using reflected light. Measurements of Hb, based on the analysis of these images at the POC, were found to compare favorably with results obtained with traditional laboratory methods. Additional advantages of these new POC technologies are that they will make possible completely new measurements that may have no direct analog with existing methods. For example, orthogonal polarization spectral imaging can give feedback regarding microvascular density, which also may be reduced in anemic subjects. This information may give earlier and different insights regarding the patient status in nutritional deficiency anemia than an Hb concentration only. However, additional research will be required to confirm the accuracy and utility of this measurement, especially in adult and pediatric populations, where anemia is more commonly encountered. The ultimate success of POC testing will require collaboration between the attending health care professional, the laboratory and institutional management to rapidly assimilate improved methodologies and new information to provide benefits to the patient.

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.

Variability in absolute lymphocyte counts obtained by automated cell counters.

There is increasing interest in the absolute lymphocyte count. This is partly driven by the need to obtain absolute values for lymphocyte subsets such as absolute CD4+ counts in human immunodeficiency virus (HIV)-infected persons. The absolute total lymphocyte count is usually determined in the routine hematology laboratory on a separate sample from the same patient specimen and then combined with percentage results from flow cytometry to obtain the absolute value of the lymphocyte subsets. We have studied analytic variability in the absolute lymphocyte determination and compared it to the variability of the total white blood count (WBC). In a series of 524 specimens, four different automated methods were compared to each other and to the traditional eye count differential. The automated methods were four widely used automated cell counters (Technicon H*1, TOA NE8000, Coulter STKS, and Abbott CD3000). The results indicate that analytic variability in the absolute lymphocyte counts, due, primarily, to method variability, is significant and is larger than the variability typically observed on interlaboratory trials of relative CD4 counts. These method biases cannot easily be reduced by calibration, since the cell classification algorithms are built-in features of the various cell counters. Analytic variability of the absolute lymphocyte counts was found to be 12.4% compared with analytic variability of only 4.9% for total WBC counts on the same samples. Our data suggest that more precise results would be obtained if flow cytometry results expressed each phenotype as a fraction of the leukocytes as well as total lymphocytes. Conversion to absolute values could then be accomplished through determination of the total WBC in the routine hematology laboratory.

Automated quantitation of cell density distribution and hyperdense cell fraction in RBC disorders.

An altered state of cell hydration is a hallmark of a number of RBC disorders. The two parameters most often used to characterize the state of cell hydration are (a) the complete cell density distribution profile, and (b) the fraction of cells with densities greater than a defined value. Buoyant density cell fractionation with a variety of polymers has long been the preferred method for obtaining data on cell density distribution. Although this method provides accurate and quantitative information on the state of RBC hydration, its applicability has been limited due to the time-consuming experimental procedure involved in generating the data. A recently developed light-scattering method has been used in the present study to quantitate RBC density distribution. This new method accurately quantitates both the cell density distribution profile and the fraction of dense, dehydrated cells in various RBC disorders. The ability of the automated method to generate this information rapidly makes possible objective testing of different hypotheses concerning the contributions of cell hydration and dehydration to the pathophysiology of various RBC disorders.

Cell membrane and volume changes during red cell development and aging.

This paper provides a summary of our understanding of cell membrane and volume changes during red cell development and aging. Cytoskeletal structures which include microtubules and microfilaments appear to play key roles in the genesis of the anucleate reticulocyte from its nucleated precursor cell, as well as in the early stages of reticulocyte development. The maturation of reticulocyte into red cell is accompanied by marked changes in cell shape and extensive remodeling of the membrane skeleton, resulting in the mature red cell acquiring a highly deformable yet remarkably stable membrane. The volume and cell density heterogeneity seen for circulating red cells also appears to be the result of the membrane changes that occur during reticulocyte maturation. Following its genesis from reticulocyte, the mature red cell undergoes further membrane and volume changes during its life span of 120 days. While it is clear that surface area loss, decrease in cell volume and cell surface modifications leading to binding of immunoglobulins accompany red cell aging, the cardinal cellular modification responsible for the removal of senescent red cells from the circulation is yet to be defined.

Accurate and independent measurement of volume and hemoglobin concentration of individual red cells by laser light scattering.

Cell volume (MCV) and hemoglobin concentration (MCHC) are the red cell indices used to characterize the blood of patients with anemia. Since the introduction of flow cytometric methods for the measurement of these indices, it has generally been assumed that the values derived by these instruments are accurate. However, it has recently been shown that a number of cellular factors, including alterations in cellular deformability, can lead to inaccurate measurement of cell volume by these automated instruments. Because cell hemoglobin concentration and hematocrit are computed from the measured values of cell volume, accuracy of these indices is also compromised by inaccurate determination of cell volume. A recently developed experimental flow cytometric method based on laser light scattering, which can independently measure volume and hemoglobin concentration, has been used in the present study to measure MCV and MCHC of density-fractionated normal and sickle red cells, hydrated and dehydrated normal red cells, and various pathologic cells. We found that the new method accurately measures both volume and hemoglobin concentrations over a wide range of MCV (30 to 120 fL) and MCHC (27 to 45 g/dL) values. This is in contrast to currently available methods in which hemoglobin concentration values are accurately measured over a more limited range (27 to 35 g/dL). In addition, as the experimental method independently measures volume and hemoglobin concentration of individual red cells, it allowed us to generate histograms of volume and hemoglobin concentration distribution and derive coefficient of variation for volume distribution and standard deviation of hemoglobin concentration distribution. We have been able to document that volume and hemoglobin concentration distributions can vary independently of each other in pathologic red cell samples.

Variability of erythrocyte size and hemoglobin content observed in man and four selected mammals.

A new measurement technique employing light scattering at different angles has been developed for analysis of blood cells. This method which is part of the Technicon H*1 system, a hematology analyzer designed for routine processing of human blood samples, allows the independent measurement of cell volume and hemoglobin content of isovolumetrically sphered red blood cells. Analysis with this instrument of the blood of humans, dogs, rabbits, rats and mice demonstrates that in addition to the expected differences in hematologic parameters, the intrasample distribution of cell hemoglobin is species dependent. In general, cell hemoglobin content is more tightly controlled for the other mammals when compared to humans. In particular, the dogs tested showed the least variability in cell hemoglobin content both within species and within sample.

Protocol for evaluation of automated blood cell counters. International Committee for Standardization in Haematology (ICSH).

This protocol is proposed for the evaluation of automated blood cell counters to assess the performance, advantages and limitations of such instruments. It is based on the International Committee for Standardization in Haematology (ICSH) 'Protocol for type testing equipment and apparatus used for haematological analysis' (1978a) and the British Committee for Standardization in Haematology 'Guidelines for the evaluation of instruments used in haematology' (Shinton, England & Kennedy, 1982). The document has been prepared by the ICSH Panel on Cytometry after discussion with colleagues. This tentative protocol will be reviewed 1 year after publication, in accordance with the ICSH rules, before it is adopted as a definitive standard.

Fully automated preparation of high-quality stained blood films.

A detailed description is given of the operation of Technicon's AutoSlide, which automatically produces a microscope-ready, precipitate-free, stained blood smear with superior cell distribution and good morphology. A 40-test-tube turntable carrying anticoagulated blood inputs samples at 40-second intervals. The blood films are drawn consecutively on a continuous Mylar substrate, with nylon mesh replacing the usual glass slide spreaders. This flexible substrate then passes through drying, fixing, staining and final drying stations. The methanol of conventional Romanowsky stains is replaced by low-volatility solvents. The fixing solution contains solvents, toluidine blue O, glutaraldehyde and a trace of water. The modified Giemsa-stain stock, when mixed with buffer, remains precipitate free for several days. Finally the blood film is imprinted with a date and identification number. Liquid monomer is dispensed onto each stained blood film, followed by a microscope slide. The monomer is then polymerized using ultraviolet light. Permanent transfer of the stained and labeled blood film occurs when the Mylar is stripped from the slide. The usable area for examination is approximately five times larger than that of a typical manual wedge smear.

New optical technique for measuring erythrocyte deformability with the ektacytometer.

The laser light scattered by erythrocytes subjected to a well-defined shear stess can be analyzed with the ektacytometer to obtain information regarding the changes in cell shape due to fluid shear. We describe an optical technique whereby an observed quantitative output derived from a mesurement of light intensity through a spatial filter is related to the change in cellular dimensions that were previously observed under similar fluid-shear conditions by use of microscopy and a cone-plate viscometer (rheoscope). We also present the predictions of a theoretical model (of the ektacytometer) based on approximations of light-scattering theory developed for nonspherical particles, and give preliminary results for the accuracy and sensitivity of this measurement of erythrocyte deformability. With this optical technique the instrumentation (ektacytometer) is made quite simple and suitable for use in the typical laboratory. This would allow a regular, quantitative assessment of this important blood cell quality, to supplement the data obtained from the complete blood count.

Characterizing blood cells by biophysical measurements in flow.

One effect of automation in the hematology laboratory has been to introduce new characterizations of blood cells. Resistive pulse sensing (Coulter) and light scatter measurements in flow provide rapid and reproducible cell counts. They also provide information about red cell size, shape, and deformability. Thus, they have provided new characterization of these cells in terms of their biophysical properties. Leukocytes have been classified by optical scatter and absorption measurements in flow after being stained cytochemically. This provides rapid and precise WBC differential counts. However, here again, additional information about relative cell-enzyme content or activity is also accessible to provide a new characterization of the leukocytes. The ultimate range of utility of this expanding technology in the automated hematology laboratory of the future will, of course, depend upon establishing relations between the biophysical parameters and the functions of the cells. This, in turn, must depend upon the use of the technology by researchers and clinicians in studying cell function and the aberrations of these functions which define disease.

Ektacytometric analysis of factors regulating red cell deformability.

Photometric analysis of laser diffraction patterns has been used to obtain quantitative measurements of deformability of specifically modified normal red cells. Variation of deformability with suspending medium osmolality and with applied shear stress was used to distinguish between changes in internal viscosity, surface area-to-volume ratio, and viscoelastic properties of the membrane in their influence on whole cell deformability.