A o t s Path. Jsp. 26(6): 633-642, 1970

FINE STRUCTURES OF THE SPLEEN IN HEREDITARY ELLIPTOCYTOSIS Noboru M~TSUMOTO*,Tokuhiro ISHIHARA**, Mutsuo TAKAHASHI**, Fumiya UCHINO**, Junichiro ONO*, Shiro MIWA*, and Yoshinori KIYOMITSU*** (Received on Dec. 10, 1976)

Light and electron microscopic studies of the spleen and liver from a patient with hereditary elllptocytosis have been reported. Characteristic findings are congestion of the cordal space, diminished variation of the erythrocyte shape in the cord, relatively empty sinuses and erythrophagocytosis by the cordal macrophages. The same basic defect of the red cell membrane as has been documented in hereditary spherocytosis might be a possible explanation on the pathogenesis of this rare hemolytic anemia. ACTA PATH. JAP. 26: 533-542, 1976.

Introduction Hereditary elliptocytosis is one of the congential red cell abnormalities characterized by the appearance of elliptical erythrocytes in the circulating blood. There is a wide variation in clinical manifestation and it is generally agreed that hereditary elliptocptosis can be divided into three categories: those with no sign of hemolysis, those with compensated hemolysis and those with non-compensated hemolysis. The precise pathogenesis of this condition is not known, but it shares many features with hereditary spherocytosis such as an abnormal cell shape and membrane permeability defectlb. The beneficial result of splenectomy in many cases with hereditary elliptocytosis indicates the sequestration and destruction of defective red cells in the spleen. The histological appearance of the spleen in this disease has been documented, but there is no report as to the ultrastructure of the spleen. In this paper, the h e structures of the spleen and liver from a patient with non-compensated hereditary elliptocytosis will be described together with a brief discussion on the role of the spleen in the development of the symptoms. Case Report

T.Y., a 57-year-old house wife was admitted to Johoku Hospital at Kanazawa city with the chief complaints of jaundice, general fatigue and anorexia. She received cholecystectomy a t the age of 48 because of gall stones, and at that time she was pointed #A!* 4% m3 i %%, &#a f@A# fi% /J* s-EL ZG #tyc; S Rrl * Third D e p a r t m t of Internal Medicine, Yamaguchi University Sehool of Medicine, ** Pirat Department of Patblogy, Yamupchi University S c h Z of Medicine, Ube *** Department of Medicine, Johlcu Hospitd, Kanmam *?$it

633

~~~

Ube

534

Acta Path. Jap.

SPLEaN IN HERlODlTARY ELLIPTOOYTOSIS

out to have moderate anemia. On admission, physical examination disclosed yellowish discoloration of the sclera, grade I1 systolic murmur a t the apex and hepatosplenomegaly (liver: 5 cm., spleen: 10 cm.). Peripheral blood smear revealed many typical elliptocytes and slight increase in reticulocyte count. Coombs test was negative. Red cell survival examined by 61Cr method was markedly 'shortened (T 1/2: 5.5 days). Examination of the red cell enzymatic activities and glycolytic intermediates was not diagnostic. No abnormal hemoglobin was detected. Other laboratory findings were consistent with hemolytic anemia. Although there was no family history of jaundice or anemia as far as we have surveyed her relatives, a diagnosis of hereditary elliptocytosis was made and splenectomy was done on Feb. 18, 1975. The spleen was 1020 g in weight and was moderately congested. Reticulocyte count examined a t the time of operation showed no significant difference among the pheripheral blood, splenic arterial blood and splenic venous blood. This result suggested that there was no sequestration of young red cells in the spleen. Splenectomy was partially effective and some hematological improvement was obtained in spite of persistent elliptocytosis in the peripheral blood. Labaratory data before and after Splenectomy are summarized in Table 1. Table 1. Laboratory Data Before and After Nplenectomy After Splenectomy Before Splenectomy _____

2.5 months 8 months _____

RBC ( x 104/mm*)

an as. 13

Ht. (%I Hb. (g/dl) Reticulocyte (yo) Indirect bilirubin (mg/dl) Red cell survival (T W, days)

8.9

10.1

304

so. a 10.3 a. 6

374 30. 9 11.a

-

1.0

3.5

5.5

18.5

-

-

Materials and Methods The peripheral blood smeara were stained with Wright stain. For scanning electron microscopy, venous blood W&B fixed in 2 per cent glutaraldehyde and was processed aa waa reported previously. The specimens were examined with JSM-Sl scanning electron microscope. Immediately after the removal of the spleen, small pieces of the tissue from several different sites were fixed in 4.16 per cent glutctraldehyde and were postfixed in 1 per cent osmium tetroxide. They were processed in a graded concentration of aloohol and finally embedded in epoxy resin by the method of Luft6. Thin sections stained with uranyl acetate and lead citrate were examined in a Hitachi HS-8 electron microscope. Small tissue fragmenta from the liver and en accessory spleen were similarly treated. For light microscopy, sections from paraffin blocka were stained with hematoxylin-eosin, Berlin blue resction for the demonstration of iron pigments, silver impregnation for the reticulum fiber and Azan-Mallory stain for the collagen fiber.

Observations 1. Elliptocytes in the Peripheral Blood Light microscopic examination of the peripheral blood stained with Wright stain

26(5): 1976

N. MATSUMOTO

61

al.

535

Fig. 1. Elliptocytes in the peripheral blood. Wright stain. x 400. Fig. 2. Scanning electron micrograph of the elliptocytes in the peripheral blood. Original magnification x 3,000.

636

S ~ E RY N EBRBIDIT~RYn m o a r r o s m

Acta Path. Jap.

revealed many typical elliptical red cells as shown in Fig. 1. Scanning electron microscopic pictures of these elliptocytes are shown in Fig. 2. Some erythrocytes showed knizocytic changes.

2. Light Microscopy The medullary cord of the spleen was moderately congested. The sinuses, on the contrary, appeared to be relatively empty (Fig. 3). Erythrophagocytosis by the cordal macrophages was difficult to be recognized because of packed red cells in the cord, Deposition of hemosiderin granules was noted in the sinus lining cells and in the cordal macrophages. The lymph follicles were slightly decreased in size. There was no foci of extramedullary erythropoiesis. The accessory spleen showed essentially similar morphology and the proliferation of collagen fibers was more prominent. The liver showed marked deposition of hemosiderin granules both in the hepatic, parenchymal cells and in the Kupffer cells. Liver cirrhosis was evident as revealed by diffuse proliferation of connective tissue separating the lobules into pseudolobules of' various sizes (Fig. 4). Erythrophagocytosis by the Kupffer cells was not seen.

3. Electron Microscopy The most characteristic finding was the marked engorgement of erythrocytes in the cordal space. The erythrocytes with less variation in shape were seen just beneath the

~.

Fig. 3. Spleen. Moderate congestion of the cordal space with distended, almost empty sinuses. H.E. x 400.

26l5): 1976

N. MATSUMOTO rl el.

537

Fig. 4. Liver. Diffuse proliferation of the connective tissue and deposition of hemosiderin granules. H.E. x 400.

cordal side of the basement membrane of the sinus (Fig. 5), suggesting the difKculty of the red cells in passing through the cord into the sinus lumen. Most of the erythrocytes packed in the cord appeared to be round or oval in shape indicating their reduced deformability. Various stages of erythrophagocytosis by the cordal macrophages were noted. Fig. 6 shows the recently engulfed red cells within the macrophage. Variation of the electron density of the engulfed erythrocytes may reflect the stages of red cell destruction and degradation of hemoglobin within the macrophages (Fig. 7). Pig. 8 also reveals phagocytosis of the red cells by the cordal macrophage and dense bodies around the engulfed erythrocyte might be a degradation product of hemoglobin. Small myelin figures are also seen. Laked red cell ghosts with empty matrix were occasionally seen (Fig. 9). Sinus lining cells characterized by the presence of many pinocytotic vesicles contained small dense granules and they also showed erythrophagocytosis in occasional areas (Fig. 10). In the hepatic parenchymal cells, deposition of hemosiderin granules with irregular shape was prominent. Erythrophagocytosis by the Kupffer cells was not noted.

Discussion Hereditary elliptocytosis is one of the rare congenital hemolytic anemias due to intrinsic defect of the red cells. Studies of the erythrocyte biochemistry showed neither primitive enzymatic defect nor an occasional coincidence of enzymatic alteratiOnS.18

538

SPLIPEN IN HBlRElDITARY ELLIPLOCYTOSIS

Acta Path. Jap.

Fig. 6 . The erythrocytesin the cord are mostly round and lack the variation in shape. x 5,500. Fig. 6. Erythrophegocytosis by the cordel macrophege. x 10,000.

26(5): 1976

539

~~

~

Fig. 7. Erythrophagocytosia by the macrophage. Note the variation in density of the erythrocyte matrix. x 10,000. Fig. 8. Erythmphagocytosia by the wrdd macrophage. The dense gresules around the red cell might be a degradation product of engulfed erythrocyte. x 12,000.

540

SPWEN IN HEREDITbRY ELLIPTO(1PTOSIS

Aeta Path. Jap.

Fig. 9. The laked red cells in the cordal space. X 8,800. Fig. 10. Erythrophegooytosis by the sinus lining cell, which contab many dense granules.

x 10,Ooo.

26(5): 1976

N. MATSUMOTO r t at.

541

The precise pathogenesis of this disease is not fully understood, but the abnormalities of the red cell membrane has been proposed as a fundamental defect.3 REBUCKand ~~ that the elliptocytes maintained their elliptical outline VAN S L Y C Kdemonstrated even after evacuation of the intracellular contents by lysis, in contrast to sickle cells, which reverted to biconcave disks and they proposed the red cel membrane abnormalities as a basic defect in hereditary elliptocytosis. Recently, RALSTON~~ has reported that the high molecular weight water-soluble membrane protein (spectrin) in the camel erythrocytes, which are elliptical in shape, appears to be very tightly bound to the cell membrane. On the contrary, this protein is easily extracted from the normal human erythrocyte membrane after incubation in low ionic strength media. Similar abnormalities of the red cell membrane protein has been reported in hereditary spherocytosis by JACOB and his associates4. In addition, hereditary elliptocytosis shares many features with hereditary spherocytosis, such as an abnormal red cell shape, active sodium efflux and effectiveness of splenectomy in most cases. Therefore, it may be reasonable to speculate that the fundamental defect in hereditary elliptocytosis resides in the abnormalities of the red cell membrane protein. In hereditary elliptocytosis, effectiveness of splenectomy has been documented by several i n ~ e ~ t i g a t o but r ~ as , ~to~the ~ ~splenic ~ ~ ~ histology ~ ~ ~ ~ ~only ~ ~ a few reports are available.215~W~The most common findings are moderate to marked congestion of the splenic cord, relatively empty sinuses and accumulation of hemosiderin granules. Erythrophagocytosis by the cordal macrophages in fresh splenic smears has been shown by BLACICBURN et al.1 However, to the best of our knowledge, the fine structures of the spleen in this disease has not been reported. Electron microscopic examination of the spleen has revealed cordal congestion of red cells, which are mostly round or oval in shape and lack the variation in shape. Such stacking of erythrocytes in the cordal space and relatively empty sinuses may imply the difficulty of elliptocytes in passing through the pore of the basement membrane into the sinus lumen due to the reduced deformability of red cells. Prolonged stay in the cordal space may facilitate erythrophagocytosis by the macrophages. In fact, the erythrocytes engulfed by the cordal macrophages are frequently seen in this case. Long standing hemolysis leads to work hypertrophy of the spleen and gives rise to more anatomical and metabolic hazards. In conclusion, light and electron microscopic studies of the spleen in hereditary elliptocytosis indicate that the spleen plays the major role in sequestration of the defective elliptocytes which have reduced deformability probably due to the abnormalities of the red cell membrane protein. Acknowledgement: This investigation waa supported in part by the Research Grant for Specific Diseases from the Ministry of Health end Welfare of the Japanese Government, and by a Grantin-aid for Scientific Research of the Ministry of Education, Science and Culture.

References

BLACKBURN, E.K. et al.: Hereditary elliptocytic hemolytic anemia. J. Clin. Path. 11: 316320, 1968. 2. CUTTING, H.O. et d.: Autosomel dominant hemolytic snemia cherscterized by ovelocytosis. 1.

542

3. 4. 5. 6. 7. 8.

9.

SPLBEN I N HIOBIODITARY ELLIPTOCWTOSIS

Acta Path. Jap.

Am. J. Med. 39: 21-34, 1965. DACIE,J.V. : The hemolytic anemias. Part 1. The congential hemolytic anemias. 2nd ed., J & A Churchill, London, pp. 151-170, 1970. JACOB,H.S. RUBY,A., OVERLAND, E.S. and MAZIA, D.: Abnormal membrane protein of red blood cells in hereditary spherocytosis. J. Clin. Invest. 50: 1800-1805, 1971. LIPTON, E.L. : Elliptocytosis with hemolytic anemia: The effect of splenectomy. Pediatrics. 15: 67-83, 1955. L u m , J.H. : Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409414, 1961. MARCHESI,S.L., STEERS,E., MARCHESI, V.T. and TILLACK,T.W.: Wysical and chemical properties of a protein isolated from red cell membranes. Biochemistry. 9: 50-57, 1970. MATSUMOTO,N. ISH~EARA, T., SHIBATA, M. et al.: Electron microscopic studies of the spleen and liver in hereditary spherocytosis. Acta Path. Jap. 23: 507-530, 1973. MOTULSKY, A.G., SINGER,K., CROSBY, W.H. et al.: The life span of elliptocyte. Hereditary elliptocytosis and its relationship to other familiar hemolytic disease. Blood. 9: 57-72,

1954. 10. PRYOR, D.S. and PITNEY,W.R.:

Hereditary elliptocytosis: A report of two families from New Guinea. Brit. J. Hmmatol. 13: 126-134, 1967. 11. RALSTON,G.B. : Proteins of the camel erythrocyte membrane. Biochim. Biophys. Acta. 401: 83-94, 1975. 12. 13. 14. 15. 16.

REBUCK,J.W. and VAN SLY^, E.J.: An unsuspected ultrastructural fault in human elliptocytes. Am. J. clin. Path. 49: 19-25, 1968. TORLONTANO, G. et al. : Hereditary elliptocytosis. Hematological and metabolic findings. Acta Haemat. 48: 1-11, 1972. WEISS, H.J.: Heredtiary elliptocytosis with hemolytic anemia. Report of six cases. Am. J. Med. 35: 455466, 1963. WILLIAMS,J.W. et al.: Hematology. McGraw-Hill Book Co. pp. 387-388, 1972. W ~ S O NH.E. , and LONG,M.J. : Hereditary Ovalocytosis (Elliptocytosis) with hypersplenism. Arch. Intarn. Med. 95: 438444, 1955.