Blut, Band 32, Seite 195-206 (1976) Service des Maladies du Sang, CHU Strasbourg (Dir. Prof. G. Mayer), Centre de Transfusion Sanguine, Strasbourg (Dir. Prof. R. Waltz), Department of Immunology, Royal Postgraduate Medical School, London (Dir. P. Lachmann)
Acquired CT-Inhibitor Deficiencies in Lymphoproliferative Diseases with Serum Immunoglobulin Abnormalities A study of three cases Georges Hauptmann, Jean-Marie Lung, Marie-Louise North, Francis Oberling, Georges Mayer and Peter Lachmann Summary Some cases os acquired Cl-inhibitor (CI-INH)* deficiencies have been reported in patients with serum immunoglobulin abnormalities. We recently discovered three additional cases in patients with lymphoproliferative disease and abnormal serum immunoglobulins: two female patients with chronic lymphocytic leukaemia and circulating 7 S IgM, one female patient with lymphosarcoma, cold urticaria and a monoclonal IgGl-• The three patients had a complement profile similar to that seen in patients having an acquired deficiency of CI-INH: Low levels os total complement and of the early complement components (C 1, C4, C2), normal levels of C3, C5, C6, C7 and normal function of the alternate pathway. Only one patient with a profound diminution of CT-INH showed symptoms of angio-oedema. Strong anticomplementary activity was present in the three sera and in vitro tests demonstrated a high capacity of the sera to activate C1. The CI-INH deficiency is most likely due to its interaction with activated C 1. The following findings indicate an acquired CI-INH depletion: absence of angiooedema in the family, absence of CI-INH deficiency in the patient's children, the finding of extremely low levels os C1 in addition to low levels of C4 and C2, presence of an abnormal serum immunoglobulin and high serum anticomplementary activity directed toward C1 in a patient suffering from a lymphoproliferative disease. These observations stress the importance of the study of the complement system in affections involving immunoglobulin abnormalities and show that identification of a reduced level in haemolytical complement ought to lead to further investigations of the complement system. Zusammenfassung F~ille yon erworbener C1-Esterase-Inhibitor (Cl-INH)-Defizienz sind im Zusammenhang mit Serum-Immunoglobulin-Ver/inderungen beschrieben worden. Wir konnten drei weitere Patienten mit einem solchen Inhibitormangel, die an einer lympho* Nomenclature used is that recommended by the World Health Organization Committee on Complement Nomenclature (Bull. W.H.O. 39, 939, 1968 or Immunochemistry 7, 137, 1970). Eingegangen am 14, 7. 1975
196
G. Haupfmann, J.-M. Lang, M.-L. ~:orth, t v. Oberling, G. Mayer and P. Lachmann
proliferativen Erkrankung mit Serum-Immunoglobin-Veriinderungen litten, untersuchen. Es handelte sich um zwei Frauen mit chronisch-lymphatischer Leuk~imie und 7 S-IgM-Immunoglobnlinen im Serum und eine dritte Patientin mit Lymphosarkom, K~ilte-Urtikaria und monoklonale IgG 1-Kryoglobulin~imie. Bei diesen drei Patienten wurde ein fihnliches Komplementprofil wie bei friiher beschriebenen Patienten mit erworbener Defizienz des CI-INH gefunden: Verminderung der gesamten Komplementkonzentration, der C1-, C4- und C2-Komponenten, normaler Weft der C3-, C5-, C6-, C7-Komponenten, und normale Funktion des ,,altemativen pathways". Nut eine Patientin mit erheblicher Defizienz des CI-INH (weniger als 10% des Normalwertes) zeigte klinische Symptome des angio-neurotischen Odems. Eine starke antikomplement~ire Serumaktivit~it wurde in allen drei F~illen nachgewiesen und in vitro-Tests demonstrierten, dab diese Aktivit~it gegen die C 1-Komponente gerichtet war. Die Cl-INH-Defizienz ist sehr wahrscheinlich die Konsequenz der hohen C 1-Aktivierung, abet andere Ursachen fiir die Verminderung des CI-INH k6nnen nicht ausgeschlossen werden: erniedrigte Synthese oder direkte Aktion der 7 S-IgM-lmmunoglobuline gegen den Inhibitor. Die folgenden Befunde sprechen f~r eine erworbene Defizienz des CI-INH: Abwesenheit einer Familiengeschichte yon angio-neurotischem Odem. Abwesenheit einer Cl-INH-Defizienz bei den Kindern der Patienten. Die Absenkung des Wertes der C1-Komponente zusittzlich einer Senkung der C4- und C2-Komponenten, w~ihrend im heredit~ren angio-neurotischen Odem die C 1-Komponente normal bleibt. Die Entdeckung abnormder Immunoglobuline im Serum und einer anti-komplement/iren Aktivitiit gegen die C 1-Komponente bei einem Patienten, der an einer lymphoproliferativen Erkrankung leidet. Diese Beobachtungen betonen die Wichtigkeit der Studie des Komplementsystems bei Erkrankungen mit Ver~tnderung der Serum-Immunoglobuline und zeigen auch, dab die Beobachtung einer Senkung der gesamten Komplementkonzentration zu einer vollstf,ndigeren Studie des Komplementsystems fiihren soil.
A congenital deficiency of the inhibitor of the first complement component (CI-INH) or the presence of its inactive form is responsible for hereditary angioneurotic oedema. Some rare cases of acquired CI-INH deficiencies have been reported in patients with serum immunoglobulin abnormalities: 7S IgM paraprotein in two patients with lymphosarcoma [3], IgG • monoclonal kryoglobulin in one patient with cold urticaria [5], IgG X monoclonal protein in one patient with diffuse normolipaemic plane xanthomatosis [13]. This paper reports studies conducted on three additional cases of CI-INH deficiency in patients with lymphoproliferative diseases, whose sera exhibited an unusual complement pattern and serum immunoglobulin abnormalities.
Acquired Cl-inhibilor deficiendes in lymphoproliferative diseases
197
Case reports
Case 1: B. J., a 66 year old caucasian w o m a n had an isolated splenomegaly since 1966. In December 1973, she was admitted to the hospital because o f anaemia and a l y m p h o i d syndrome. Physical examination revealed lymphadenopathies in the cervical and inguinal area and a striking splenomegaly.
Laboralory dala: Hb 10 g/100 mI, RBC 3.0•
haematocrit 33%, WBC 54,200/bd, differential count: 27% segmented neutrophils, 1% eosinophils, 1% basophils and 69% lymphocytes. There were numerous Gumprecht's shadows on the blood smear. Platelets 39,400/bd. Bone marrow differential count showed an increase of tymphocytes (43~o) and histological examination revealed a lymphocytic hyperplasia with numerous lymphocytic monomorph islands. Erythrocyte sedimentation rate was 8/18. Serum protein titration and electrophoresis revealed a decrease in total serum proteins (56 g/l) with a distinct decrease in the level of y-globulins (3.4 g/l) which was confirmed by quantitative titration of immunoglobulins: IgG = 340 rag/100 ml, IgA = 20 mg/100 ml and IgM = 40 rag/100 ml. Whilst in hospital, the patient had a sudden attack of hemiplegia and died some days later. HistologicaI examination o f the spleen, l y m p h nodes, bone m a r r o w and other organs (liver, kidneys) revealed a generalized lymphocytic infiltration. This patient had never shown clinical symptoms of angioneurotic oedema. There is no family history o f angioneurotic oedema. The patient's two sons are free o f this disease.
Case 2: B. G., a 61 year old caucasian w o m a n was admitted to a hospital in the region of Strasbourg in March 1974 on account o f a severe attack o f anemia involving jaundice. Findings showed an auto-immune hemolytic anemia, a x I g M dysglobulinemia and a bone marrow hyperlymphocytosis. Haemolysis was stopped by means o f corticotherapy and the patient was admitted to the Haematological Clinic for complementary investigations. The patient reported oedema attacks of the upper and lower limbs and the face from several years onwards. These attacks occur almost one or two times monthly and are mainly triggered by exposure to cold. Generalized attacks of oedema have occurred twice. The oedemas are typical for angio-oedema, painless and nor~ pruriginous. U p o n admission, the patient showed a hepato-splenomegaly without adenopathies.
Laboratory dala: Hb 12 g/100 ml, RBC 3.7 X 106/ptl, haematocrit 43~o, WBC 3,400/~,1, differential count: 35~o segmented neutrophils, 2% eosinophils, 2% basophils, 8% monocytes and 53% lymphocytes. Platelets 60,000/~,I. There was a high reticulocytosis (42%o). Bone marrow examination revealed a cellular hyperplasia including lymphocytes (18~ and erythroblasts. Coombs's direct test was positive upon admission, then became negative at several controis. Attempts to find irregular anti-erythrocyte antibodies proved negative. Erythrocyte sedimentation rate was 65/76. The level of serum proteins was 65 g/1. Immunoelectrophoresis revealed a IgM • dysglobulinemia. Increase in the level of IgM (520 mg/100 ml) was confirmed by radial immuno-diffusion titration of immunoglobulins whilst IgG (800 rag/100 ml) and IgA (100 mg/100 ml) were slightly reduced. Ultracentrifugation did not reveal a significant increase of the 19 S globulins. There was no kryoglobulin in the serum.
198
G. Haudoimann,J.-M. Lang, M.-L. INrorlh, t v. Oberling, G. Mayer and P. Lachmann
During the hospitalization, the patient contracted jaundice due to post-transfusional hepatitis with overall disturbance of h~patic functions and the presence of anti-Australia antibodies was noted. There is no family story of angio-neurotic oedema. The patient had a daughter of 26, in g o o d health. The patient was treated by cortisone and chloraminophene and no recurrence of the disease has since occurred.
Case 3: G. M., a 53 year old caucasian w o m a n was admitted to the Haematological Clinic in October 1973 for weakness, anemia, ]eukopenia, elevated erythrocyte sedimentation rate and hyper-y-globulinemia. The patient complained of urticaria on her face and extremities after cold exposure. She had no known allergies and there was no family history of cold urticaria. Physical examination revealed clinical signs of anemia but no other abnormalities.
Laboralory data: Hb 8.5 g/100 ml, RBC 4.1 • 10~/bd, haematocrit 33%, WBC 2,200/hal, differential count: 64% segmented neutrophils, 30% lymphocytes, 6% monocytes. Platelet count: 210,000/bd. Erythrocyte sedimentation rate was 70/110 mm. The total serum protein was 84 g/l: albumin 37.8 g/l, m-globulins 9.3 g/l, ~-globulins 8.4 g/1, y-globulins 28.5 g/1. The formation of a cloudy precipitate was noted in the patient's serum cooled at 4 ~ C; this precipitate readily dissolved on rewarming. Immunoelectrophoresis revealed an IgG • monoclonal protein. Serum level of IgG determined by radial immunodiffusion was in excess of 3,200 mg/100 ml, serum IgM level was normal (160 mg/100 ml) and serum IgA level was also normal (230 rag/100 ml). Serum ultracentrifugation revealed an increase of the 7 S globulins. Bone marrow examination showed 5% plasma ceils which appeared to be normal. Roentgenograms of the bones showed no abnormalities. Lymphangiography revealed an abnormal volume of pelvic and paraaortic lymph nodes which appeared non homogenous with irregular filling defects. A n exploratory laparotomy was performed and two enlarged lymph nodes were removed for histological study. The spleen and liver appeared to be normal. Microscopic examination of the lymph nodes showed post-lymphangiographic modifications and a proliferation of medium-sized, poorly differentiated lymphocytes and the presence of plasma cells. The histological picture was compatible with the diagnosis of a nodular lymphocytic lymphosarcoma without extranodal localisation. The liver biopsy was normal. The patient was treated by radiotherapy and no extension of the disease has since occurred. Material and methods Blood taken by venepuncture was allowed to clot at room temperature and the serum separated by centrifugation. Serum was stored at --70 ~ C.
Complement studies Haemolytic complement (CH 50) was measured by a modification of Mayer's method [17]. Concentrations of C1, C4, C2, C5, C6 and C7 were measured by haemolytical tests [10, 15]. C3, C4, C5, factor B (GBG)were estimated by radial immunodiffusion [18] using commercial monospecific antisera.
Acquired Cl-inhibitor deficienciesin Iymph@roliferalivediseases
199
Two methods were used to titrate C]--INH: Functional titration [9] and radial immunodiffusion with an anti-Cl-INH specific serum (Behring Lab.). Fong and Good's test [6] was carried out on the descendant's sera in order to ascertain the absence of CT-INH deficiency in the course of family investigations. Serum anticomplementary activity was measured by a modification of Pickering's method [21, 7] : anticomplementary activity of fresh and heated serum (560 C for 30 rain) was expressed as percentage inactivation of guinea pig complement [7]. A kinetic flow analysis of the mixture of the patient's sera and guinea pig serum incubated at 37 ~ C for 1 h was performed using Hoffmann's method [11]. The capacity of the patient's sera to inhibit haemolytic C1 was judged by the following test: equal volumes of patient's sera and normal control serum were incubated at 37 ~ C (1 h) and at 0 ~ C (1 h) followed by incubation at 37 ~ C (20 rain). Haemolytical C 1 activity was measured before and after incubation. The radiolabelled C l q binding test (19) was kindly performed by Prof. P. H. Lambert (WHO Research Unit, Geneva).
Immunoglobulin studies In cases 1 and 2, different techniques were used to find low molecular weight I g M (IgM 7 S) : a) immunoelectrophoresis in a 1% agarose gel, b) double immunodiffusion (Ouchterlony) in 4% acrylamide gel according to Caldwell's technique [3], c) gel filtration through a column of Sephadex G200 in order to detect the presence o f material with heavy I g M chain specificity in the eluted peaks. Antiserum against I g M (specific for ha-chain) was purchased from Behring Lab. I n case 3, the monoclonal I g G kryoglobulin was isolated and purified by several precipitations at 0 ~ C. Dr. L. Rivat (Bois-Guillaume, France) kindly defined the subclass of the isolated protein by study of the genetic markers. Results
Results of the titrations of comyement components and C I - I N H The results are given in Table 1. The level of total complement (CH50) and of the fractions C1, C4 and C2 is much reduced whilst the level of fractions C3, C5, C6 and C 7 is normal. The results are almost similar for the three patients. In case 1, the level of CT-INH is reduced to 15 to 20% o f normal value and in case 2 is even further reduced. In case 3, the concentration of C I - I N H was clearly low on one occasion and low normal on two other occasions. We were not able to pursue subsequent modifications in the levels of case 1 who died at the beginning of January 1974. As regards case 2 the reappearance o f haemolytical activity of the serum within three week's evolution and a distinct increase in C I - I N H level is noted. At that particular time the patient contracted a post-transfusional hepatitis. At the very outbreak of this hepatitis a temporary fall in C3 and C5 level was observed. Subsequent tests revealed that the C1--INH level had again dropped to 15% o f normal value. The serum of all patients showed a strong anticomplementary activity (Table 2). This anticomplementary activity was found to be completely heat-labile in case 2 and partially heat-labile in the two other cases.
600
18,000
to
26,000
Acquired CT-Inhibitor Deficiencies in Lymphoproliferative Diseases with Serum Immunoglobulin Abnormalities A study of three cases Georges Hauptmann, Jean-Marie Lung, Marie-Louise North, Francis Oberling, Georges Mayer and Peter Lachmann Summary Some cases os acquired Cl-inhibitor (CI-INH)* deficiencies have been reported in patients with serum immunoglobulin abnormalities. We recently discovered three additional cases in patients with lymphoproliferative disease and abnormal serum immunoglobulins: two female patients with chronic lymphocytic leukaemia and circulating 7 S IgM, one female patient with lymphosarcoma, cold urticaria and a monoclonal IgGl-• The three patients had a complement profile similar to that seen in patients having an acquired deficiency of CI-INH: Low levels os total complement and of the early complement components (C 1, C4, C2), normal levels of C3, C5, C6, C7 and normal function of the alternate pathway. Only one patient with a profound diminution of CT-INH showed symptoms of angio-oedema. Strong anticomplementary activity was present in the three sera and in vitro tests demonstrated a high capacity of the sera to activate C1. The CI-INH deficiency is most likely due to its interaction with activated C 1. The following findings indicate an acquired CI-INH depletion: absence of angiooedema in the family, absence of CI-INH deficiency in the patient's children, the finding of extremely low levels os C1 in addition to low levels of C4 and C2, presence of an abnormal serum immunoglobulin and high serum anticomplementary activity directed toward C1 in a patient suffering from a lymphoproliferative disease. These observations stress the importance of the study of the complement system in affections involving immunoglobulin abnormalities and show that identification of a reduced level in haemolytical complement ought to lead to further investigations of the complement system. Zusammenfassung F~ille yon erworbener C1-Esterase-Inhibitor (Cl-INH)-Defizienz sind im Zusammenhang mit Serum-Immunoglobulin-Ver/inderungen beschrieben worden. Wir konnten drei weitere Patienten mit einem solchen Inhibitormangel, die an einer lympho* Nomenclature used is that recommended by the World Health Organization Committee on Complement Nomenclature (Bull. W.H.O. 39, 939, 1968 or Immunochemistry 7, 137, 1970). Eingegangen am 14, 7. 1975
196
G. Haupfmann, J.-M. Lang, M.-L. ~:orth, t v. Oberling, G. Mayer and P. Lachmann
proliferativen Erkrankung mit Serum-Immunoglobin-Veriinderungen litten, untersuchen. Es handelte sich um zwei Frauen mit chronisch-lymphatischer Leuk~imie und 7 S-IgM-Immunoglobnlinen im Serum und eine dritte Patientin mit Lymphosarkom, K~ilte-Urtikaria und monoklonale IgG 1-Kryoglobulin~imie. Bei diesen drei Patienten wurde ein fihnliches Komplementprofil wie bei friiher beschriebenen Patienten mit erworbener Defizienz des CI-INH gefunden: Verminderung der gesamten Komplementkonzentration, der C1-, C4- und C2-Komponenten, normaler Weft der C3-, C5-, C6-, C7-Komponenten, und normale Funktion des ,,altemativen pathways". Nut eine Patientin mit erheblicher Defizienz des CI-INH (weniger als 10% des Normalwertes) zeigte klinische Symptome des angio-neurotischen Odems. Eine starke antikomplement~ire Serumaktivit~it wurde in allen drei F~illen nachgewiesen und in vitro-Tests demonstrierten, dab diese Aktivit~it gegen die C 1-Komponente gerichtet war. Die Cl-INH-Defizienz ist sehr wahrscheinlich die Konsequenz der hohen C 1-Aktivierung, abet andere Ursachen fiir die Verminderung des CI-INH k6nnen nicht ausgeschlossen werden: erniedrigte Synthese oder direkte Aktion der 7 S-IgM-lmmunoglobuline gegen den Inhibitor. Die folgenden Befunde sprechen f~r eine erworbene Defizienz des CI-INH: Abwesenheit einer Familiengeschichte yon angio-neurotischem Odem. Abwesenheit einer Cl-INH-Defizienz bei den Kindern der Patienten. Die Absenkung des Wertes der C1-Komponente zusittzlich einer Senkung der C4- und C2-Komponenten, w~ihrend im heredit~ren angio-neurotischen Odem die C 1-Komponente normal bleibt. Die Entdeckung abnormder Immunoglobuline im Serum und einer anti-komplement/iren Aktivitiit gegen die C 1-Komponente bei einem Patienten, der an einer lymphoproliferativen Erkrankung leidet. Diese Beobachtungen betonen die Wichtigkeit der Studie des Komplementsystems bei Erkrankungen mit Ver~tnderung der Serum-Immunoglobuline und zeigen auch, dab die Beobachtung einer Senkung der gesamten Komplementkonzentration zu einer vollstf,ndigeren Studie des Komplementsystems fiihren soil.
A congenital deficiency of the inhibitor of the first complement component (CI-INH) or the presence of its inactive form is responsible for hereditary angioneurotic oedema. Some rare cases of acquired CI-INH deficiencies have been reported in patients with serum immunoglobulin abnormalities: 7S IgM paraprotein in two patients with lymphosarcoma [3], IgG • monoclonal kryoglobulin in one patient with cold urticaria [5], IgG X monoclonal protein in one patient with diffuse normolipaemic plane xanthomatosis [13]. This paper reports studies conducted on three additional cases of CI-INH deficiency in patients with lymphoproliferative diseases, whose sera exhibited an unusual complement pattern and serum immunoglobulin abnormalities.
Acquired Cl-inhibilor deficiendes in lymphoproliferative diseases
197
Case reports
Case 1: B. J., a 66 year old caucasian w o m a n had an isolated splenomegaly since 1966. In December 1973, she was admitted to the hospital because o f anaemia and a l y m p h o i d syndrome. Physical examination revealed lymphadenopathies in the cervical and inguinal area and a striking splenomegaly.
Laboralory dala: Hb 10 g/100 mI, RBC 3.0•
haematocrit 33%, WBC 54,200/bd, differential count: 27% segmented neutrophils, 1% eosinophils, 1% basophils and 69% lymphocytes. There were numerous Gumprecht's shadows on the blood smear. Platelets 39,400/bd. Bone marrow differential count showed an increase of tymphocytes (43~o) and histological examination revealed a lymphocytic hyperplasia with numerous lymphocytic monomorph islands. Erythrocyte sedimentation rate was 8/18. Serum protein titration and electrophoresis revealed a decrease in total serum proteins (56 g/l) with a distinct decrease in the level of y-globulins (3.4 g/l) which was confirmed by quantitative titration of immunoglobulins: IgG = 340 rag/100 ml, IgA = 20 mg/100 ml and IgM = 40 rag/100 ml. Whilst in hospital, the patient had a sudden attack of hemiplegia and died some days later. HistologicaI examination o f the spleen, l y m p h nodes, bone m a r r o w and other organs (liver, kidneys) revealed a generalized lymphocytic infiltration. This patient had never shown clinical symptoms of angioneurotic oedema. There is no family history o f angioneurotic oedema. The patient's two sons are free o f this disease.
Case 2: B. G., a 61 year old caucasian w o m a n was admitted to a hospital in the region of Strasbourg in March 1974 on account o f a severe attack o f anemia involving jaundice. Findings showed an auto-immune hemolytic anemia, a x I g M dysglobulinemia and a bone marrow hyperlymphocytosis. Haemolysis was stopped by means o f corticotherapy and the patient was admitted to the Haematological Clinic for complementary investigations. The patient reported oedema attacks of the upper and lower limbs and the face from several years onwards. These attacks occur almost one or two times monthly and are mainly triggered by exposure to cold. Generalized attacks of oedema have occurred twice. The oedemas are typical for angio-oedema, painless and nor~ pruriginous. U p o n admission, the patient showed a hepato-splenomegaly without adenopathies.
Laboratory dala: Hb 12 g/100 ml, RBC 3.7 X 106/ptl, haematocrit 43~o, WBC 3,400/~,1, differential count: 35~o segmented neutrophils, 2% eosinophils, 2% basophils, 8% monocytes and 53% lymphocytes. Platelets 60,000/~,I. There was a high reticulocytosis (42%o). Bone marrow examination revealed a cellular hyperplasia including lymphocytes (18~ and erythroblasts. Coombs's direct test was positive upon admission, then became negative at several controis. Attempts to find irregular anti-erythrocyte antibodies proved negative. Erythrocyte sedimentation rate was 65/76. The level of serum proteins was 65 g/1. Immunoelectrophoresis revealed a IgM • dysglobulinemia. Increase in the level of IgM (520 mg/100 ml) was confirmed by radial immuno-diffusion titration of immunoglobulins whilst IgG (800 rag/100 ml) and IgA (100 mg/100 ml) were slightly reduced. Ultracentrifugation did not reveal a significant increase of the 19 S globulins. There was no kryoglobulin in the serum.
198
G. Haudoimann,J.-M. Lang, M.-L. INrorlh, t v. Oberling, G. Mayer and P. Lachmann
During the hospitalization, the patient contracted jaundice due to post-transfusional hepatitis with overall disturbance of h~patic functions and the presence of anti-Australia antibodies was noted. There is no family story of angio-neurotic oedema. The patient had a daughter of 26, in g o o d health. The patient was treated by cortisone and chloraminophene and no recurrence of the disease has since occurred.
Case 3: G. M., a 53 year old caucasian w o m a n was admitted to the Haematological Clinic in October 1973 for weakness, anemia, ]eukopenia, elevated erythrocyte sedimentation rate and hyper-y-globulinemia. The patient complained of urticaria on her face and extremities after cold exposure. She had no known allergies and there was no family history of cold urticaria. Physical examination revealed clinical signs of anemia but no other abnormalities.
Laboralory data: Hb 8.5 g/100 ml, RBC 4.1 • 10~/bd, haematocrit 33%, WBC 2,200/hal, differential count: 64% segmented neutrophils, 30% lymphocytes, 6% monocytes. Platelet count: 210,000/bd. Erythrocyte sedimentation rate was 70/110 mm. The total serum protein was 84 g/l: albumin 37.8 g/l, m-globulins 9.3 g/l, ~-globulins 8.4 g/1, y-globulins 28.5 g/1. The formation of a cloudy precipitate was noted in the patient's serum cooled at 4 ~ C; this precipitate readily dissolved on rewarming. Immunoelectrophoresis revealed an IgG • monoclonal protein. Serum level of IgG determined by radial immunodiffusion was in excess of 3,200 mg/100 ml, serum IgM level was normal (160 mg/100 ml) and serum IgA level was also normal (230 rag/100 ml). Serum ultracentrifugation revealed an increase of the 7 S globulins. Bone marrow examination showed 5% plasma ceils which appeared to be normal. Roentgenograms of the bones showed no abnormalities. Lymphangiography revealed an abnormal volume of pelvic and paraaortic lymph nodes which appeared non homogenous with irregular filling defects. A n exploratory laparotomy was performed and two enlarged lymph nodes were removed for histological study. The spleen and liver appeared to be normal. Microscopic examination of the lymph nodes showed post-lymphangiographic modifications and a proliferation of medium-sized, poorly differentiated lymphocytes and the presence of plasma cells. The histological picture was compatible with the diagnosis of a nodular lymphocytic lymphosarcoma without extranodal localisation. The liver biopsy was normal. The patient was treated by radiotherapy and no extension of the disease has since occurred. Material and methods Blood taken by venepuncture was allowed to clot at room temperature and the serum separated by centrifugation. Serum was stored at --70 ~ C.
Complement studies Haemolytic complement (CH 50) was measured by a modification of Mayer's method [17]. Concentrations of C1, C4, C2, C5, C6 and C7 were measured by haemolytical tests [10, 15]. C3, C4, C5, factor B (GBG)were estimated by radial immunodiffusion [18] using commercial monospecific antisera.
Acquired Cl-inhibitor deficienciesin Iymph@roliferalivediseases
199
Two methods were used to titrate C]--INH: Functional titration [9] and radial immunodiffusion with an anti-Cl-INH specific serum (Behring Lab.). Fong and Good's test [6] was carried out on the descendant's sera in order to ascertain the absence of CT-INH deficiency in the course of family investigations. Serum anticomplementary activity was measured by a modification of Pickering's method [21, 7] : anticomplementary activity of fresh and heated serum (560 C for 30 rain) was expressed as percentage inactivation of guinea pig complement [7]. A kinetic flow analysis of the mixture of the patient's sera and guinea pig serum incubated at 37 ~ C for 1 h was performed using Hoffmann's method [11]. The capacity of the patient's sera to inhibit haemolytic C1 was judged by the following test: equal volumes of patient's sera and normal control serum were incubated at 37 ~ C (1 h) and at 0 ~ C (1 h) followed by incubation at 37 ~ C (20 rain). Haemolytical C 1 activity was measured before and after incubation. The radiolabelled C l q binding test (19) was kindly performed by Prof. P. H. Lambert (WHO Research Unit, Geneva).
Immunoglobulin studies In cases 1 and 2, different techniques were used to find low molecular weight I g M (IgM 7 S) : a) immunoelectrophoresis in a 1% agarose gel, b) double immunodiffusion (Ouchterlony) in 4% acrylamide gel according to Caldwell's technique [3], c) gel filtration through a column of Sephadex G200 in order to detect the presence o f material with heavy I g M chain specificity in the eluted peaks. Antiserum against I g M (specific for ha-chain) was purchased from Behring Lab. I n case 3, the monoclonal I g G kryoglobulin was isolated and purified by several precipitations at 0 ~ C. Dr. L. Rivat (Bois-Guillaume, France) kindly defined the subclass of the isolated protein by study of the genetic markers. Results
Results of the titrations of comyement components and C I - I N H The results are given in Table 1. The level of total complement (CH50) and of the fractions C1, C4 and C2 is much reduced whilst the level of fractions C3, C5, C6 and C 7 is normal. The results are almost similar for the three patients. In case 1, the level of CT-INH is reduced to 15 to 20% o f normal value and in case 2 is even further reduced. In case 3, the concentration of C I - I N H was clearly low on one occasion and low normal on two other occasions. We were not able to pursue subsequent modifications in the levels of case 1 who died at the beginning of January 1974. As regards case 2 the reappearance o f haemolytical activity of the serum within three week's evolution and a distinct increase in C I - I N H level is noted. At that particular time the patient contracted a post-transfusional hepatitis. At the very outbreak of this hepatitis a temporary fall in C3 and C5 level was observed. Subsequent tests revealed that the C1--INH level had again dropped to 15% o f normal value. The serum of all patients showed a strong anticomplementary activity (Table 2). This anticomplementary activity was found to be completely heat-labile in case 2 and partially heat-labile in the two other cases.
600
18,000
to
26,000
