Red Blood Cell Morphology in Sickle Cell Anemia as Determined by Image Processing Analysis: The Relationship to Painful Crises

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1 Red Blood Cell orphology in Sickle Cell Anemia as Determined by Image Processing Analysis: The Relationship to Painful Crises AXWELL P. WESTERAN,.D. AND JAES W. BACUS, PH.D. Red blood cell morphology was studied in the peripheral blood of adults with sickle cell anemia to determine if changes occur during painful crises. Image processing of the cells with an automated system of red blood cell analysis was used. our groups of cells were observed: normocytes, macrocytes, target cells, and cells with the shape of irreversibly sickled cells. During asymptomatic periods, the percentages of these cells differed in each individual but were typical for that individual and generally were stable. During crises, macrocytosis occurred and the concentration of irreversibly sickled cells showed greater fluctuation. The macrocytosis most likely reflected a marrow response to increased hemolysis and demonstrated that the increased red blood cell destruction observed during pain crises may be more extensive than previously considered. Changes in the concentration of irreversibly sickled cells during crises-were not consistent and could not be used as an indicator of a crisis. Image processing with automated red blood cell analysis allows for accurate assessment of all the morphologic groups of red blood cells in patients with sickle cell anemia and compares well with standard methods for measuring the concentration of irreversibly sickled cells. (Key words: Red blood cell morphology; Sickle cell anemia; Image processing; Painful crises) Am J Clin Pathol 98; 79: PATIENTS WITH SICKLE CELL ANEIA have numerous clinical problems, among which are recurrent painful crises. The crises are most likely related to occlusion of small blood vessels by aggregated sickle red blood cefls because of rheologic and morphologic changes in the cells. Various abnormalities, such as changes in the gelation time of hemoglobin, ' cellular dehydration and abnormalities in the cell membrane, "' 9 irreversible sickling of cells, or increased adherence of the cells to vascular endothelium 9 may contribute singly or together to this process: The changes could be associated with alterations in red blood cell morphology and might provide important clinical and pathogenetic information about crises. To determine whether such changes do occur and to evaluate their significance, we have examined the red Received August, 98; received revised manuscript and accepted for publication September 7, 98. Address reprint requests to Dr. Westerman: Hematology-Oncology Unit, ount Sinai Hospital, 5th Street and California Avenue, Chicago, Illinois Department of edicine and edical Automation Research Unit, Rush edical College, ount Sinai Hospital and Presbyterian St. Luke's edical Center, Chicago, Illinois blood cells in peripheral blood smears of adults with sickle cell anemia using an image processing system with an automated method for analysis of the cells. The system identifies cells according to shape, size, and hemoglobin content and provides concentrations of the different morphologic types of red blood cells. In the present study we have () examined red blood cell morphology in the peripheral blood smears of patients during painful crises, and during asymptomatic periods () compared the counts of irreversibly sickle shaped cells obtained by image processing analysis with counts obtained by more traditional methods of examination. ethods ifteen patients with sickle cell anemia as confirmed by hemoglobin electrophoresis and by family study, when indicated, were examined. The age range was from to 5 years. The patients had no evidence by physical examination of enlarged spleens. Studies were obtained while patients were asymptomatic or during painful crises that were sufficiently severe to require hospitalization. Representative clinical and laboratory data for each patient are shown in Table. Image processing analysis of red blood cells was done as described previously. Whole blood samples were obtained in Vacutainer EDTA tubes. Preparation of films of cells was done by a centrifugal "spinner." This was followed by image processing analysis which describes morphologic characteristics of the cells and measures hemoglobin absorption of the cells at 5 n using a system of high speed processing. Six measurements, consisting of () size, () hemoglobin content; () central pallor, () shape (circularity), (5) shape (elongation), and (6) shape (spicularity), were obtained with subsequent separation of the cells into possible classes, i.e., the red blood cell differential count.' The classes are shown in Table. or the purposes of this study, classes /8/0600/0667 $0.00 American Society of Clinical Pathologists 667

2 I 668 WESTERAN AND BACUS A.J.C.P. June 98 Patient Table. Representative Clinical and Laboratory Data and Red Blood Cell Differential Counts in Patients with Sickle Cell Anemia during Asymptomatic Periods Age (Years) Sex Hemoglobin (g/dl) ean corpuscular hemoglobin concentration, t ean corpuscular volume. Hematocrit Reticulocyte count CHC* CVf r Normocytes (fl) ' RBC Differential:): ISCs Target Cells X Abbreviated red blood cell differential count as described in text. Cells with irreversibly sickled shapes. acrocytes """' (elongated cells) and 8 (irregular shaped cells) were morphologically similar to cells that are considered to be native irreversibly sickled cells and were counted as such. One thousand cells were counted. To determine if macrocytosis was present in the slides analyzed by image processing, the macrocyte/normocyte ratio was calculated for each film. To obtain the ratio, the percentage of macrocytes was divided by the percentage of normocytes. Normocytes included classes,, and, and macrocytes included classes,, and 9. Target cells (class 9) were included in the macrocytic group because they were large and more closely resembled the size of macrocytes (target cells 6.0 /i ±.; macrocytes 70.0 n ± 0.6; normocytes 5.7 p ± 0.07; ± SE). A mean macrocyte/normocyte ratio then was determined for each patient. This was derived from all the films obtained during crises or asymptom- Class Number Table. Classes of Red Blood Cells Class Name Normocytes Normocytes without central pallor acrocytes without central pallor Spherocytes Elongated spiculed cells Spiculed, irregular shaped cells Elongated cells Irregular shaped cells Target cells Hypochromic microcytes Hypochromic normocytes Hypochromic macrocytes Normochromic microcytes Normochromic macrocytes atic periods. The use of the macrocyte/normocyte ratio, i.e., comparison between macrocytes and normocytes in the same patient, avoided artifactual errors that might have occurred if comparisons in the concentration of macrocytes had been made between different patients. The mean corpuscular volume (CV) of red blood cells was obtained on a Coulter odel S and the mean corpuscular hemoglobin concentration (CHC) was derived from measurements of the hemoglobin concentration and the hematocrit. orphologic study of red blood cells using standard visual microscopic methods was done on glutaraldehyde-fixed preparations of cells (% glutaraldehyde in buffered saline; 0 m CI, 0 m sodium phosphate, ph 7.) and on films stained with Wright-Giemsa stain. The glutaraldehyde fixed and the Wright-stained preparations were obtained from blood samples that had been prepared like those used for image processing. Whole blood was diluted to % hematocrit with 0.9% NaCl prior to preparation of the samples for the glutaraldehyde-fixed and Wright-stained preparations. The criteria for morphologic diagnosis of the various types of red blood cells followed standard practice. Red blood cells that were "sweet potato" shaped with one or more pointed extremity were considered to be irreversibly sickled cells. 5 One thousand cells were counted on these preparations. Results our morphologic groups of red blood cells were obtained in the image processing analysis of the peripheral blood cells of the patients. These consisted of normocytes, macrocytes, target cells, and cells with the shape

3 Vol. 79-No. 6 RBC ORPHOLOGY IN SICKLE CELL ANEIA 669 of irreversibly sickled cells. Wide variation in the concentration of these groups of cells was observed among patients as shown in the red blood cell differential count (Table ). Sequential measurements were made during asymptomatic periods on eight patients on 7 separate occasions to determine the stability of red blood cell morphology during these periods. Variation in morphology was generally moderate. Wider variations were observed in approximately 0% of the slides during the asymptomatic periods. luctuation was greater in normocyte and macrocyte concentrations, while lesser changes occurred in the concentration of irreversibly shaped sickle cells and target cells. Seven patients were examined during painful crises on separate occasions. The results were compared with those obtained in the same patients examined on 6 separate occasions during symptom-free periods. During crises, macrocytosis occurred. The macrocyte/ normocyte ratio was greater during crises in six of the seven patients than that observed during asymptomatic periods (Table ). Analysis of variance to test the difference in the macrocyte/normocyte ratio between crises and asymptomatic periods was significant (<0.000). Examination of histograms for changes of cell size during crises also showed a marked rightward shift of the area under the curve compatible wjth macrocytosis. arked fluctuation in the percentages of the different cell types regularly occurred during crises. The mean percentage of irreversibly shaped sickle cells did not show a consistent change during crises as compared with asymptomatic periods, but individual counts did show greater fluctuation during crises. The mean percentage of target cell concentrations remained the same during painful and asymptomatic periods. To find out if the concentration of irreversibly shaped sickle cells determined by image processing accurately reflects the ISC concentrations as determined by standard methods, comparisons were made between ISC counts obtained by image processing and by visual examination of glutaraldehyde-fixed red blood cells. Both types of cell preparations were prepared simultaneously. Comparisons were done on five patients on 5 separate occasions and showed a close correlation between the percentages of irreversibly shaped sickle cells obtained by both methods (ig. ) (r = 0.95, P = 0.000). To determine the reproducibility of results obtained by image processing, nine pajrs of duplicate, simultaneously prepared slides from four patients were examined and the results compared with each other. A high correlation between the two slides for the cell types (irreversibly shaped sickle ce}ls ; normocytes and macrocytes) was observed (r = 0.8, P = 0.008; r = 0.86, P = 0.00; r = 0.68, P = 0.0, respectively) (igs. A-C). Table. acrocyte/normocyte Ratios in Patients during Crises and Asymptomatic Periods Patient "5 6 7 Crises.98* (8)t.9 (6). () 0.7 (5).00 (6).5 (5). () Asymptomatic 0. () 0.89() 0.59(5) 0.8 (7).05(8) 0.5 (5).06() * ean ratio of all specimens obtained during crises or asymptomatic periods. f Number of specimens from which mean macrocyte/normocyte ratios were calculated. Each specimen was taken on a day of crisis or an asymptomatic day. Studies of the effects of brief aeration that occurs during film preparation for image processing were done to determine whether the percentage of cells with irreversibly sickled shapes was affected by aeration. Analyses were made on eight occasions from six patients in whom smears were prepared in the standard manner, i.e., without added aeration of the blood sample, and on smears that were prepared from blood after one hour of aeration. The results showed no change in irreversibly shaped sickle cell concentrations after aeration (Table ). Since formalin fixing also might cause reduction of hemoglobin to the deoxy form with consequent sickling of cells and is necessary for preparation of films for image processing, comparisons of ISC counts in Wrightstained formalin-fixed and in Wright-stained unfixed smears were made. ormalin fixing had no effects on 60r o 50 z g «a. o. $ 0 < I > «o O 0 So "Tt ~ % ISC's BY VISUAL EXAINATION IG.. Comparisons between the image processing method and visual examination of glutaraldehydefixedred blood cells for determining the concentration of irreversibly shaped sickle cells (ISCs).

4 WESTERAN AND BACUS 80r A.J.C.P. June UJ g - 0l- _l < t Z 0 z p 0 (0 70 ^60 si P 50 0 * % ISC's ON DUPLICATE SLIDE % NOROCYTES ON DUPLICATE SLOE SO LU Q w 50 _i < t Z 0 z O UJ H 0 O cr IG.. Comparisons of the concentration of {A, upper left) irreversibly shaped sickle cells (B, upper right) normocytes, and (C. lower left) macrocytes in duplicate slides as obtained by image processing. ^ % ACROCYTES ON DUPLICATES SLIDE 70 the concentration of ISCs in four patients who were studied on five occasions (Table 5). Discussion In our study, we show that peripheral blood red blood cells in patients with sickle cell anemia are divided into four morphologic groups: () normal cells, () macrocytes, () target cells, and () cells with the shape of irreversibly sickled cells. During asymptomatic periods the percentage of each type of cell is relatively typical and constant for each patient but varies considerably from patient to patient. The least change in concentration of cell types during these periods occurs in cells with irreversibly sickled shapes and in target cells, while more variability is observed in the percentage of normocytes and macrocytes. Red blood cell production appears to be relatively stable during these periods since neither the hematocrit nor the percentage of the various types of peripheral red blood cells show significant

5 Vol. 79 No. 6 RBC ORPHOLOGY IN SICKLE CELL ANEIA 67 changes. Occasionally, however, an abrupt and marked change in the concentration of the various groups of red blood cells did occur, although the hematocrit remained unchanged. This suggests that the factors affecting the morphology of peripheral red blood cells during asymptomatic periods may, at times, change although they are generally stable. Whether these occasional, marked alterations in peripheral red blood cell morphology during asymptomatic periods represent the effects of less extensive vasocclusive episodes than those observed in typical severe painful crises is not certain. This would, however, be a likely explanation since similar but more protracted changes in red blood cell morphology were observed during severe crises. The variations in morphology also suggest that a single measurement of the concentration of any one cell type may not accurately represent the concentration of that particular cell type during an entire asymptomatic period. During crises, several changes occurred in the concentrations of the various groups of cells. acrocytosis was observed by the increase in the macrocyte/normocyte ratio in six of seven patients and by the increase in the number of large cells seen in histograms that showed the size distribution of cells. The concentration of irreversibly shaped sickle cells was not consistently increased or decreased as compared with the concentration observed during asymptomatic periods. Target cell concentrations were unchanged. The significance of these changes could be several. The increase in macrocytes in association with a stable hematocrit most likely represents the delivery of a greater number of young red blood cells to the periphery in response to more marked red blood cell destruction. Increased hemolysis during pain crises has been described previously in patients with sickle cell anemia; however, it was thought to occur only to a minor extent and did not indicate a widespread increase in hemolysis. Also, the previously described increase in hemolysis did not appear to induce a marrow response since a reticulocyte rise above an already elevated level was not observed. 6 Our results show that the hemolysis that occurs during vasocclusive episodes is sufficiently severe to induce a marrow response, as shown by peripheral macrocytosis, and thus may be more marked than previously noted. This finding which differs from the earlier result also may be related to the differences in methods of measurement. Reticulocyte counts, although very useful, have limited reproducibility and accuracy 5 and may be relatively insensitive to moderate changes in hemolysis. easurement of cell size by image processing is an accurate, reproducible method and would more precisely reflect an increased concentration of large red cells in the periphery. The finding of a marrow response is compatible with the Table. Concentrations of Irreversibly Shaped Sickle Cells Observed on ilms Prepared Before or After Aeration* of Blood Samplesf ISC Patient Before After Blood samples were exposed to room air for 60 minutes prior to preparation of films. t Image processing analysis was used. concept that further marrow compensation could occur in these patients. The relationship between red blood cell morphology and vasocclusive pain crises in patients with sickle cell anemia has had limited study. Irreversibly sickled cells have been considered to be an important precipitating factor in the development of painful crises since they contribute significantly to the increased viscosity of oxygenated sickle blood. Because of this, and since ISC concentrations in peripheral blood may reflect the degree of in vivo sickling, 6 " 8 it would seem that the concentration of peripheral blood ISCs might change during vasocclusive episodes. Our studies show that the concentration of ISCs does fluctuate much more widely during crises; however, a consistent increase or decrease does not occur. Previous studies had similarly shown that the percentage of ISCs does not show a consistent change during crises, 6 although the percentage of sickled cells in venous blood did increase during crises.-' These results suggest that the relationship between in vivo sickling and ISC concentration may not be as close as considered. The findings would not negate the observations Table 5. Concentrations of Irreversibly Shaped Sickle Cells Observed in ilms Prepared with or without ormalin ixing* Patient " Without ormalin ixing ISC With ormalin ixing 0 * Counts were done manually on Wright-Gicsma-stained smears.

6 67 WESTERAN AND BACUS A.J.C.P.-June 98 that irreversibly sickled cells may be precipitating events in painful vasocclusive crises but show that peripheral ISC counts are not useful as an indicator of painful crises. The variation in individual ISC counts during crises is also sufficiently large to suggest that single measurements are not a satisfactory indicator of the concentration of ISCs that are present during crises. Image processing is very appropriate for evaluating the red blood cells from patients with sickle cell anemia. This is of some interest since the stickiness of Hb SS red blood cells, the changes that occur during oxygenation, and the necessity for cell fixation prior to image processing could create difficulties. The reproducibility of the method is very good, however, and it does not appear that significant changes of the cells occur during formalin fixation or slide preparation. Image processing also readily permits analysis of all types of red blood cells and not just one type such as the ISC. Acknowledgment. The authors would like to thank Nijole Dumbrys and argaret Sheedy for their excellent technical assistance. References. Bacus JW, Weens JH: An automated method of differential red blood cell classification with application to the diagnosis of anemia. J Histochem Cytochem 977; 5:6-6. Bacus J W: Quantitative morphological analysis of red blood cells. Blood Cells 980;6:95-. Barreras L, Diggs LW: Bicarbonatcs, ph and percentage of sickled cells in venous blood of patients in sickle cell crisis. Am J ed Sci 96; 7:7-78. Chien S, Usami S, Bertles J: Abnormal rheology of oxygenated blood in sickle cell anemia. J Clin Invest 970; 9: Clark R, Guatelli JC, ohandas N, Shohet SB: Influence of red cell water content on the morphology of sickling. Blood 980; 55: Diggs LW: The crises in sickle cell anemia: hematologic studies. Am J Clin Pathol 956;6: Eaton JW, Skelton TD, SwofTord HS, Kolpin CE, Jacob HS: Elevated erythrocyte calcium in sickle cell disease. Nature 97; 6: Glader BE, Nathan DG: Cation permeability alterations during sickling: relationship to cation composition and cellular hydration of irreversibly sickled cells. Blood 978: 5: Hebbel RP, Boogaerts AB, Eaton JW, Steinberg H: Erythrocyte adherence to endothelium in sickle cell anemia: a possible determinant of disease severity. N Engl J ed 980; 0: Hofrichter J, Ross PD, Eaton WA: Kinetics and mechanism of deoxyhemoglobin S gelation: a new approach to understanding sickle cell disease. Proc Natl Acad Sci USA 97; 7: Lux SE, John K, Karnovsky J: Irreversible deformation of the spectrin-actin lattice in irreversibly sickled cells. J Clin Invest 976; 58: ccurdy PR: Erythrokinetics in abnormal hemoglobin syndromes. Blood 96; 0: Naumann HN, Diggs LW, Barreras L, Williams BJ: Plasma hemoglobin and hemoglobin fractions in sickle cell crisis. Am J Clin Pathol 97; 56:7-7. Noguchi CT, Schechter AN: The intracellular polymerization of sickle hemoglobin and its relevance to sickle cell disease. Blood 98;58: Peebles DA, Hochberg A, Clarke TD: Analysis of manual reticulocyte counting. Am J Clin Pathol 98; 76: Serjeant GR, Serjeant BE, ilner P: The irreversibly sickled cell; a determinant of haemolysis in sickle cell anaemia. Br J Haematol 969; 7: Serjeant GR: Irreversibly sickled cells and splenomegaly in sickle cell anemia. Br J Haematol 970; 9: Serjeant GR, Serjeant BE, Condon PI: The conjunctival sign in sickle cell anemia. JAA 97; 9:8-9. Shaklai N, Sharma VS, Ranney H: Interaction of sickle cell hemoglobin with erythrocyte membranes. Proc Natl Acad Sci USA 98; 78:65-68