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The spleen: A correlative overview of normal and pathologic anatomy
T H E SPLEEN: A Correlative Overview of Normal and Pathologic Anatomy Monica B. Bishop, MB, ChB, MD, and Lawrence S. Lansing, MD The human spleen, an organ o f unique anatomic and functional importance, is the largest component of the reticuloendothelial system, with direct interposition between systemic and portal circulation, and yet the morphologic correlates o f its various functions remain somewhat mysterious. The contributions o f transmission and scanning electron microscopy to the understanding o f splenic structure have been considerable. They have helped clarify the three fundamental sites of structural alteration and specialization that are defined and discussed: 1) the white pulp with its two variable c o m p o n e n t s - - t h e lymphoid follicle and periarteriolar s h e a t h ~ w h i c h , with the marginal zone of the red pulp, is the primary site o f lymphoproliferative activity; 2) the cords o f the red pulp, the functionally slow component o f the splenic circulation, which sequester senescent or structurally altered red cells and effect their removal by means o f scavenging macrophages (and which may be secondarily involved by the accumulation of platelets or certain types of leukemic ceils, resulting in chronic cordal distention, or by the accumulation o f collagen in fibrocongestive splenomegaly); and 3) the splenic sinuses, the unique structure of which determines that only healthy red cells with normally plastic and flexible membranes pass through to the venous circulation. Abnormal transiting cells such as sickle cells frequently clog the apertures to these sinuses. Direct arteriocapillary sinus terminations provide the anatomic basis for a fast component o f the red pulp circulation, the existence o f which was questioned for many years and the extent o f which is still unknown in pathologic states. H u m Pathol 13:334-342, 1982.
lineating tile morphologic correlates o f altered function. This new knowledge has been gleaned from studies o f animal and human spleens in experimental models ~-6 and specific disease states, 7-~4 but much o f this information is widely scattered in the literature and is not readily accessible. The purpose of this study is to summarize the current view o f the normal splenic anatomy and to discuss structural alterations in selected pathologic conditions using illustrative material from our own transmission and scanning electron microscopy studies. MATERIALS AND METHODS In our diagnostic ser,)ice we h a v e studied 28 spleens by transmission electron microscopy (TEM), 18 by scanning electron microscopy (SEM), and 12 by both modalities; our stndies have included spleens affected by such disorders as sickle cell disease, hairy ,,
pER]AR|[RIAL tYMPH&IIC SHEATH
T h e spleen has always been something o f an enigma to the physiologist, anatomist, and pathologist alike. It is the largest component of the reticuloendothelial system, and it plays important roles in the body defenses, yet it is an organ that is not essential to life. Many features of its unique anatomy have eluded understanding for centnries, and the morphologic correlates of its various functions have remained somewhat mysterious. For pathologists, a particular Source of frustration has been the seemingly banal and nonspecific changes revealed by the light microscope when there is known to be severe functional derangement. During the past two decades, notable advances ltave been made in the u n d e r s t a n d i n g o f splenic function. Many factors have contributed to this understanding, including more precise knowledge of the nature of cell membranes and their contents and the availability of radioisotopic methods for in vivo study. The use of transmission and scanning electron microscopy, however, has contributed most to deReceived from the Electron MicroscopySection of Laboratory Service, Veterans Administration Medical Center #500, (113A), Albany, NY 12208. Address correspondence and reprint requests to Dr. Bishop Veterans AdministrationMedicalCenter #500 (113A), Albany,NY 12208.
334
Figure l. Schematicrepresentation of normal splenic anatomy and blood flow. (Adapted from Weiss.z6)
ANATOMY OF T H E SPLEEN--Btsuov AnD LANSING
3
Figure 2. Reticulin stain ltigbligbting circumferential reticulin fibers of periarterial lymphatic sheath (PAS) and adjacent lymphoid nodules (LN). (Scheuer reticulin stain, x250.) Figure 3. Scanning electron micrograph of splenic lymphoid nodule (LN). Marginal sinuses (hiS) present laterally. (x425.) At this low magnification, individual cells within and around the nodule cannot be precisely identified. cell leukemia, l y m p h o m a a n d leukemia, H o d g k i n disease, idiopathic thrombocytopenic purpura, t h r o m b o t i c t h r o m b o c y t o p e n i c p u r p u r a , and congestive splenomegaly, and some spleens (serving as controls) that were excised for reasons not related to a splenic d i s o r d e r per se. T h e fresh splenic tissue was immediately minced into 1.0-mrn and 2.0-mm pieces for T E M a n d SEM, respectively. Tissue for T E M was fixed in cold 3.8 per cent p a r a f o r m a l d e h y d e in Millonig buffer, postfixed
for 15 min in 2 per cent osmium tetroxide, dehydrated in g r a d e d alcohol, and e m b e d d e d in E p o n 812. O n e - m i c r o n sections were cut on an LKB UIt r o t o m e III a n d stained with toluidine blue and basic fuchsin methylene blue. T h i n sections were selected, cut on an U l t r o t o m e III, doubly stained with lead citrate and uranyl acetate, and viewed with a Philips 3000 transmission e l e c t r o n microscope. Tissue for SEM was selected u n d e r a dissecting microscope and p r e p a r e d by tile n o n c o a t i n g m e t h o d o f Sweney and
335
HUMAN PATHOLOGY--VOLUME 13, NUMBER 4 April 1982
4
5 Figure 4.
Periarteriolar lymphatic sheath expanded by proliferated plasma cells. (Scheuer reticulin counter-stained with trichrome.
Figure 5.
Uhrastructural counterpart of sheath shown in figure 4. Splenic cord (C) expanded by plasma cells (PC) borders splenic
•
sinuses. (• Shapiro, ~5 critically point-dried, m o u n t e d on alumin u m stubs by means o f conductive tape, and viewed with the ISI 60A scanning electron microscope. DISCUSSION
Outline of Splenic Structural and Functional
Relationships Classically, the splenic pulp is divided into two basic components, tile red and the white pulps, the f o r m e r designation derived from the rich vascularity o f this component, the latter from the appearance o f the l y m p h o i d content, the traditional m a l p i g h i a n corpuscle. T h e complex biologic inter-relationship a n d the dynamic nature o f these components is a freq u e n t source o f misunderstanding, misinterpretation, a n d even empiric oversimplification. T h e situation is m a d e more complex by the unique microanatomy o f the pulp subcomponents, the peculiar interposition o f the organ between the systemic and portal circulations, a n d the multiple functions o f an o r g a n of the reticuloendothelial system. It is customary and con-
336
venient to consider the red and the white pulps separately, but they are intimately and precisely related to the splenic vasculature as a whole. Blood enters tile spleen via the splenic artery, a vessel of disproportionately large caliber in relation to the mass o f the n o r m a l spleen. T h e artery ramifies into distributing trabecular arteries, which in turn enter the white pulp a n d the marginal zone via central arteries (fig. 1). Blood flow then proceeds in one o f two ways--directly from the central artery to tile marginal zone or via a germinal follicle o f the white pulp. T h e arteries terminate either in a sinus o f the red pulp, a functionally fast c o m p a r t m e n t or in the cords, a functionally slow compartment. T h e extent o f the fast c o m p a r t m e n t is not known with certainty; indeed, its very existence as a "closed circulation" was questioned for m a n y years. T h e functionally slow c o m p o n e n t is the circuitous, relatively stagnant circulation t h r o u g h the reticular f r a m e w o r k o f the cords. Both routes ultimately allow cells to pass into the splenic sinuses a n d from there into the venous circulation. T o interpret the pathology of this organ intelli-
ANATOMY OF TIIE SPLEEN--IhsnoP ANn LANSING
6
Figure 6. l,ight micrograph of periarteriolar lymphatic sheath (I'AS) expanded b)' acid-phosphatase- (tartrate-resistant) positive cells from a case of hairy cell leukemia. LN: lymphoid nodule. (x250.) Figure 7. Transmission electron micrograph of splenic cord (C) from a case of hairy cell leukemia. Note the lack of distinctive elongated cellular processes of infiltrating neoplastic cells. (x6,400.) gently, o n e must appreciate tile significance o f this vascular f r a m e w o r k and the relationship o f tile cellular c o m p o n e n t s to it. It then becomes a p p a r e n t that t h e r e are three f u n d a m e n t a l sites o f structural alteration and specialization--the white pulp, the splenic cords, and the splenic sinuses. The White
Pulp
T h e white pulp consists o f two variable c o m p o n e n t s - t h e periarterial l y m p h a t i c sheaths a n d tile lymphatic nodules. Tile cellular constitution and tile extent o f these c o m p o n e n t s is dynamic, reflecting the antigenic milieu o f the spleen. T h e periarterial lymplmtic sheaths are cylinders that s u r r o u n d the arterial vessels and are recognizable structurally in silver-stained sections by the circtnnferential a r r a n g e m e n t o f the retictdar fibers (fig. 2). T h e y contain the lymphatics and freely circulating
small lymphocytes, some plasma cells, and variable n u m b e r s o f m a c r o p h a g e s , t6 T h e m a c r o p h a g e s are usually m o r e n u m e r o u s in relation to the m o r e finely b r a n c h e d retictdum, with its associated reticular cells adjacent to the red pulp, which constitutes tile marginal zone. Figure 3 shows tile SEM a p p e a r a n c e o f the l y m p h o i d n o d u l e arising within the p e r i a r t e r i o l a r sheath and b o r d e r e d by the marginal sinuses. Many o f the smaller arteries t e r m i n a t e in this m a r g i n a l zone, but some c o n t i n u e into the pulp cords a n d curve back to e m p t y inio the marginal zone. T o what extent the marginal zone o f the red pulp and tlm arteriolar sheaths function as a trait is not clear, but the a r r a n g e m e n t o f the reticular fibers serves to de= llneate its limits. T h e periarterial sheath and the marginal zone reflect the reaction o f tile spleen to chronic antigenic challenge by e x p a n s i o n o f the l y m p h o i d content o f the sheath and proliferation o f plasma cells along its course. T h i s relationship is clearly d e m o n -
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14UMAN PATttOLOGY~VOLUME 13, NUMBER 4 April 1982
Figure 8. Scanningelectron micrographof splenic sinus (S) and cord (C), illustratingtypicalappearance of sinus liningcells(SLC)and cordal macrophage (M). strated in states of abnormal globulin production by means of counterstained silver preparations (fig. 4), in which plasma cells can be seen expanding the periarteriolar sheath. T h e hyperplastic nature of these plasma cells is best appreciated by TEM; a representative micrograph from a patient with amyloidosis is shown in fig. 5. The lymphoid nodules usually arise at arterial bifurcations within the arterial sheath; in tile inactive state they are loose lymphoid aggregates composed predominantly of small lymphocytes. Under stimulation, tile nodules become more discrete and hang like grapes from the arteries. Three zones have been defined by LukestV----a central dark zone of small lymphocytes, a middle zone of larger lymphocytes with more abundant cytoplasm, and an outer zone of smaller lymphocytes similar to those elsewhere in tile lymphatic shizath. In nodules that develop germinal centers, this outer rim of lymphocytes constitutes a mantle similar to that seen about the lymphoid follicles of lymph nodes. It is probable, however, that most such nodules do have germinal centers ~ but are not ahvays visible because of tile plane of section, so a target-like arrangement is more commonly seen. T h e changes in the white pulp in pathologic states have received very little detailed attention, but our own studies indicate tlmt they are predictable and that diagnosis is facilitated by ultrastructural study, which allows for better appreciation o f the degree of differentiation of the cells involved than does light microscopy alone. In non-Hodgkin lymphoma the neoplastic nodules pre-empt the location o f normal follicles and are most obvious at the bifurcation of vessels. However, satellite nodules may arise anywhere along the periarterial sheaths, hut even when
338
they appear abundant in the red pulp, they maintain the same relationship to the arterial and arterlolar branches in the cords. In Hodgkin disease the initial location seems to be within the periarteriolar sheaths giving rise to nodules with the usual cellular components anywhere along their course. The lymphoid leukemias should be included in a discussion of the white pulp because, although they ultimately involve all parts o f the spleen, it seems that their primary site o f involvement is in the periarteriolar sheath and that extension into the cords is a secondary phenomenon. This would certainly seem to be the case with hairy cell leukemia and prolymphocytic leukemia, both of which involve the s p l e e n massively. Carefifl study o f appropriately stained sections shows the greatest concentration of these cells in the region of the periarteriolar sheaths, with their extension along the cords in this relationship (fig. 6). It is interesting to note that when hairy cells become tightly packed in the cords, they lose their characteristic cytoplasmic projections, an indication that their hairiness is an environmental phenomenon
(fig. 7). The Red Pulp The red pulp is composed of the sinuses and the tissue between them, the cords. The terminal arterial branches extend for a variable distance into the red pulp as the straight penicilliary arteries and terminate either in the sinuses as a functionally fast compartment or in the cords as a functionally slow compartment. Rapid transport o f blood.elements tln'ough the spleen dictates a dual red-pulp circulation and in all likelihood is the result o f direct arterial capillary sinus
A N A T O M Y O F T H E S l ' L E E N - - B l s l l o e AND LA,~SI,~C.
10
11 Figure 9.
Distinctive hoop-like arrangement of basement membrane of splenic sinuses. (Scheuer reticulin. •
Figure I0. Scanning ultrastructural counterpart of material shown in figure 9. Partial autolysis and desquamation of sinus lining cells permits visualization of subjacent rcticulin (arrow). S: splenic sinus. (• 1,900.) Figure I1. Transmission electron micrograph of splenic cord (C) and splenic sinuses (S). Note the basally situated aggregates of tnicrofilaments (arrows) of sinus lining cells. (x5,500.)
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HUMAN PATHOLOGY--VOLUME 13, NUMBER 4 April 1982
12
13
Figure 12. Nucleated red cell (RC) traversing aperture between splenic cord (C) and sinus (S). Note the marked cytoplasmic deformation encountered in normal cellular transit from cord to sinus. SLC: sinus lining cells. (x9,600.) Figure 13. Scanning electron micrograph of normal anisocytosis of red cells traversing splenic sinus. (• t e r m i n a t i o n , a l t h o u g h tile n u m b e r s a r e p r o b a b l y small u n d e r n o r m a l circumstances. ~G It is the circuitous, relatively stagnant circulation t h r o u g h the cords that sequesters senescent o r structurally altered r e d cells a n d effects their r e m o v a l by m e a n s o f scavenging m a c r o p h a g e s . T h e cords themselves are a vascular space irregularly t r a v e r s e d by reticular cells a n d their extracellular reticulum, f o r m i n g i n t e r c o m m u n i c a t i n g channels. M a c r o p h a g e s sense relatively m i n o r structural c h a n g e s in red cells a n d a c c u m u l a t e within the c o r d s in a clinging association with the walls o f the
340
cord. T o the m a c r o p h a g e s is attributed the ability not only to cull a b n o r m a l cells such as sickle cells a n d s p h e r o c y t e s ~~ b u t also to pit such i n c l u s i o n s as H e i n z bodies ~9"2~a n d malarial parasites a n d to induce f r a g m e n t a t i o n o f red cells in a u t o i m m u n e hemolytic a n e m i a , s thalassemia, ~2 a n d h e m o g l o b i n H disease. 2~ By SEM, m a c r o p h a g e s are frequently seen a l o n g the c o r d s in close p r o x i m i t y to the a p e r t u r e s o f the splenic sinuses. W h e t h e r the m a c r o p h a g e s o r the a p e r t u r e s o f the splenic sinus are the passive m o v e r s in the r e m o v a l o f d a m a g e d cells is p e r h a p s debatable,
ANATOMY OF THE SPLEEN--BzsnoP AND LaNSINC
Figure 14. Splenicsinus in sickle cell disease. Note the marked distortion of red cells within the sinus (S) and the macrophages (M) along the splenic cord (C). (• but certainly the relationship between splenic cord, sinus, and macrophage is central to the understanding o f splenic p a t h o l o g y . T h i s u n i q u e t h r e e dimensional relationship is demonstrated to advantage by scanning ultrastructure (fig. 8). The sinuses of the red pulp have a distinctive anatomy by both light and electron microscopy. Although apparent by light microscopy only by means of silver impregnation (fig. 9), the unique sieve-like reticular network of the sinus is readily apparent by SEM. The lining endothelial cells of the sinus are s u r r o u n d e d in a hoop-like arrangement by interrupted ring fibers of basement membrane (fig. 10). The space between the overlying littoral cells, real or potential, defines the aperture that the red cell must traverse in its excursion from cord to sinus. A further specialization is the presence of clusters of line fibrils basally located and running the length of the sinus lining cells on either side of the interendothelial slits, which have the staining characteristics of xnyofibrils,2" implying a contractile function (fig. 1 1). Whether this arrangement serves to restore the shape of the sinus endothelial cells and close the aperture or to act as a sphincter, as was suggested by Knisely,23 is still open to question. It is evident that this path of egress from cord to sinus is precisely engineered and even in the normal spleen is not easily traversed. Considerable deformity of the traveling red cell is evident by scanning and transmission ultrastructure (figs. 12 and 13). What is less clear is what impels the appropriate red cell to make this passage. Predictably, in cases o f d e c r e a s e d red-cellmembrane plasticity or altered cellular configuration, the cords are distended by the affected cells. This is particularly apparent in elliptocytosis and sickle cell
disease. In sickle cell disease, red cells subjected to tile inimical biochemical inilieu of the cords sickle. By SEM the sickled cells are f r e q u e n t l y o b s e r v e d occluding or hridging the apertures (fig. 14). The ultimate fate o f the individual sequestered cell is phagocytosis, for the most part by cordal macrophages but to a lesser extent by the sinus lining cells. The long-term effect of chronic cordal distention, which is decreased flexibility of the cord itself, serves as an additional impediment to the transit of less-than-plastic red cells. In infiltrative hematologic processes in which the cords become distended with neoplastic cells and effectively increase the distance between central arteriole and sinus, these cells also induce, as they do elsewhere, the development of reticulin. In fibrocongestive splenomegaly, however, the increased portal pressure transmitted through predominantly the network of sinuses and cords induces the production of collagen, in the basement membrane of the cordal vessels, which impart abnormal rigidity to the cordal framework and fibrosis, resulting in dilatation of the sinuses. The result is impeded flow o f blood elements from cords to sinuses, increased exposure to scavenger m a c r o p h a g e s , and r e s u l t a n t h y p e r splenism. The normal anatomy o f the spleen and its alteration in Certain hematologic disorders has been described and illuminated by the techniques o f TEM and SEM. Although these modalities have added another dimension to our understanding of an inherently complex organ, numerous physiologic processes and recently generated ultrastructural findings still await functional and structural correlations. More enlightenment can be expected from the further application of TEM and SEM supplemented hy enzy-
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13, N U M B E R 4
April 1982
matic and immunologic techniques to cases that have bccn well studied clinically. ACKNOWLEDGMENTS
We gratefully acknowledge the encouragement of Dr. Wallace Jcnsen, who also provided some o f the case material for our studies, Ronie Jacobsen and Mary Alice Egy fi)r their technical help, and Frances Radzyminski for typing the manuscript. REFERENCES I. Weiss L, Chert I.T: The role of the sinus wall in the passage of erythrocytes through the spleen. Blood 41:4, 1973. 2. tlirasawa Y, Tokuhiro 1I: Electron microscopic studies on the normal human spleen, especially on the red pulp and the reticuluendothelial cells. Blood 35:201, 1970. 3. Burke JS, Simon GT: Electron microscopy of the spleen. I. Anatomy and microcirculation. Am J Pathol 58:127, 1970. 4. Barnhardt MI, Lusher JM: The human spleen as revealed by scanning elcctron microscopy. Am J ltematol 1:243, 1976. 5. Fujita "I': A scanning electron microscope stud)" of the human spleen. Arch llistol J 37:187, 1974. 6. Weiss I.: A scanning clcctron microscopic study of the spleen. Blood ,t3:665, 197,t. 7. Brietfeld V, Lee RE: l'athology of the spleen in hematologic disease. Surg Clin North Am 55:233, 1975. 8. Bowdler AJ: The role of the spleen and sl)lenectoiny in autoimmune hemolytic disease. Sere Hematol 13:335, 1976. 9. Mohandas N, l'hillips WM, Bessis M: Red blond cell deformability and heinolytic aiaemias. Sere 1lenmtnl 16:95, 1979.
342
10. Molnar Z, Rappal)ort 11: Fine structure of the red pul l) of the spleen in hereditary spheroc)tosis. Blnod 39:81, 1972. 11. Bnwman tiE, l'ettit VD, Caldwell F, et ah Morphology of tile spleen in idiopathic thrombocytope,lic purpura. Lab Invest 9t:206, 1955. 12. Sen Gupta PC, Chatterjea JB, Mjkherjee AM, et ah Observations of foam cell in thalassemia. Blood 10:1039, 1960. 13. Tavassoli M, Weiss L: An electron microscopic stud)" of spleen in myclofibrosis with myeloid metaplasia. Blood 42:267, 1973. 14. Rappaport II: The pathologic anatomy of the splenic red pulp. In Leimert K, Harms D (eds): The Spleen. New York, Springer-Verlag, 1970, p 24. 15. Sweney LR, Shapiro BL: Rapid preparation of uncoated hlological specimens fi~r SEM. Stain Technol 52:22I, 1977. 16. Weiss L: Spleen. In Greep RO, Weiss I. (eds): Histology, 3rd Ed. New York, McGraw-Hill Book Co, 1973. 17. Lukes RJ: The patholog)" of the white pulp of the spleen. In L e n n e r t K, l l a r m s D (eds): T h e Spleen. New York, Springer-Verlag, 1970, p 130. 18. Crosby WII: Siderocytes and the spleen. Blood 12:165, 1957. 19. Schuitzer B, Soderman "I', Mead ML, et al: Pitting functions of the spleen in malaria: ultrastructural observations. Science 177:175, 1972. 20. Rifkind RA: Heinz body anemia: an uhrastructural study. II. Red cell sequestration and destruction. Blood 26:433, 1965. 21. Wennberg E, Weiss L: Splenic er)throclasia: an electron microscopic stud)' of hemoglnbin H disease. Blood 31:778, 1968. 22. Meloan SN, Waldrop FS, Puchtler 11, et al: Cross-striated nlyoendothelial cells in sl)lenic venous sinuses. J Reticulnendothel Soc 11:566, 1972. 23. KniselyMtl: Spleen studies. I. Microscopic observations of the circulatory system of living unstimulated nmmnmlian spleen. Anat Rec 65:23, 1936.
lineating tile morphologic correlates o f altered function. This new knowledge has been gleaned from studies o f animal and human spleens in experimental models ~-6 and specific disease states, 7-~4 but much o f this information is widely scattered in the literature and is not readily accessible. The purpose of this study is to summarize the current view o f the normal splenic anatomy and to discuss structural alterations in selected pathologic conditions using illustrative material from our own transmission and scanning electron microscopy studies. MATERIALS AND METHODS In our diagnostic ser,)ice we h a v e studied 28 spleens by transmission electron microscopy (TEM), 18 by scanning electron microscopy (SEM), and 12 by both modalities; our stndies have included spleens affected by such disorders as sickle cell disease, hairy ,,
pER]AR|[RIAL tYMPH&IIC SHEATH
T h e spleen has always been something o f an enigma to the physiologist, anatomist, and pathologist alike. It is the largest component of the reticuloendothelial system, and it plays important roles in the body defenses, yet it is an organ that is not essential to life. Many features of its unique anatomy have eluded understanding for centnries, and the morphologic correlates of its various functions have remained somewhat mysterious. For pathologists, a particular Source of frustration has been the seemingly banal and nonspecific changes revealed by the light microscope when there is known to be severe functional derangement. During the past two decades, notable advances ltave been made in the u n d e r s t a n d i n g o f splenic function. Many factors have contributed to this understanding, including more precise knowledge of the nature of cell membranes and their contents and the availability of radioisotopic methods for in vivo study. The use of transmission and scanning electron microscopy, however, has contributed most to deReceived from the Electron MicroscopySection of Laboratory Service, Veterans Administration Medical Center #500, (113A), Albany, NY 12208. Address correspondence and reprint requests to Dr. Bishop Veterans AdministrationMedicalCenter #500 (113A), Albany,NY 12208.
334
Figure l. Schematicrepresentation of normal splenic anatomy and blood flow. (Adapted from Weiss.z6)
ANATOMY OF T H E SPLEEN--Btsuov AnD LANSING
3
Figure 2. Reticulin stain ltigbligbting circumferential reticulin fibers of periarterial lymphatic sheath (PAS) and adjacent lymphoid nodules (LN). (Scheuer reticulin stain, x250.) Figure 3. Scanning electron micrograph of splenic lymphoid nodule (LN). Marginal sinuses (hiS) present laterally. (x425.) At this low magnification, individual cells within and around the nodule cannot be precisely identified. cell leukemia, l y m p h o m a a n d leukemia, H o d g k i n disease, idiopathic thrombocytopenic purpura, t h r o m b o t i c t h r o m b o c y t o p e n i c p u r p u r a , and congestive splenomegaly, and some spleens (serving as controls) that were excised for reasons not related to a splenic d i s o r d e r per se. T h e fresh splenic tissue was immediately minced into 1.0-mrn and 2.0-mm pieces for T E M a n d SEM, respectively. Tissue for T E M was fixed in cold 3.8 per cent p a r a f o r m a l d e h y d e in Millonig buffer, postfixed
for 15 min in 2 per cent osmium tetroxide, dehydrated in g r a d e d alcohol, and e m b e d d e d in E p o n 812. O n e - m i c r o n sections were cut on an LKB UIt r o t o m e III a n d stained with toluidine blue and basic fuchsin methylene blue. T h i n sections were selected, cut on an U l t r o t o m e III, doubly stained with lead citrate and uranyl acetate, and viewed with a Philips 3000 transmission e l e c t r o n microscope. Tissue for SEM was selected u n d e r a dissecting microscope and p r e p a r e d by tile n o n c o a t i n g m e t h o d o f Sweney and
335
HUMAN PATHOLOGY--VOLUME 13, NUMBER 4 April 1982
4
5 Figure 4.
Periarteriolar lymphatic sheath expanded by proliferated plasma cells. (Scheuer reticulin counter-stained with trichrome.
Figure 5.
Uhrastructural counterpart of sheath shown in figure 4. Splenic cord (C) expanded by plasma cells (PC) borders splenic
•
sinuses. (• Shapiro, ~5 critically point-dried, m o u n t e d on alumin u m stubs by means o f conductive tape, and viewed with the ISI 60A scanning electron microscope. DISCUSSION
Outline of Splenic Structural and Functional
Relationships Classically, the splenic pulp is divided into two basic components, tile red and the white pulps, the f o r m e r designation derived from the rich vascularity o f this component, the latter from the appearance o f the l y m p h o i d content, the traditional m a l p i g h i a n corpuscle. T h e complex biologic inter-relationship a n d the dynamic nature o f these components is a freq u e n t source o f misunderstanding, misinterpretation, a n d even empiric oversimplification. T h e situation is m a d e more complex by the unique microanatomy o f the pulp subcomponents, the peculiar interposition o f the organ between the systemic and portal circulations, a n d the multiple functions o f an o r g a n of the reticuloendothelial system. It is customary and con-
336
venient to consider the red and the white pulps separately, but they are intimately and precisely related to the splenic vasculature as a whole. Blood enters tile spleen via the splenic artery, a vessel of disproportionately large caliber in relation to the mass o f the n o r m a l spleen. T h e artery ramifies into distributing trabecular arteries, which in turn enter the white pulp a n d the marginal zone via central arteries (fig. 1). Blood flow then proceeds in one o f two ways--directly from the central artery to tile marginal zone or via a germinal follicle o f the white pulp. T h e arteries terminate either in a sinus o f the red pulp, a functionally fast c o m p a r t m e n t or in the cords, a functionally slow compartment. T h e extent o f the fast c o m p a r t m e n t is not known with certainty; indeed, its very existence as a "closed circulation" was questioned for m a n y years. T h e functionally slow c o m p o n e n t is the circuitous, relatively stagnant circulation t h r o u g h the reticular f r a m e w o r k o f the cords. Both routes ultimately allow cells to pass into the splenic sinuses a n d from there into the venous circulation. T o interpret the pathology of this organ intelli-
ANATOMY OF TIIE SPLEEN--IhsnoP ANn LANSING
6
Figure 6. l,ight micrograph of periarteriolar lymphatic sheath (I'AS) expanded b)' acid-phosphatase- (tartrate-resistant) positive cells from a case of hairy cell leukemia. LN: lymphoid nodule. (x250.) Figure 7. Transmission electron micrograph of splenic cord (C) from a case of hairy cell leukemia. Note the lack of distinctive elongated cellular processes of infiltrating neoplastic cells. (x6,400.) gently, o n e must appreciate tile significance o f this vascular f r a m e w o r k and the relationship o f tile cellular c o m p o n e n t s to it. It then becomes a p p a r e n t that t h e r e are three f u n d a m e n t a l sites o f structural alteration and specialization--the white pulp, the splenic cords, and the splenic sinuses. The White
Pulp
T h e white pulp consists o f two variable c o m p o n e n t s - t h e periarterial l y m p h a t i c sheaths a n d tile lymphatic nodules. Tile cellular constitution and tile extent o f these c o m p o n e n t s is dynamic, reflecting the antigenic milieu o f the spleen. T h e periarterial lymplmtic sheaths are cylinders that s u r r o u n d the arterial vessels and are recognizable structurally in silver-stained sections by the circtnnferential a r r a n g e m e n t o f the retictdar fibers (fig. 2). T h e y contain the lymphatics and freely circulating
small lymphocytes, some plasma cells, and variable n u m b e r s o f m a c r o p h a g e s , t6 T h e m a c r o p h a g e s are usually m o r e n u m e r o u s in relation to the m o r e finely b r a n c h e d retictdum, with its associated reticular cells adjacent to the red pulp, which constitutes tile marginal zone. Figure 3 shows tile SEM a p p e a r a n c e o f the l y m p h o i d n o d u l e arising within the p e r i a r t e r i o l a r sheath and b o r d e r e d by the marginal sinuses. Many o f the smaller arteries t e r m i n a t e in this m a r g i n a l zone, but some c o n t i n u e into the pulp cords a n d curve back to e m p t y inio the marginal zone. T o what extent the marginal zone o f the red pulp and tlm arteriolar sheaths function as a trait is not clear, but the a r r a n g e m e n t o f the reticular fibers serves to de= llneate its limits. T h e periarterial sheath and the marginal zone reflect the reaction o f tile spleen to chronic antigenic challenge by e x p a n s i o n o f the l y m p h o i d content o f the sheath and proliferation o f plasma cells along its course. T h i s relationship is clearly d e m o n -
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Figure 8. Scanningelectron micrographof splenic sinus (S) and cord (C), illustratingtypicalappearance of sinus liningcells(SLC)and cordal macrophage (M). strated in states of abnormal globulin production by means of counterstained silver preparations (fig. 4), in which plasma cells can be seen expanding the periarteriolar sheath. T h e hyperplastic nature of these plasma cells is best appreciated by TEM; a representative micrograph from a patient with amyloidosis is shown in fig. 5. The lymphoid nodules usually arise at arterial bifurcations within the arterial sheath; in tile inactive state they are loose lymphoid aggregates composed predominantly of small lymphocytes. Under stimulation, tile nodules become more discrete and hang like grapes from the arteries. Three zones have been defined by LukestV----a central dark zone of small lymphocytes, a middle zone of larger lymphocytes with more abundant cytoplasm, and an outer zone of smaller lymphocytes similar to those elsewhere in tile lymphatic shizath. In nodules that develop germinal centers, this outer rim of lymphocytes constitutes a mantle similar to that seen about the lymphoid follicles of lymph nodes. It is probable, however, that most such nodules do have germinal centers ~ but are not ahvays visible because of tile plane of section, so a target-like arrangement is more commonly seen. T h e changes in the white pulp in pathologic states have received very little detailed attention, but our own studies indicate tlmt they are predictable and that diagnosis is facilitated by ultrastructural study, which allows for better appreciation o f the degree of differentiation of the cells involved than does light microscopy alone. In non-Hodgkin lymphoma the neoplastic nodules pre-empt the location o f normal follicles and are most obvious at the bifurcation of vessels. However, satellite nodules may arise anywhere along the periarterial sheaths, hut even when
338
they appear abundant in the red pulp, they maintain the same relationship to the arterial and arterlolar branches in the cords. In Hodgkin disease the initial location seems to be within the periarteriolar sheaths giving rise to nodules with the usual cellular components anywhere along their course. The lymphoid leukemias should be included in a discussion of the white pulp because, although they ultimately involve all parts o f the spleen, it seems that their primary site o f involvement is in the periarteriolar sheath and that extension into the cords is a secondary phenomenon. This would certainly seem to be the case with hairy cell leukemia and prolymphocytic leukemia, both of which involve the s p l e e n massively. Carefifl study o f appropriately stained sections shows the greatest concentration of these cells in the region of the periarteriolar sheaths, with their extension along the cords in this relationship (fig. 6). It is interesting to note that when hairy cells become tightly packed in the cords, they lose their characteristic cytoplasmic projections, an indication that their hairiness is an environmental phenomenon
(fig. 7). The Red Pulp The red pulp is composed of the sinuses and the tissue between them, the cords. The terminal arterial branches extend for a variable distance into the red pulp as the straight penicilliary arteries and terminate either in the sinuses as a functionally fast compartment or in the cords as a functionally slow compartment. Rapid transport o f blood.elements tln'ough the spleen dictates a dual red-pulp circulation and in all likelihood is the result o f direct arterial capillary sinus
A N A T O M Y O F T H E S l ' L E E N - - B l s l l o e AND LA,~SI,~C.
10
11 Figure 9.
Distinctive hoop-like arrangement of basement membrane of splenic sinuses. (Scheuer reticulin. •
Figure I0. Scanning ultrastructural counterpart of material shown in figure 9. Partial autolysis and desquamation of sinus lining cells permits visualization of subjacent rcticulin (arrow). S: splenic sinus. (• 1,900.) Figure I1. Transmission electron micrograph of splenic cord (C) and splenic sinuses (S). Note the basally situated aggregates of tnicrofilaments (arrows) of sinus lining cells. (x5,500.)
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HUMAN PATHOLOGY--VOLUME 13, NUMBER 4 April 1982
12
13
Figure 12. Nucleated red cell (RC) traversing aperture between splenic cord (C) and sinus (S). Note the marked cytoplasmic deformation encountered in normal cellular transit from cord to sinus. SLC: sinus lining cells. (x9,600.) Figure 13. Scanning electron micrograph of normal anisocytosis of red cells traversing splenic sinus. (• t e r m i n a t i o n , a l t h o u g h tile n u m b e r s a r e p r o b a b l y small u n d e r n o r m a l circumstances. ~G It is the circuitous, relatively stagnant circulation t h r o u g h the cords that sequesters senescent o r structurally altered r e d cells a n d effects their r e m o v a l by m e a n s o f scavenging m a c r o p h a g e s . T h e cords themselves are a vascular space irregularly t r a v e r s e d by reticular cells a n d their extracellular reticulum, f o r m i n g i n t e r c o m m u n i c a t i n g channels. M a c r o p h a g e s sense relatively m i n o r structural c h a n g e s in red cells a n d a c c u m u l a t e within the c o r d s in a clinging association with the walls o f the
340
cord. T o the m a c r o p h a g e s is attributed the ability not only to cull a b n o r m a l cells such as sickle cells a n d s p h e r o c y t e s ~~ b u t also to pit such i n c l u s i o n s as H e i n z bodies ~9"2~a n d malarial parasites a n d to induce f r a g m e n t a t i o n o f red cells in a u t o i m m u n e hemolytic a n e m i a , s thalassemia, ~2 a n d h e m o g l o b i n H disease. 2~ By SEM, m a c r o p h a g e s are frequently seen a l o n g the c o r d s in close p r o x i m i t y to the a p e r t u r e s o f the splenic sinuses. W h e t h e r the m a c r o p h a g e s o r the a p e r t u r e s o f the splenic sinus are the passive m o v e r s in the r e m o v a l o f d a m a g e d cells is p e r h a p s debatable,
ANATOMY OF THE SPLEEN--BzsnoP AND LaNSINC
Figure 14. Splenicsinus in sickle cell disease. Note the marked distortion of red cells within the sinus (S) and the macrophages (M) along the splenic cord (C). (• but certainly the relationship between splenic cord, sinus, and macrophage is central to the understanding o f splenic p a t h o l o g y . T h i s u n i q u e t h r e e dimensional relationship is demonstrated to advantage by scanning ultrastructure (fig. 8). The sinuses of the red pulp have a distinctive anatomy by both light and electron microscopy. Although apparent by light microscopy only by means of silver impregnation (fig. 9), the unique sieve-like reticular network of the sinus is readily apparent by SEM. The lining endothelial cells of the sinus are s u r r o u n d e d in a hoop-like arrangement by interrupted ring fibers of basement membrane (fig. 10). The space between the overlying littoral cells, real or potential, defines the aperture that the red cell must traverse in its excursion from cord to sinus. A further specialization is the presence of clusters of line fibrils basally located and running the length of the sinus lining cells on either side of the interendothelial slits, which have the staining characteristics of xnyofibrils,2" implying a contractile function (fig. 1 1). Whether this arrangement serves to restore the shape of the sinus endothelial cells and close the aperture or to act as a sphincter, as was suggested by Knisely,23 is still open to question. It is evident that this path of egress from cord to sinus is precisely engineered and even in the normal spleen is not easily traversed. Considerable deformity of the traveling red cell is evident by scanning and transmission ultrastructure (figs. 12 and 13). What is less clear is what impels the appropriate red cell to make this passage. Predictably, in cases o f d e c r e a s e d red-cellmembrane plasticity or altered cellular configuration, the cords are distended by the affected cells. This is particularly apparent in elliptocytosis and sickle cell
disease. In sickle cell disease, red cells subjected to tile inimical biochemical inilieu of the cords sickle. By SEM the sickled cells are f r e q u e n t l y o b s e r v e d occluding or hridging the apertures (fig. 14). The ultimate fate o f the individual sequestered cell is phagocytosis, for the most part by cordal macrophages but to a lesser extent by the sinus lining cells. The long-term effect of chronic cordal distention, which is decreased flexibility of the cord itself, serves as an additional impediment to the transit of less-than-plastic red cells. In infiltrative hematologic processes in which the cords become distended with neoplastic cells and effectively increase the distance between central arteriole and sinus, these cells also induce, as they do elsewhere, the development of reticulin. In fibrocongestive splenomegaly, however, the increased portal pressure transmitted through predominantly the network of sinuses and cords induces the production of collagen, in the basement membrane of the cordal vessels, which impart abnormal rigidity to the cordal framework and fibrosis, resulting in dilatation of the sinuses. The result is impeded flow o f blood elements from cords to sinuses, increased exposure to scavenger m a c r o p h a g e s , and r e s u l t a n t h y p e r splenism. The normal anatomy o f the spleen and its alteration in Certain hematologic disorders has been described and illuminated by the techniques o f TEM and SEM. Although these modalities have added another dimension to our understanding of an inherently complex organ, numerous physiologic processes and recently generated ultrastructural findings still await functional and structural correlations. More enlightenment can be expected from the further application of TEM and SEM supplemented hy enzy-
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matic and immunologic techniques to cases that have bccn well studied clinically. ACKNOWLEDGMENTS
We gratefully acknowledge the encouragement of Dr. Wallace Jcnsen, who also provided some o f the case material for our studies, Ronie Jacobsen and Mary Alice Egy fi)r their technical help, and Frances Radzyminski for typing the manuscript. REFERENCES I. Weiss L, Chert I.T: The role of the sinus wall in the passage of erythrocytes through the spleen. Blood 41:4, 1973. 2. tlirasawa Y, Tokuhiro 1I: Electron microscopic studies on the normal human spleen, especially on the red pulp and the reticuluendothelial cells. Blood 35:201, 1970. 3. Burke JS, Simon GT: Electron microscopy of the spleen. I. Anatomy and microcirculation. Am J Pathol 58:127, 1970. 4. Barnhardt MI, Lusher JM: The human spleen as revealed by scanning elcctron microscopy. Am J ltematol 1:243, 1976. 5. Fujita "I': A scanning electron microscope stud)" of the human spleen. Arch llistol J 37:187, 1974. 6. Weiss I.: A scanning clcctron microscopic study of the spleen. Blood ,t3:665, 197,t. 7. Brietfeld V, Lee RE: l'athology of the spleen in hematologic disease. Surg Clin North Am 55:233, 1975. 8. Bowdler AJ: The role of the spleen and sl)lenectoiny in autoimmune hemolytic disease. Sere Hematol 13:335, 1976. 9. Mohandas N, l'hillips WM, Bessis M: Red blond cell deformability and heinolytic aiaemias. Sere 1lenmtnl 16:95, 1979.
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10. Molnar Z, Rappal)ort 11: Fine structure of the red pul l) of the spleen in hereditary spheroc)tosis. Blnod 39:81, 1972. 11. Bnwman tiE, l'ettit VD, Caldwell F, et ah Morphology of tile spleen in idiopathic thrombocytope,lic purpura. Lab Invest 9t:206, 1955. 12. Sen Gupta PC, Chatterjea JB, Mjkherjee AM, et ah Observations of foam cell in thalassemia. Blood 10:1039, 1960. 13. Tavassoli M, Weiss L: An electron microscopic stud)" of spleen in myclofibrosis with myeloid metaplasia. Blood 42:267, 1973. 14. Rappaport II: The pathologic anatomy of the splenic red pulp. In Leimert K, Harms D (eds): The Spleen. New York, Springer-Verlag, 1970, p 24. 15. Sweney LR, Shapiro BL: Rapid preparation of uncoated hlological specimens fi~r SEM. Stain Technol 52:22I, 1977. 16. Weiss L: Spleen. In Greep RO, Weiss I. (eds): Histology, 3rd Ed. New York, McGraw-Hill Book Co, 1973. 17. Lukes RJ: The patholog)" of the white pulp of the spleen. In L e n n e r t K, l l a r m s D (eds): T h e Spleen. New York, Springer-Verlag, 1970, p 130. 18. Crosby WII: Siderocytes and the spleen. Blood 12:165, 1957. 19. Schuitzer B, Soderman "I', Mead ML, et al: Pitting functions of the spleen in malaria: ultrastructural observations. Science 177:175, 1972. 20. Rifkind RA: Heinz body anemia: an uhrastructural study. II. Red cell sequestration and destruction. Blood 26:433, 1965. 21. Wennberg E, Weiss L: Splenic er)throclasia: an electron microscopic stud)' of hemoglnbin H disease. Blood 31:778, 1968. 22. Meloan SN, Waldrop FS, Puchtler 11, et al: Cross-striated nlyoendothelial cells in sl)lenic venous sinuses. J Reticulnendothel Soc 11:566, 1972. 23. KniselyMtl: Spleen studies. I. Microscopic observations of the circulatory system of living unstimulated nmmnmlian spleen. Anat Rec 65:23, 1936.