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Red Cell Morphology and the Peripheral Blood Film

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Red Cell Morphology and the Peripheral Blood Film INTERPRETATION OF THE PERIPHERAL 00. ם BLOOD FILM 0272–2712/02 $15.00 RED CELL MORPHOLOGY AND THE PERIPHERAL BLOOD FILM Robert V. Pierre, MD Leeuwenhoek17 first observed red blood cells with his primitive microscope while examining his own blood. It is not clear who first prepared a blood smear. Early smears were air dried; fixed by heating them over a flame or treatment by strong fixatives, such as picric acid; and stained by a variety of available stains. It was Hoppe-Seyer15 in 1866 who demonstrated that the function of the hemo- globin in red cells was to take up and discharge oxygen. Gulliver12 made extensive study of the red corpuscles in vertebrates and published a remarkable figure showing the varied size range and shapes of the red cells (Fig. 1). It was Ehrlich9 who introduced the triacidic stain to blood films in 1877, revolutionizing hematology. His stain made it possible for the first time to define the nucleus and cytoplasm and many other cellular features, and set the stage for modern hematology. Morphologic features of red cells in numerous disease states were described by Hayem,8 Sabin,26 and Downey,35 and many others, but the determi- nation of the cause or mechanisms of these morphologic abnormalities had to wait for nearly half a century. The era of modern hematology opened in 1926 with the announcement by Minot and Murphy19 that the addition of 120 to 240 g of lightly cooked beef liver to the diet of patients with pernicious anemia caused improvement and was life saving. This was the first clinical study leading to definition of the cause of pernicious anemia. Before the introduction of automated cell counting by the Coulter Counter Model A (Coulter Electronics, Hialeah, FL) in 1958,7 several indices of red cells derived from manual techniques were introduced to aid physicians in the interpretation of red cell morphology. The first was the red cell color index. The color index was proposed by Laache16 in 1883. Hayem13 had noted that there was a relatively greater reduction of red cells than of hemoglobin in pernicious anemia. Laache16 proposed the color index, which was determined by the ratio From the Department of Pathology, Los Angeles County/University of Southern California Medical Center, Los Angeles, California CLINICS IN LABORATORY MEDICINE VOLUME 22 • NUMBER 1 • MARCH 2002 25 26 PIERRE Figure 1. Variations in red cell size and shape from many vertebrates. (From Gulliver G: Proc Zoological Soc. London, 1875, p 474; with permission.) RED CELL MORPHOLOGY AND THE PERIPHERAL BLOOD FILM 27 of the hemoglobin percentage to the red cell percentage compared with normal. The normal result was 1. In pernicious anemia the ratio was greater than 1 and in iron deficiency it was less than 1. This ratio was still being reported to clinicians at the Mayo Clinic in 1967; the author had it discontinued at that time. A significant and useful advance was made in 1933 by Wintrobe37 following his introduction of the simple method of determining the hematocrit. Wintrobe37 introduced the red blood cell indices mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentra- tion (MCHC). These indices proved very helpful to the classification of anemia and clinicians’ understanding of anemia.36 Wintrobe’s indices have survived to today and are reported by all automated hematology analyzers. Additional instrument-derived indices have appeared. The red cell distribution width (RDW) was introduced by Bessman et al3 in 1983. The RDW has proved to be a useful tool in evaluating automated analyzer results to determine whether a blood film should be reviewed. The ability to measure individual red cell hemoglobin content by the Technicon hematology system has also led to new ways of looking at red cell classification.32 Despite the advances in technology of automated hematology analyzers, specialized flow cytometry, molecular diagnostics, cytogenetics, and chemical studies of the blood cells, it is still necessary to examine a blood film in the patient presenting with anemia for three simple reasons. (1) Many morphologic abnormalities of red cells, which are quickly and easily recognized by experi- enced morphologists, cannot currently be recognized by these advanced tech- niques. For example, current automated flow cytometric hematology analyzers using impedance or light scattering principles cannot recognize most red cell shape changes. (2) Technical limitations of the analytic methods can cause errors. In impedance instruments, red cells are deformed by the electrical force field as they pass through the measuring aperture. Calculation of their volume assumes a typical deformation of the cell and a shape factor is used in the calculations transforming the voltage change and the shape of the voltage curve to a red cell volume measurement. Red cells with alteration in their membranes leading to increased rigidity or decreased rigidity may have falsely low or high MCV results, respectively.33 (3) Even if the red cell abnormalities could be recognized by one or more of the specialized procedures (e.g., detection of high oxygen affinity hemoglobin to explain erythrocytosis), routine screening of all patients by these specialized procedures is not cost effective as an initial laboratory investigation step. Morphologic screening can help to determine whether any of these techniques are indicated to establish or confirm a diagnosis. The morphologic evaluation of a blood film for red cell abnormalities should no longer accompany the routine complete blood count if the red cell parameters, leukocyte parameters, and platelet parameters are within reference limits and there are no instrument flags. This does not imply that such patients do not have any red cell abnormalities (e.g., a patient with trait of hereditary ellipto- cytosis may show no abnormalities on the complete blood count). If there are clinical indications for a blood film review for any reason, the red cell morphol- ogy should be examined and commented on. Reasons for examining a blood film, even in the presence of a normal automated complete blood count include the following: 1. Clinical information that the patient had a prior red cell abnormality and its current status is questioned 2. A family history of a morphologic red cell abnormality 28 PIERRE 3. Determine whether the patient has a hyposplenic or postsplenectomy blood picture 4. Determine the presence of parasites EVALUATION OF RED BLOOD MORPHOLOGY ON A STAINED BLOOD FILM The quality of the blood film and blood film staining is of paramount importance in evaluation of red cell morphology. A well-made wedge blood film consists of a thick region, a region of good morphology, and a thin region (Fig. 2). The thick region next to the starting point of the film has red cells piled on top of one another and in contact with one another. The cells are thicker and rounded up and usually have no central area of pallor. The red cells appear smaller in diameter than in the region of good morphology. The red cells in the thin region or end of the film are widely separated and generally appear larger. They are thinner and may not show the normal area of central pallor. Small numbers of cells, which resemble spherocytes, may also be seen in the thin area. The area of good morphology is fairly small. In this area about 50% of red cells may be touching one another. This is the best area in which to determine red cell size and shape.20 A spun blood film prepared by a slide centrifuge should have uniform distribution of red cells, so that red cell morphology can be evaluated anywhere on the slide. The proper method of preparing high-quality blood films is dis- cussed in the article by Houwen elsewhere in this issue. If scanning of the blood film shows it to be of acceptable quality and staining, red cell morphology can be evaluated. Figure 2. Suitable examination area of a blood film by a battlement scan. Note that the blood film is narrower than the glass slide. RED CELL MORPHOLOGY AND THE PERIPHERAL BLOOD FILM 29 Figure 3. Comparison of red cell diameter and volume. Macrocytes and leptocytes have an increased diameter, but only the macrocyte has an increased volume. The codocyte or hypochromic microcytic red cell and the spherocyte both have a decreased diameter but the spherocyte has a normal volume. (Adapted from Bessis M: Living Blood Cells and Their Ultrastructure. New York, Springer Verlag, 1972, p 197; with permission.) EVALUATION OF RED CELL MORPHOLOGY ON A STAINED BLOOD FILM There are four features of red cells that should be evaluated on a peripheral blood film: (1) size, (2) shape, (3) color, and (4) inclusions. Red Cell Size Evaluation Determination of red cell size on a blood film is difficult and affected by many factors. Red cell size is a measure of red cell diameter, not of red cell volume like the MCVas determined by the automated hematology analyzer. The two parameters may disagree. If one does not keep this difference in mind, errors in determining red cell size are made. Figure 3 illustrates this critical difference.2 Spherocytes have a decreased diameter but have a normal volume. Leptocytes have an increased diameter but have a normal volume. Some lepto- cytes, such as those seen in the postsplenectomy state, may produce an elevated MCVbecause their increased deformability causes them to have a falsely high MCVdetermination in impedance instruments. See the article by Dalal and Brigden on red cell artifacts elsewhere in this issue for a more complete discus- sion. Effect of Hematocrit Red cell size cannot be estimated accurately from blood films prepared from patients with an abnormally high or low hematocrit. Blood films with a region of good morphology cannot be prepared from blood samples with hematocrits above 50%.
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