the ruputure spleen. a hisological morphometrical an immunohistochemical study

7/30/2019 The Ruputure Spleen. a Hisological Morphometrical an Immunohistochemical Study

1/6

Histol Histopath (1990)5:299-304 HistologyandHistopathology

The ruptured spleen. A histological, morphometricaland immunohistochemical studyH. Barnard, E.J. Dreef and J.H.J.M. van KriekenDepartment of Patho logy , University Hospital Leiden, The N etherlands

Summary. A traumatically ruptured spleen is regardedas a proper co ntrol in man y histological and im muno lo-gical s tudies on the human spleen. This paper comparesspleens that ruptured due to t rauma and spleens whichwere remo ved d uring surgery in patients without splenicpathology. Based o n a histological, mo rphom etrical, andimmu nohistochemcial description of the c ontrol spleensit is shown that the traumatically ruptured spleenscontain alterations in the lymphoid tissue. Th e amo unt ofwhite pulp is increased du e to a larger amo unt of CD4 -positive lymphocytes. Furth erm ore there are alterationsin lymphocyte populations in the different spleniccompartments. I t is concluded that spleens that rupturemay be predisposed due to immunological s t imulat ion.Key words: Spleen, Rupture , His tology, Imm unohis to-chemis try, Morphom etry

lntroductionT h e s p le e n m a y r u p t u r e d u e t o t r a u m a o rspontaneous ly, i .e . w i thout ap paren t t rauma (Mitchelland Morris . 1983). Traumatical ly ru ptured spleens aremostly regarded as no rm al. Althou gh it is diffucult todef ine what an adequate t rauma is , there are severa1

disorders which may facili tate spontaneous splenicrupture ; infectious mononucleosis is one of the most wellknown examples (Andrews et al . , 1980; Rogers andShah, 1980; Armitage et al . , 1981; Berlin et al . , 1982;Adouin and D iebold, 1983; Bennet t e t al . , 1984; Carusoand Hal l , 1984; Chamber lain et al . , 1984; Cochrane,1984; Doe hrma nn and Scheele, 1984; Cooperberg et al . ,1985; Fallingborg e t al . , 1985; Mirchadan i e t al . , 1985;Birenbaum et al . . 1986; Majewski and Stahlknecht ,1986).Offprint requests to: J.H.J.M. van Krieken, MD, PhD, Laboratory ofPathology, University of Leiden, PO Bo x 9603, 2300RC Leiden, TheNetherlands

A previous study has shown tha t there is an increase inthe amount of white pulp in traumatically rupturedspleens compared to spleens which were removed inci-dentally during abdominal surgery without apparentsplenic pathology (van Krieken et a l . , 1983). This leadsto the suggestion that in many cases of so-calledtraumatic rupture of the spleen there might be apredisposing factor, such as, for instance, a vira1infection.In o rder to investigate the lymphoid tissue in rupture dspleens in more detail , i t was compared with thelymphoid tissue of spleens without splenic pathologyusing morphological, immunohistochemical, and mor-phometr ical m ethods .Materials and methods

Al1 spleens removed were processed immediately,which included methylmethacrylate embedding andfreezing of tissue blocks as described before (vanKrieken et al . , 19 83,1986) . From ruptured spleen blockswere taken from macroscopical n orm al -t issue at adistance from the rupture. Extensively crushedspecimens were excluded f rom s tudy.For this study ten spleens with m inor rupture d ue tot rauma were avai lable and compared to the ten mostrecent spleens removed during cancer surgery (mainlypartial gastrectomy for early gastric cance r), and to ninespleens removed during surgery for non-malignantdisease without splenic pathology. The latter groupconsists mainly of cases of highly selective vagotomy orimplantation of aortic prothesis.Immunohistochemistry

Cryostat sections were stained using a three-stepimmunoperoxidase technique with the followingantibodies: Leu 4 (CD3) , Leu 3 (CD 4) , Leu 1 (CD5) ,Leu 2 (CDZ), Le u 14 (CD22) al1 Becton & Dickinson; B1(CD20) , B2 (CD21) , bo th Or thoc lone ; Tul (CD23) ,Biotes t .


2/6

Ruptured spleen

MorphometryAl1 measurements were performed by one of theauthors (H.B.) without knowing the reason for removal

of the specimens as described before (van Krieken et al .,1983). In short, with a light-bearing cursor areas of asection were encircled using a Leitz Prism-microscope;perimeter and surface area were calculated usinga Kontron MOP-amo 3. This procedure gives thepercentage of the measured compartment in the spleen.The amount of a compartment was calculated bymultiplying this percentage and the weight of thespleen. In methylmethacrylate sections, stained withmethenaminesilver/H&E, white pulp and perifollicularzone were measured (Fig. 1): n cryostat sections stainedfor CD3 the T-cell areas (Fig. 2) , and in sections stainedfor CD22 the B-cell follicles were measured.In the cryostat sections stained for the differentantigens the number of positive cells for each lymphoidcompartment (Van Krieken and Te Velde, 1989) (Figs.1, 3) was semiquantitatively scored in four categories(O = no positive cell; + + + = more than 90% ofthe cells positive).Statistical methods

The morphometrical data were analysed bycomparing two groups using Students's t-test; thesemiquantitative data were compared using the square-chi test. P-values < 0.05 were considered statisticallysignificant.ResultsHistology

Al1 specimens showed a normal splenic architecture,with the normal variation in the prominence of whitepulp (van Krieken and Te Velde, 1988).lrnrnunohistochemistry (Lable 1)

There were no differences between spleensremoved during cancer surgery and those removed innon-malignant disorders. The results of these groups arecombined and given in Table 1 . The white pulp consistedof T-cell areas, mainly CD4-positive (Fig. 2), and B-cellfollicles. The B-cell follicles may contain a germinalcentre . There was no difference between primary follicleand mantle zone lymphocytes: CD19, CD22, CD23, al1positive, CD5 weakly positive. The marginal zoneconsisted of somewhat larger lymphocytes with thefollowing phenotype: CD19- and CD22-positive, CD23-and CD5-negative. Severa1 CD4-positive cells were alsopresent (Fig. 4a). In the red pulp CD19-, CD22- andCD23-positive lymphocytes were intermingled withCD8-positive lymphocytes (Fig. 5a). In the non-filteringareas the B-cells dominated, and there were also someCD4 cells; in the perivascular rim the CD8 cellsdominated.

Fig. 1.Traumatically ruptured spleen. Methylmethacrylateembedding. This figure shows at small magnificationthe compartments of the white pulp. Note thepresence of the relatively large perifollicular zone.1 = germinal centre2 = mantle zone3 = marginal zone4 = perifollicular zone5 = T-cell area6 = red pulp

Fig. 2. Spleen removed during highly selectivevagotomy procedure. Three-step immunoperoxidasetechnique using Leu3 as antibody (CD4). Stronglypositive T-cell areas and scattered positive cells inthe red pulp.


3/6

Ruptured spleen

Table 1.Summary of the results of the immunohistochemical analysis. Semiquantitative score of the presence of cells positive for each antibodyused in each splenic compartment. O = no positive cell; + + + = almost al1 cells positive.B-Cell antigens

CD20 CD21 CD22 CD23Germinal centre + + + + +++ + +Mantlezone +++ + + + + + + +Marginal zone + + + + + + + +T-cell area O O O OPerifollicular zone + + + +Red pulp O O O ONon-filtering area + + + +

T-cell antigensCD3 CD4 CD8

Fig. 3. Same specimens as figure 1. Detail of the red pulpshowing sheathed capillary (S) and groups oflymphocytes iri non-filtering areas (N).

The main distribution of lymphocyte subsets was thesame in the ruptured spleens, but some differences werefound. In the marginal zone there was a significantincrease of CD23- positive cells, whereas the number ofCD4- positive cells decreased (Fig. 4b). CD21 hasdisappeared from a part of the cells in the follicles.In the red pulp there was a decrease of the number ofCD8-positive lyn~phocytes Fig. 5b).

Morphometrical results (Figs. 6, 7)Again no differences were found between spleensremoved for malignant vs non-malignant disease.Ruptured spleens showed a significant increase in thepercentage of the perifollicular zone compared to bothother groups, and the percentage of white pulp slightlyincreased.Due to the inclusion of a part of the perifollicularzone, which could not be recognized in cryostat sections,the summation of the percentage of CD23- and CD3-

positive areas exceeds the percentage of white pulp asmeasured in methylmethacrylate-embedded sections.As this was the case in al1 three groups, it does notinfluence the comparison. Th e CD22-positive areas werein al1 three groups similar, but there was more T-cell areain ruptured spleens compared to both other groups. Thisbecomes especially clear after correction for splenicweight. That means that there is an absolute increase inthe amount of perifollicular zone and T-cell area inruptured spleens compared to the other groups.Discussion

Known causes of splenic rupture include: perforatingtrauma, blunt trauma (Sherman, 1980; Seufert, 1983).infectious mononucleosis (Chamberlain et al., 1984;Cochrane, 1984; Majewski and Stahlknecht, 1986),AIDS (Mirchani et al ., 1985), malignancies (Andrews etal., 1980; Rogers and Shah, 1980; Armitage et al., 1981;Bennet et al., 1984; Cooperberg et al., 1985). peliosis(Berlin et al.. 1982; Audoin and Diebold, 1983) andextra-uter ine pregnancy (Caruso and Hall, 1984). Sple-nomegaly enhances the changes of splenic rupturefollowing blunt trauma (Sherman, 1980; Bisshop andLansaing, 1982; Cochrane, 1984). In many studies atraumatically ruptured spleen is considered to be normaland the ruptured spleen is, for instance, not discussed in arecent textbook on the pathology of the spleen (Wolf and


4/6

Ruptured spleen

Fig. 4. Comparison of Leu 2 (CD8) staining of marginal zone between ruptured (a) and non-ruptured (bi spleen

Fig. 5. Comparison of Leu (CD8) staining of red pulp between ruptured (a) and non-ruptured (b) spleen

Neiman, 1989). A previous study has shown thatruptured spleens contain more lymphoid tissue thanspleens which can be considered as normal (vanKrieken, 1983). Similar results were found in thisstudy which comprises different, more recentlyobtained specimens, of which frozen tissue wasavailable. There are some differences between thepresent group of ruptured spleens and the previousseries. Namely, less white pulp and more perifolli-cular zone. Although these differences are not sta-tistically significant (p 0.05) one possible explanationneeds mentioning. In the period when we obtained ourfirst series (1975-1980) spleens with small superficial

ruptures were always removed, and splenic repair wasnot attempted. This is contrasts with the present lessagressive approach, and this may have influenced thecomposition of the present series of specimens.However, the differences compared to our controlgroup, for which the same difference in approach mayalso have had some influence, is not altered.Although the number of specimens is inevitably low(spleens with minor rupture nowadays rarely reach ourdepartment of pathology), this immunohistochemicalstudy made it possible to show that the increase in whitepulp in ruptured spleens is solely due to an increasedamount of CD4 (helper1inducer)-lymphocytes. Although


5/6

Ruptured spleen

Fig. 7. Surnrnary of rnorphornetrical results. Cornparison of rupturedand non-ruptured spleens regarding rnean and standard deviation ofthe arnount of white pulp, perifollicularzone,Leu 4-positive areas, andR M NM Leu 14-positive areas.6.Weight of the spleen, rnean and standard deviation, of the three R - ruptured groupC - cornbined rnalignant and non-rnalignant group

- ruptured group (rnean weight: 0.191 kg)- rnalignant group (rnean weight: 0.1 73 kg)M - non-malignant group (rnean weight: 0.1 72 kg)

he amount of B-cell follicles is not altered, theomposition changes, with more mantle zone cells andess marginal zone cells (van Krieken et al., 1989). Inhe red pulp there are less CD8 (suppressor/cytotoxic)It is difficult in a system as complex as the immune

utoimmune disorders (van Krieken and Te Velde,1986; van Krieken et al., 1988), Hodgkin's disease(Timens et a l., 1982) and some other malignancies. Thefindings in ruptured spleens are completely differentfrom those data and a re difficult to interpret. Anyway,the present results point to an activation of the lymphoidsystem and fit into the hypothesis that spleens thatrupture are predisposed to the event du e to (subclinical)infections in a similar way as is already well known ininfectious mononucleosis. In immunological studies onthe human spleen one has to be cautious when usingruptured spleens as controls.

WP - white pulp: rnean R: 0.023 kgrnean C: 0.067 kgPZ - perifollicular zone: mean R: 0.027 kgmean C: 0.018 g4 - LEU 4-positive: mean R: 0.01 0 kgmean C: 0.006 kg14- LEU 14-positive: mean R: 0.01 9 kgrnean C: 0.022 kgReferencesAndrews D.F., Grafton W., Hernndez R. and Williams D.M.(1980). Pathologic rupture of the spleen in non-Hodgkin's

lymphoma. Arch. Int. Med. 140, 119-120.Armitage J.O., Bauer T.W. and Haskins G.E. (1981). Splenic

rupture in patients with hematologic malignancies. Cancer48,2729-2733.

Audouin J. and Diebold J. (1983). Peliosis of the spleen. Am. J.Surg. Pathol. 197-204.

Bennett R.R., Collins R.D., Cousar J.B., Glick A.D., Greer J.P. andStein R.S. (1 984). Death due to splenic rupture in suppressorcell mycosis fungodes; a case report. Am. J. Clin. Pathol. 82,104-1 09.

Berlin R.B., Garca R.L. and Kahn M.K. (1982). Peliosis of thespleen with rupture. Hum. Pathol. 13, 177-179.


6/6

Ruptured spleen

Birenbaum H.J., Harris M.D., Macaraeg A.L. and Silverman B.M.(1986). Splenic rupture in a very low birth weight infant.Maryland Med. J. 35,399-400.

Bishop M.B. and Lansing L.S. (1982). The spleen: a correlativeoverview of normal and pathological anatomy. Hum. Pathol.13,334-342.

Caruso V. and Hall W.H.J. (1984). Primary abdominal pregnancyin the spleen; a case report. J. Pathol. 16,93-94.

Chamberlain M.J., Cohen P., Nicholson R.L. and Vezina W.C.(1 984). Radionucleotide diagnosis of splenic rupture ininfectious mononucleosis. Clin. Nucl. Med. 9,341 -344.

Cochrane J.P.S. (1984). Ruptured spleen. Brit. J. Hosp Med. 24,398-404.

Cooperberg A., Kienzle G.D., Osborne C.A. and Stern J. (1985).Spontaneous rupture of the spleen in primary plasma cellleukemia; scintigraphic-pathologic correlation. Clin. Nucl.Med. 10,639-641.

Doehrmann D. and Scheele C. (1984). Spontanruptur der Milz beilnfectioeser Mononucleose. Chirurg. 55,480-481.Fallingborg J., Jacobsen J., Lautsen J., Svanhol H. and Winther P.(1985). Atraumatic rupture of the spleen in periarteritis nodosa.Acta Chirg. Scand. 151,85-87.

Majewski A. and Stahlknecht T. (1986). Das asymptomatischesubkapsulaere Milzhaematom bei der lnfectiosenMononukleose (M. Pfeiffer). Roentgenblaetter 39,264-265.

Mirchandani H.G., Mirchandani I.H. and Pak M.S.Y. (1985).Spontaneous rupture of the spleen due to acquiredimmunodeficiency syndrome in an intravenous drug abuser.Arch. Pathol. Lab. Med. 109,1114-1 116.

Mitchell A. and Morris P.J. (1983). Surgery of the spleen. In:Clincis in hameatology 12; the spleen. Lewis S.M. (ed), WBSaunders, London. 565-590.

Rogers J.S. and Shah S. (1980). Spontaneous splenic rupture inplasma cell leukemia. Cancer 46,212-214.

Seufert R.M. (1983). Chirurgie der Milz. Ferdinand Enke Verlag.Stuttgart.

Sherman R. (1980). Percpectives in management of trauma to thespleen. J. Trauma 20, 1-13.Timens W., Koundstaal J. and Poppema S. (1982).

Morphometrical analysis of T- and B-cell compartments ofthe spleen in Hodgkin's disease. Virchows Arch (B); 38,291 -296.

Van Krieken J.H.J.M., Velde J. te, Hermans J., Cornelisse C.J.,Welvaart K. and Ferrari M. (1 983). The amount of white pulp inthe spleen; a morphometrical study done in mzthylmethacry-late embedded splenectomy specimens. Histopathology 7,767-782.

Van Krieken J.H.J.M. and Velde J. te. (1986). lmmunohistology ofthe spleen. An inventory of the localization of lymphocytesubpopulation. Histopathology 10,285-294.

Van Krieken J.H.J.M. and Velde J. te (1988). Normal histology ofthe human spleen. Am. J. Surg. Pathol. 12,777-785.Van Krieken J.H.J.M., Breedveld P.C. and Velde J. te (1988). The

spleen in Felty's syndrome: a histological, morphometricaland immunohistochemical study. Eur. J. Haematol. 40,58-64.

Van Krieken J.H.J.M., Schilling C. von, Kluin Ph.M. and Lennert K.(1989). Splenic marginal zone lymphocytes and related cells inthe lymph node: a morphologic and immunohistochemicalstudy. Hum. Pathol. 20,320-325.

Wolf B.C. and Neiman R.S. (1989). Disorders of the spleen. W.B.Saunders Company. Philadelphia.

Accepted January 15,1990

the ruputure spleen. a hisological morphometrical an immunohistochemical study

Documents