266 CASE REPORTS: THROMBOCYTOPENIC PURPURA Canad. Med. Ass. 3.Aug. 10, 1963, vol. 89

pECENTLY the association of a peculiar typeLI of lipidic histiocytosis and thrombocytopenicpurpura has been observed and reported6'7 inspleens removed for the latter disease. A review of14 spleens removed at the Jewish General Hospitalin cases of thrombocytopenic purpura revealedin three of the 14 spleens a histologic picture andhistochemical findings of lipidic histiocytosis similarto those previously reported. The clinical and histo-logic features of our cases are described in whichattempts to identify the lipids were made.

CASE 1.-S.M., a 2-year-old white Jewish male,was admitted because of generalized purpura and ec-chymosis of one week's duration. The laboratory find-ings on admission were: hemoglobin 10.6 g. %, hemato-crit 31%, platelets 52,000/c.mm., bleeding time 9 mm.,clotting time 7 mm., no clot retraction after 24 hours,and Rumpel-Leede test ++ ±. The bone marrowshowed increased megakaryocytes but no abnormalcells. A diagnosis of idiopathic thrombocytopenia wasmade and the patient was started on prednisone 10mg., four times daily. After one month of steroidtherapy the platelets increased to 164,000/c.mm.The patient was discharged on steroid therapy but

was readmitted 10 days later with recurrence ofbruising and a platelet count of 40,000/c.mm. Heshowed the characteristics of excessive and prolongedcortisone therapy. Because of persistent thrombocyto-penia, a splenectomy was performed three months afterthe onset of purpura.

Following operation the platelets increased to amaximum level of 589,O00/c.mm. four days post-operatively. Subsequently, there was a gradual fall inthe number of platelets to 100,000/c.mm. The corti-costeroids were progressively reduced and the patientwas followed up in the clinic. When he was last seen,four months after the operation, the platelets count was400,000/c.mm. and no purpura was noted.

Pathologic FindingsThe spleen weighed 60 g. and on section appeared to

be congested. The follicles and trabeculae could notbe identified grossly. After formalin fixation, routinestains revealed a somewhat altered splenic architecturedue to a definite increase in the number of cells ofreticuloendothelial type which occurred in sheets andclusters. These cells were large and pale, and hadcentrally placed, slightly vesicular nuclei and poorlydefined, somewhat foamy cytoplasm, which at timesappeared to show tiny vacuoles (Fig. 1). The Mal-pighian corpuscles were prominent and showed hyper-plasia with well-defined secondary nodule formation.

From the Hematology Laboratory of the Department ofMedicine and the Department of Pathology, Jewish GeneralHospital, Montreal, Que.

Fig. I .--Case 1-routIne stain. Large cells of reticuloendo-thelial type with foamy and granular cytoplasm occurringin clusters.

In addition, there was moderate dilatation of thesinusoids throughout the spleen, without excessivehyperemia.

Formalin-fixed frozen sections when stained for fatfailed to reveal the presence of sudanophilic material.Examination of frozen sections under polarized lightconfirmed the absence of any significant amount of fatwithin these cells. After chromating forinalin-fixedtissue with Zenker's solution and restaining with sudanIII and sudan IV, a remarkable degree of sudanophilicmaterial was demonstrable within the reticiiloendo-thelial cells. Further staining with sudan black B onparaffin section revealed that most of the vacuoles ofthe foamy large cells were filled with black, fine andcoarse granules. The vacuoles were periodic-acid-Schiff(PAS) negative.

CASE 2.-S.R., an 11-year-old white Jewish female,was admitted to hospital with a history of purpura andeasy bruising of six months' duration. On admission thehemoglobin was 11.6 g. %, hematocrit 36%, platelets36,000/c.mm., bleeding time 5'/2 mm., clotting time14 mm., clot retraction present, prothrombin time 14.2seconds, Rumpel-Leede's test positive +, prothrombinconsumption time 15.6 seconds. The bone marro.v

Canad. Med. Ass. J.Aug. 10. 1963. vol. 89 CAsE REPORTS: THROMBOCYTOPENIC PuRPu1. 267

section material showed the deposition of a small butdefinitu and recognizable amount of sudanophilicmaterial within these large reticuloendothelial cells.Following chromatization, frozen section material, whensimilarly stained, again demonstrated the presence ofsudanophiic droplets. Staining with sudan black Bshowed definite positive fine granules in most of thevacuolated cells. The periodic acid-Schiff stain wasslightly positive in some of the vacuolated cells(Fig. 2).

CASE 3.-K.N.H., a 22-year-old white female, wasrecognized as having idiopathic thrombocytopenia pur-pura because of a platelet count of 90,000/c.mm. anda history of frequent nose bleeds in childhood, markedbleeding after tooth extraction two years previously,and easy bruising for one year. Three years after thediagnosis of idiopathic thrombocytopenic purpura wasestablished, the patient was admitted to hospital be-cause of a miscarriage followed by persistent vaginalbleeding. At this time the platelets were 60,O0O/c.mm.The patient was started on prednisone 12.5 mg. daily.Although the purpura and other bleeding subsidedcompletely, splenectomy was performed four month9later because of persistent thrombocytopenia. At thetime of splenectomy the hemoglobin was 12.4 g. %,hematocrit 39%, sedimentation rate 10 mm./hr., icterusindex 5, WBC 7400/c.mm., bleeding time 6 minutes,clotting time 9. minutes, clot retraction present, pro-thrombin time 15 seconds, Rumpel-Leede's test nega-tive, prothrombin consumption time 22 seconds, plate-lets 160,000/c.mm., and increased megakaryocytes inthe bone marrow.

After splenectomy the platelets rose to 700,000/c.mm. and gradually fell to normal levels. Two yearsfollowing splenectomy the patient gave birth to ahealthy baby. At present, three years after splenectomy,she is well and her platelet count is 290,000/c.mm.

Pathological FindingsThe spleen weighed 140 g. and the accessory spleen

found at operation weighed 2 g. Both were describedas slightly congested. The microscopic examination ofboth spleens showed essentially similar changes. TheMalpighian corpuscles were larger than usual andmany revealed distinct hyperplasia with well-definedsecondary nodule formation. The most striking changenoted, in addition to the hyperplasia described above,was the presence of considerable numbers of largepolyhedral cells with small, eccentric, pyknotic nucleiand granular to foamy cytoplasm. Frozen formalin-fixed sections stained for neutral fat with sudan IVshowed only occasional cells which stained red. Ex-amination of unstained frozen sections showed nooptically active crystalloid structures within these cells.No unblocked material was available for chromatiza-tion. Staining with sudan black B showed granules andvacuoles which stained black (Fig. 3). Sections stainedby the P.A.S. method revealed periodic-acid-Schiffpositive granules in a considerable number of theselarge polyhedral cells.

DIscussIoN

The three patients described all presented withthe symptoms and usual laboratory findings ofthrombocytopenic purpura. All had been subjected

268 CAsE REPORTS: THROMBOCYTOPENIC PURPURA

to steroid therapy3 and later required splenectomy.The clinical and histopathologic findings arestrikingly similar to those in the nine cases de-scribed by Landing et al.,6 in the previous casereported by Marshall and Adams7 and probably inthose of other authors.2' .The spleens were normal in weight and all

showed a similar picture characterized by histio-cytosis. The histiocytes contained lipids whichshowed common histochemical features in all threecases. It is noteworthy that the vacuoles andgranules present in the histiocytes stained eitherpoorly or not at all with mixtures of sudan LII andIV when formalin-fixed material alone was used.However, when Zenker's solution was used as asecond fixative and such frozen sections werestained with mixtures of sudan III and IV, a re-markable degree of sudanophilia was demonstratedin the splenic histiocytes of Cases 1 and 2. No un-blocked material for secondary chromatization wasavailable in Case 3.When paraffin sections were stained with sudan

black B, they showed a remarkable amount ofpositive black-staining granules in all three cases.These findings suggest the presence of formalin-fixed lipoproteins, the lipid components of whichmay by phosphatides or cerebrosides. While sudanblack B staining is not specffic for phosphatides,they occur more commonly and hence are the lipidsmost likely to be present, particularly if neutralfats are excluded9 by negative or only faint stainingreaction with sudan III and IV, as occurred in ourcases.The periodic-acid-Schiff (PAS ) -positive granules

and vacuoles in the sections from Cases 1 and2 indicate the presence of phospholipids or poly-saccharides. The main substances in the phospho-lipid group are sphingosine, fatty acids, phosphoricacid and choline. Since sphingosine contains aprimary acetylated amine adjacent to a hydroxylgroup, it is capable of reacting with periodic acid(H104) to give an aldehyde and thus a positivePAS-reaction.

All three cases showed absence of optically activecrystalloid structures within the histiocytes, but thisnegative result does not exclude the presence offat.9 From these histochemical properties one mayspeculate that we are dealing with histiocytes con-taining phospholipids. This was confirmed by Land-ing and Strauss with chemical studies in their cases;unfortunately, no tissue was available for chemicalextraction and study of lipids in the cases reportedhere.

Other known lipidoses having phospholipids asthe principal lipid are amaurotic familial idiocyand Neimann-Pick disease. Our patients did notshow evidence of central nervous system involve-ment; thus amaurotic familial idiocy was excluded.Neimann-Pick disease is differentiated by theabsence of splenomegaly, adenopathy and otherclinical features, together with absence of thecharacteristic cells diagnostic of Neimann-Pick

Canad. Med. Ass. J.Aug. 10, 1963, vol. 89

Fig. 3.-Case 3-sudan tdack B stain. Black-stained finegranules are present in most of the vacuolated cells.

disease.4'" Gaucher's disease is a cerebrosidelipidosis characterized by typical cells in themarrow and spleen, and by hepatosplenomegaly,features not found in our cases.

Because all of our patients were treated withsteroids, the question is raised whether these casesrepresent cholesterol lipidosis (xanthomatosis)secondary to steroid therapy.8 The case describedby Marshall and Adams, and the fifth case reportedby Landing and Strauss, did not receive steroidtherapy, and the relatively low incidence of thistype of histiocytosis encountered in patients onsteroid therapy, splenectomized because of idio-pathic thrombocytopenic purpura, suggests thatthere is probably no cause-and-effect relationshipbetween the steroid therapy and this type of spleniclipidosis.The histologic picture in the presently reported

cases exhibit some similarity to the foamy splenichistiocytosis seen in some cases of thalassemia.However, the strong positivity in thalassemia forperiodic-acid-Schiff stain, and the slight reactionswith lipid-staining dyes, suggest the presence of anacid mucopolysaccharide of the chondroitin- sulfuricacid type in the cytoplasms of the histiocytic cells'0in thalassemia. This is in contrast to the strong posi-tivity for sudan black B, and to a lesser degree forperiodic acid-Schiff, of the foamy material in thehistiocytes of our cases.

Canad. Med. Ass. 3. CASE REPORTS: THIROMBOCYTOPENIC Pulu'URA 269Aug. 10, 1963. vol. 89

Other authors5 have observed foamy histiocytosisin three of 47 spleens removed from chronic idio-pathic thrombocytopenic purpura. They concluded,as a result of the negative fat staining, that thefoamy histiocytes contained mucopolysaceharides,and not lipid. It should be noted that they failedto mordant their tissue in Zenker's solution and torestain with sudan black B.

All of the cases described in this report did wellafter splenectomy, with a return to normal plateletcounts. One month follow-up in the first case, fivemonths in the second case, and three years in thethird case failed to reveal any hemorrhagic tend-ency or platelet deficiency. The findings of threecases of histiocytosis among 14 patients withthrombocytopenic purpura who underwent splen-ectomy at the Jewish General Hospital, Montreal,would suggest a higher incidence than that reportedin the literature. The number of cases described todate is not sufficiently large to postulate any ageor sex incidence.

SUMMARY

Three additional cases of thrombocytopenic purpurawith unusual histiocytosis of the spleen are described.It is felt that the material present in the foamy cells ismost likely a phospholipid because of a positive stain-ing with sudan black B and PAS.The clinical features of these cases are indistinguish-

able from those of idiopathic thrombocytopenic pur-pura which do not have lipidic histiocytosis of thespleen.

REFERENCES

1. BOWMAN, H. E. et al.: Lab. Invest., 4: 206, 1955.2. CARPENTER, A. F. et al.: J. A. M. A., 171: 1911, 1959.3. COOPERBERG, A. A.: Canad. Med. Ass. J., 80: 937, 1959.4. CROCKER, A. C. AND FARBER, S.: Medicine (Bait.), 37: 1,

1958.5. CZERNOBILSKY, B., FREEDMAN, H. H. AND FRUMIN, A. M.:

Blood, 19: 99, 1962.6. LANDING, B. H. et al.: New Engi. J. Med., 265: 572, 1961.7. MARSHALL, A. H. E. AND ADAMS, C. W. M.: J. Path. Bact.,

76: 159, 1958.8. OPPENHEIMER, E. H. AND GUILD, H. A.: Bull. Johns

Hopkins Hasp., 106: 368. 19609. PEARSE A. G. E.: Histochemistry: theoretical and ap-

pliec1., Little, Brown and Co., Boston, 1960. p. 234, 299-300, 307.

10. SEN GUPTA P C et al.: Blood, 16: 1039, 1960.11. TERRY, R. 5 SPERRY, W. M. AND BRODOFF, B.: Amer. J.

Path., 30: 263, 1954.

CANADIAN JOURNAL OF SURGERY

The July 1963 issue of the Canadian Journal of Surgery contains the following original articles, casereports and experimental surgery:

Original Articles: Pheochromocytoma: an important type of "curable" hypertension: a report of 11 casesand a review of the literature-N. C. Delarue and D. O'Brien. Hemiacidrin (Renacidin): a study of its use in 40patients with bladder calculi-R. G. Hepworth. The prognostic significance of gross venous invasion of carcinomaof the rectum-S. E. Carroll. Severe tissue damage in intra-arterial perfusion-R. W. Hakstian, E. J. Tabah andF. M. Woolhouse. Non-insulin-producing islet cell tumours of the pancreas: a review of five cases including fourwith Zollinger-Ellison syndrome-J. W. Martyn and W. K. Welsh. Herniation through congenital diaphragmaticdefects in adults-J. T. MacDougall, A. C. Abbott and T. K. Goodhand. The local control of carcinoma of thebreast-J. E. Devitt. Thoracotomy for the diagnosis of intrathoracic lesions-J. J. Quinlan, V. D. Schaffner andJ. E. Hiltz. Central nervous system emboli in open heart surgery-P. Allen.

Review Article: Perforation of jejunal and ileal neoplasms: a survey of the literature and case reports-P. M.Higgins, G. Lehman and H. S. Morton.

Case Reports: Pyelon6phrite lithiasique tuberculofde-L. Bernier et A. B.dard. Recovery from a cerebralAspergillus abscess metastatic from a primary focus in the lung-E. B. Hendrick and P. E. Conen. Osteochondritisdissecans: a report of two cases-D. A. Gibson. Enterolithiasis with multiple tuberculous .B. C. Bapna, B. S. Rao and R. C. Chawla. Melanotic freckle as a precursor of malignant melanoma: report of acase-W. E. Austin.

Experimental Surgery: Further studies on methylcholanthrene-induced tumours of the gallbladder and liverin the golden hamster-G. 0. Bain, J. Siegenberg and K. Kowalewski. An investigation of polyurethane foam inexperimental animals-C. Hollenberg, R. K. Macoomb, J. R. Trott and W. Zingg. An experimental study of thegrowth of arterial anastomoses-C. Bull, T. Hoeksema, J. A. Duckworth and W. T. Mustard. Perfusate tempera-ture-a factor influencing leakage in isolated regional perfusion using extracorporeal circulation-R. Beaudry,R. Poisson, L. G. Stephens-Newsham, E. J. Tabah and J. R. Moore.

The Canadian Journal of Surgery is published quarterly by The Canadian Medical Association. Subscriptionrates are $10.00 a year ($5.00 a year for recognized postgraduate trainees in surgery) or $2.50 for individual copies.Yearly subscriptions and back issues are available on request from the Canadian Journal of Surgery, C.M.A. House,150 St. George Street, Toronto 5, Ontario.