American Journal of Hematology 28:37-39 (1988)

Erythrocytes in Hb SC Disease Are Microcytic and Hyperchromic

Samir K. Ballas and William Kocher Cardeza Foundation for Hematologic Research (S.K.B.) and Departments of Medicine (S.K.B.) and Pathology (W.K.), Thomas Jefferson University, Philadelphia

We have used the new Technicon H.1 Hematology Analyzer to determine the indices of erythrocytes obtained from 18 nonthalassemic (OLOLIOLOL genotype) adult patients with Hb SC disease. Controls were 14 normal black adults and 29 white adults. The data showed that SC erythrocytes are significantly smaller than normal RBC (P < .001) with a signifi- cantly higher MCHC value (P < .001) than controls. These features of SC RBC could not be reproduced by an Ortho ELT-8 electonic counter. Hb SC erythrocytes have unique indices which are best demonstrated either by semimanual methods or by the H.1 system.

Key words: red cell indices, MCV, MCHC

INTRODUCTION

Hb SC disease is characterized by mild anemia which is often described as normochromic and normocytic [ 1,2]. Few reports [3-51, however, have indicated that the red cells in this disease are typically microcytic with an elevated mean corpuscular hemoglobin concentration (MCHC). These measurements were determined by semi- manual methods for the determination of hemoglobin (Hb) and hematocrit (Hct) and by the Coulter Counter Model F for the estimation of the red cell count. Other investigators who used fully automated electronic Coul- ter S or Coulter S-plus counters did not emphasize the microcytic hyperchromic indices in this disorder [6,7]. Recently Technicon has introduced its Ha 1 Hematology Analyzer, which operates as a flow cytometer using light- scattering measurements from two sources [8-lo]. This instrument has the unique advantage of directly and in- dependently measuring the MCV and the Hb concentra- tion. The results are displayed as histograms showing the distribution of cell size and Hb concentration. Moreover, the width of volume distribution (RDW) and the width of hemoglobin concentration distribution (HDW) are com- puted from the histograms. RDW is derived as the coef- ficient of variation of the volume histogram, whereas HDW is derived as the standard deviation of the hemo- globin concentration histogram [ll]. The advent of this technology gave us the opportunity to reevaluate the red cell indices in Hb SC disease. Our findings confirm that

erythrocytes in this disorder are indeed typically micro- cytic and hyperchromic in nature.

MATERIALS AND METHODS Patients

The procedures described in this study were performed on blood obtained from 18 individuals with acwlaa ge- notype and Hb SC disease who were being followed in our sickle cell center while this study was in progress. All patients were adult American blacks. Black control subjects were adult university personnel or normal vol- unteer blood donors. White control subjects were adult patients who were admitted to the hospital for elective surgery and who had no hematologic disorders.

Hematological Data Red cell counts, red cell indices, red cell distribution

width (RDW), and hemoglobin concentration distribution width (HDW) were determined with Technicon H.1 (Technicon Instruments Corp., Tarrytown, NY) analyzer [8-lo]. Some samples were also analyzed by the Ortho ELT-8 (Ortho Diagnostic Systems, Inc., Westwood, MA)

Received for publication October 15, 1987; accepted January 14, 1988.

Address reprint requests to Samir K. Ballas, M.D., Cardeza Founda- tion, 1015 Walnut Street, Philadelphia, PA 19107.

0 1988 Alan R. Liss, Inc.

38 Ballas and Kocher

- TABLE 1. Hematological Values by Laser Light Scattering in Hb SC Disease and Control Subjectst

Controls SC disease Black White (n = 18) (n = 14) (n = 29)

3.88 f 0.45* 4.55 k 0.29 4.46 t 0.34 RBC, lO3/~l 13.0 f 0.99 13.7 f 1.03 Hb, gldl 11.2 + 1.13*

PCV, liter/litera 31.8 i- 3.07* 40.1 f 2.26 40.9 f 3.06 81.4 f 5.28* 88.1 f 3.75 91.7 rt 3.81 MCV, fl

MCH, pg 28.9 f 2.73 28.5 f 1.55 30.8 f 1.40 MCHC, g/dl 35.5 f 2.20* 32.4 f 1.61 33.6 f 0.76 RDW 16.5 f 1.60* 13.3 f 0.62 13.2 f 0.69

2.3 f 0.26 HDW 3.1 f 0.44* 2.3 f 0.23

?Numbers shown are mean SD. *Value significantly different ( P < .001) from corresponding value in controls. aPCV: packed cell volume.

B A

RBC VOLUME (0-200 f L )

. l . l . I . l

HCB CONC

RBC VOLUME (0-200 f L )

1 . 1 . 1 . . 1 . 1 . 1 . l 1 . 1 .

(0-50 g/dL) . f

Ha3 CONC c 0-50 g/dL >

1 . 1 . 1 . 1 . 1 . 1 1 . 1 t . 1 . 1 . 1 . 1 . 1

Fig. 1. Typical examples of panels from a screen printer gram. The numbers in parentheses indicate the minimum showing the RBC and HGB histograms. A: Normal black and maximum values on the X-axis of the adjacent histo- control. B: A patient with Hb SC disease. The vertical lines gram. HGB = hemoglobin. indicate the boundaries of the range of the normal histo-

electronic cell counter [ 111. Reticulocyte count was made by routine methods. The diagnosis of Hb SC disease was confirmed by hemoglobin electrophoresis on starch gel, starch block, or agar gel on membrane-free hemolysate by established methods [ 121. Individual globin chains were differentiated by cellulose acetate electrophoresis of membrane-free hemolysate as described by Ueda and Schneider [ 131. Alpha-globin genotypes were determined by Southern blot hybridization of genomic DNA as was described previously [5]. Statistical significance was cal- culated by the two-tailed Student’s t-test for paired data.

RESULTS

Table I compares the hematological data of Hb SC disease with those of normal black and of white controls. Noteworthy is that the MCV in Hb SC disease is signifi- cantly lower than that in controls despite a mildly ele- vated (6.4 %) reticulocyte count. Coexistent a-thalassemia

was ruled out by the presence of 4 a genes in all patients studied. The MCHC in Hb SC disease, however, is significantly increased. Both the RDW and HDW are considerably higher in Hb SC disease than in controls. Figure 1 depicts a typical volume and hemoglobin con- centration histograms in Hb SC and a normal black control. It is clear that in Hb SC the red cell size is shifted to low values whereas hemoglobin concentration is shifted to high values. It must be emphasized that these characteristics of RBC in Hb SC were not apparent when the Ortho ELT-8 cell counter was used. Thus, the values generated by this instrument were MCV = 88.1 6.4 and MCHC = 32.3 1.37 in 16 determinations. These were not significantly different (P > .05) from controls.

DISCUSSION

Our findings clearly show that patients with Hb SC disease and no co-existent a-thalassemia typically have microcytic and hyperchromic indices as measured by the

Hb SC Disease 39

Technicon H.1 system. This confirms previous reports of liams WJ, Beutler E, Erslev kl, Lichtman MA (eds): “Hematol- these characteristics of SC RBc obtain4 by using semi-

3. Sergeant GR: “Sickle Cell Disease.” Oxford: Oxford University manual (spun hematocrit) methodology [3-51. Moreover, Press, 1985, 299. the data generated by the system show that the 4. Ballas SK, Lewis CN, Noone AM, Krasnow SH, Kamarulzaman dispersion of RBC (RDW) and of Hb (HDW) vary inde- E, Burka ER: Clinical, hematological, and biochemical features pendently from each other [14]. These unique features of of Hb SC disease. Am J Hematol 13:37-51, 1982. sc erythrocytes seem not to be readily demonstrable by 5 . Ballas SK, Larner J, Smith Ed, Surrey S, Schwartz E, Rappaport

EF: The xerocytosis of Hb SC disease. Blood 69: 124-130, 1987. Other are 6. Steinberg MH, Coleman MD, Adams JG, Platica 0, Gillette P, dehydrated and less deformable than ‘Orma’ RBC Rider RF: The effects of alpha-thalassemia in Hb SC disease. [5,15,16]. Both the Coulter and the Ortho ELT-8 counters compute the Hct and MCHC values indirectly from the 7. Webster P, Castro 0: Red cell distribution width in sickle cell measured MCV [ll]. Previous studies have shown clearly that cellular dehvdration and decreased M C deformaba- 8. Mohandas N, Kim YR, Tycko DH, Orlik J, Wyatt J, Groner W:

OgY,” Ed 3. New YOrk: McGraw-Hill, 1983, P 583.

counters because these

Br J Haematol55:487-492, 1983.

disease. Ann Clin Lab Sci 16:274-277, 1986.

ity compro~se ;he accuracy of he determination of he MCV and, hence, the accuracy of other red cell indices by these instillments [11,171. Because of the high Hb

Accurate and independent measurement of volume and hemoglo- bin concentration of individual red cells by laser light scattering. Blood 68:506-513, 1986,

9. Tvcko DH, Metz MH, EDstein EA, Grinbaum A: Flow-cytomet- concentration in Sc RBC, these machines will give a falsely high level of MCV and, consequently, a falsely low value for MCHC.

ric light scattering measurement of red blood cell volume and hemoglobin concentration APPI opt *4:1355-13651 1985,

10. Bollinger PB, Drewinko B, Brailas CD, Smeeton NA, Trujllo JM: The Technicon H* P-An automated hematology analyzer for today and tomorrow. Am J Clin Pathol 87:71-78, 1987.

11. Mohandas N, Clark MR, Kissinger S, Bayer C, Shohet SB: Inaccuracies associated with the automated measurement of mean cell hemoglobin concentration in dehydrated cells. Blood 56: 125- 128, 1980.

CONCLUSIONS

RBC in Hb SC disease are typically microcytic and hyperchromic’ These features are best ’pun hematocrit method Or by the the 12. Huisman THJ, Jonxis JHP: ‘,The Hemoglob,nopathies: Tech- system. niques of Identification.” New york: Defier, 1977, p 94. Moreover, the generation of such data by these methods 13. Ueda S, Schneider I: Rapid differentiation of polypeptide chains should alert one to the possible diagnosis of Hb SC of hemoglobin by cellulose acetate electrophoresis of hemoly- disease or other disorders characterized by xerocytosis sates. Blood 34230-235, 1969.

erythrocyte size and hemoglobin content observed in man and indices of pathologic cells are best determined by these four selected mammals. Blood cells 12:65-80, 1986. 15. Fabry ME, Kaul DK, Raventos-Suarez C, Chang H, Nagel RL:

SC erythrocytes have an abnormally high intracellular concentra-

and decreased RBC deformability. Conversely accurate 14. Groner w , Boyett J, Johnson A, ScantlebuV M: Variability of

methods.

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