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The microvascular architecture of spleen in congestive splenomegaly
Path. Res. Pract. 174, 131-146 (1982) * Cattedra dl SemelOtica MedICa-DlVlSlOne dl Medlclna generale dell' Ospedale Bellana
* * Istltuto di IstologIa ed Embnologla generale * ** I Cattedra dl Statlstlca, UmvefSlty of Bologna, Bologna, Italy
The Microvascular Architecture of Spleen in Congestive Splenomegaly A Morphological-Histometric Study G. CAVALLI*, G. RE*, ANNA MARIA CASALI ** and PAOLA MONARI***
Summary Spleens obtained from patients suffering from chromc active hepatitis, hepatic cirrhosIs, Idiopathic portal hypertension and splemc vein thrombOSIS have been subjected to morphological observation associated with histometncal analysIs and compared to 3 control spleens surgically removed for trauma. All the pathological spleens show the picture of congestive splenomegaly. Moreover, In chromc active hepatitis small non-specific granulomas are observed In the red pulp, along with lymphocytic-plasma cellular sheaths surrounding the terminal artenal branches. The latter are also encountered, less frequently, In bver cirrhosIs. In Idiopathic portal hypertension the presence of artenolar-slnusal shunts can be emphasized. The stereology of the same spleens shows In chromc active hepatitis, and more markedly In liver cirrhosIs, higher values of SinUS length and a lower mean sectional area of the sinuses In companson with controls. In IdIOpathIC portal hypertensIOn the slnusallength IS also greater and the mean slnusal sectIOnal area IS lower. However, companson of the statistical with the geometnc determination of the external surface area of sinuses allows us to hypotheSize that there IS a nch interconnection between sinuses In bver cmhosls, but not In idIOpathiC portal hypertension. In the latter spleens the statistical and geometrical determinatIOns show values qUite Similar to each other, as well as in control spleens, In spite of the Important structural rearrangement presented here. In splemc vein thrombOSIS the hlstometnc findings are consistent with SinUS dilatation. The microvascular architecture of red pulp, especially when studied with hlstometnc methods, shows charactenstic features In IdIOpathiC portal hypertension which permit us to separate this form of congestive splenomegaly from all the others examined.
132 . G. CavallI, G. Re, Anna Mana CasalI, and Paola Monan
Introduction Splenic enlargement is a frequent finding in the course of portal hypertension due either to intra-hepatic causes or to main portal vein obstruction. Splenomegaly of some degree is observed in almost 50% of cases of liver cirrhosis (Snell, 1931; Armas-Cruz et aI., 1951) and its incidence is still higher at necropsy examlOation (Klopstock, 1907; Armas-Cruz et aI., 1951), although a syndrome of severe portal hypertension expressed by bleeding from oesophageal varices can occur without spleen enlargement (Liebowitz, 1963). However, splenomegaly does not show the same incidence in the different types of cirrhosis, being much less frequent in alcoholic cirrhosis than in the other forms. The splemc enlargement in the course of liver disease may be regarded as a direct consequence of "congestion" caused by long-standing portal hypertension, but it is also currently explained as resulting from a generic reaction to toxic or infectIOUS agents, from an immune response to the hepatocyte damage, or from ill-defined liver damage (Tumen, 1970). Besides those cases determined by intra- or extra-hepatic block, infrequent forms of portal hypertension with splenomegaly are described, in which no morphological changes capable of obstructing the blood flow can be demonstrated in the liver or in the splenoportal trunk (Rousselot, 1936, 1940; Hunt, 1955; Cacciari et aI., 1957; Leger et aI., 1966; Sotgiu, 1977). These forms show a geographical distribution, with a higher incidence in tropical or subtropical zones. They are clinically characterized by conspicuous enlargement of the spleen, oesophageal and gastric varices with tendency to haemorrhages. Such cases of portal hypertension have been variously named (idiopathic portal hypertension, essential portal hypertension, primary portal hypertension, portal hypertensIOn without obstacle) and have been related to excessive blood flow through the spleen. However, it must be pointed out that an increased blood flow proportional to the increased splenic volume (Patrassi et aI., 1961) is not found exclusively in idiopathic portal hypertension, but is also seen in splenomegalic liver cirrhosis. The conception of impaired hepatic distensibility in face of portal hyperflow as a factor of hypertension has been improved by Sotgiu (1977), who introduced the concept of a defective compliance of the liver and portal vascular bed to explain the presence of portal hypertension in cases with no detectable obstacle. The decreased compliance might represent the relative obstacle that initiates the syndrome of portal hypertension due to increased flow and might then result in a fibrotic rearrangement of the vascular structures. Alternatively, the decreased compliance itself might depend on the vascular structure alterations induced by the increase in flow. The classification of idiopathic portal hypertension (IPH) as a distinct
Microvascular Architecture of Spleen
In
Congestive Splenomegaly . 133
nosographic form of congestive splenomegaly remains a matter for debate. In fact the existence of a primary condition of splenic "hyperflow" is not admitted by all authors, since an increased blood flow can also occur in splenomegalIc lIver cirrhosis (Patrassi et al., 1961) and the various primary syndromes reported in different geographIc zones might have a different aetiopathogenesis (Iber, 1970). The study of the vascular formations may contribute elements useful to a better understanding of the congesttve splenomegalIes and to their nosography. In this belief we have exammed the splemc mIcrovascular architecture in some different types of splenomegalies. To thIs purpose histological observation has been associated with a mathemattcal-geometncal study of the structures under investigation, since the histometric analysis has already proved to be profitable also when apphed to the spleen (Sekl, 1965; Stutte and Heusermann, 1972; Cavalli et al., 1978; Yamamoto, 1978, 1979).
Material The study was carned out on 20 spleens that had been removed surgICally and fixed Immediately, with the exceptIOn ot one that was removed at necropsy. Three spleens from persons operated for accidental trauma served as controls. The other spleens were obtained from patients showmg marked splenomegaly and have been selected for this study because the clinIco-pathologIcal picture allowed the patient to be ascrIbed With certamty to one of the followmg groups of cases. Chrome actIVe hepatltts All these patients (4 men and 3 women) presented the serum fmdmgs and the hepatIC histopathological aspects consistent With the diagnOSIs of moderately aggressive chronic hepatitis (both m needle and m surgically pertormed lIver bIOpSies). The search for Auantigen m the serum was negative tor all of them. Moreover, every patient had a conspicuously enlarged spleen With associated signs of hypersplenism, such as anaemia and especially leukopenia and thrombocytopenia. The mtrasplenIc pressure (at splenomanometry) was normal or slightly mcreased, but the presence of collateral portacaval circulatIOn was not eVidenced by splenoportography or dunng the venous phase of selective coelIac angIOgraphy m any of these cases. Splenectomy was done because ot the disturbance due to the greatly enlarged spleen and the syndrome of hypersplenism; It was followed m all cases by Improvement of the blood cell counts. Follow-up after the operatIOn mdlcated that splenectomy did not affect the clInical course of the chrOniC hepatitis. HepatIC CIrrhOSIS The dlagnosl~ of non-alcoholic lIver CirrhOSIS was made m all these cases (3 men and 1 woman) on the baSIS of the clInical history, the laboratory data and the histopathology of the lIver (both m percutaneous and surgical bIOpSies). All the patients presented a very enlarged spleen, signs of hypersplenism and the syndrome of portal hypertensIOn from mtra-hepatlc obstructIOn, eVidenced also by splenomanometry, splenoportography, and/or selective coelIac angiography. The splenectomy wa, performed at the time of porta-caval shunt operation for repeatmg gastro-mtestmal haemorrhages
134 . G. Cavalli, G. Re, Anna Mana Casali, and Paola Monan
Id,opath,c portal hypertensIOn. This group consists of 5 patients (3 men and 2 women). 4 of them had eVidence of marked splenomegaly, hypersplenism and severe portal hypertension with gastro-mtestinal haemorrhages The laboratory data and the liver histology were normal. The study of portal circulation demonstrated, m addItIon to mcreased values of splenomanometry, the patency of the splenoportal trunk mJected by splenoportography, the mcreased translational speed of the medIUm from spleen to liver, the absence of hepatofugal circulation. After splenectomy, three patients showed a clear clinical Improvement, disappearance of gastro-mtestmal haemorrhages and normal blood cell counts III follow-up made for over 15 years. The fourth patient died of other causes shortly after the mterventlon. The fIfth case of thiS group concerns a man 65 years old who 30 years before had the diagnosIs made of "pnmary congestive splenomegaly" (according to Greppl, 1928) and who died from prostatic carcmoma; thiS case also could be ascnbed to primary portal hypertensIOn on the basIs of the exammations performed. The spleen of thiS patient IS the only one of our study obtamed at necropsy. The histology of the liver showed only a slight portal fIbroSIS Splentc vein thrombosIs. Fmally we have considered the case of a girl 13 years old. The onset of her clinical history dated back to only 7 months before, when she presented high fever, eplgastnc pam, haematemesls, melaena and transitory aSCites, followed by splenomegaly and hypochromic anaemia. At the splenoportographlc exammatlon the splenoportal trunk did not appear mJected; the bulk of radIO-opaque dye was stopped at the hilus of spleen and diverted through the gastnc and oesophageal vems At splenectomy, the presence of spleniC vem thrombosIs at the hilus was confirmed.
Methods For histological exammatlOn, tissue blocks obtamed from different zones of every spleen (the poles and deep central regions) were fixed With Boum's flUid and embedded m paraffm. 4 spleens (2 normal and 2 of IdIOpathic portal hypertensIOn) were perfUSIOn-fixed. They were artenally perfused With Rmger solution contammg heparm at 37°C before fixatIOn, takmg care that the mJectlon was made m an mtermIttent way (about 50 pulses/mm) and With vanatlon of pressure from 110 mm Hg to 70 mm Hg. After washmg, the perfusIOn solutIOn was replaced With Boum's flUid. The subsequent procedure was the same as for all other spleens. Senes of 5 Ilm thick sectIOns were stamed With haematoxylin and eosm, Azan, PAS reactIOn, orcem, Achucarro after deammatlon. The hlstometnc mvestlgatlOns were carned out on the same microSCOPIC preparatIOns. For measurements, nets of pomts have been used to record the number of pomts falling on red pulp, white pulp and trabeculae respectively and three groups of elementary data: the number of test pomts fallmg on smuses; the number of mtersectlons of the test hnes With the traces of smus walls; the number of smus profties per test net. From these, by applymg current formulas for stereology (Weibel et aI., 1966; Elias et aI., 1971), the followmg quantities have been determmed m every spleen: percentage of red pulp, of white pulp and of trabeculae per Unit volume; per cent volume of smuses and of cords m the red pulp, volume ratio of smuses to cords m the red pulp; mean Width of the spleniC cords; sectional surface area of smuses; length of sinuses per Unit volume; smusOldal surface area per Unit volume. The groups of values were then subjected to statistical analYSIS.
MIcrovascular Architecture of Spleen III Congestive Splenomegaly . 135
Results Morphological observations The common feature of all the pathological spleens examined 1S the general microscopic picture of fibrocongestive splenomegaly. No perisplenitis is observed. A more or less marked hyalinosis of follicular arteries and fibrosis of Malpighian corpuscles are more frequent in the cases of liver cirrhos1s than in idiopathic portal hypertension, where they are present, although moderately, in only two cases (F. T. and S. B.). A slight perivascular fibrosis is occasionally observed in chronic hepatitis. The periarterial fibrosis, already described by Diirr (1924) in so called Banti's spleens, can also be detected in idiopathic portal hypertension. At scanning electron microscope observation (Yamamoto, 1979) th1s proved to be composed of bundles of channels formed by reticulum cells. In idiopathic portal hypertension the Schweigger-Se1del sheaths may give the appearance of hyalinosis of the wall bes1des the increase of the surrounding fibrillar network (Fig. 1 c, d, e). Periarterial sheaths of lymphocytes, and especially plasma cells, surround the terminal arterial branches such as penicillar arteries and sheathed capillaries in chronic hepatltls (F1g. 1 a, f, g). Such penartenal sheaths are thinner and rarer in liver cirrhosis, and are virtually absent in idIOpathic portal hypertension and in splenic vein thrombosis. In longitudinal sections they appear to extend from the penicillar artery to the whole sheathed capillary. In chronic hepatitis, small granulomas (150 to 250 Ilm in diameter) formed by a core of histIOcytes and a mantle of lymphocytes can be encountered in the red pulp (Fig. 1 b) close to the terminal arterial branches. In idiopathic portal hypertension, channels are sometimes observed to originate from branches of follicular arteries and empty directly into large venous sinuses (Fig. 2) after a slightly sinuous or almost stra1ght course. Their wall remains fairly thick along the entire course (Cavalli et aI., 1972) and appears to be endowed with intensely PAS-positive fibrils with intermingled smooth muscle cells arranged longitudinally or obliquely to the major axis of the channel. Gandi-Gamna nodules are present m liver cirrhosis and occasionally in idiopathic portal hypertension, while are not observed in our cases of chronic hepatitis. In all the pathological spleens, the Malpighian corpuscles frequently show a large clear center rich in blast cells, in which mitoses are rare. In chronic hepatitis the wide clear center often contains clumps of PAS-pos1tive material.
Fig. 1. a) Chromc hepatitis. A termmal artenal branch IS touched by the section plane and appears to be surrounded by a mamly plasma cellular sheath. FIx.: Boum. Stam: PAS-haematoxy1m. 220 x. b) Chromc hepatitis. Small granuloma m the red pulp. Its core mamly consists of histiocytic cells and flbnls; lymphocytes and plasma cells form a thm halo. FIx.: Boum. Stam:
Fig. 2. Idiopathic portal hypertensIOn. a) A small arterial branch (arrows) runs from the white pulp towards a large SinUS of red pulp. Collagen bundles can also be seen, which are Increased around the follicular artery. FIx.: Bouln. Stain' PAS. 100 x. b) Enlargement of a). The arteriolar Inlet Into the sinus appears here touched by the plane sectIOn. c) Histologic sectIOn adJOining that shown In b). Here the arteriolar Inlet Into the SinUS IS contained Within the sectIOn. FIx.: Bouln. Stain: PAS-haematoxylln. 400 x. d) A small arterial branch of the perifollicular zone emptIes Into a SinUS after a short, almost straight tract. fix.: Bouln. Stain: PAS. 400 x. Fig. 1. continued· PAS-haematoxylln. 150 x. c) d) e) IdIOpathIC portal hypertensIOn Sheathed capillaries shOWing a wall thickening due to Increase of the fibrillar network. FIx: Bouln. Stain: PAS-haematoxylin. c) 150 x; d) 200 x; e) 250 x. f) g) Chromc hepatitis. Pemcillar arteries shOWing infiltratIOn of the wall by lymphocytes and plasma cells FIX.: Alcohol-formol-acetlc aCid. Stain: Orcelnhaematoxylln. 400 x.
138 . G. Cavalli, G. Re, Anna Mana Casali, and Paola Monan
In the only case of splenic vein thrombosis a picture of vascular congestion and dilatation prevailed.
Stereo logy The percentage of red pulp per unit volume is higher in all the pathological spleens in comparison with controls (Table 1). The different groups do not present substantial differences in red pulp volume from 80 to about 88%; the only exception is one case of lIver cmhosis (A. D. S.) where it is 74.2%.
Table 1. Per cent volume of red pulp, white pulp and trabeculae m mdlVldual abnormal spleens
Controls 1 2 3
Sex
Age
M M F
25 38 34
F M M F F M
1250 820 730 980 1500 1750 940
20 25 27 38 18 25 15
Splemc Weight (g)
Intra splemc Pressure (cm H 2 O)
White Pulp Red Pulp Trabecular Volume Volume Volume (%) (%) (%)
} pool 22-27
63-66
7-12
11.7 12.0 9.7 8.0 7.6 7.7 8.2
81.0 80.0 84.8 84.1 87.5 87.6 84.3
7.3 8.0 5.5 7.9 4.9 4.7 7.5
ACH M.B. M.R. S. P. V. V. R. B. A.D. M.D.V.
M
32 45 31 38 26 18 35
HC A. C. M.P. G. C. A. D. S.
M F M M
51 39 54 61
590 480 870 630
40 42 48 33
8.7 10.0 8.1 18.4
83.4 82.0 82.3 74.2
7.9 8.0 9.6 7.4
IPH A.M.M. F. T. P. F. M.G. S. B.
F M M F M
26 65 21 31 16
2100 870 1830 920 1270
40 36 30 65 45
8.6 9.5 10.9 7.1 5.1
85.5 85.7 82.6 84.6 80.8
5.9 4.8 6.5 8.3 14.1
SVThr A.A.
F
13
980
32
12.7
79.5
7.8
ACH = Active Chromc Hepatitis; HC = Hepatic CIrrhoSIS; IPH sIOn; SVThr = Splemc Vem ThrombOSIS.
= Idiopathic Portal Hyperten-
MICrovascular Architecture of Spleen m Congestive Splenomegaly . 139
Consequently, the white pulp and trabeculae appear to be lower as a percentage per unit volume, although their effective quantities are not reduced since the pathological spleens exceed three to ten times the controls 10 weight. The white pulp volume varies from about 5% to about 13% in all the pathologIcal spleens but one, in which it IS 18.4% (A. D. S.); the control values are between 22 and 27%. In reckoning the relative volume of connective tissue, the sub-
Table 2. IndlVldual values of hlstometncally determmed quantltles m normal and abnormal spleens Smus Pulp Smus Smus Pulp cord Mean Volume cord VollPulpWldth SectIOnal Length Volume cord Vol Area of smgle Smuses (%)
(%)
(Il)
(Ill)
Smus Smus Surface Surface -statlstlcal-geometnc model- model-
(mlcm 3 )
(cm2/cm 3) (cm 2/cm3 )
Controls 1 41.38 2 39.13 3 36.16
58.62 60.87 63.84
0.70 0.64 0.57
22.77 20.77 23.80
643.87 623.53 508.75
642.67 627.55 710.76
514.89 586.13 537.46
578.08 555.49 568.30
ACH M.B. M.R. S. P. V. V. R. B. A.D. M.D.V.
32.69 39.50 40.69 41.50 36.72 41.61 32.11
67.31 60.50 59.31 58.50 63.28 58.39 67.89
0.49 0.65 0.69 0.71 0.58 0.71 047
23.41 18.04 17.59 16.44 27.38 14.84 2754
339.26 378.19 418.05 415.00 686.21 490.17 472.21
963.56 1044.44 973.32 1001.00 535.11 848.88 679.98
575.05 670.73 674.36 711.67 462.23 786.92 493.02
629.14 720.01 705.46 722.15 496.90 666.23 523.81
HC A. C. M.P. G. C. A. D. S.
39.56 38.38 40.27 37.36
60.44 61.62 59.73 62.64
0.65 0.62 0.67 0.60
13.71 13.78 14.81 13 22
327.97 288.81 430.64 370.61
1206.21 1328.20 935.12 992.01
881.69 894.33 806.61 947.65
774.36 800.58 686.90 682.44
IPH A. M. M. 56.19 F. T. 57.31 P. F. 48.94 M.G. 46.13 S. B. 47.69
43.81 42.69 51.06 53.87 52.31
1.28 1.34 0.96 0.86 0.91
9.26 9.09 12.37 12.73 12.71
423.40 457.59 476.28 415.84 505.67
1327.11 1252.43 1027.55 1109.21 943.10
946.22 939.27 825.55 846.35 823.13
968.02 949.72 794.94 801.82 751.79
Svrhr A.A.
43.75
1.28
15.92
1228.76
457.77
549.62
442.23
56.25
ACH = Acnve Chromc Hepatltls; HC = Hepatic Cmhosls; IPH sIOn; Svrhr = Splemc Vem ThrombOSIS
= Idiopathic Portal Hyperten-
140 . G. CavallI, G. Re, Anna Mana CasalI, and Paola Monan
capsular zones have been excluded, where thin connective bands run into the red pulp from the capsule. So the trabecular volume varies in normal spleens from 7 to 12%, and in pathological spleens from a little less than 5% to 9.6% reaching 14.1 % 10 only one case of idiopathIc portal hypertension (S. B.). All the other values determined 10 every spleen are shown in detail in Table 2. Table 3 illustrates the arithmetical mean and standard deviation of all the quantities investigated for every group of pathological spleens. The results obtained in the various groups have been statistically analyzed by the Student's t-test for the sigmficance of the observed differences from the control group (Table 3).
Table 3. Arithmetical mean and standard deViatIOn of the hlstometnc quantities determmed m the smgle groups of spleens C M
SD
ACH M SD
HC M
IPH SD
M
SVThr SD
Smus Volume (%)
38.89
2.62
37.83 (n. s.)
4.06
38.89 (n. s.)
1.29
51.25 5.13 (p < 0.001)
56.25
Pulp cord Volume (%)
61.11
2.62
62.17 (n. s.)
4.06
61.11 (n. s.)
1.29
48.75 5.13 (p < 0.01)
43.75
Smus Vol/Pulp cord Vol
0.64
0.07
0.61
0.10
0.64
0.03
0.22
1.29
1.07
2075 5.29 13.88 0.67 11.23 1.88 15.92 (n. s.) (p < 0.001) (p < 0.001) Mean SectIOnal Area of 592.05 72.85 457.01113.45 354.51 60.76 455.76 37.271228.76 smgle Smuses (Ill) (n. s.) (p < 0.01) (p < 0.01) Pulp cord Width (~)
22.45
1.54
Smus Length (m/em 3)
660.33 44.33 867.76189.491115.39183.721131.88157.86 356.05 (p < 0.01) (p < 0.05) (p < 0.01)
Sinus Surface-S' statistical model
546.16 36.41 624.85118.79 882.57 58.15 876.10 61.55 549.62
(cm 2/em 3 )
(n. s.) Smus Surface-S" geometric model
(p < 0.001)
(p < 0.001)
567.29 11.33 637.67 93.26 736.07 60.34 853.26 98.51 442.23
(cm 2/cm 3 )
(n. s.)
(p
< 0.01)
(p
< 0.01)
C = Control; ACH = Active Chromc Hepatitis; HC = HepatiC CirrhOSIS; IPH = Idiopathic Portal HypertensIOn; SVThr = Splemc Vem ThrombOSIS; M = Mean; SD = Standard DeViatIOn. Under every mean IS mdlcated between brackets the slgmflcance of the difference from the control values; n. s. = not slgmflcant
Microvascular Architecture of Spleen
In
Congestlve Splenomegaly . 141
The per cent volume of splenIc smuses m chronic hepatitis (37.83%) and in liver cirrhosis (38.89%) appears to be virtually the same as in controls (38.89%). Similarly, no significant differences are shown for the per cent volume of splenic cords in comparison with controls. In idiopathic portal hypertension, splenic sinuses are 51.25% and splenic cords 48.75% in volume, with reversal of their volume ratio. The mean sectional area of splenic sinuses appears to be lesser in chronic hepatitis (457.76 Ilm 2), in liver cirrhosis (354.51 Ilm2 ) , and in idiopathic portal hypertension (455.76 Ilml), when compared with controls (592.05 Ilm2 ). On the other hand, the smus length per UnIt volume in chronic hepatitis (867.76 mlcm 3 ) and especially m lIver CIrrhOSIS (1115.39 mlcm3 ) and in idiopathic portal hypertension (1131.88 mlcm3 ) is higher than in controls (660.33 mlcm3). Such results point out the diverse increase in number of the small venous sinuses per unit volume in the different pathological situations. In controls the mean width of splenIC cords IS 22.45 Ilm. It is smaller in chronic hepatitis (20.75 Ilm), but especially in lIver CIrrhosis (13.88 Ilm) and in idiopathic portal hypertensIOn (11.23 Ilm). The sinus surface per unit volume has been determined both directly from histometric data ("statistical model") (S') and indirectly by a "geometric model" (S"). In the latter the sinuses are represented as cylinders, the sections of which have regular profiles. In controls the values obtained by the two different methods are coincident, so indirectly supporting a structural model in which sinuses have circular outlines. The mean values of sinus surface per unit volume are fairly similar to normal in chronic hepatitis, while they are higher than normal in liver cirrhosis and in idiopathic portal hypertension. However, the results obtained by the two different methods are coincident in idiopathic portal hypertension only, whereas in liver cirrhosis the values, although both higher than normal, are discordant. No statistical analysis could be performed for the only case of splenic vein thrombosis.
Discussion Microscopic observation illustrated some morphological features which prevail in the spleens in chronic hepatitis, such as sheaths of lymphocytes and plasma cells surrounding the terminal arterial branches and the occurrence of small non-specific granulomas. These findings might suggest active chronic inflammation. In idiopathic portal hypertension thickening of the wall is frequently observed in the terminal arterial branches. Moreover, shunts between small
142 . G. Cavalli, G. Re, Anna Mana Casali, and Paola Monan
arterial branches and venous sinuses were encountered only in these cases; they are fairly infrequent and may result from a marked rearrangement of preexistent formatlOns rather than from newly formed structures. Such shunts m the splenic circulation might convey into the sinuses a blood volume greater than that permitted by the usual vascular structures. However, it is difficult to define what a role they play in portal hypertenslOn. The series of histometric values obtamed in control spleens served for comparison with the data determined m the different groups of pathological spleens. Among normal spleens a certain variability occurs, probably due to the continuous changes the spleen undergoes as part of its role in Immunity and its position in the portal circulation. In chronic active hepatitIS, the mean value of sinus length is slightly higher than that of controls, while the mean sectional area of sinuses is slightly lower. The mean per cent volumes of sinuses and cords and the mean cord width are similar to control values, but theIr standard deviation is very much greater than that of controls, indicating a great scatter in the values. Such differences, although they are not significant from a statistical point of view, may suggest, when related to the clmlcal situation, an intermediate stage in the possible evolution to liver cirrhosis. The spleens m liver cirrhosis show a per cent volume of sinuses consistent with normal, but the sinus sectional area is smaller and the smus length per unit volume IS higher than in controls, as in chronic active hepatitis. In the case of liver cirrhOSIS however the observed differences are statistically signiftcant. Although the per cent volume of cords is the same as normal, the cord mean width shows a highly significant decrease. In idiopathIc portal hypertenslOn all the histometric quantities differ significantly from controls. The per cent volume of venous sinuses is higher, the mean sectional area of sinuses IS reduced, while the mean sinus length is increased. The per cent volume of splenic cords is dimimshed, as is the cord mean width. In particular, the volume ratlO of sinuses to cords IS higher than 1 (1.07), whereas in controls and in all the other groups of spleens considered it is always less than 1. In our only case of splenic vein thrombosis there is a hIgher percentage of red pulp per umt volume and the volume occupied by sinuses is greater than that occupied by cords. Consequently, the volume ratlO of sinuses to cords is also higher than 1 m this case (1.29). The mean sectional area of sinuses IS very much hIgher than normal, while the total length of sinuses is less. Therefore, the prevailing features are dtlatation and congestion of sinuses, in both the morphological and the histometnc study. Our findmgs differ from those shown by Stutte and Heusermann (1972) In cases of splenic vein thrombosis with associated haemocytopenia. It IS possIble that thIS depends upon the time
Microvascular Architecture of Spleen
In
Congestive Splenomegaly· 143
the splenectomy was made m our case (7 months after the acute incident); at this time signs of spleen congestion may prevail and a suitable collateral circulation may not yet be formed completely. Moreover, this case did not present signs of hyperspleOlsm. On the basis of the companson of the histometnc data obtained from normal spleens with those obtained from spleens of the d1fferent pathological groups, we tried to develop a morphological model of the congestive splenomegalies studied. For this purpose it must be postulated that sinuses are tubular structures with d1stensible walls wh1ch under hydrostatic pressure tend to assume regularly circular outlmes. Moreover, the correct evaluation of the external sinus surface area must be cons1dered to be that obtained by applying statistical stereo logic methods based on theorems of solid geometry and probability theory. A geometric model of spleen may be suggested in which the sinuses are variously oriented cylindrical formations, having a variable length and convoluted course. In this case every section of the cylindrical formations produces regular profiles (circular or elliptical). Therefore, the external surface area of sinuses can be correctly calculated by applying geometric formulas. The estimate so obtained from the volume and the mean cross-sectional surface is indicated as S" (= extrapolated evaluation of the external surface area of sinuses, according to the geometric model). Here S" is, by defiOltion, coincident with the stereometncal evaluation of the same surface mdicated as S'. However, the normal spleen has a more complex structure because of the presence of connections between the smuses. The reticular meshwork of splenic cords in normal red pulp is a plastic structure and, in some places, the processes of reticular cells can adopt a tubular conformation makmg possible the communicat1on between sinuses (Weiss, 1977). Such communications in normal spleens were also recently demonstrated by scanning electron microscopy (Yamamoto, 1979). Nevertheless, these connections between sinusesespecially for their relative Slze - ought not to mfluence the geometnc representation of the above model, since on stereometnc analysis the S' and S" values are almost coinc1dent m the control spleens. The suggested model can therefore be applied to the normal spleen. When the number and/or the size of connectlOns between sinuses is markedly higher than 10 the above model, a cons1derable number of abnormal profiles will appear in the secttons. Such irregularities of tubular profiles will introduce a systematic error m the evaluation of the external sinusal surface area by geometric extrapolation. Thus S" 1S different, m this case, from S'. S" will appear considerably less than S', smce It IS calculated from a model in which the external surface area is the lowest, the volume and the mean cross section being equal. Therefore, the only sltuatlOn that justify geometrically a
144 . G. CavallI, G. Re, Anna Mana CasalI, and Paola Monan
disagreement between S' and S" is a plentiful interconnection of the tubular structures. In the spleens of liver CIrrhosis both S' and S" differ significantly (p < 0.001 and p < 0.01, respectively) from the values of controls, but they also differ considerably from each other, being S" less than S'. Therefore, in these spleens a great number of irregular profiles may be suggested, produced by numerous and wide connections between sinuses. The occurrence of many interconnecting sinuses is a feature of the progressive morphologic rearrangement induced by the portal hypertensIOn. Moreover, in these spleens the sinuses are narrower and very much longer than those of controls. Such a "lengthening" of the sinusoidal system is probably also due to newly formed slender anastomoses between sinuses, lO addition to stretching of the preexistent sinuses (see Stutte and Heusermann, 1972). Therefore, in the spleens of cases of liver cirrhosis the sinuses present more numerous lOterconnections than in normal spleen, in contrast with what was stated by Yamamoto (1978). The red pulp is involved as a whole in the morphologic rearrangement, since the relative per cent volume of SlOuses and cords, as well as theIr ratio, are the same as lO control spleens. In chronic idiopathic hypertensIOn the spleen shows significant differences from controls for all the quantities analyzed stereometrically. The relative per cent volume of sinuses and cords and their ratio are also different from normal. Both the estimates S' and S" differ significantly from controls, but they are almost coincident With each other. Therefore, in these spleens the number of connections between sinuses is not higher than in the model of normal spleen, in contrast with the modifications observed in the spleens of liver cirrhosis and in the case of splenic vein thrombosis. Little communication between the venous SlOuses running parallel with each other has been found on the scanning electron microscope by Yamamoto (1979). The microvascular architecture of red pulp thus presents, in idIOpathic portal hypertensIOn, distinctive features that facilitate the blood flow from the arterious to venous passages. On the basis of morphologic and especially histometric findings, the congested spleens in idiopathic portal hypertension can be separated from the other types of congestive splenomegaly we have studied. In fact these spleens, when compared with controls, exhibit a set of modifications that is unique to this group among other forms of congestive splenomegaly.
MICrovascular Architecture of Spleen m Congestive Splenomegaly· 145
References Armas-Cruz, R., Yazlgl, R, Lopez, 0, Montero, E., Cabell a, ]., and Lobo, G.: Portal CirrhOSIS: analYSIS of 208 cases, With correlatIOn of clinical, laboratory and autopsy fmdmgs. Gastroenterology. 17, 327-343 (1951) Cacclan, c., PlsI, E., and Cavalli, G. Splenoportografla e splenomanometna. RlVlsta MedICa Bologna 1957 Cavalli, G., Casali, A. M., and Bianchi, F. Boo Aspettl dl mlcroanglOarchltettura della mliza m corso dl spienomegalia congestlZla. BlOCh. Exp. BIOI. 1, 137-146 (1972) Cavalli, G., Monan, P., Casali, A. M., LambertlnI, F., Fedeli, F., and DelflnI, E.: ApprocclO stereometnco allo studIO delle spienomegalie congestlZle. Bull. SCI. Med. 4, 293-319 (1978) Durr, R.: Bantlmliz und hepatolienale Fibrose. Beltr. Path. Anat. 72,418-455 (1924) Elias, H., Hennig, A., and Schwartz, 0 E.: Stereology: ApplicatIOns to bIOmedICal research. PhyslOl. Rev. 51, 158-200 (1971) GreppI, E.: II tumor dl mllza contrattlle emorraglparo, come probablle forma dl splenoanglOpatia pnmltlva. Mmerva Med. 8, 644-655 (1928) Hunt, A. H.: An mvestigatlOn of the pressures and speeds m portal Circulation. IV Congr. europ. Gastroenterol., Pans 1954; 1, P 27; 2, p. 79. Masson, Pans 1955 Iber, F. L.: Portal hypertensIOn m the presence of normal liver morphology. Ann. N.Y. Acad. SCI.
170, 115-126 (1970) Klopstock, F.: Uber Mliztumor, Icterus und ascites bel Leberclrrhose. Vlrchows Arch. path. Anat.
187,111-124 (1907) Leger, L., Lemalgre, G., Rlcharme, ]., and ChapUls, Y.: L'hypertenslOn portale essentlelle. A propos de dlx observatIOns personelles. Pre sse Med. 74,313-318 (1966) LiebOWitz, H. R.: Splenomegaly and hypersplenism pre- and postportacaval shunt. New York]. Med. 63,2631-2638 (1963) Patrassl, G., Dal Palu', c., Ruol, A La pletora portale. LXIII Congr. Soc. Ital. Med. Int., Tormo 1961; POZZI, Roma 1961 Rousselot, L. M.: Role of congestIOn (Portal HypertensIOn) m so called Bantl's syndrome; clinical and pathologiC study of 31 cases With late results followmg splenectomy. ].A.M.A. 107,
1788-1793 (1936) Rousselot, L. M.: Late phase of congestive splenomegaly (Bantl's syndrome) with hematemesls but Without CirrhOSIS of liver; further observatIOns on etIOlogy of Bantl's syndrome and effect on prognOSIS of certam vanatlOns m portal venous pattern. Surg. 8 , 34-42 (1940) Sekl, K.: Hlstometncal studies of the spleen m Bantl's syndrome With reference to clinicopathologic correlatIOns. Tohoku J. Exp. Med. 87,222-243 (1965) Snell, A. M.: Clinical aspects of portal CIrrhOSIS. Ann. Int. Med. 5, 338-357 (1931) SOtglU, G.: RevlSlone ed mquadramento nosologlco dell'lpertenslOne portale. Mm. Med. 68,
3751-3760 (1977) Stutte, H. ]., Heusermann, U. Splenomegaly and red blood cell destructIOn: a morphometric study on the human spleen. Vlrchows Arch. Abt. B Zellpath. 12, 1-21 (1972) Tumen, H. J.: Hypersplenism and portal hypertensIOn. Ann. N.Y. Acad. SCI. 170, 332-344
(1970) Weibel, E. R., Kistler, G. S., and Scherle, W. F.' Practical stereologlcal methods for morphometnc cytology. ]. Cell BIOI. 30, 23-38 (1966) WeiSS, L.: The spleen. In: L. Weiss and R. O. Greep, Histology, p. 545. McGraw-HIli, New York
1977
10 Path Res Pract Vol 174
146 . G. Cavalli, G Re, Anna Mana Casali, and Paola Monan Yamamoto, K.. Morphological studies of the spleen In splenomegalic hver cirrhoSIS comparing with the spleen In Idiopathic portal hypertensIOn (so-called Bantl's Syndrome without liver cirrhosIs). Acta Path. Jap. 28, 891-905 (1978) Yamamoto, K.· Morphological studies of the spleen In Idiopathic portal hypertensIOn (so-called Bantl's Syndrome without liver cirrhosIs) uSing light microscopy, scanmng electron microscopy and hlstometry Acta Path Jap 29, 1-19 (1979)
Received November 3, 1981
Accepted February 20, 1982
Key words: Spleen - Splenomegaly - Portal Hypertension - Stereo logy Prof. Glancarlo Cavalli, vlale XII GlUgno, 13, 140124, Bologna, Italy
* * Istltuto di IstologIa ed Embnologla generale * ** I Cattedra dl Statlstlca, UmvefSlty of Bologna, Bologna, Italy
The Microvascular Architecture of Spleen in Congestive Splenomegaly A Morphological-Histometric Study G. CAVALLI*, G. RE*, ANNA MARIA CASALI ** and PAOLA MONARI***
Summary Spleens obtained from patients suffering from chromc active hepatitis, hepatic cirrhosIs, Idiopathic portal hypertension and splemc vein thrombOSIS have been subjected to morphological observation associated with histometncal analysIs and compared to 3 control spleens surgically removed for trauma. All the pathological spleens show the picture of congestive splenomegaly. Moreover, In chromc active hepatitis small non-specific granulomas are observed In the red pulp, along with lymphocytic-plasma cellular sheaths surrounding the terminal artenal branches. The latter are also encountered, less frequently, In bver cirrhosIs. In Idiopathic portal hypertension the presence of artenolar-slnusal shunts can be emphasized. The stereology of the same spleens shows In chromc active hepatitis, and more markedly In liver cirrhosIs, higher values of SinUS length and a lower mean sectional area of the sinuses In companson with controls. In IdIOpathIC portal hypertensIOn the slnusallength IS also greater and the mean slnusal sectIOnal area IS lower. However, companson of the statistical with the geometnc determination of the external surface area of sinuses allows us to hypotheSize that there IS a nch interconnection between sinuses In bver cmhosls, but not In idIOpathiC portal hypertension. In the latter spleens the statistical and geometrical determinatIOns show values qUite Similar to each other, as well as in control spleens, In spite of the Important structural rearrangement presented here. In splemc vein thrombOSIS the hlstometnc findings are consistent with SinUS dilatation. The microvascular architecture of red pulp, especially when studied with hlstometnc methods, shows charactenstic features In IdIOpathiC portal hypertension which permit us to separate this form of congestive splenomegaly from all the others examined.
132 . G. CavallI, G. Re, Anna Mana CasalI, and Paola Monan
Introduction Splenic enlargement is a frequent finding in the course of portal hypertension due either to intra-hepatic causes or to main portal vein obstruction. Splenomegaly of some degree is observed in almost 50% of cases of liver cirrhosis (Snell, 1931; Armas-Cruz et aI., 1951) and its incidence is still higher at necropsy examlOation (Klopstock, 1907; Armas-Cruz et aI., 1951), although a syndrome of severe portal hypertension expressed by bleeding from oesophageal varices can occur without spleen enlargement (Liebowitz, 1963). However, splenomegaly does not show the same incidence in the different types of cirrhosis, being much less frequent in alcoholic cirrhosis than in the other forms. The splemc enlargement in the course of liver disease may be regarded as a direct consequence of "congestion" caused by long-standing portal hypertension, but it is also currently explained as resulting from a generic reaction to toxic or infectIOUS agents, from an immune response to the hepatocyte damage, or from ill-defined liver damage (Tumen, 1970). Besides those cases determined by intra- or extra-hepatic block, infrequent forms of portal hypertension with splenomegaly are described, in which no morphological changes capable of obstructing the blood flow can be demonstrated in the liver or in the splenoportal trunk (Rousselot, 1936, 1940; Hunt, 1955; Cacciari et aI., 1957; Leger et aI., 1966; Sotgiu, 1977). These forms show a geographical distribution, with a higher incidence in tropical or subtropical zones. They are clinically characterized by conspicuous enlargement of the spleen, oesophageal and gastric varices with tendency to haemorrhages. Such cases of portal hypertension have been variously named (idiopathic portal hypertension, essential portal hypertension, primary portal hypertension, portal hypertensIOn without obstacle) and have been related to excessive blood flow through the spleen. However, it must be pointed out that an increased blood flow proportional to the increased splenic volume (Patrassi et aI., 1961) is not found exclusively in idiopathic portal hypertension, but is also seen in splenomegalic liver cirrhosis. The conception of impaired hepatic distensibility in face of portal hyperflow as a factor of hypertension has been improved by Sotgiu (1977), who introduced the concept of a defective compliance of the liver and portal vascular bed to explain the presence of portal hypertension in cases with no detectable obstacle. The decreased compliance might represent the relative obstacle that initiates the syndrome of portal hypertension due to increased flow and might then result in a fibrotic rearrangement of the vascular structures. Alternatively, the decreased compliance itself might depend on the vascular structure alterations induced by the increase in flow. The classification of idiopathic portal hypertension (IPH) as a distinct
Microvascular Architecture of Spleen
In
Congestive Splenomegaly . 133
nosographic form of congestive splenomegaly remains a matter for debate. In fact the existence of a primary condition of splenic "hyperflow" is not admitted by all authors, since an increased blood flow can also occur in splenomegalIc lIver cirrhosis (Patrassi et al., 1961) and the various primary syndromes reported in different geographIc zones might have a different aetiopathogenesis (Iber, 1970). The study of the vascular formations may contribute elements useful to a better understanding of the congesttve splenomegalIes and to their nosography. In this belief we have exammed the splemc mIcrovascular architecture in some different types of splenomegalies. To thIs purpose histological observation has been associated with a mathemattcal-geometncal study of the structures under investigation, since the histometric analysis has already proved to be profitable also when apphed to the spleen (Sekl, 1965; Stutte and Heusermann, 1972; Cavalli et al., 1978; Yamamoto, 1978, 1979).
Material The study was carned out on 20 spleens that had been removed surgICally and fixed Immediately, with the exceptIOn ot one that was removed at necropsy. Three spleens from persons operated for accidental trauma served as controls. The other spleens were obtained from patients showmg marked splenomegaly and have been selected for this study because the clinIco-pathologIcal picture allowed the patient to be ascrIbed With certamty to one of the followmg groups of cases. Chrome actIVe hepatltts All these patients (4 men and 3 women) presented the serum fmdmgs and the hepatIC histopathological aspects consistent With the diagnOSIs of moderately aggressive chronic hepatitis (both m needle and m surgically pertormed lIver bIOpSies). The search for Auantigen m the serum was negative tor all of them. Moreover, every patient had a conspicuously enlarged spleen With associated signs of hypersplenism, such as anaemia and especially leukopenia and thrombocytopenia. The mtrasplenIc pressure (at splenomanometry) was normal or slightly mcreased, but the presence of collateral portacaval circulatIOn was not eVidenced by splenoportography or dunng the venous phase of selective coelIac angIOgraphy m any of these cases. Splenectomy was done because ot the disturbance due to the greatly enlarged spleen and the syndrome of hypersplenism; It was followed m all cases by Improvement of the blood cell counts. Follow-up after the operatIOn mdlcated that splenectomy did not affect the clInical course of the chrOniC hepatitis. HepatIC CIrrhOSIS The dlagnosl~ of non-alcoholic lIver CirrhOSIS was made m all these cases (3 men and 1 woman) on the baSIS of the clInical history, the laboratory data and the histopathology of the lIver (both m percutaneous and surgical bIOpSies). All the patients presented a very enlarged spleen, signs of hypersplenism and the syndrome of portal hypertensIOn from mtra-hepatlc obstructIOn, eVidenced also by splenomanometry, splenoportography, and/or selective coelIac angiography. The splenectomy wa, performed at the time of porta-caval shunt operation for repeatmg gastro-mtestmal haemorrhages
134 . G. Cavalli, G. Re, Anna Mana Casali, and Paola Monan
Id,opath,c portal hypertensIOn. This group consists of 5 patients (3 men and 2 women). 4 of them had eVidence of marked splenomegaly, hypersplenism and severe portal hypertension with gastro-mtestinal haemorrhages The laboratory data and the liver histology were normal. The study of portal circulation demonstrated, m addItIon to mcreased values of splenomanometry, the patency of the splenoportal trunk mJected by splenoportography, the mcreased translational speed of the medIUm from spleen to liver, the absence of hepatofugal circulation. After splenectomy, three patients showed a clear clinical Improvement, disappearance of gastro-mtestmal haemorrhages and normal blood cell counts III follow-up made for over 15 years. The fourth patient died of other causes shortly after the mterventlon. The fIfth case of thiS group concerns a man 65 years old who 30 years before had the diagnosIs made of "pnmary congestive splenomegaly" (according to Greppl, 1928) and who died from prostatic carcmoma; thiS case also could be ascnbed to primary portal hypertensIOn on the basIs of the exammations performed. The spleen of thiS patient IS the only one of our study obtamed at necropsy. The histology of the liver showed only a slight portal fIbroSIS Splentc vein thrombosIs. Fmally we have considered the case of a girl 13 years old. The onset of her clinical history dated back to only 7 months before, when she presented high fever, eplgastnc pam, haematemesls, melaena and transitory aSCites, followed by splenomegaly and hypochromic anaemia. At the splenoportographlc exammatlon the splenoportal trunk did not appear mJected; the bulk of radIO-opaque dye was stopped at the hilus of spleen and diverted through the gastnc and oesophageal vems At splenectomy, the presence of spleniC vem thrombosIs at the hilus was confirmed.
Methods For histological exammatlOn, tissue blocks obtamed from different zones of every spleen (the poles and deep central regions) were fixed With Boum's flUid and embedded m paraffm. 4 spleens (2 normal and 2 of IdIOpathic portal hypertensIOn) were perfUSIOn-fixed. They were artenally perfused With Rmger solution contammg heparm at 37°C before fixatIOn, takmg care that the mJectlon was made m an mtermIttent way (about 50 pulses/mm) and With vanatlon of pressure from 110 mm Hg to 70 mm Hg. After washmg, the perfusIOn solutIOn was replaced With Boum's flUid. The subsequent procedure was the same as for all other spleens. Senes of 5 Ilm thick sectIOns were stamed With haematoxylin and eosm, Azan, PAS reactIOn, orcem, Achucarro after deammatlon. The hlstometnc mvestlgatlOns were carned out on the same microSCOPIC preparatIOns. For measurements, nets of pomts have been used to record the number of pomts falling on red pulp, white pulp and trabeculae respectively and three groups of elementary data: the number of test pomts fallmg on smuses; the number of mtersectlons of the test hnes With the traces of smus walls; the number of smus profties per test net. From these, by applymg current formulas for stereology (Weibel et aI., 1966; Elias et aI., 1971), the followmg quantities have been determmed m every spleen: percentage of red pulp, of white pulp and of trabeculae per Unit volume; per cent volume of smuses and of cords m the red pulp, volume ratio of smuses to cords m the red pulp; mean Width of the spleniC cords; sectional surface area of smuses; length of sinuses per Unit volume; smusOldal surface area per Unit volume. The groups of values were then subjected to statistical analYSIS.
MIcrovascular Architecture of Spleen III Congestive Splenomegaly . 135
Results Morphological observations The common feature of all the pathological spleens examined 1S the general microscopic picture of fibrocongestive splenomegaly. No perisplenitis is observed. A more or less marked hyalinosis of follicular arteries and fibrosis of Malpighian corpuscles are more frequent in the cases of liver cirrhos1s than in idiopathic portal hypertension, where they are present, although moderately, in only two cases (F. T. and S. B.). A slight perivascular fibrosis is occasionally observed in chronic hepatitis. The periarterial fibrosis, already described by Diirr (1924) in so called Banti's spleens, can also be detected in idiopathic portal hypertension. At scanning electron microscope observation (Yamamoto, 1979) th1s proved to be composed of bundles of channels formed by reticulum cells. In idiopathic portal hypertension the Schweigger-Se1del sheaths may give the appearance of hyalinosis of the wall bes1des the increase of the surrounding fibrillar network (Fig. 1 c, d, e). Periarterial sheaths of lymphocytes, and especially plasma cells, surround the terminal arterial branches such as penicillar arteries and sheathed capillaries in chronic hepatltls (F1g. 1 a, f, g). Such penartenal sheaths are thinner and rarer in liver cirrhosis, and are virtually absent in idIOpathic portal hypertension and in splenic vein thrombosis. In longitudinal sections they appear to extend from the penicillar artery to the whole sheathed capillary. In chronic hepatitis, small granulomas (150 to 250 Ilm in diameter) formed by a core of histIOcytes and a mantle of lymphocytes can be encountered in the red pulp (Fig. 1 b) close to the terminal arterial branches. In idiopathic portal hypertension, channels are sometimes observed to originate from branches of follicular arteries and empty directly into large venous sinuses (Fig. 2) after a slightly sinuous or almost stra1ght course. Their wall remains fairly thick along the entire course (Cavalli et aI., 1972) and appears to be endowed with intensely PAS-positive fibrils with intermingled smooth muscle cells arranged longitudinally or obliquely to the major axis of the channel. Gandi-Gamna nodules are present m liver cirrhosis and occasionally in idiopathic portal hypertension, while are not observed in our cases of chronic hepatitis. In all the pathological spleens, the Malpighian corpuscles frequently show a large clear center rich in blast cells, in which mitoses are rare. In chronic hepatitis the wide clear center often contains clumps of PAS-pos1tive material.
Fig. 1. a) Chromc hepatitis. A termmal artenal branch IS touched by the section plane and appears to be surrounded by a mamly plasma cellular sheath. FIx.: Boum. Stam: PAS-haematoxy1m. 220 x. b) Chromc hepatitis. Small granuloma m the red pulp. Its core mamly consists of histiocytic cells and flbnls; lymphocytes and plasma cells form a thm halo. FIx.: Boum. Stam:
Fig. 2. Idiopathic portal hypertensIOn. a) A small arterial branch (arrows) runs from the white pulp towards a large SinUS of red pulp. Collagen bundles can also be seen, which are Increased around the follicular artery. FIx.: Bouln. Stain' PAS. 100 x. b) Enlargement of a). The arteriolar Inlet Into the sinus appears here touched by the plane sectIOn. c) Histologic sectIOn adJOining that shown In b). Here the arteriolar Inlet Into the SinUS IS contained Within the sectIOn. FIx.: Bouln. Stain: PAS-haematoxylln. 400 x. d) A small arterial branch of the perifollicular zone emptIes Into a SinUS after a short, almost straight tract. fix.: Bouln. Stain: PAS. 400 x. Fig. 1. continued· PAS-haematoxylln. 150 x. c) d) e) IdIOpathIC portal hypertensIOn Sheathed capillaries shOWing a wall thickening due to Increase of the fibrillar network. FIx: Bouln. Stain: PAS-haematoxylin. c) 150 x; d) 200 x; e) 250 x. f) g) Chromc hepatitis. Pemcillar arteries shOWing infiltratIOn of the wall by lymphocytes and plasma cells FIX.: Alcohol-formol-acetlc aCid. Stain: Orcelnhaematoxylln. 400 x.
138 . G. Cavalli, G. Re, Anna Mana Casali, and Paola Monan
In the only case of splenic vein thrombosis a picture of vascular congestion and dilatation prevailed.
Stereo logy The percentage of red pulp per unit volume is higher in all the pathological spleens in comparison with controls (Table 1). The different groups do not present substantial differences in red pulp volume from 80 to about 88%; the only exception is one case of lIver cmhosis (A. D. S.) where it is 74.2%.
Table 1. Per cent volume of red pulp, white pulp and trabeculae m mdlVldual abnormal spleens
Controls 1 2 3
Sex
Age
M M F
25 38 34
F M M F F M
1250 820 730 980 1500 1750 940
20 25 27 38 18 25 15
Splemc Weight (g)
Intra splemc Pressure (cm H 2 O)
White Pulp Red Pulp Trabecular Volume Volume Volume (%) (%) (%)
} pool 22-27
63-66
7-12
11.7 12.0 9.7 8.0 7.6 7.7 8.2
81.0 80.0 84.8 84.1 87.5 87.6 84.3
7.3 8.0 5.5 7.9 4.9 4.7 7.5
ACH M.B. M.R. S. P. V. V. R. B. A.D. M.D.V.
M
32 45 31 38 26 18 35
HC A. C. M.P. G. C. A. D. S.
M F M M
51 39 54 61
590 480 870 630
40 42 48 33
8.7 10.0 8.1 18.4
83.4 82.0 82.3 74.2
7.9 8.0 9.6 7.4
IPH A.M.M. F. T. P. F. M.G. S. B.
F M M F M
26 65 21 31 16
2100 870 1830 920 1270
40 36 30 65 45
8.6 9.5 10.9 7.1 5.1
85.5 85.7 82.6 84.6 80.8
5.9 4.8 6.5 8.3 14.1
SVThr A.A.
F
13
980
32
12.7
79.5
7.8
ACH = Active Chromc Hepatitis; HC = Hepatic CIrrhoSIS; IPH sIOn; SVThr = Splemc Vem ThrombOSIS.
= Idiopathic Portal Hyperten-
MICrovascular Architecture of Spleen m Congestive Splenomegaly . 139
Consequently, the white pulp and trabeculae appear to be lower as a percentage per unit volume, although their effective quantities are not reduced since the pathological spleens exceed three to ten times the controls 10 weight. The white pulp volume varies from about 5% to about 13% in all the pathologIcal spleens but one, in which it IS 18.4% (A. D. S.); the control values are between 22 and 27%. In reckoning the relative volume of connective tissue, the sub-
Table 2. IndlVldual values of hlstometncally determmed quantltles m normal and abnormal spleens Smus Pulp Smus Smus Pulp cord Mean Volume cord VollPulpWldth SectIOnal Length Volume cord Vol Area of smgle Smuses (%)
(%)
(Il)
(Ill)
Smus Smus Surface Surface -statlstlcal-geometnc model- model-
(mlcm 3 )
(cm2/cm 3) (cm 2/cm3 )
Controls 1 41.38 2 39.13 3 36.16
58.62 60.87 63.84
0.70 0.64 0.57
22.77 20.77 23.80
643.87 623.53 508.75
642.67 627.55 710.76
514.89 586.13 537.46
578.08 555.49 568.30
ACH M.B. M.R. S. P. V. V. R. B. A.D. M.D.V.
32.69 39.50 40.69 41.50 36.72 41.61 32.11
67.31 60.50 59.31 58.50 63.28 58.39 67.89
0.49 0.65 0.69 0.71 0.58 0.71 047
23.41 18.04 17.59 16.44 27.38 14.84 2754
339.26 378.19 418.05 415.00 686.21 490.17 472.21
963.56 1044.44 973.32 1001.00 535.11 848.88 679.98
575.05 670.73 674.36 711.67 462.23 786.92 493.02
629.14 720.01 705.46 722.15 496.90 666.23 523.81
HC A. C. M.P. G. C. A. D. S.
39.56 38.38 40.27 37.36
60.44 61.62 59.73 62.64
0.65 0.62 0.67 0.60
13.71 13.78 14.81 13 22
327.97 288.81 430.64 370.61
1206.21 1328.20 935.12 992.01
881.69 894.33 806.61 947.65
774.36 800.58 686.90 682.44
IPH A. M. M. 56.19 F. T. 57.31 P. F. 48.94 M.G. 46.13 S. B. 47.69
43.81 42.69 51.06 53.87 52.31
1.28 1.34 0.96 0.86 0.91
9.26 9.09 12.37 12.73 12.71
423.40 457.59 476.28 415.84 505.67
1327.11 1252.43 1027.55 1109.21 943.10
946.22 939.27 825.55 846.35 823.13
968.02 949.72 794.94 801.82 751.79
Svrhr A.A.
43.75
1.28
15.92
1228.76
457.77
549.62
442.23
56.25
ACH = Acnve Chromc Hepatltls; HC = Hepatic Cmhosls; IPH sIOn; Svrhr = Splemc Vem ThrombOSIS
= Idiopathic Portal Hyperten-
140 . G. CavallI, G. Re, Anna Mana CasalI, and Paola Monan
capsular zones have been excluded, where thin connective bands run into the red pulp from the capsule. So the trabecular volume varies in normal spleens from 7 to 12%, and in pathological spleens from a little less than 5% to 9.6% reaching 14.1 % 10 only one case of idiopathIc portal hypertension (S. B.). All the other values determined 10 every spleen are shown in detail in Table 2. Table 3 illustrates the arithmetical mean and standard deviation of all the quantities investigated for every group of pathological spleens. The results obtained in the various groups have been statistically analyzed by the Student's t-test for the sigmficance of the observed differences from the control group (Table 3).
Table 3. Arithmetical mean and standard deViatIOn of the hlstometnc quantities determmed m the smgle groups of spleens C M
SD
ACH M SD
HC M
IPH SD
M
SVThr SD
Smus Volume (%)
38.89
2.62
37.83 (n. s.)
4.06
38.89 (n. s.)
1.29
51.25 5.13 (p < 0.001)
56.25
Pulp cord Volume (%)
61.11
2.62
62.17 (n. s.)
4.06
61.11 (n. s.)
1.29
48.75 5.13 (p < 0.01)
43.75
Smus Vol/Pulp cord Vol
0.64
0.07
0.61
0.10
0.64
0.03
0.22
1.29
1.07
2075 5.29 13.88 0.67 11.23 1.88 15.92 (n. s.) (p < 0.001) (p < 0.001) Mean SectIOnal Area of 592.05 72.85 457.01113.45 354.51 60.76 455.76 37.271228.76 smgle Smuses (Ill) (n. s.) (p < 0.01) (p < 0.01) Pulp cord Width (~)
22.45
1.54
Smus Length (m/em 3)
660.33 44.33 867.76189.491115.39183.721131.88157.86 356.05 (p < 0.01) (p < 0.05) (p < 0.01)
Sinus Surface-S' statistical model
546.16 36.41 624.85118.79 882.57 58.15 876.10 61.55 549.62
(cm 2/em 3 )
(n. s.) Smus Surface-S" geometric model
(p < 0.001)
(p < 0.001)
567.29 11.33 637.67 93.26 736.07 60.34 853.26 98.51 442.23
(cm 2/cm 3 )
(n. s.)
(p
< 0.01)
(p
< 0.01)
C = Control; ACH = Active Chromc Hepatitis; HC = HepatiC CirrhOSIS; IPH = Idiopathic Portal HypertensIOn; SVThr = Splemc Vem ThrombOSIS; M = Mean; SD = Standard DeViatIOn. Under every mean IS mdlcated between brackets the slgmflcance of the difference from the control values; n. s. = not slgmflcant
Microvascular Architecture of Spleen
In
Congestlve Splenomegaly . 141
The per cent volume of splenIc smuses m chronic hepatitis (37.83%) and in liver cirrhosis (38.89%) appears to be virtually the same as in controls (38.89%). Similarly, no significant differences are shown for the per cent volume of splenic cords in comparison with controls. In idiopathic portal hypertension, splenic sinuses are 51.25% and splenic cords 48.75% in volume, with reversal of their volume ratio. The mean sectional area of splenic sinuses appears to be lesser in chronic hepatitis (457.76 Ilm 2), in liver cirrhosis (354.51 Ilm2 ) , and in idiopathic portal hypertension (455.76 Ilml), when compared with controls (592.05 Ilm2 ). On the other hand, the smus length per UnIt volume in chronic hepatitis (867.76 mlcm 3 ) and especially m lIver CIrrhOSIS (1115.39 mlcm3 ) and in idiopathic portal hypertension (1131.88 mlcm3 ) is higher than in controls (660.33 mlcm3). Such results point out the diverse increase in number of the small venous sinuses per unit volume in the different pathological situations. In controls the mean width of splenIC cords IS 22.45 Ilm. It is smaller in chronic hepatitis (20.75 Ilm), but especially in lIver CIrrhosis (13.88 Ilm) and in idiopathic portal hypertensIOn (11.23 Ilm). The sinus surface per unit volume has been determined both directly from histometric data ("statistical model") (S') and indirectly by a "geometric model" (S"). In the latter the sinuses are represented as cylinders, the sections of which have regular profiles. In controls the values obtained by the two different methods are coincident, so indirectly supporting a structural model in which sinuses have circular outlines. The mean values of sinus surface per unit volume are fairly similar to normal in chronic hepatitis, while they are higher than normal in liver cirrhosis and in idiopathic portal hypertension. However, the results obtained by the two different methods are coincident in idiopathic portal hypertension only, whereas in liver cirrhosis the values, although both higher than normal, are discordant. No statistical analysis could be performed for the only case of splenic vein thrombosis.
Discussion Microscopic observation illustrated some morphological features which prevail in the spleens in chronic hepatitis, such as sheaths of lymphocytes and plasma cells surrounding the terminal arterial branches and the occurrence of small non-specific granulomas. These findings might suggest active chronic inflammation. In idiopathic portal hypertension thickening of the wall is frequently observed in the terminal arterial branches. Moreover, shunts between small
142 . G. Cavalli, G. Re, Anna Mana Casali, and Paola Monan
arterial branches and venous sinuses were encountered only in these cases; they are fairly infrequent and may result from a marked rearrangement of preexistent formatlOns rather than from newly formed structures. Such shunts m the splenic circulation might convey into the sinuses a blood volume greater than that permitted by the usual vascular structures. However, it is difficult to define what a role they play in portal hypertenslOn. The series of histometric values obtamed in control spleens served for comparison with the data determined m the different groups of pathological spleens. Among normal spleens a certain variability occurs, probably due to the continuous changes the spleen undergoes as part of its role in Immunity and its position in the portal circulation. In chronic active hepatitIS, the mean value of sinus length is slightly higher than that of controls, while the mean sectional area of sinuses is slightly lower. The mean per cent volumes of sinuses and cords and the mean cord width are similar to control values, but theIr standard deviation is very much greater than that of controls, indicating a great scatter in the values. Such differences, although they are not significant from a statistical point of view, may suggest, when related to the clmlcal situation, an intermediate stage in the possible evolution to liver cirrhosis. The spleens m liver cirrhosis show a per cent volume of sinuses consistent with normal, but the sinus sectional area is smaller and the smus length per unit volume IS higher than in controls, as in chronic active hepatitis. In the case of liver cirrhOSIS however the observed differences are statistically signiftcant. Although the per cent volume of cords is the same as normal, the cord mean width shows a highly significant decrease. In idiopathIc portal hypertenslOn all the histometric quantities differ significantly from controls. The per cent volume of venous sinuses is higher, the mean sectional area of sinuses IS reduced, while the mean sinus length is increased. The per cent volume of splenic cords is dimimshed, as is the cord mean width. In particular, the volume ratlO of sinuses to cords IS higher than 1 (1.07), whereas in controls and in all the other groups of spleens considered it is always less than 1. In our only case of splenic vein thrombosis there is a hIgher percentage of red pulp per umt volume and the volume occupied by sinuses is greater than that occupied by cords. Consequently, the volume ratlO of sinuses to cords is also higher than 1 m this case (1.29). The mean sectional area of sinuses IS very much hIgher than normal, while the total length of sinuses is less. Therefore, the prevailing features are dtlatation and congestion of sinuses, in both the morphological and the histometnc study. Our findmgs differ from those shown by Stutte and Heusermann (1972) In cases of splenic vein thrombosis with associated haemocytopenia. It IS possIble that thIS depends upon the time
Microvascular Architecture of Spleen
In
Congestive Splenomegaly· 143
the splenectomy was made m our case (7 months after the acute incident); at this time signs of spleen congestion may prevail and a suitable collateral circulation may not yet be formed completely. Moreover, this case did not present signs of hyperspleOlsm. On the basis of the companson of the histometnc data obtained from normal spleens with those obtained from spleens of the d1fferent pathological groups, we tried to develop a morphological model of the congestive splenomegalies studied. For this purpose it must be postulated that sinuses are tubular structures with d1stensible walls wh1ch under hydrostatic pressure tend to assume regularly circular outlmes. Moreover, the correct evaluation of the external sinus surface area must be cons1dered to be that obtained by applying statistical stereo logic methods based on theorems of solid geometry and probability theory. A geometric model of spleen may be suggested in which the sinuses are variously oriented cylindrical formations, having a variable length and convoluted course. In this case every section of the cylindrical formations produces regular profiles (circular or elliptical). Therefore, the external surface area of sinuses can be correctly calculated by applying geometric formulas. The estimate so obtained from the volume and the mean cross-sectional surface is indicated as S" (= extrapolated evaluation of the external surface area of sinuses, according to the geometric model). Here S" is, by defiOltion, coincident with the stereometncal evaluation of the same surface mdicated as S'. However, the normal spleen has a more complex structure because of the presence of connections between the smuses. The reticular meshwork of splenic cords in normal red pulp is a plastic structure and, in some places, the processes of reticular cells can adopt a tubular conformation makmg possible the communicat1on between sinuses (Weiss, 1977). Such communications in normal spleens were also recently demonstrated by scanning electron microscopy (Yamamoto, 1979). Nevertheless, these connections between sinusesespecially for their relative Slze - ought not to mfluence the geometnc representation of the above model, since on stereometnc analysis the S' and S" values are almost coinc1dent m the control spleens. The suggested model can therefore be applied to the normal spleen. When the number and/or the size of connectlOns between sinuses is markedly higher than 10 the above model, a cons1derable number of abnormal profiles will appear in the secttons. Such irregularities of tubular profiles will introduce a systematic error m the evaluation of the external sinusal surface area by geometric extrapolation. Thus S" 1S different, m this case, from S'. S" will appear considerably less than S', smce It IS calculated from a model in which the external surface area is the lowest, the volume and the mean cross section being equal. Therefore, the only sltuatlOn that justify geometrically a
144 . G. CavallI, G. Re, Anna Mana CasalI, and Paola Monan
disagreement between S' and S" is a plentiful interconnection of the tubular structures. In the spleens of liver CIrrhosis both S' and S" differ significantly (p < 0.001 and p < 0.01, respectively) from the values of controls, but they also differ considerably from each other, being S" less than S'. Therefore, in these spleens a great number of irregular profiles may be suggested, produced by numerous and wide connections between sinuses. The occurrence of many interconnecting sinuses is a feature of the progressive morphologic rearrangement induced by the portal hypertensIOn. Moreover, in these spleens the sinuses are narrower and very much longer than those of controls. Such a "lengthening" of the sinusoidal system is probably also due to newly formed slender anastomoses between sinuses, lO addition to stretching of the preexistent sinuses (see Stutte and Heusermann, 1972). Therefore, in the spleens of cases of liver cirrhosis the sinuses present more numerous lOterconnections than in normal spleen, in contrast with what was stated by Yamamoto (1978). The red pulp is involved as a whole in the morphologic rearrangement, since the relative per cent volume of SlOuses and cords, as well as theIr ratio, are the same as lO control spleens. In chronic idiopathic hypertensIOn the spleen shows significant differences from controls for all the quantities analyzed stereometrically. The relative per cent volume of sinuses and cords and their ratio are also different from normal. Both the estimates S' and S" differ significantly from controls, but they are almost coincident With each other. Therefore, in these spleens the number of connections between sinuses is not higher than in the model of normal spleen, in contrast with the modifications observed in the spleens of liver cirrhosis and in the case of splenic vein thrombosis. Little communication between the venous SlOuses running parallel with each other has been found on the scanning electron microscope by Yamamoto (1979). The microvascular architecture of red pulp thus presents, in idIOpathic portal hypertensIOn, distinctive features that facilitate the blood flow from the arterious to venous passages. On the basis of morphologic and especially histometric findings, the congested spleens in idiopathic portal hypertension can be separated from the other types of congestive splenomegaly we have studied. In fact these spleens, when compared with controls, exhibit a set of modifications that is unique to this group among other forms of congestive splenomegaly.
MICrovascular Architecture of Spleen m Congestive Splenomegaly· 145
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170, 115-126 (1970) Klopstock, F.: Uber Mliztumor, Icterus und ascites bel Leberclrrhose. Vlrchows Arch. path. Anat.
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1788-1793 (1936) Rousselot, L. M.: Late phase of congestive splenomegaly (Bantl's syndrome) with hematemesls but Without CirrhOSIS of liver; further observatIOns on etIOlogy of Bantl's syndrome and effect on prognOSIS of certam vanatlOns m portal venous pattern. Surg. 8 , 34-42 (1940) Sekl, K.: Hlstometncal studies of the spleen m Bantl's syndrome With reference to clinicopathologic correlatIOns. Tohoku J. Exp. Med. 87,222-243 (1965) Snell, A. M.: Clinical aspects of portal CIrrhOSIS. Ann. Int. Med. 5, 338-357 (1931) SOtglU, G.: RevlSlone ed mquadramento nosologlco dell'lpertenslOne portale. Mm. Med. 68,
3751-3760 (1977) Stutte, H. ]., Heusermann, U. Splenomegaly and red blood cell destructIOn: a morphometric study on the human spleen. Vlrchows Arch. Abt. B Zellpath. 12, 1-21 (1972) Tumen, H. J.: Hypersplenism and portal hypertensIOn. Ann. N.Y. Acad. SCI. 170, 332-344
(1970) Weibel, E. R., Kistler, G. S., and Scherle, W. F.' Practical stereologlcal methods for morphometnc cytology. ]. Cell BIOI. 30, 23-38 (1966) WeiSS, L.: The spleen. In: L. Weiss and R. O. Greep, Histology, p. 545. McGraw-HIli, New York
1977
10 Path Res Pract Vol 174
146 . G. Cavalli, G Re, Anna Mana Casali, and Paola Monan Yamamoto, K.. Morphological studies of the spleen In splenomegalic hver cirrhoSIS comparing with the spleen In Idiopathic portal hypertensIOn (so-called Bantl's Syndrome without liver cirrhosIs). Acta Path. Jap. 28, 891-905 (1978) Yamamoto, K.· Morphological studies of the spleen In Idiopathic portal hypertensIOn (so-called Bantl's Syndrome without liver cirrhosIs) uSing light microscopy, scanmng electron microscopy and hlstometry Acta Path Jap 29, 1-19 (1979)
Received November 3, 1981
Accepted February 20, 1982
Key words: Spleen - Splenomegaly - Portal Hypertension - Stereo logy Prof. Glancarlo Cavalli, vlale XII GlUgno, 13, 140124, Bologna, Italy