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Virus-associated hemophagocytic syndrome due to epstein-barr virus
HUMAN PATHOLOGY
Volume 15, No. 3 (March 1984)
VIRUS-ASSOCIATED HEMOPHAGOCYTIC SYNDROME DUE TO EPSTEIN-BARR VIRUS ROBERT P. REISMAN,MD,* AND M. ALBA GRECO,MDf
The dinical and autopsv findings in a case of virus-associated hemophagocytic syndrome in a pra,iously health)' 17-year-old g~rl are presented. Serum titers to Epstein-Barr viral antigens were umtsually elevated, h~ addition to widespread proliferation of hemophagoo.tic histioo'tes and lymphoid depletion, there were areas of non-suppurative necrosis in lymph nodes and spleen, which have not been pra,iously reported. HUM PArHOL 15:290--293, 1984. T h e virus-associated hemophagocytic syndrome (VAHS) is a benign disorder cbaracterized by the proliferation of cytologically benign hemophagocytic histiocytes in the bone marrow, lymph nodes, spleen, liver, and leptomeninges in association with an acute systemic viral infection. Of the 19 patients described in the original report in 1979 by Risdall et al.,' 13 were receiving immunosuppressive therapy for renal transplantation and one for systemic lupus erythematosus. The remaining five had previously been well. Documentation of acute infection with cytomegalovirus was present in ten patients, Epstein-Barr virus (EBV) in two, and herpes simplex, varicella-zoster, and adenovirus in one each. Subsequently, two additional fatal cases of EBV-associated VAHS have been reported: one in a previously well 16-year-old boy e and the other in a 9-year-old boy with acute lymphoblastic leukemia in remission. ~ We present the clinical and autopsy findings in the case of a previously well 17-year-old girl with EBV-related VAHS, in which the lymph nodes and spleen revealed marked lymphoid depletion and extensive necrosis. T o the best of our knowledge, the presence of necrosis in lymph nodes and spleen has not been previously reported in VAHS. In addition, titers of antibodies to viral capsid antigen and early antigen-D were unusually elevated. REPORT OF A CASE A 17-year-old white girl was transferred to the New York University Hospital Pediatric Service for evaluation of neutropenia and hepatosplenomegaly. She had been well until two weeks previously, when a sore throat and fever developed, for which she was given oral amoxicillin and intramuscular penicillin. A throat culture and Monospot test were negative. A purified protein derivative tuberculin test was negative, One week later, on admission to another hospital, hepatosplenomegaly was noted. The leukocyte count was 2,200/mm 3, the platelets 68,000/ram s, and the hemoglobin 10.6 g/dl. A bone marrow aspirate and biopsy were performed two days prior to the patient's transfer to NYU. This material and a peripheral blood smear obtained the same day were reviewed by the authors and revealed a normocellular bone marrow. Neither histiocytosis nor he-
mophagocytosis were present. Atypical lymphocytes were not increased in tile peripheral blood. A chest radiograph was normal. During tiffs hospitalization, the patient experienced nausea, vomiting, diarrhea, arthralgia, easy bruising, and a dry cough. The patient had remote histories of measles, mumps, varicella, and intmunization with DPT. Carefully elicited family and social itistories revealed no pertinent information. Upon admission to NYU.Hospital, the patient's temperature was 105.5 ~ F, and she was alert. Tender bilateral enlarged cervical iympl, nodes, hepatosplenomegaly, erythema of tile tonsils, and ecchymoses of lower extremities were observed. Neither rash nor jaundice was present. Hemoglobin was 9.8 g/dl, and hematocrit was 28 per cent. The leukocyte count was 2,300/ram? with 35 per cent segmented neutrophils, 21 per cent bands, 43 per cent lymphocytes, and 1 per cent monocytes. Atypical lymphocytes were not noted. The platelets were 6 7 , 0 0 0 / m m j, reticulocytes 0.5 per cent, and erythrocyte sedimentation rate 4 ram/hr. The prothrombin time was 38.8/11.5 and the partial thromboplastin time was greater than two minutes. The total bilirubin was 6.3 mg/dl, direct 3.8 mg/dl, alkaline phosphatase 326 U/liter (normal, I00 U/liter), aspartate aminotransferase (SGOT) 795 U/liter (normal, 40 U/I), lactate dehydrogenase 2,374 U/liter (normal, 225 U/liter), and creatinine phospbokinase 2,215 U/liter (normal, 225 U/liter). Intravenous fluids and ampicillin were given. Transfusions of fresh frozen plasma, erythrocytes, and cryoprecipitate were administered following the passage of bright red blood by rectunt. Ascites and a right pleural effusion appeared. Fibrin split products were measured in the plasma at 27.04 ng/ml (normal, 5.6 ng/ml). Intravenous gentamicin and corticosteroids were given. Antinuclear antibody, a serologic test for syphilis (VDRL), and Monospot tests were negative. On the third hospital day, the plasma ammonia was elevated at 120 itmoles/liter (normal, 11 to 35 itmol/liter). Lactulose was given for asterixis, lethargy, and confusion. On the fourth hospital day the prothrombin time was 79/11.7. On the fifth hospital day, papilledema and obtundation were observed. Despite therapy with intravenous fluids, pressor agents, and blood transfusions, the patient died on the fifth day. Four sets of blood cultures and a culture of pleural fluid were negative. Viral Diagnostic Findings* Serologic tests for EBV-related antibodies performed on a specimen drawn on the second hospital day were as follows: the titer of IgM antibody to viral capsid antigen (VCA) was 1:20; IgG-VCA antibody 1:10,240; early antigen-D component (EA-D) 1:2,560; early antigen-R component (EA-R) less than 1:5; and Epstein-Barr nuclear antigen (EBNA) 1:5. Antibodies to cytomegalovirus were present in a titer of 1:8. In a blood sample obtained on the third hospital day, the titer of IgM-VCA was the same, IgG-VCA was 1:5,120,
Received September 24, 1982, from the Department of Pathology, New York University School of Medicine, New York City. Accepted for publication October 29, 1982. * Resident in Pathology. I" Assistant Professor of Pathology and Pediatrics. Address correspondence and reprint requests to Dr. Greco: Department of Pathology~MSB 611, New York University Medical Center, 550 First Ave, New York, NY 10016.
* Tests performed by Virolab Inc, Emeryville, California.
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FIGURE i (left). Peripancreatic lymph node with broad zone of non-suppurative necrosis and generalized lymphoid cell depletion. Germinal centers are absent. (Hematoxylin-eosin stain, x40.) FIGURE2 (right). Phagocytasis of erythrocytes and nucleated cells in bone marrow. (Hematoxylin-eosin stain. •
EA-D 1:1,280, EA-R 1:5, EBNA 1:20, and antibody to CMV was unmeasurable.
Gross Pathologic Findings A complete autopsy was performed 16 hours after death. Cutaneous ecchymoses and jaundice were evident. The abdominal and pleural cavities contained hemorrhagic fluid. The heart was normal and the lungs were congested and edematous. The liver was mildly enlarged at 1,900 g, brown to orange in color, and flabby in consistency. After formalin fixation, scattered light tan punctate foci 2 to 5 mm in diameter became enhanced against the deep green color of tile parenchyma. Tile spleen was diffusely enlarged at 900 g, deeply congested, and, though solid in consistency, friable. Patchy tan-gray areas were distributed throughout the parenchyma. Lymph nodes in the cervical, mediastinal, para-aortic, and celiac groups measured up to 2.5 cm in diameter. On cut section, the homogeneous gray lymphoid tissue showed irregular areas of necrosis with hemorrhage. The thymus gland weighed 9 g (normal for age, 15.9 to 49.7 g4) and was fatty in consistency. The gastric mucosa was congested, and there was focal mucosal hemorrhage in the.cecum. The mucosal surfaces of the pharynx, larynx, trachea, and proximal bronchi were diffusely ulcerated with friable superficial exudate. Other organs ivere unremarkable.
Histopathologic Findings Histiocytes with bland nuclei and abundant eosinophilic cytoplasm containing erythrocytes and nucleated blood cells were present throughout hepatic sinusoids and portal areas. Portal triads contained a lymphohistiofiytic infilti'ate. There 291
were diffuse fatty change and occasional loci of hepatocellular necrosis. Splenic cords, sinusoids, and veins contained abundant histiocytes with ingested nucleated blood ceils and erythrocytes. Extensive necrosis of the periarterial lymphoid sheaths as well as of broad areas of red pulp was present. The wall of a large splenic blood vessel as well as capsular and trabecular connective tissue was infiltrated by mononuclear cells. Neutrophils and plasma cells were absent. Lymph nodes revealed broad irregular zones of necrosis in cortex and medullae containing eosinophilic material, ghosts of cells, karyorrhectic debris, and occasional mononuclear cells. These areas were surrounded by narrow rims of viable cells adjacent to sinusoids and capsule (fig. 1). Dilated subcapsular, trabecular, and medullary sinuses were filled with bemophagocytic histiocytes. Phagocytosis of nucleated cells resembling lymphocytes was most prominent, and ingested erythrocytes were less conspicuous. The cortex, paracortex, and medulla showed loss of distinction and marked cellular depletion with sparse small iymphocytes and hemophagocytic histiocytes distributed throughout. Follicles were not discernible. Bacterial, fungal, and acid-fast stains of spleen, liver, and lymph nodes were negative. The bone marrow was generally hypercellular; however, erythrocyte precursors were diminished and there was a leftward shift in the myeloid series. Megakaryocytes were increased. Numerous histiocytes were packed with erythrocytes and with numerous nucleated forms of either erythroid or lymphoid origin (fig. 2). Hemophagocytic histiocytes were also found in the pericapsular fat of lymph nodes and adrenal glands, in the cardiac interstitium, and perivascularly in the brain. The thymus revealed lymphoid depletion, loss of distinction between cortex and medulla, and fatty replacement. The larynx showed diffuse ulceration of mucosa with infiltration by neutrophils and mononuclear cells. The surface was covered by fibrinous exudate containing clusters of gram-positive cocci.
HUMAN PATHOLOGY
Volume '15, No. 3 [March "1984]
Viral inclusions were not observed in any tissue. Electron microscopic studies of liver and brain specimens failed to demonstrate viral particles. Ingested erythrocytes were seen in histiocytes in hepatic sinusoids. DISCUSSION The patient's clinical course was typical of that described in the original series ~of VAHS, in which a prodrome of a nonspecific viral illness is followed within two to six weeks by an abrupt increase in severity of constitutional symptoms, higher fever, lymphadenopathy, hepatosplenomegaly with hepatocellular dysfunction, pancytopenia, and disseminated int~vascular coagulation. The less typical features of pulmonary infiltrates and skin rash were absent in our patient. Six of the 19 patients in the original series died in the acute episode. The remainder recovered within two to eight w e e k s . I In the original series, well-differentiated histiocytes containing phagocytosed erythrocytes, platelets, and nucleated blood cells were found in the bone marrow; in the sinusoids and red pulp of the spleen; in the sinuses, medullae, and cortex of lymph nodes; and in the leptomeninges and in hepatic sinusoids in association with a portal lymphohistiocytic infiltrate. Focal hepatic necrosis was consistently present. In our case, hemophagocytic histiocytes were also found in the cardiac interstitium, in perilymph-nodai and periadrenal fat, and perivascularly in the brain rather than in the meninges. The non-suppurative necrosis observed in the lymph nodes and spleen of our patient has not been previously reported in VAHS. In the spleen, necrosis involved both red and white pulp; however, in many loci the necrosis was confined to the periarterial lymphoid sheaths. Splenic necrosis confined to the periarterial lymphoid sheath and lymph nodal necrosis have been reported in cases of the X-linked iymphoproliferative syndrome and other immunodeficiency states with EBV infection, s'6 Focal splenic necrosis lms been observed in familial erythrophagocytic lymphohistiocytosis.7 Also included in the differential diagnosis of non-suppurative necrosis of lymph node parenchyma are influenza, 8 systemic lupus erythematosus 9 diphenyl-hydantoin toxicity, l~ congenital herpes simplex viral infection, ~ familial erythrophagocytic lymphohistiocytosis,7 typical infectious mononucleosis, ~ and the subacute necrotizing iymphadenitis that has been described in Japan) s Necrosis in the presence of lymphoid depletion rather than proliferation and hemophagocytic histiocytosis distinguish VAHS from these diseases, with the exception of familial erythrophagocytic lymphohistiocytosis, which may be morphologically indistinguishable from VAHS but differs in its clinical features. In our patient, lymphoid cells were markedly depleted in B and T cell areas of lymph nodes and spleen, and germinal centers were absent. Lymphoid depletion was a feature of the previous cases of VAHS) '~ Some germinal centers were present in lymph nodes in previously reported cases, but these had a "burned-out" appearance. Lymphoid depletion was variably present in fatal infectious mononucleosis of the X-linked lymphoproliferative syndrome; in contrast, many such cases showed immunoblastic and plasmacytic proliferation rather than depletion, s The thynms gland in our case was also depleted oflymphocytes and was subnormal in size. Again, this tins been seen in familial erythrophagocytic lymphohistiocytosis,7 the X-linked lymphoproliferative syndrome, and other immunodeficiency states, s Whether this represents a primary fea-
ture of V A H S or secondary involution due to acute illness has not been determined. Bone marrow examination is likely to provide the diagnosis in VAHSI; however, in our case, the bone marrow aspiration and biopsy performed five days before death showed neither hemophagocytosis nor histiocytosis at a time when lymphadenopathy, hepatosplenomegaly, and cytopenia were already present. This observation is in agreement with the view of Risdall et al. t ttmt the cytopenias reflect, at least in part, a direct depression of hematopoiesis rather than consumption of formed elements by histiocytes. This also shows that a negative bone marrow examination does not exclude the diagnosis of VAHS. Detection of antibodies to EBV-specific antigens, i.e., VCA, EA-D, and EBNA, permit the diagnosis of EBV infection in the absence of heterophil antibody or the typical features of infectious mononucleosis. In typical infectious mononucleosis, VCA and EA-D antibodies reach their peak early in the course of clinical illness. Titers greater than 1:320 are in the high range for both antibodies. Convalescence is accompanied by a progressive fall in these species and a progressive rise in anti-EBNA, which then persists for decades or for life) 4 Our patient's sera contained antibodies to VCA and EA-D in extraordinarily high titers, accompanied by a proportionately low titer of antibody to EBNA. Atypical patterns of antibody response to EBV infection with high titers of lgG antibodies to VCA and EA and delayed or impaired production ofanti-EBNA t5 have been observed m immunosuppressive states. For example, in a report of two cases, one of Hodgkin's disease in remission and one of chronic lymphatic leukemia in remission) ~ titers were as high as 1:20,480 for IgG-anti-VCA and 1:1,280 for antiEA-D. Anti-EBNA, although present, was proportionately much lower~less than 1:80-1:160. Defects in a number of cell-mediated immune fimctions were demonstrated. Another reported c a s e 17 w a s that of a 5-year-old girl who died from prolonged EBV infection associated with hypergammaglobulinemia and systemic immunoblastic proliferation. In this case titers were as high as 1:160,000 for IgG-anti VCA and 1:80,000 for anti-EA. Anti-EBNA was present, but again in the proportionately much lower titer of I : 1,280. Defects in hmnorai and cell-mediated immunity were investigated but not demonstrated, but a defect in immune interferon secretion was observed. In summary, we have presented the previously unreported findings of non-suppurative necrosis in the lymph nodes and spleen of a patient with virus-associated hemophagocytic syndrome in the presence of extraordinarily ifigh titers of antibodies to EB viral antigens. Although the clinical history did not indicate an immunosuppressive state prior to the onset of clinical illness, the lymphoid depletion and necrosis, the atypical EBV antibody response, and the previously known association of VAHS with immunosuppression implicate an altered immunologic status as a contributory factor in the pathogenesis of this patient's fatal illness.
Acknowledgments.
Tile a u t h o r s t h a n k Dr. Q. Valensi for reviewing the hlstologic material.
REFERENCES 1. Risdall RJ, McKenna RW, Nesbit ME, et al: Virus-associated hemophagocytlc syndrome. Cancer 44:993, 1979 2. Wilson ER, Malluh A, Stagno S, et ah Fatal Epstein-Barr virus-associated hemophagocytic syndrome. J Pedlatr 98:260, 1981 3. Look AT, Naegele RF, CalIihan T, et al: Fatal Epstein-Barr virus infection in a child with acute lymphoblastic leukemia in remission. Cancer Res 41:4280, 1981
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CASE STUDIES 4. Rosai J, Levine GD: Tumors of the thymus. Atlas of Tumor Pathology, second series, fascicle 13. Washington, DC, Armed Forces Institute of Pathology, 1976 5. Purtilo DT, Sakamoto K, Saemundsen AK, et al: Documentation of EpsteinBarr virus infection in immunodeficient patients with life-threatening lymphoproliferative diseases by clinical, virological, and immunopatbological studies. Cancer Res 41:4226, 1981 6. Provisor AJ, lacuoneJJ, Chilcote RR, et al: Acquired agammaglobulinemia after a life-threatening illness with clinical and laboratory features of infectious mononucleosis in three related male children. N Engl J Med 293:62, 1975 7. Perry MC, Ilarrison EG, Burgert EO, et al: Familial erythrophagocytic lymphohistiocytosis. Cancer 38:209, 1976 8. Tindle BtI: Lymphadenopathy in influenza. In Coulson WF (ed): Surgical Pathology, vol 2. Plfiladelphia, JB Lippincott Co, 1978, p 904 9. RosaiJ: Ackerman's Surgical Pathology, sixth edition. St. Louis, CV Mosby Co, 1981, p 1168 10. Krasznai G, Gyory GY: ilydantoin lymphadenopathy. J Pathol Bacteriol 95:314, 1968
1I. Singer DB: Pathology of neonatal herpes simplex virus infection. In Rosenberg 11S, Bernstein J (eds): Perspectives in Pediatric Pathology, vol 6. New York, Masson Publistfing USA, 1981, p 243 12. Tindle BII, Parker JW, Lukes RJ: "Reed-Sternberg cells" in infectious mononucleosis? Am J Clin Pathol 58:607, 1972 13. lmamuca M, Ueno H, Matsuura A, et al: An ultrastructural stud)' of subacute necrotizing lymphadenitis. Am J Pathol 107:292, 1982 14. Henle W, llenle GE, llorwitz CA: Epstein-Barr virus specific diagnostic tests in infectious mononucleosis. I1UM PATIIOL5:551, 1974 15. Henle W, }tenle G: Epstein-Barr virus specific serology in immunologlcally compromised individuals. Cancer Res 41:4222, 1981 16. Masucci MG, Szigeti R, Ernberg I, et al: Cell-mediated immune reactions in three patients with malignant lymphoproliferative disease in remission and abnormally high Epsteln-Barr virus antibody titers. Cancer Res 41:4292, 1981 17. Virelizler J-L, Lenoir G, Griscelli C: Persistent Epstein-Barr virus infection in a child with hypergammaglobulinemia and immunoblastic proliferation associated with a selective defect in immune interferon secretion. Lancet 2:231, 1978
SIMULTANEOUS ALPHA-I-ANTITRYPSIN A C C U M U L A T I O N IN LIVER A N D PANCREAS FRANCESCO CALLEA, M D , * J O t l A N FEVERY, A H O , t AND VALEER
Inclusions posith,e for periodic acid-Schiff,, resistant to diastase, and imm unoreacth,e to alpha-l-antitrypsin (AA T)werefouad in hepatoc)'tes and pancreatic islet cells of a patient with clinical and pathologic features of AAT deficieno'. Alpha-l-antitrypsin was detected in all pancreatic islets, and AAT-positk,e cells were observed in the excretory pancreatic ducts. Thesefindings suggest that the pancreas synthesizes AAT and possibly serves as a "storage" place in AAT deficieno'. Intercalated cells in the excretory pancreatic ducts may be an additional source of AAT. Hu, xI PATHOL 15:293--295, 1984.
Alptm-l-antitrypsin (AAT) is the major protein of the alplm-I globulin fraction. Its functions are only partially known, but serum deficiency of this protein is associated with pulmonary emphysema and hver orrhosls. ' By a highresolution technique (isoelectric focusing) several phenotypes have been identified. The presence of one or two pi Z alleles is associated with the so-called A A T deficiency state. It has long been thought that A A T was only synthetized by the liver and that it accumulated in the hepatocytic endoplasmic retictdum of subjects with AAT deficiency. However, by use ofimmunofluorescence techniques with specific antibodies, A A T was recently demonstrated in pancreatic islets in humans 3'4 and mice. 5 Moreover, mouse pancreatic islets have been shown to synthetize A A T in vitro. 5 We wish to report the first case of AAT accumulation occurring simultaneously in liver and pancreas of a patient with clinical and pathologic features of AAT deficiency. 9
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REPORT OF A CASE Ascites developed in a 54-year-old woman who had recurrent thrombopldebitis since the age of 23. Liver cirrhosis Received from the Department Medical Research, Department of Internal Medicine, Catholic University Leuven, Leuven, Belgium. Accepted for publication N o v e m b e r 1, 1982. * Research Assistant, Department o f Medical Research. Professor o f Medicine, Department o f Internal Medicine. Professor of Pathology, Chief Laboratory of Histochemistry and Cytochemistry. Address correspondence and reprint requests to Dr. Callea: Labo Cyto en Histochemie, K.U.L., Minderbroersstraat 12, B-3000 Leuven, Belgium.
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was diagnosed on peritoneoscopy, but biopsy was not performed at tltat time because of Itypoprotitrombinemia. In 1978, jaundice, ascites, and edema developed. Splenomegaly was present. Serum bilirubin was 3.9 mg/dl, aspartate aminotransferase 28 U/liter, alanine aminotransferase 27 U/liter, alkaline phosphatase 143 U/liter, IgA 538 mg/dl, IgM 235 mg/dl, and IgG 1,203 mg/dl. Results of tests for HBsAg, HBsAb, HBcAb, ANF, and AMA were negative. On serum electrophoresis, tile alplta-1 globulin peak was absent9 Serum AAT was less than 53 m g / d l (normal, 200 mg/dl). Owing to recurrent massive ascites, a peritoneojugular shunt by the method of Le Veen was performed. A few days later, tile patient died in shock because of bleeding from gastric ulcerations. At autopsy tile liver weighed 750 g and bad a nodular appearance. A well-demarcated 3-cm tumor nodule was found on liver sections. Stones were present in the gallbladder but not in the bile ducts. The pancreas had a normal appearance; neither signs of pancreatitis nor tumor were observed. Tile lungs weighed 330 and 300 g, respectively. They had obvious features of emphysema. An emphysetnatous bulla 10 cm in diameter was present in the right lung. On Itistologic examination, the liver showed features of advanced multilobular cirrhosis. Lymphoid infiltration and a striking proliferation of bile ductules in the connective tissue septa were observed. Hepatocytes contained numerous PAS-positive, diastase-resistant inclusions of variable size. The globules were unevenly distributed throughout the cirrhotic nodules, and in some were absent. T h e inclusions, which were identified as AAT on specific immunostaitfing, showed more pronounced reactivity at tbeir peripberies (fig. 1). At the ultrastructural level, a semi-electron-dense amorphous material was observed within dilated cisternae or the rough endoplasmic reticulum of the hepatocytes. The tumor nodule had features of a well-differentiated hepatocellular carcinoma. T h e tumor cells did not have PAS-positiv'e inclusions. Bile pigments and copper granules were present in both tumoral and non-tumoral hepatocytes. The pancreas showed an impressive increase in the number and size of islets, a microadenoma, and occasional islet cell dysplasia. Many islet cells contained PAS-positive,
Volume 15, No. 3 (March 1984)
VIRUS-ASSOCIATED HEMOPHAGOCYTIC SYNDROME DUE TO EPSTEIN-BARR VIRUS ROBERT P. REISMAN,MD,* AND M. ALBA GRECO,MDf
The dinical and autopsv findings in a case of virus-associated hemophagocytic syndrome in a pra,iously health)' 17-year-old g~rl are presented. Serum titers to Epstein-Barr viral antigens were umtsually elevated, h~ addition to widespread proliferation of hemophagoo.tic histioo'tes and lymphoid depletion, there were areas of non-suppurative necrosis in lymph nodes and spleen, which have not been pra,iously reported. HUM PArHOL 15:290--293, 1984. T h e virus-associated hemophagocytic syndrome (VAHS) is a benign disorder cbaracterized by the proliferation of cytologically benign hemophagocytic histiocytes in the bone marrow, lymph nodes, spleen, liver, and leptomeninges in association with an acute systemic viral infection. Of the 19 patients described in the original report in 1979 by Risdall et al.,' 13 were receiving immunosuppressive therapy for renal transplantation and one for systemic lupus erythematosus. The remaining five had previously been well. Documentation of acute infection with cytomegalovirus was present in ten patients, Epstein-Barr virus (EBV) in two, and herpes simplex, varicella-zoster, and adenovirus in one each. Subsequently, two additional fatal cases of EBV-associated VAHS have been reported: one in a previously well 16-year-old boy e and the other in a 9-year-old boy with acute lymphoblastic leukemia in remission. ~ We present the clinical and autopsy findings in the case of a previously well 17-year-old girl with EBV-related VAHS, in which the lymph nodes and spleen revealed marked lymphoid depletion and extensive necrosis. T o the best of our knowledge, the presence of necrosis in lymph nodes and spleen has not been previously reported in VAHS. In addition, titers of antibodies to viral capsid antigen and early antigen-D were unusually elevated. REPORT OF A CASE A 17-year-old white girl was transferred to the New York University Hospital Pediatric Service for evaluation of neutropenia and hepatosplenomegaly. She had been well until two weeks previously, when a sore throat and fever developed, for which she was given oral amoxicillin and intramuscular penicillin. A throat culture and Monospot test were negative. A purified protein derivative tuberculin test was negative, One week later, on admission to another hospital, hepatosplenomegaly was noted. The leukocyte count was 2,200/mm 3, the platelets 68,000/ram s, and the hemoglobin 10.6 g/dl. A bone marrow aspirate and biopsy were performed two days prior to the patient's transfer to NYU. This material and a peripheral blood smear obtained the same day were reviewed by the authors and revealed a normocellular bone marrow. Neither histiocytosis nor he-
mophagocytosis were present. Atypical lymphocytes were not increased in tile peripheral blood. A chest radiograph was normal. During tiffs hospitalization, the patient experienced nausea, vomiting, diarrhea, arthralgia, easy bruising, and a dry cough. The patient had remote histories of measles, mumps, varicella, and intmunization with DPT. Carefully elicited family and social itistories revealed no pertinent information. Upon admission to NYU.Hospital, the patient's temperature was 105.5 ~ F, and she was alert. Tender bilateral enlarged cervical iympl, nodes, hepatosplenomegaly, erythema of tile tonsils, and ecchymoses of lower extremities were observed. Neither rash nor jaundice was present. Hemoglobin was 9.8 g/dl, and hematocrit was 28 per cent. The leukocyte count was 2,300/ram? with 35 per cent segmented neutrophils, 21 per cent bands, 43 per cent lymphocytes, and 1 per cent monocytes. Atypical lymphocytes were not noted. The platelets were 6 7 , 0 0 0 / m m j, reticulocytes 0.5 per cent, and erythrocyte sedimentation rate 4 ram/hr. The prothrombin time was 38.8/11.5 and the partial thromboplastin time was greater than two minutes. The total bilirubin was 6.3 mg/dl, direct 3.8 mg/dl, alkaline phosphatase 326 U/liter (normal, I00 U/liter), aspartate aminotransferase (SGOT) 795 U/liter (normal, 40 U/I), lactate dehydrogenase 2,374 U/liter (normal, 225 U/liter), and creatinine phospbokinase 2,215 U/liter (normal, 225 U/liter). Intravenous fluids and ampicillin were given. Transfusions of fresh frozen plasma, erythrocytes, and cryoprecipitate were administered following the passage of bright red blood by rectunt. Ascites and a right pleural effusion appeared. Fibrin split products were measured in the plasma at 27.04 ng/ml (normal, 5.6 ng/ml). Intravenous gentamicin and corticosteroids were given. Antinuclear antibody, a serologic test for syphilis (VDRL), and Monospot tests were negative. On the third hospital day, the plasma ammonia was elevated at 120 itmoles/liter (normal, 11 to 35 itmol/liter). Lactulose was given for asterixis, lethargy, and confusion. On the fourth hospital day the prothrombin time was 79/11.7. On the fifth hospital day, papilledema and obtundation were observed. Despite therapy with intravenous fluids, pressor agents, and blood transfusions, the patient died on the fifth day. Four sets of blood cultures and a culture of pleural fluid were negative. Viral Diagnostic Findings* Serologic tests for EBV-related antibodies performed on a specimen drawn on the second hospital day were as follows: the titer of IgM antibody to viral capsid antigen (VCA) was 1:20; IgG-VCA antibody 1:10,240; early antigen-D component (EA-D) 1:2,560; early antigen-R component (EA-R) less than 1:5; and Epstein-Barr nuclear antigen (EBNA) 1:5. Antibodies to cytomegalovirus were present in a titer of 1:8. In a blood sample obtained on the third hospital day, the titer of IgM-VCA was the same, IgG-VCA was 1:5,120,
Received September 24, 1982, from the Department of Pathology, New York University School of Medicine, New York City. Accepted for publication October 29, 1982. * Resident in Pathology. I" Assistant Professor of Pathology and Pediatrics. Address correspondence and reprint requests to Dr. Greco: Department of Pathology~MSB 611, New York University Medical Center, 550 First Ave, New York, NY 10016.
* Tests performed by Virolab Inc, Emeryville, California.
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FIGURE i (left). Peripancreatic lymph node with broad zone of non-suppurative necrosis and generalized lymphoid cell depletion. Germinal centers are absent. (Hematoxylin-eosin stain, x40.) FIGURE2 (right). Phagocytasis of erythrocytes and nucleated cells in bone marrow. (Hematoxylin-eosin stain. •
EA-D 1:1,280, EA-R 1:5, EBNA 1:20, and antibody to CMV was unmeasurable.
Gross Pathologic Findings A complete autopsy was performed 16 hours after death. Cutaneous ecchymoses and jaundice were evident. The abdominal and pleural cavities contained hemorrhagic fluid. The heart was normal and the lungs were congested and edematous. The liver was mildly enlarged at 1,900 g, brown to orange in color, and flabby in consistency. After formalin fixation, scattered light tan punctate foci 2 to 5 mm in diameter became enhanced against the deep green color of tile parenchyma. Tile spleen was diffusely enlarged at 900 g, deeply congested, and, though solid in consistency, friable. Patchy tan-gray areas were distributed throughout the parenchyma. Lymph nodes in the cervical, mediastinal, para-aortic, and celiac groups measured up to 2.5 cm in diameter. On cut section, the homogeneous gray lymphoid tissue showed irregular areas of necrosis with hemorrhage. The thymus gland weighed 9 g (normal for age, 15.9 to 49.7 g4) and was fatty in consistency. The gastric mucosa was congested, and there was focal mucosal hemorrhage in the.cecum. The mucosal surfaces of the pharynx, larynx, trachea, and proximal bronchi were diffusely ulcerated with friable superficial exudate. Other organs ivere unremarkable.
Histopathologic Findings Histiocytes with bland nuclei and abundant eosinophilic cytoplasm containing erythrocytes and nucleated blood cells were present throughout hepatic sinusoids and portal areas. Portal triads contained a lymphohistiofiytic infilti'ate. There 291
were diffuse fatty change and occasional loci of hepatocellular necrosis. Splenic cords, sinusoids, and veins contained abundant histiocytes with ingested nucleated blood ceils and erythrocytes. Extensive necrosis of the periarterial lymphoid sheaths as well as of broad areas of red pulp was present. The wall of a large splenic blood vessel as well as capsular and trabecular connective tissue was infiltrated by mononuclear cells. Neutrophils and plasma cells were absent. Lymph nodes revealed broad irregular zones of necrosis in cortex and medullae containing eosinophilic material, ghosts of cells, karyorrhectic debris, and occasional mononuclear cells. These areas were surrounded by narrow rims of viable cells adjacent to sinusoids and capsule (fig. 1). Dilated subcapsular, trabecular, and medullary sinuses were filled with bemophagocytic histiocytes. Phagocytosis of nucleated cells resembling lymphocytes was most prominent, and ingested erythrocytes were less conspicuous. The cortex, paracortex, and medulla showed loss of distinction and marked cellular depletion with sparse small iymphocytes and hemophagocytic histiocytes distributed throughout. Follicles were not discernible. Bacterial, fungal, and acid-fast stains of spleen, liver, and lymph nodes were negative. The bone marrow was generally hypercellular; however, erythrocyte precursors were diminished and there was a leftward shift in the myeloid series. Megakaryocytes were increased. Numerous histiocytes were packed with erythrocytes and with numerous nucleated forms of either erythroid or lymphoid origin (fig. 2). Hemophagocytic histiocytes were also found in the pericapsular fat of lymph nodes and adrenal glands, in the cardiac interstitium, and perivascularly in the brain. The thymus revealed lymphoid depletion, loss of distinction between cortex and medulla, and fatty replacement. The larynx showed diffuse ulceration of mucosa with infiltration by neutrophils and mononuclear cells. The surface was covered by fibrinous exudate containing clusters of gram-positive cocci.
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Volume '15, No. 3 [March "1984]
Viral inclusions were not observed in any tissue. Electron microscopic studies of liver and brain specimens failed to demonstrate viral particles. Ingested erythrocytes were seen in histiocytes in hepatic sinusoids. DISCUSSION The patient's clinical course was typical of that described in the original series ~of VAHS, in which a prodrome of a nonspecific viral illness is followed within two to six weeks by an abrupt increase in severity of constitutional symptoms, higher fever, lymphadenopathy, hepatosplenomegaly with hepatocellular dysfunction, pancytopenia, and disseminated int~vascular coagulation. The less typical features of pulmonary infiltrates and skin rash were absent in our patient. Six of the 19 patients in the original series died in the acute episode. The remainder recovered within two to eight w e e k s . I In the original series, well-differentiated histiocytes containing phagocytosed erythrocytes, platelets, and nucleated blood cells were found in the bone marrow; in the sinusoids and red pulp of the spleen; in the sinuses, medullae, and cortex of lymph nodes; and in the leptomeninges and in hepatic sinusoids in association with a portal lymphohistiocytic infiltrate. Focal hepatic necrosis was consistently present. In our case, hemophagocytic histiocytes were also found in the cardiac interstitium, in perilymph-nodai and periadrenal fat, and perivascularly in the brain rather than in the meninges. The non-suppurative necrosis observed in the lymph nodes and spleen of our patient has not been previously reported in VAHS. In the spleen, necrosis involved both red and white pulp; however, in many loci the necrosis was confined to the periarterial lymphoid sheaths. Splenic necrosis confined to the periarterial lymphoid sheath and lymph nodal necrosis have been reported in cases of the X-linked iymphoproliferative syndrome and other immunodeficiency states with EBV infection, s'6 Focal splenic necrosis lms been observed in familial erythrophagocytic lymphohistiocytosis.7 Also included in the differential diagnosis of non-suppurative necrosis of lymph node parenchyma are influenza, 8 systemic lupus erythematosus 9 diphenyl-hydantoin toxicity, l~ congenital herpes simplex viral infection, ~ familial erythrophagocytic lymphohistiocytosis,7 typical infectious mononucleosis, ~ and the subacute necrotizing iymphadenitis that has been described in Japan) s Necrosis in the presence of lymphoid depletion rather than proliferation and hemophagocytic histiocytosis distinguish VAHS from these diseases, with the exception of familial erythrophagocytic lymphohistiocytosis, which may be morphologically indistinguishable from VAHS but differs in its clinical features. In our patient, lymphoid cells were markedly depleted in B and T cell areas of lymph nodes and spleen, and germinal centers were absent. Lymphoid depletion was a feature of the previous cases of VAHS) '~ Some germinal centers were present in lymph nodes in previously reported cases, but these had a "burned-out" appearance. Lymphoid depletion was variably present in fatal infectious mononucleosis of the X-linked lymphoproliferative syndrome; in contrast, many such cases showed immunoblastic and plasmacytic proliferation rather than depletion, s The thynms gland in our case was also depleted oflymphocytes and was subnormal in size. Again, this tins been seen in familial erythrophagocytic lymphohistiocytosis,7 the X-linked lymphoproliferative syndrome, and other immunodeficiency states, s Whether this represents a primary fea-
ture of V A H S or secondary involution due to acute illness has not been determined. Bone marrow examination is likely to provide the diagnosis in VAHSI; however, in our case, the bone marrow aspiration and biopsy performed five days before death showed neither hemophagocytosis nor histiocytosis at a time when lymphadenopathy, hepatosplenomegaly, and cytopenia were already present. This observation is in agreement with the view of Risdall et al. t ttmt the cytopenias reflect, at least in part, a direct depression of hematopoiesis rather than consumption of formed elements by histiocytes. This also shows that a negative bone marrow examination does not exclude the diagnosis of VAHS. Detection of antibodies to EBV-specific antigens, i.e., VCA, EA-D, and EBNA, permit the diagnosis of EBV infection in the absence of heterophil antibody or the typical features of infectious mononucleosis. In typical infectious mononucleosis, VCA and EA-D antibodies reach their peak early in the course of clinical illness. Titers greater than 1:320 are in the high range for both antibodies. Convalescence is accompanied by a progressive fall in these species and a progressive rise in anti-EBNA, which then persists for decades or for life) 4 Our patient's sera contained antibodies to VCA and EA-D in extraordinarily high titers, accompanied by a proportionately low titer of antibody to EBNA. Atypical patterns of antibody response to EBV infection with high titers of lgG antibodies to VCA and EA and delayed or impaired production ofanti-EBNA t5 have been observed m immunosuppressive states. For example, in a report of two cases, one of Hodgkin's disease in remission and one of chronic lymphatic leukemia in remission) ~ titers were as high as 1:20,480 for IgG-anti-VCA and 1:1,280 for antiEA-D. Anti-EBNA, although present, was proportionately much lower~less than 1:80-1:160. Defects in a number of cell-mediated immune fimctions were demonstrated. Another reported c a s e 17 w a s that of a 5-year-old girl who died from prolonged EBV infection associated with hypergammaglobulinemia and systemic immunoblastic proliferation. In this case titers were as high as 1:160,000 for IgG-anti VCA and 1:80,000 for anti-EA. Anti-EBNA was present, but again in the proportionately much lower titer of I : 1,280. Defects in hmnorai and cell-mediated immunity were investigated but not demonstrated, but a defect in immune interferon secretion was observed. In summary, we have presented the previously unreported findings of non-suppurative necrosis in the lymph nodes and spleen of a patient with virus-associated hemophagocytic syndrome in the presence of extraordinarily ifigh titers of antibodies to EB viral antigens. Although the clinical history did not indicate an immunosuppressive state prior to the onset of clinical illness, the lymphoid depletion and necrosis, the atypical EBV antibody response, and the previously known association of VAHS with immunosuppression implicate an altered immunologic status as a contributory factor in the pathogenesis of this patient's fatal illness.
Acknowledgments.
Tile a u t h o r s t h a n k Dr. Q. Valensi for reviewing the hlstologic material.
REFERENCES 1. Risdall RJ, McKenna RW, Nesbit ME, et al: Virus-associated hemophagocytlc syndrome. Cancer 44:993, 1979 2. Wilson ER, Malluh A, Stagno S, et ah Fatal Epstein-Barr virus-associated hemophagocytic syndrome. J Pedlatr 98:260, 1981 3. Look AT, Naegele RF, CalIihan T, et al: Fatal Epstein-Barr virus infection in a child with acute lymphoblastic leukemia in remission. Cancer Res 41:4280, 1981
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CASE STUDIES 4. Rosai J, Levine GD: Tumors of the thymus. Atlas of Tumor Pathology, second series, fascicle 13. Washington, DC, Armed Forces Institute of Pathology, 1976 5. Purtilo DT, Sakamoto K, Saemundsen AK, et al: Documentation of EpsteinBarr virus infection in immunodeficient patients with life-threatening lymphoproliferative diseases by clinical, virological, and immunopatbological studies. Cancer Res 41:4226, 1981 6. Provisor AJ, lacuoneJJ, Chilcote RR, et al: Acquired agammaglobulinemia after a life-threatening illness with clinical and laboratory features of infectious mononucleosis in three related male children. N Engl J Med 293:62, 1975 7. Perry MC, Ilarrison EG, Burgert EO, et al: Familial erythrophagocytic lymphohistiocytosis. Cancer 38:209, 1976 8. Tindle BtI: Lymphadenopathy in influenza. In Coulson WF (ed): Surgical Pathology, vol 2. Plfiladelphia, JB Lippincott Co, 1978, p 904 9. RosaiJ: Ackerman's Surgical Pathology, sixth edition. St. Louis, CV Mosby Co, 1981, p 1168 10. Krasznai G, Gyory GY: ilydantoin lymphadenopathy. J Pathol Bacteriol 95:314, 1968
1I. Singer DB: Pathology of neonatal herpes simplex virus infection. In Rosenberg 11S, Bernstein J (eds): Perspectives in Pediatric Pathology, vol 6. New York, Masson Publistfing USA, 1981, p 243 12. Tindle BII, Parker JW, Lukes RJ: "Reed-Sternberg cells" in infectious mononucleosis? Am J Clin Pathol 58:607, 1972 13. lmamuca M, Ueno H, Matsuura A, et al: An ultrastructural stud)' of subacute necrotizing lymphadenitis. Am J Pathol 107:292, 1982 14. Henle W, llenle GE, llorwitz CA: Epstein-Barr virus specific diagnostic tests in infectious mononucleosis. I1UM PATIIOL5:551, 1974 15. Henle W, }tenle G: Epstein-Barr virus specific serology in immunologlcally compromised individuals. Cancer Res 41:4222, 1981 16. Masucci MG, Szigeti R, Ernberg I, et al: Cell-mediated immune reactions in three patients with malignant lymphoproliferative disease in remission and abnormally high Epsteln-Barr virus antibody titers. Cancer Res 41:4292, 1981 17. Virelizler J-L, Lenoir G, Griscelli C: Persistent Epstein-Barr virus infection in a child with hypergammaglobulinemia and immunoblastic proliferation associated with a selective defect in immune interferon secretion. Lancet 2:231, 1978
SIMULTANEOUS ALPHA-I-ANTITRYPSIN A C C U M U L A T I O N IN LIVER A N D PANCREAS FRANCESCO CALLEA, M D , * J O t l A N FEVERY, A H O , t AND VALEER
Inclusions posith,e for periodic acid-Schiff,, resistant to diastase, and imm unoreacth,e to alpha-l-antitrypsin (AA T)werefouad in hepatoc)'tes and pancreatic islet cells of a patient with clinical and pathologic features of AAT deficieno'. Alpha-l-antitrypsin was detected in all pancreatic islets, and AAT-positk,e cells were observed in the excretory pancreatic ducts. Thesefindings suggest that the pancreas synthesizes AAT and possibly serves as a "storage" place in AAT deficieno'. Intercalated cells in the excretory pancreatic ducts may be an additional source of AAT. Hu, xI PATHOL 15:293--295, 1984.
Alptm-l-antitrypsin (AAT) is the major protein of the alplm-I globulin fraction. Its functions are only partially known, but serum deficiency of this protein is associated with pulmonary emphysema and hver orrhosls. ' By a highresolution technique (isoelectric focusing) several phenotypes have been identified. The presence of one or two pi Z alleles is associated with the so-called A A T deficiency state. It has long been thought that A A T was only synthetized by the liver and that it accumulated in the hepatocytic endoplasmic retictdum of subjects with AAT deficiency. However, by use ofimmunofluorescence techniques with specific antibodies, A A T was recently demonstrated in pancreatic islets in humans 3'4 and mice. 5 Moreover, mouse pancreatic islets have been shown to synthetize A A T in vitro. 5 We wish to report the first case of AAT accumulation occurring simultaneously in liver and pancreas of a patient with clinical and pathologic features of AAT deficiency. 9
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REPORT OF A CASE Ascites developed in a 54-year-old woman who had recurrent thrombopldebitis since the age of 23. Liver cirrhosis Received from the Department Medical Research, Department of Internal Medicine, Catholic University Leuven, Leuven, Belgium. Accepted for publication N o v e m b e r 1, 1982. * Research Assistant, Department o f Medical Research. Professor o f Medicine, Department o f Internal Medicine. Professor of Pathology, Chief Laboratory of Histochemistry and Cytochemistry. Address correspondence and reprint requests to Dr. Callea: Labo Cyto en Histochemie, K.U.L., Minderbroersstraat 12, B-3000 Leuven, Belgium.
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DESMET, A H O *
was diagnosed on peritoneoscopy, but biopsy was not performed at tltat time because of Itypoprotitrombinemia. In 1978, jaundice, ascites, and edema developed. Splenomegaly was present. Serum bilirubin was 3.9 mg/dl, aspartate aminotransferase 28 U/liter, alanine aminotransferase 27 U/liter, alkaline phosphatase 143 U/liter, IgA 538 mg/dl, IgM 235 mg/dl, and IgG 1,203 mg/dl. Results of tests for HBsAg, HBsAb, HBcAb, ANF, and AMA were negative. On serum electrophoresis, tile alplta-1 globulin peak was absent9 Serum AAT was less than 53 m g / d l (normal, 200 mg/dl). Owing to recurrent massive ascites, a peritoneojugular shunt by the method of Le Veen was performed. A few days later, tile patient died in shock because of bleeding from gastric ulcerations. At autopsy tile liver weighed 750 g and bad a nodular appearance. A well-demarcated 3-cm tumor nodule was found on liver sections. Stones were present in the gallbladder but not in the bile ducts. The pancreas had a normal appearance; neither signs of pancreatitis nor tumor were observed. Tile lungs weighed 330 and 300 g, respectively. They had obvious features of emphysema. An emphysetnatous bulla 10 cm in diameter was present in the right lung. On Itistologic examination, the liver showed features of advanced multilobular cirrhosis. Lymphoid infiltration and a striking proliferation of bile ductules in the connective tissue septa were observed. Hepatocytes contained numerous PAS-positive, diastase-resistant inclusions of variable size. The globules were unevenly distributed throughout the cirrhotic nodules, and in some were absent. T h e inclusions, which were identified as AAT on specific immunostaitfing, showed more pronounced reactivity at tbeir peripberies (fig. 1). At the ultrastructural level, a semi-electron-dense amorphous material was observed within dilated cisternae or the rough endoplasmic reticulum of the hepatocytes. The tumor nodule had features of a well-differentiated hepatocellular carcinoma. T h e tumor cells did not have PAS-positiv'e inclusions. Bile pigments and copper granules were present in both tumoral and non-tumoral hepatocytes. The pancreas showed an impressive increase in the number and size of islets, a microadenoma, and occasional islet cell dysplasia. Many islet cells contained PAS-positive,