Case Reports R.G. (198 7) Exaggerated spontaneous release of platelet-derived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis. New England Journal of Medicine, 31 7, 202-209. Raccuglia, G. (1971 ) Gray platelet syndrome. A variety of qualitative platelet disorder. American Journal of Medicine, 5 1, 818. Slater. D.N.. Trowbridge, E.A. &Martin,J.F. (1983) The megakaryo-
543
cyte in thrombocytopenia: a microscopic study which supports the theory that platelets are produced in the pulmonary circulation. Thrombosis Research. 31, 163-1 7 6 . Trowbridge. E.A.. Martin, J.F. & Slater. D.N.(1 982) Evidence for a theory of physical fragmentation of megakaryocytes, implying that all platelets are produced in the pulmonary circulation. Thrombosis Research, 28, 461-475.
ANTI-ERYTHROPOIETIN ANTIBODIES I N HYPERVISCOSITY SYNDROME ASSOCIATED W I T H GIANT LYMPH NODE HYPERPLASIA (GLNH; CASTLEMAN'S DISEASE) Giant lymph node hyperplasia (GLNH) was first described i n adults (Castleman et al, 1 9 5 6 ) , but children have been affected (Keller et al, 1972). Originally this disease, resembling a thymoma, w a s believed t o affect only t h e mediastinum, b u t later reports prove occurrence in other areas (Keller et al, 1 9 7 2 ; Burgert et al, 1 9 7 5 ; Dorfmann & Cibull, 1978; Cohen, 1 9 5 7). T h e morphological characteristics of t h e variants of GLNH are hyaline-vascular and plasma cell type, which comprise approximately 9 1 % and 9% of cases. respectively. Unique to the plasma cell variant are t h e occasional systemic manifestations of hypergammaglobulinaemia, anaemia, and hyperviscosity (Burgert et al, 1975). A n anti-erythropoietin
(Ep) factor demonstrated via a mouse j9Fe uptake bioassay has been reported in t h e plasma cell variant of GLNH (Burgert et al, 1975).These anti-erythropoietin antibodies m a y be o n e component responsible for anaemia in GLNH aside from chemotherapy and/or a chronic disease state. T h e authors report a case of GLNH, plasma cell type, with demonstration of anti-Ep antibodies, hyperviscosity, anaemia, and subsequent response to steroid therapy. A 10-year-old black male presented with a s t h m a and a submandibular mass. Examination revealed proptosis, enlarged kidneys, and generalized lymphadenopathy. Right axillary lymph node biopsy demonstrated t h e plasma cell
B
A 1
2
3
4
5
r w t
IC
4 8 12 16 20 Serial twofold dilution
24
Fig 1. (A) Sodium dodecylsulphate polyacrylamide gel electrophoresis. Silver stain of protein standards (lane l ) , pure human and recombinant Ep (lanes 2 and 3), and crude Ep (lane 4). (B) Autoradiograph of an immunoblot of pure and crude Ep with patient's IgG containing antiBp antibodies. I4C protein molecular weight standards (lane I ) , pure human Ep (lane 2), pure human recombinant Ep (lane 3 ) , unlabelled protein molecular weight standards (lane 4). and crude human Ep (lane 5). Immunoreactive proteins were identified by ''1 labelled affinity purified sheep antihuman IgG as the second antibody. These results indicate that the IgG purified from pre-steroid treated serum recognizes human Ep from both pure and crude preparations. Corresponding blots with IgG from post-steroid treatment and normal control showed no detectable Ep bound. (C) Solid phase radioimmunoassay of patient's serum with pure Ep. Anti-Ep binding was detected by lL'I-labelled sheep anti-human Igs. Titration of binding was carried out by serial twofold dilution. Curve A represents the binding results of serum from pre-steroid treatment stage, curves B and C correspond to the binding results of serum from post-steroid treatment and from normal controls. respectively. These results demonstrate that pre-steroid treatment serum from this patient with Castleman's disease contains anti-Ep antibodies.
544
Case Reports
variant of GLNH with an unremarkable bone marrow biopsy. On admission his haematocrit was 0.24; blood viscosity 14.0 (normal range 1.4-1.8; Ostwald viscosimeter: viscosity = flow time of serum through viscosimeter divided by flow time of water through viscosimeter);and total protein was 100.0 g/l (albumin 18 g/l; IgG 12.2 g/l; IgM ~ 0 . g/l; 1 IgA 0.24 g/l). Red cell smear, indices, Coombs test, white blood cell count, and chemistries were not remarkable. Chest X-ray was significant for bilateral hilar adenopathy. The patient received plasmapheresis with a resulting diminution in viscosity to 5.5. Two months later the patient returned due to epistaxis, oral haemorrhage, occasional fatigue, and myopia. Laboratory results were: haematocrit 0.26, viscosity 14.2, total protein 165 g/l, albumin 24 g/l, platelet count 212 x 109/1. prothrombin time 12. Ss, and partial thromboplastin time 53 s. Plasmapheresis and steroid treatment resulted in a drop in total protein to 90 g/1 and viscosity of 4.5. Concomitantly, the haematocrit rose to 0.29, probably due to haemoconcentration. After 1 month of this regimen, his total protein and viscosity had dropped 26% and 71% respectively. This is in contrast to a 10%drop in total protein and a 36% drop in viscosity attributable to plasmapheresis. Haematocrit increased from 0.25 on admission to 0.43, a 71% increase. Over the past 2 years he has undergone periodic plasmapheresis and steroid therapy while remaining in a reasonably good state of health. Presently, he is at the 50th and 75th percentile for height and weight, respectively. The presence of anti-Ep antibodies in the patient’s serum was studied by four methods: (1)sodium dodecylsulphate polyacrylamide gel electrophoresis (Fig 1A) with molecular weight markers, and pure human Ep, and recombinant control Ep; (2) immunoblot (Fig 1B) with 14C labelled molecular weight markers, crude and pure human Ep, and recombinant Ep (Lee-Hnang, 1984); ( 3 )solid phase radioimmunoassay (Fig 1C); (4) the in vivo exhypoxic polycythaemic mice assay, including control (Camiscoli & Gordon, 1470), with data presented in the text. Sera from the child was positive for anti-Ep antibodies via ‘*jI radioimmunoassay (Fig 1C) and immunoblot techniques (Figs 1A and 1B). Whole sera was fractionated via a Protein A Sepharose CL-4B column, and both Protein A included and excluded immunoglobulin fractions inhibited jYFeuptake in the exhypoxic polycythaemic mouse bioassay by - 22.9% and - 32.5%, respectively, from normal serum fractions
( - 3.5% and
- 3.7% inhibition of 5YFe uptake). After 5 weeks ofsteroid therapy, serum from the child (total protein = 80 g/l, viscosity = 1.8. haematocrit =0.42) was again tested via mouse bioassay and had a + 6%increase in 5yFeuptake from standard control values, suggesting a compensatory increased level of erythropoietin. Steroid treatment, via reducing lymph node size and plasma cell number, reduces anti-Ep antibodies with preservation of free and biologically active Ep. In summary, this case report specifically demonstrates anti-Ep antibodies in Castleman’s disease via in-vivo and in-vitro techniques and subsequent physiologic response to therapy.
‘Department of Pathology, J. J. STEIN BERG^ Albert Einstein College of PHILIPL. HIJANG~ Medicine, PAUL LJUBICH3 Departments of 3Pathology SYLVIA LEE-HUANG4 and *Biochemistry, New York University Medical Center, New York, zM.D. -Fh.D. Program, Harvard Medical School, Boston. Massachusetts
REFERENCES Burgert, E.O., Jr. Gilchrist, G.S.. Fairbanks, V.F., Lynn, H.B.. Dukes, P.P. & Harrison, E.G., Jr, (1 975) Intraabdominal angio-follicular lymph node hyperplasia (plasma cell) with an anti-erythropoietic factor. Mayo Clinic Proceedings, 50, 542-546. Camiscoli. J.F. & Gordon, A S . (1970) Bioassay and standardization of erythropoietin. Regulation ofHematopoiesis (ed. by A. S. Gordon), Volume 1 , pp. 370-396. Meredith. New York. Castleman, B., Iverson, L. & Menendex, V. (1956)Localized mediastinum lymph-node hyperplasia resembling thymoma. Cancer. 9, 822-830. Cohen, H. (1957) Tumor-like proliferation of lymphoid tissue. Occurrence in deltoid muscle and mediastinum. Journal of the Mount Sinai Hospital, 24, 750-760. Dorfman, R.F. & Cibull, M. ( I 978) Castleman’s disease. Recent Results in Cancer Research. 64, 284-287. Keller. A., Hochholzer. L. & Castleman, B. (1972) Hyaline-vascular and plasma-cell types of giant lymph node hyperplasia of the mediastinum and other locations. Cancer, 29, 670-683. Lee-Huang, S. (1984) Cloning and expression of human erythropoietin cDNA in Esherichiu coli. Proceedings ofthe National Academy ofSriences of the United States of America. 81, 2708-2712.
543
cyte in thrombocytopenia: a microscopic study which supports the theory that platelets are produced in the pulmonary circulation. Thrombosis Research. 31, 163-1 7 6 . Trowbridge. E.A.. Martin, J.F. & Slater. D.N.(1 982) Evidence for a theory of physical fragmentation of megakaryocytes, implying that all platelets are produced in the pulmonary circulation. Thrombosis Research, 28, 461-475.
ANTI-ERYTHROPOIETIN ANTIBODIES I N HYPERVISCOSITY SYNDROME ASSOCIATED W I T H GIANT LYMPH NODE HYPERPLASIA (GLNH; CASTLEMAN'S DISEASE) Giant lymph node hyperplasia (GLNH) was first described i n adults (Castleman et al, 1 9 5 6 ) , but children have been affected (Keller et al, 1972). Originally this disease, resembling a thymoma, w a s believed t o affect only t h e mediastinum, b u t later reports prove occurrence in other areas (Keller et al, 1 9 7 2 ; Burgert et al, 1 9 7 5 ; Dorfmann & Cibull, 1978; Cohen, 1 9 5 7). T h e morphological characteristics of t h e variants of GLNH are hyaline-vascular and plasma cell type, which comprise approximately 9 1 % and 9% of cases. respectively. Unique to the plasma cell variant are t h e occasional systemic manifestations of hypergammaglobulinaemia, anaemia, and hyperviscosity (Burgert et al, 1975). A n anti-erythropoietin
(Ep) factor demonstrated via a mouse j9Fe uptake bioassay has been reported in t h e plasma cell variant of GLNH (Burgert et al, 1975).These anti-erythropoietin antibodies m a y be o n e component responsible for anaemia in GLNH aside from chemotherapy and/or a chronic disease state. T h e authors report a case of GLNH, plasma cell type, with demonstration of anti-Ep antibodies, hyperviscosity, anaemia, and subsequent response to steroid therapy. A 10-year-old black male presented with a s t h m a and a submandibular mass. Examination revealed proptosis, enlarged kidneys, and generalized lymphadenopathy. Right axillary lymph node biopsy demonstrated t h e plasma cell
B
A 1
2
3
4
5
r w t
IC
4 8 12 16 20 Serial twofold dilution
24
Fig 1. (A) Sodium dodecylsulphate polyacrylamide gel electrophoresis. Silver stain of protein standards (lane l ) , pure human and recombinant Ep (lanes 2 and 3), and crude Ep (lane 4). (B) Autoradiograph of an immunoblot of pure and crude Ep with patient's IgG containing antiBp antibodies. I4C protein molecular weight standards (lane I ) , pure human Ep (lane 2), pure human recombinant Ep (lane 3 ) , unlabelled protein molecular weight standards (lane 4). and crude human Ep (lane 5). Immunoreactive proteins were identified by ''1 labelled affinity purified sheep antihuman IgG as the second antibody. These results indicate that the IgG purified from pre-steroid treated serum recognizes human Ep from both pure and crude preparations. Corresponding blots with IgG from post-steroid treatment and normal control showed no detectable Ep bound. (C) Solid phase radioimmunoassay of patient's serum with pure Ep. Anti-Ep binding was detected by lL'I-labelled sheep anti-human Igs. Titration of binding was carried out by serial twofold dilution. Curve A represents the binding results of serum from pre-steroid treatment stage, curves B and C correspond to the binding results of serum from post-steroid treatment and from normal controls. respectively. These results demonstrate that pre-steroid treatment serum from this patient with Castleman's disease contains anti-Ep antibodies.
544
Case Reports
variant of GLNH with an unremarkable bone marrow biopsy. On admission his haematocrit was 0.24; blood viscosity 14.0 (normal range 1.4-1.8; Ostwald viscosimeter: viscosity = flow time of serum through viscosimeter divided by flow time of water through viscosimeter);and total protein was 100.0 g/l (albumin 18 g/l; IgG 12.2 g/l; IgM ~ 0 . g/l; 1 IgA 0.24 g/l). Red cell smear, indices, Coombs test, white blood cell count, and chemistries were not remarkable. Chest X-ray was significant for bilateral hilar adenopathy. The patient received plasmapheresis with a resulting diminution in viscosity to 5.5. Two months later the patient returned due to epistaxis, oral haemorrhage, occasional fatigue, and myopia. Laboratory results were: haematocrit 0.26, viscosity 14.2, total protein 165 g/l, albumin 24 g/l, platelet count 212 x 109/1. prothrombin time 12. Ss, and partial thromboplastin time 53 s. Plasmapheresis and steroid treatment resulted in a drop in total protein to 90 g/1 and viscosity of 4.5. Concomitantly, the haematocrit rose to 0.29, probably due to haemoconcentration. After 1 month of this regimen, his total protein and viscosity had dropped 26% and 71% respectively. This is in contrast to a 10%drop in total protein and a 36% drop in viscosity attributable to plasmapheresis. Haematocrit increased from 0.25 on admission to 0.43, a 71% increase. Over the past 2 years he has undergone periodic plasmapheresis and steroid therapy while remaining in a reasonably good state of health. Presently, he is at the 50th and 75th percentile for height and weight, respectively. The presence of anti-Ep antibodies in the patient’s serum was studied by four methods: (1)sodium dodecylsulphate polyacrylamide gel electrophoresis (Fig 1A) with molecular weight markers, and pure human Ep, and recombinant control Ep; (2) immunoblot (Fig 1B) with 14C labelled molecular weight markers, crude and pure human Ep, and recombinant Ep (Lee-Hnang, 1984); ( 3 )solid phase radioimmunoassay (Fig 1C); (4) the in vivo exhypoxic polycythaemic mice assay, including control (Camiscoli & Gordon, 1470), with data presented in the text. Sera from the child was positive for anti-Ep antibodies via ‘*jI radioimmunoassay (Fig 1C) and immunoblot techniques (Figs 1A and 1B). Whole sera was fractionated via a Protein A Sepharose CL-4B column, and both Protein A included and excluded immunoglobulin fractions inhibited jYFeuptake in the exhypoxic polycythaemic mouse bioassay by - 22.9% and - 32.5%, respectively, from normal serum fractions
( - 3.5% and
- 3.7% inhibition of 5YFe uptake). After 5 weeks ofsteroid therapy, serum from the child (total protein = 80 g/l, viscosity = 1.8. haematocrit =0.42) was again tested via mouse bioassay and had a + 6%increase in 5yFeuptake from standard control values, suggesting a compensatory increased level of erythropoietin. Steroid treatment, via reducing lymph node size and plasma cell number, reduces anti-Ep antibodies with preservation of free and biologically active Ep. In summary, this case report specifically demonstrates anti-Ep antibodies in Castleman’s disease via in-vivo and in-vitro techniques and subsequent physiologic response to therapy.
‘Department of Pathology, J. J. STEIN BERG^ Albert Einstein College of PHILIPL. HIJANG~ Medicine, PAUL LJUBICH3 Departments of 3Pathology SYLVIA LEE-HUANG4 and *Biochemistry, New York University Medical Center, New York, zM.D. -Fh.D. Program, Harvard Medical School, Boston. Massachusetts
REFERENCES Burgert, E.O., Jr. Gilchrist, G.S.. Fairbanks, V.F., Lynn, H.B.. Dukes, P.P. & Harrison, E.G., Jr, (1 975) Intraabdominal angio-follicular lymph node hyperplasia (plasma cell) with an anti-erythropoietic factor. Mayo Clinic Proceedings, 50, 542-546. Camiscoli. J.F. & Gordon, A S . (1970) Bioassay and standardization of erythropoietin. Regulation ofHematopoiesis (ed. by A. S. Gordon), Volume 1 , pp. 370-396. Meredith. New York. Castleman, B., Iverson, L. & Menendex, V. (1956)Localized mediastinum lymph-node hyperplasia resembling thymoma. Cancer. 9, 822-830. Cohen, H. (1957) Tumor-like proliferation of lymphoid tissue. Occurrence in deltoid muscle and mediastinum. Journal of the Mount Sinai Hospital, 24, 750-760. Dorfman, R.F. & Cibull, M. ( I 978) Castleman’s disease. Recent Results in Cancer Research. 64, 284-287. Keller. A., Hochholzer. L. & Castleman, B. (1972) Hyaline-vascular and plasma-cell types of giant lymph node hyperplasia of the mediastinum and other locations. Cancer, 29, 670-683. Lee-Huang, S. (1984) Cloning and expression of human erythropoietin cDNA in Esherichiu coli. Proceedings ofthe National Academy ofSriences of the United States of America. 81, 2708-2712.
