CASE REPORT Macroglobulinemia in a Dog E.B. MEJIA, S. CARMAN AND J.H. LUMSDEN*
Summarv This report describes a case of macroglobulinemia in a six year old castrate male Collie cross dog with clinical signs of epistaxis, anemia, retinopathy and high serum viscosity. The highest total serum protein was 12 g/dl with approximately 60% monoclonal beta globulin. Proteinuria, Bence Jones protein and osteolytic lesions were not detected. Chemotherapy and partial removal of the plasma protein by withdrawal of whole blood and transfusion with packed red cells from a DEA negative donor resulted in transient clinical remission. Resume Un cas de macroglobulinemie canine Les auteurs decrivent un cas de macroglobulinemie, chez un chien Collie croise et castre, qui presentait les signes cliniques suivants: epistaxis, anemie, retinopathie et grande viscosite du serum. Le taux le plus eleve de proteines seriques totales atteignit 12 g/dl, dont environ 60% de betaglobuline monoclonale. Ils ne decelerent pas de proteinurie, de proteine de Bence Jones ou de lesions osteolytiques. La chimiotherapie et l'enlevement partiel des proteines plasmatiques, par le moyen d'une saignee et d'une transfusion d'hematies provenant d'un donneur DEA negatif, resulterent en une disparition transitoire des signes cliniques.
Introduction Macroglobulinemia is included in a group of disorders called monoclonal gammopathies (10, 13), monoclonal immunoproliferative disorders (26) or plasma cell dyscrasias (7). Two major features are shared by these disorders: an uncontrolled proliferation of a clone of cells of the plasma cell or lymphocytic series and production by these cells of an excessive amount of homogenous immunoglobulin or its subunits or frag-
ments (7). The nature of the protein produced serves as a convenient way to classify these diseases such as IgG, IgA, IgD, or IgE myeloma, IgM macroglobulinemia, light chain disease or heavy chain disease (7). IgM molecules are macroglobulins with a molecular weight of approximately 1,000,000 daltons. They have a sedimentation coefficient of 15 Svedberg units or greater with a peak in the range of 17 S or 21 S (26). Due to their size and shape macroglobulins increase the serum viscosity and in excess can produce a series of physiological and pathological changes (3, 6, 7, 8, 10, 12, 13, 21, 23, 25, 26). Several clinical manifestations are common to monoclonal gammopathies but there is often an increased association with certain disorders. Osteolysis, bone marrow infiltration with plasma cells, Bence Jones proteinuria, and renal failure are frequently associated with multiple myeloma (5, 7, 8, 10, 12, 22, 23, 26) while bleeding tendencies, serum hyperviscosity, retinopathy, mental depression and bone marrow infiltration with lymphoid or lymphocytoid plasma cells are most often found with macroglobulinemia (3, 6, 7, 8, 10, 11, 13, 15, 18, 22, 23, 26). It should be emphasized that a few cases are reported in which these diseases overlap both clinically and biochemically, e.g. macroglobulinemia with bone destruction (24), the hyperviscosity syndrome in multiple myeloma (21), macroglobulinemia and myelomatosis (1). Dogs (8, 10) and humans (4, 5, 7, 13, 22, 23, 26) with monoclonal gammopathies develop an increased susceptibility to infections due to acquired immunodeficiency associated with decreased ability to synthesize specific antibody and increased rate of immunoglobulin catabolism. Susceptibility to bacterial sepsis is further increased if cytotoxic chemotherapy is used.
Case Report On July 15, a six year old castrate male Collie cross weighing 22 kg was presented to the Teaching Hospital because of persistent unilateral epistaxis and increased rate and depth of respiration. Two weeks prior to admission the dog experienced the initial and most severe bout of epistaxis from the right nostril. At that time the nasal sinuses were scoped and the skull radiographed. No abnormalities were detected. On admission the dog was found to be obese, depressed, had increased respiration rate, pale mucous membranes and mild epistaxis occurring from the right nostril. No other clinical abnormalities were detected. Laboratory parameters were obtained on admission (Table I). A severe anemia (PCV 15.6%) with a marked erythroid response and marked rouleaux formation was found. A direct antiglobulin test was negative. Blood platelet levels were within low normal limits but were con-
*Department of Pathology (Mejia and Lumsden) and Department of Clinical Studies (Carman), Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1.
28
Can. vet. J. 20: 28-33 (January 1979)
TABLE I VARIATION OF HEMATOLOGICAL PARAMETERS AT DIFFERENT STAGES OF THE DISEASE Sate RBC
x
PCV
%
Julv 16
July 26
July 28
2.01
2.80
2.96
4.57
15.6
Hgb
gm/dl
WBC
x
Neutrophils Segmented
July 15 10 6/ul
103/ul 3
x
10 /ul
20.9
26.2
Aug. 9
Aug. 13
3.94
29.9
Aug. 16
4.21
29.0
Sept. 9
3.28
29.1
3.95
Oct. 14 2.48
22.2
24.6
14.7
4.45
24.7
29.9
7.1
10.2
9.9
10.0
8.2
8.4
5.7
7.6
7.7
8.6
9.7
10.2
18.0
10.0
12.7
8.6
7.3
4.2
2.0
10.8
4.3
10.8
13.6
8.55
10.2
6.02
6.42
2.85
1.16
9.5
0.69
15.20
7.17
200
180
----
510
170
150
250
200
280
3100
820
1450
300
1270
1800
290
970
600
280
90
1090
1820
640
240
440
120
40
760
----
260
---
---
---
9.18
1220
420
190
420
560
Monocytes
/ul
300
1570
90
---
Eosinophils
/ul
----
----
----
---
Rubricytes
/ul
1670
1220
----
+
444
4444.
+
+
+
+
-
+
+
230
21
187
63
42
16
110
12
147
140
103/ul
Nov. 8
3.79
9.4
1220
x
28.7
Nov. 1
6.8
/ul
103/ul
3.85
17.4
/ul
x
21.1
Oct. 28
4.8
Lymphocytes
Reticulocyte
3.46
15.2
Neutrophils Bands
Polychromasia
Oct. 22
200
180 ----.
200
.
----
40
430
..
Rouleaux Formation
Platelets
125
227
91
Platelet
aggultinatton
+++
...
++-+
+4-+
+++
+
++
+++
++
si
Shift Platelets LN.
-
to
++-+
+++
+
+
+++
low norma l.
+4.4.-
range
of
presence.
sistently clumped making accurate counting difficult. In vitro erythrocyte rouleaux and platelet agglutination were constant features through the course of the disease. Blood urea nitrogen and urinalysis findings were within normal limits. Total serum protein was markedly elevated (10.6 g/dl) (Table II). Serum electrophoresis demonstrated a monoclonal peak in the B2 globulin region of the electrophoretic pattern (6.2 g/dl) (Figure 1 and Table II). This protein was later identified to be an IgM macroglobulin by immunoelectrophoresis using monospecific antisera.' Serum ultracentrifugation on two different occasions determined the paraprotein to be a macromolecule
with a sedimentation coefficient of 17 Svedburg units.2 The serum viscosity was found to be 3.5 cps, a value three times greater than that of the normal control.3 Bone marrow aspiration and examination demonstrated the presence of increased numbers of mature plasma cells in a hypercellular population of normal myeloid and erythroid marrow cells. These plasma cells had oval to round eccentrically located nuclei with a heavy chromatin pattern and abundant pale to dark blue cytoplasm. Prothrombin time (28.5s vs 17s normal control), partial thromboplastin time (28.5s vs 21s normal control), fibrinogen levels and platelet numbers
TABLE II TOTAL PROTEIN. ELECTROPHORESIS AND VISCOSITY VALUES Total Protein Date 7 20 7 30 8 5 8 17 9 9 10 12 10 14 10 15
(g/dl) 10.6 7.7 9.1 9.6 10.4 12.2 10.9 9.8
alb = albumen, glob
=
alb (g/dl) 2.6 2.5 3.0 3.1 2.8 2.9 2.5 2.6
glob (g/dl) 8.0 5.3 6.5 6.5 7.6 9.3 8.4 7.2
al glob (g/dl) 0.4 0.3 0.4 0.52 0.6 0.4 0.5 0.5
a2 glob (g/dl) 1.2 1.2 1.6 0.91 0.6 1.25 0.6 0.7
/ glob (g/dl) 6.2 3.5 4.2 4.9 6.2 7.2 7.1 5.8
y glob (g/dl) 0.2 0.2 0.3 0.2 0.2 0.4 0.2 0.2
alb/glob 0.3 0.5 0.5 0.5 0.4 0.3 0.3 0.4
Viscosity cps 3.5 2.9 3.6 5.5 4.4
globulin, cps = centipoises.
'Immunoelectrophoresis was performed by Dr. Arthur Hurvitz, Animal Medical Centre, 510 E 62nd. St.. New York. N.Y. 1002 1. 2Ultracentrifugation studies were performed by Dr. R.J. Douglas, Professor of Microbiology, University of Guelph. 3Viscosity measurements were performed by S. Al-Izzi, Graduate Student Clinical Pathology, Department of Pathology, O.V.C. 29
la&6- Iocl
(+
FIGURE 3. Thoracic radiograph taken at the time of initial presentation.
5,/2IYI(-
02
Repeated urinalysis failed to demonstrate proteinuria nor were Bence Jones proteins detectable on three separate occasions (9) using the heat precipitation method. Thoracic radiographs demonstrated the presence of expansile rib lesions on the second left rib and on the mid region of the fourth and fifth right ribs (Figure 3). Repeated radiographs at a later date showed the lesion in the fifth right rib to be expanding. A radiographic scan of the skeleton did not reveal the classical myeloma "punched-out" bony lesions which are suggestive of osteolysis by invading tumor cells (17, 24). Old fractures were identified in the area of the left pubis and right femoral neck. The radiographic cardiac silhouette was within normal limits. Later repeated radiographic examinations displayed the development
FIGURE 1. Serum electrophoretic pattern during the course of the disease. Notice homogeneous peak in beta 2 globulin zone. alb = albumin, a, = alpha 1 globulin, a2 = alpha 2 globulin, ,f1 = beta I globulin, 182 = beta 2 globulin and y = gamma globulin.
were examined and monitored throughout the course of the disease. Blood clotting times remained moderately prolonged while fibrinogen levels remained at low normal limits (105 mg/dl vs 100-300 mg/dl reference values). The average platelet counts (145,000/,l) were low. (Table 1). Serum calcium, phosphorous, magnesium, enzyme levels and electrolyte values were within normal limits and remained so throughout the course of the disease.
35F
35
30k
30
25-
25 I
201
I, II'
15[
~ \ ~~~ ~
~
I10--f-,
I I
I
w
,I
I,
I
20
0
~ , II% c I
I , I~~~~~~~~~c
'tIb
15
I
101
10 "cr
5
5
I*IlIlfl*fIfI
-LEUKERAN
0
2mg/day 25mg/dayr PREDNIZONE h
40
r
-U---ALKERAN Jo 6.5mg/day
/ay[
O]
L
AMPICILLIN 1500mg DAILY il
014
Wl
I
18
22 JULY
26
30
l
I
7 3 11 AUGUST
PREDNIZONE 50mg/day :30g/ay
|AMPICILLIN I
I
!CEPOREX
I
15
15
19 23 27 OCTOBER
31
FIGURE 2. Graphic representation of PCV, WBC and total serum protein values related to the therapy. = Total serum protein g/dl. o o = microhematocrit %. o - ---- -0 = leukocytes x IO3/ ml. Phlebotomy and transfusions were made on July 26 and Oct. 14.
30
FIGURE 4. Retinal vessel sacculations detected at the time of initial presentation.
of generalized cardiac enlargement (Figure 3). Electrocardiograph findings were normal. Retinal examination showed the presence of dilated vessels with saccular enlargements. No retinal hemorrhages were present (Figure 4). A diagnosis was made of macroglobulinemia with hyperviscosity syndrome and defective hemostasis. Treatment was initiated and continued over a four month period. Management With the absence of secondary kidney disease, skeletal pain or concurrent infection, the collie was considered a good candidate for chemotherapy. In preparation the dog was given systemic antibiotic,4 vitamin B complex with iron5 and anabolic steroid therapy6 to prevent superinfection and exacerbation of the anemia problem. In an attempt to correct the anemia, decrease the serum protein levels, and decrease blood loss by epistaxis, a crude plasmaphoresis was performed as previously described (10) under halothane anaesthesia. One liter of whole blood was removed and replaced simultaneously by 650 ml of DEA negative packed red blood cells suspended in 500 ml of an isotonic electrolyte solution. Following this procedure total serum protein levels decreased from 10.6 g/dl to 7.7 g/dl and serum globulin levels decreased from 6.2 g/dl to 3.5 g/dl. The hematocrit increased from 15% to 32% and the serum viscosity decreased from 3.5 cps to 2.9 cps (Table II). The epistaxis ceased. Chlorambucil7 therapy (1 mg/ 12.5 kg every second day) as previously described (20) in combination with prednisone8 therapy (2.2
FIGURE 5. The second bone marrow aspiration biopsy contained many lymphocytoid plasma cells. Wrights X240.
mg/kg/d) was initiated. Chlorambucil therapy was continued for a total of eight doses. During this time serum protein levels continued to rise while leukocyte levels fell only mildly. Because of the ineffective response, therapy was discontinued. Further chemotherapy was postponed because of the nonresponsive anemia. At the time of discharge serum protein levels had risen to 9.6 g/ dl yet the serum viscosity remained at 3.9 cps (Table II). The dog was represented two months following initial discharge. A large hematoma was present on the right elbow. Total serum protein levels had risen (12.2 g/dl), the hematocrit had fallen (22%), the anemia was unresponsive and blood viscosity levels had risen (5.5 cps) (Table II). Severe epistaxis began two days following admission causing the hematocrit to fall to 14.7%. A decrease in serum protein levels occurred as well (10.9 g/dl) due to the blood loss (Table II). Plasmaphoresis was repeated with the dog under halothane anaesthesia. Eleven hundred and fifty ml of whole blood were removed and replaced by 650 ml of packed DEA negative red blood cells suspended in one liter of isotonic electrolyte solution. The hematocrit increased (30.4%) and total serum protein levels decreased (9.8 g/dl) although not as much as previously (Table II). Again epistaxis ceased. Maintenance therapy with systemic antibiotics,9 vitamin B complex with iron'0 and anabolic steroids" was reinstituted. Another bone marrow biopsy was examined
4Ampicillin, Ayerst Laboratories, Montreal, Quebec. 5Beminal with iron, Ayerst Laboratories, Montreal, Quebec. 6Winstrol, Winthrop Laboratories, Aurora, Ontario. 'Leukeran, Burroughs Wellcome Ltd., LaSalle, Quebec. XDeltacortisone, Upjohn Company of Canada, Orangeville, Ontario. 9Cephalexin monohydrate, Ceporex Glaxo Laboratories, Toronto, Ontario. '°Beminal with iron, Ayerst Laboratories, Montreal, Quebec. Winstrol, Winthrop Laboratories, Aurora, Ontario.
31
(Figure 5). The previously identified mature plasma cells were replaced by very immature plasmacytoid cells with a detectable maturation sequence. These cells now composed 60% of the marrow population. Normal leukocyte precursors were present but there was a relative depression of the red cell series. The hematocrit was 35%, leukocyte count 9.8 x 103/,ul and the serum protein 12 g/dl. L-phenylalanine mustard'2 therapy (0.25 mg/kg/day) was begun and continued for a total of seven doses (Figure 3). During this course of therapy the total serum protein level decreased and the blood leukocyte count fell to 2,000/,ul. Prednisone'3 therapy was initiated at the same time (2.2 mg/ kg/day) and continued after the withdrawal of the alkylating agent. Leukocyte levels returned to normal levels after discontinuation of alkeran therapy while the serum protein levels remained stable (Figure 2). The dog was discharged one week later with antibiotic therapy'4 to be continued at home and prednisone therapy to be gradually withdrawn. L-phenylalanine mustard therapy was to be repeated at six week intervals for a period of four days in an attempt to maintain remission. Two weeks following discharge the dog was presented in septic shock. A massive soft tissue swelling of the left hind limb was present. Escherichia coli was cultured from the peripheral blood. Euthanasia was performed because of the poor prognosis.
Gross and Histological Findings The dog was in good body condition with large amounts of both subcutaneous and intra-abdominal fat. The left hind limb was markedly swollen distal to the stifle joint with both subcutaneous and interfacial edema fluid. The spleen was congested and contained only limited amounts of follicular tissue. The previously identified rib lesions were easily located and consisted of areas of marrow cavity enlargement in the mid rib region. The bony cortex at these locations was thin, brittle and easily cut and marrow cells filled the cavity. The ratio of red to yellow marrow was within expected limits. The kidneys appeared to be normal. The heart appeared dilated due to the presence of a thickened and dilated left ventricle. On microscopic examination the edema fluid of the limb contained moderate numbers of mature neutrophils. The spleen contained a few karyorrhectic lymphocytes and a few differentiated plasma cells. This decreased number of plasma cells may be a reflection of previous chemotherapy. The liver histology was unremarkable with only periacinar lipidosis present. The bone marrow appeared hypercellular with the majority of the cells poorly differentiated medium-size mononuclear cells be-
longing to the lymphocytic series. The erythroid series was markedly reduced. Mature plasma cells were found in a few areas only. The medullary collecting ducts of the kidneys were found to contain proteinaceous casts. Many of the glomeruli had membranes thickened by eosinophilic material. Some of the Bowman's spaces contained proteinaceous material as well. Proteinuria was not evident in previous urinalysis. These kidney changes may have been due to secondary disease or directly related to the primary disease process.
Discussion Epistaxis may be associated with localized tissue destruction due to tumor masses, trauma, infection or primary blood coagulation defects. Macroglobulinemia or other dysproteinurias should be considered as a cause of epistaxis and be included in the differential diagnosis. Epistaxis was the presenting problem in this animal. The defective hemostasis is not believed to be due to one parameter derangement but to combined alterations in the clotting system. Precipitation of blood clotting factors (II, V, VII), interference of the thrombin-fibrinogen conversion reaction, inadequate platelet adhesiveness with altered platelet function due to platelet surface coating, Ca++ chelation, and vascular wall damage with the activation of the fibrinolytic system by the presence of the abnormal tumor product are believed to be responsible for the overall bleeding disorder in man (18). A routine total serum or plasma protein determination is important in the early recognition of dysproteinemias. A diagnosis of monoclonal gammopathy can only be reached by the evaluation of a serum electrophoretic pattern and the detection of a monoclonal protein peak (19). Immunoelectrophoresis with specific reagents and ultracentrifugation can be used to identify the protein involved and define its molecular weight. Identification of the specific paraprotein produced is important in human medicine for therapeutic and prognostic purposes (2). Only as this information is accumulated will the clinical importance be determined for animal species. Examination of bone marrow aspirate smears will often reveal the presence of an abnormal number or type of lymphocytic or plasmacytic cells. The cellular morphology and level of cellular maturity cannot alone be used to distinguish myeloma from macroglobulinemia. The course of the disease, the presence or absence of osteolysis, and the type of protein being produced do not correlate well with the morphological form of lymphocytic cell observed (24). Proteinuria, if present may be due to Bence Jones proteinuria or due to glomerular membrane
'2Alkeran, Burroughs Wellcome Ltd., LaSalle, Quebec. '3Deltacortisone, Upjohn Company of Canada, Orangeville, Ontario. '4Cephalexin monohydrate, Ceporex Glaxo Laboratories, Toronto, Ontario. 32
damage induced by circulating macroglobulins. In these cases impaired renal function is closely associated with greatly reduced patient survival time (2). Blood or serum viscosity measurements allow the identification of the hyperviscosity syndrome. Retinal vessel distention and sacculation, hematological defects and congestive heart failure due to the presence of an expanded blood volume may be present as a result of increased serum viscosity ( 14). Plasmaphoresis can serve as an important method of therapy not only in times of acute crisis during massive epistaxis, but also as a method of maintenance therapy during prolonged treatment of the disease (16). It is an effective method in reducing elevated serum protein levels and serum viscosity and halting continued bleeding episodes without the toxicity associated with chemotherapeutic agents. When used correctly and in conjunction with the alkylating agents, the patients' lifespan may be effectively lengthened.
Veterinary Therapy V. R.W. Kirk, Editor. pp. 372374. Philadelphia: W.B. Saunders Co. 1974. 11. KOK, D.A.. D.N. WHITMORE and R.W. AINWORTH. Four
cases of Waldenstrom's macroglobulinemia. J. clin. Path. 76: 351-361. 12. KYLE. R.A. Diagnosis of monoclonal gammopathies. In Manual of Clinical Immunology. N.R. Rose and H. Friedman, Editors. pp. 734-752. Washington, D.C.: American Society for Microbiology. 1976. 13. KYLE, R.A. and E.A. BAYRD. The Monoclonal Gammopathies. Springfield, Illinois: Charles C. Thomas. 1976. 14. MacKENZIE, M.R., E. BROWN, H.H. FUNDENBERG and L.
GOODENDAY. Waldenstrom's macroglobulinemia: Correlation between expanded plasma volume and increased serum viscosity. Blood 35: 394-408. 1970. 15. MacKENZIE, M.R. and H.H. FUNDEMBERG. Macroglobulinemia: An analysis for forty patients. Blood 39: 874-889. 1972. 16. ORENTREICH, N., S. ETTINGER, R.J. TASHJIAN, A.A.S.
STANISLOUISKI and R. HENKIN. Intensive chronic plasmaphoresis in dogs. Am. J. vet. Res. 29: 19291933. 1968. 17. OSBORNE, C.A., V. PERMAN, J.H. SAUTTER, J.B. STEVENS
Refi lelicel.S
and G.F. HANLON. Multiple myeloma in the dog. J. Am. vet. med. Ass. 153: 1300-1319. 1968.
1. ADNER, P.L., G. WALLENIUS and I. WERNER. Macro-
18. PERKINS, H.A., M.R. MacKENZIE and H.H. FUNDEMBERG.
globulinemia and myelomatosis. Acta med. scand. 168: 431-437. 1960. 2. ANONYMOUS. A comparative study by acute Leukemia Group B: Correlation of abnormal immunoglobulins with clinical features of myeloma. Archs intern. Med. 135: 46-52. 1975. 3. BLOCH, K.J. and D.G. MAKI. Hyperviscosity syndrome associated with immunoglobulin abnormalities. Semin. Hematol. 10: 113-124. 173. 4. DUTCHER, T.F. and J.F. FAHEY. The histopathology of the macroglobulinemia of Waldenstrom. J. natn. Cancer Inst. 22: 887-917. 1959.
Hemostatic defects in dysproteinemias. Blood 35: 695-707. 1970. 19. PUTMAN, F.W. Plasma-cell myeloma and macroglobulinemia. I. Physiochemical immunochemical and isotopic turnover studies of the abnormal serum and urinary proteins. New Engl. J. Med. 18: 902-908. 1959.
5. ENGLE. R.L. Jr. and L.A. WALLIS. Immunoglobulinopathies; Immunoglobulins, Immune Deficiency Syndromes, Multiple Myeloma and Related Disorders. Springfield, Illinois: C.C. Thomas. 1969. 6. FAHEY. T.L.. W.F. BARTH and A. SOLOMON. Serum
hyperviscosity syndrome. J. Am. med. Ass. 192:120133. 1965. 7. HARVEY, A. Mc., R.T. JOHNS, A.H. OWENS Jr. and R.S.
ROSS. Immunoglobulins and plasma cell dyscrasias. In The Principles and Practice of Medicine. 19th Edition. pp. 1324-1343. New York: Appelton Century Crofts. 1976. 8. HURVITZ, Al.., S.C. HASKINS and C.A. FISHER. Macro-
globulinemia with hyperviscosity syndrome in a dog. J. Am. vet. med. Ass. 157: 455-460. 1970. 9. HURVITZ, A.l., M. KEHOE, J.D. CAPRA and R. PRATA.
Bence Jones protein and proteinuria in a dog. J. Am. vet. med. Ass. 159: 1112-1116. 1971. 10. HURVITZ, A.l. Monoclonal Gammopathies in Current
20. SHEPARD, V.J., W.J. DODDS-LAFFIN and R.J. LAFFIN.
Gamma A myeloma in a dog with defective hemostasis. J. Am. vet. med. Ass. 1121-1127. 1972. 21. SMITH, E., S. KOCHWA and L.R. WASSERMAN. Aggregation of IgG globulin in vivo. I. The hyperviscosity syndrome in multiple myeloma. Am. J. Med. 39: 3548. 1965. 22. WALDENSTROM, J.C. Macroglobulinemia In Adv. Metab. Disord. 2: 115-153. New York: Academic Press. 1965. 23. WALDENSTROM, J.C. Monoclonal and Polyclonal Hypergammaglobulinemia. Clinical and Biological Significance. Nashville: Vanderbilt University Press. 1968. 24. WELTON, T., S.R. WALKER, G.C. SHARP, L.A. HERREMBERG, R. WISLAR and W.P. GREGORY. Macroglobu-
linemia with bone destruction. Am. J. Med. 44: 280288. 1968. 25. WINTROBE, M.M. Clinical Hematology, 7th Edition. pp. 1625-1626. Philadelphia: Lea and Febiger. 1974. 26. ZAWADZKI, Z.A. and G.A. EDWARDS. Nonmyelomatosis monoclonal immunoglobulinemia. In Progress in Clinical Immunology. R.S. Schwartz, Editor. pp. 105-156. New York: Grune and Stratton. 1972.
33
Summarv This report describes a case of macroglobulinemia in a six year old castrate male Collie cross dog with clinical signs of epistaxis, anemia, retinopathy and high serum viscosity. The highest total serum protein was 12 g/dl with approximately 60% monoclonal beta globulin. Proteinuria, Bence Jones protein and osteolytic lesions were not detected. Chemotherapy and partial removal of the plasma protein by withdrawal of whole blood and transfusion with packed red cells from a DEA negative donor resulted in transient clinical remission. Resume Un cas de macroglobulinemie canine Les auteurs decrivent un cas de macroglobulinemie, chez un chien Collie croise et castre, qui presentait les signes cliniques suivants: epistaxis, anemie, retinopathie et grande viscosite du serum. Le taux le plus eleve de proteines seriques totales atteignit 12 g/dl, dont environ 60% de betaglobuline monoclonale. Ils ne decelerent pas de proteinurie, de proteine de Bence Jones ou de lesions osteolytiques. La chimiotherapie et l'enlevement partiel des proteines plasmatiques, par le moyen d'une saignee et d'une transfusion d'hematies provenant d'un donneur DEA negatif, resulterent en une disparition transitoire des signes cliniques.
Introduction Macroglobulinemia is included in a group of disorders called monoclonal gammopathies (10, 13), monoclonal immunoproliferative disorders (26) or plasma cell dyscrasias (7). Two major features are shared by these disorders: an uncontrolled proliferation of a clone of cells of the plasma cell or lymphocytic series and production by these cells of an excessive amount of homogenous immunoglobulin or its subunits or frag-
ments (7). The nature of the protein produced serves as a convenient way to classify these diseases such as IgG, IgA, IgD, or IgE myeloma, IgM macroglobulinemia, light chain disease or heavy chain disease (7). IgM molecules are macroglobulins with a molecular weight of approximately 1,000,000 daltons. They have a sedimentation coefficient of 15 Svedberg units or greater with a peak in the range of 17 S or 21 S (26). Due to their size and shape macroglobulins increase the serum viscosity and in excess can produce a series of physiological and pathological changes (3, 6, 7, 8, 10, 12, 13, 21, 23, 25, 26). Several clinical manifestations are common to monoclonal gammopathies but there is often an increased association with certain disorders. Osteolysis, bone marrow infiltration with plasma cells, Bence Jones proteinuria, and renal failure are frequently associated with multiple myeloma (5, 7, 8, 10, 12, 22, 23, 26) while bleeding tendencies, serum hyperviscosity, retinopathy, mental depression and bone marrow infiltration with lymphoid or lymphocytoid plasma cells are most often found with macroglobulinemia (3, 6, 7, 8, 10, 11, 13, 15, 18, 22, 23, 26). It should be emphasized that a few cases are reported in which these diseases overlap both clinically and biochemically, e.g. macroglobulinemia with bone destruction (24), the hyperviscosity syndrome in multiple myeloma (21), macroglobulinemia and myelomatosis (1). Dogs (8, 10) and humans (4, 5, 7, 13, 22, 23, 26) with monoclonal gammopathies develop an increased susceptibility to infections due to acquired immunodeficiency associated with decreased ability to synthesize specific antibody and increased rate of immunoglobulin catabolism. Susceptibility to bacterial sepsis is further increased if cytotoxic chemotherapy is used.
Case Report On July 15, a six year old castrate male Collie cross weighing 22 kg was presented to the Teaching Hospital because of persistent unilateral epistaxis and increased rate and depth of respiration. Two weeks prior to admission the dog experienced the initial and most severe bout of epistaxis from the right nostril. At that time the nasal sinuses were scoped and the skull radiographed. No abnormalities were detected. On admission the dog was found to be obese, depressed, had increased respiration rate, pale mucous membranes and mild epistaxis occurring from the right nostril. No other clinical abnormalities were detected. Laboratory parameters were obtained on admission (Table I). A severe anemia (PCV 15.6%) with a marked erythroid response and marked rouleaux formation was found. A direct antiglobulin test was negative. Blood platelet levels were within low normal limits but were con-
*Department of Pathology (Mejia and Lumsden) and Department of Clinical Studies (Carman), Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1.
28
Can. vet. J. 20: 28-33 (January 1979)
TABLE I VARIATION OF HEMATOLOGICAL PARAMETERS AT DIFFERENT STAGES OF THE DISEASE Sate RBC
x
PCV
%
Julv 16
July 26
July 28
2.01
2.80
2.96
4.57
15.6
Hgb
gm/dl
WBC
x
Neutrophils Segmented
July 15 10 6/ul
103/ul 3
x
10 /ul
20.9
26.2
Aug. 9
Aug. 13
3.94
29.9
Aug. 16
4.21
29.0
Sept. 9
3.28
29.1
3.95
Oct. 14 2.48
22.2
24.6
14.7
4.45
24.7
29.9
7.1
10.2
9.9
10.0
8.2
8.4
5.7
7.6
7.7
8.6
9.7
10.2
18.0
10.0
12.7
8.6
7.3
4.2
2.0
10.8
4.3
10.8
13.6
8.55
10.2
6.02
6.42
2.85
1.16
9.5
0.69
15.20
7.17
200
180
----
510
170
150
250
200
280
3100
820
1450
300
1270
1800
290
970
600
280
90
1090
1820
640
240
440
120
40
760
----
260
---
---
---
9.18
1220
420
190
420
560
Monocytes
/ul
300
1570
90
---
Eosinophils
/ul
----
----
----
---
Rubricytes
/ul
1670
1220
----
+
444
4444.
+
+
+
+
-
+
+
230
21
187
63
42
16
110
12
147
140
103/ul
Nov. 8
3.79
9.4
1220
x
28.7
Nov. 1
6.8
/ul
103/ul
3.85
17.4
/ul
x
21.1
Oct. 28
4.8
Lymphocytes
Reticulocyte
3.46
15.2
Neutrophils Bands
Polychromasia
Oct. 22
200
180 ----.
200
.
----
40
430
..
Rouleaux Formation
Platelets
125
227
91
Platelet
aggultinatton
+++
...
++-+
+4-+
+++
+
++
+++
++
si
Shift Platelets LN.
-
to
++-+
+++
+
+
+++
low norma l.
+4.4.-
range
of
presence.
sistently clumped making accurate counting difficult. In vitro erythrocyte rouleaux and platelet agglutination were constant features through the course of the disease. Blood urea nitrogen and urinalysis findings were within normal limits. Total serum protein was markedly elevated (10.6 g/dl) (Table II). Serum electrophoresis demonstrated a monoclonal peak in the B2 globulin region of the electrophoretic pattern (6.2 g/dl) (Figure 1 and Table II). This protein was later identified to be an IgM macroglobulin by immunoelectrophoresis using monospecific antisera.' Serum ultracentrifugation on two different occasions determined the paraprotein to be a macromolecule
with a sedimentation coefficient of 17 Svedburg units.2 The serum viscosity was found to be 3.5 cps, a value three times greater than that of the normal control.3 Bone marrow aspiration and examination demonstrated the presence of increased numbers of mature plasma cells in a hypercellular population of normal myeloid and erythroid marrow cells. These plasma cells had oval to round eccentrically located nuclei with a heavy chromatin pattern and abundant pale to dark blue cytoplasm. Prothrombin time (28.5s vs 17s normal control), partial thromboplastin time (28.5s vs 21s normal control), fibrinogen levels and platelet numbers
TABLE II TOTAL PROTEIN. ELECTROPHORESIS AND VISCOSITY VALUES Total Protein Date 7 20 7 30 8 5 8 17 9 9 10 12 10 14 10 15
(g/dl) 10.6 7.7 9.1 9.6 10.4 12.2 10.9 9.8
alb = albumen, glob
=
alb (g/dl) 2.6 2.5 3.0 3.1 2.8 2.9 2.5 2.6
glob (g/dl) 8.0 5.3 6.5 6.5 7.6 9.3 8.4 7.2
al glob (g/dl) 0.4 0.3 0.4 0.52 0.6 0.4 0.5 0.5
a2 glob (g/dl) 1.2 1.2 1.6 0.91 0.6 1.25 0.6 0.7
/ glob (g/dl) 6.2 3.5 4.2 4.9 6.2 7.2 7.1 5.8
y glob (g/dl) 0.2 0.2 0.3 0.2 0.2 0.4 0.2 0.2
alb/glob 0.3 0.5 0.5 0.5 0.4 0.3 0.3 0.4
Viscosity cps 3.5 2.9 3.6 5.5 4.4
globulin, cps = centipoises.
'Immunoelectrophoresis was performed by Dr. Arthur Hurvitz, Animal Medical Centre, 510 E 62nd. St.. New York. N.Y. 1002 1. 2Ultracentrifugation studies were performed by Dr. R.J. Douglas, Professor of Microbiology, University of Guelph. 3Viscosity measurements were performed by S. Al-Izzi, Graduate Student Clinical Pathology, Department of Pathology, O.V.C. 29
la&6- Iocl
(+
FIGURE 3. Thoracic radiograph taken at the time of initial presentation.
5,/2IYI(-
02
Repeated urinalysis failed to demonstrate proteinuria nor were Bence Jones proteins detectable on three separate occasions (9) using the heat precipitation method. Thoracic radiographs demonstrated the presence of expansile rib lesions on the second left rib and on the mid region of the fourth and fifth right ribs (Figure 3). Repeated radiographs at a later date showed the lesion in the fifth right rib to be expanding. A radiographic scan of the skeleton did not reveal the classical myeloma "punched-out" bony lesions which are suggestive of osteolysis by invading tumor cells (17, 24). Old fractures were identified in the area of the left pubis and right femoral neck. The radiographic cardiac silhouette was within normal limits. Later repeated radiographic examinations displayed the development
FIGURE 1. Serum electrophoretic pattern during the course of the disease. Notice homogeneous peak in beta 2 globulin zone. alb = albumin, a, = alpha 1 globulin, a2 = alpha 2 globulin, ,f1 = beta I globulin, 182 = beta 2 globulin and y = gamma globulin.
were examined and monitored throughout the course of the disease. Blood clotting times remained moderately prolonged while fibrinogen levels remained at low normal limits (105 mg/dl vs 100-300 mg/dl reference values). The average platelet counts (145,000/,l) were low. (Table 1). Serum calcium, phosphorous, magnesium, enzyme levels and electrolyte values were within normal limits and remained so throughout the course of the disease.
35F
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25 I
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I I
I
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I
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~ , II% c I
I , I~~~~~~~~~c
'tIb
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I
101
10 "cr
5
5
I*IlIlfl*fIfI
-LEUKERAN
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2mg/day 25mg/dayr PREDNIZONE h
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r
-U---ALKERAN Jo 6.5mg/day
/ay[
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AMPICILLIN 1500mg DAILY il
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7 3 11 AUGUST
PREDNIZONE 50mg/day :30g/ay
|AMPICILLIN I
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!CEPOREX
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15
15
19 23 27 OCTOBER
31
FIGURE 2. Graphic representation of PCV, WBC and total serum protein values related to the therapy. = Total serum protein g/dl. o o = microhematocrit %. o - ---- -0 = leukocytes x IO3/ ml. Phlebotomy and transfusions were made on July 26 and Oct. 14.
30
FIGURE 4. Retinal vessel sacculations detected at the time of initial presentation.
of generalized cardiac enlargement (Figure 3). Electrocardiograph findings were normal. Retinal examination showed the presence of dilated vessels with saccular enlargements. No retinal hemorrhages were present (Figure 4). A diagnosis was made of macroglobulinemia with hyperviscosity syndrome and defective hemostasis. Treatment was initiated and continued over a four month period. Management With the absence of secondary kidney disease, skeletal pain or concurrent infection, the collie was considered a good candidate for chemotherapy. In preparation the dog was given systemic antibiotic,4 vitamin B complex with iron5 and anabolic steroid therapy6 to prevent superinfection and exacerbation of the anemia problem. In an attempt to correct the anemia, decrease the serum protein levels, and decrease blood loss by epistaxis, a crude plasmaphoresis was performed as previously described (10) under halothane anaesthesia. One liter of whole blood was removed and replaced simultaneously by 650 ml of DEA negative packed red blood cells suspended in 500 ml of an isotonic electrolyte solution. Following this procedure total serum protein levels decreased from 10.6 g/dl to 7.7 g/dl and serum globulin levels decreased from 6.2 g/dl to 3.5 g/dl. The hematocrit increased from 15% to 32% and the serum viscosity decreased from 3.5 cps to 2.9 cps (Table II). The epistaxis ceased. Chlorambucil7 therapy (1 mg/ 12.5 kg every second day) as previously described (20) in combination with prednisone8 therapy (2.2
FIGURE 5. The second bone marrow aspiration biopsy contained many lymphocytoid plasma cells. Wrights X240.
mg/kg/d) was initiated. Chlorambucil therapy was continued for a total of eight doses. During this time serum protein levels continued to rise while leukocyte levels fell only mildly. Because of the ineffective response, therapy was discontinued. Further chemotherapy was postponed because of the nonresponsive anemia. At the time of discharge serum protein levels had risen to 9.6 g/ dl yet the serum viscosity remained at 3.9 cps (Table II). The dog was represented two months following initial discharge. A large hematoma was present on the right elbow. Total serum protein levels had risen (12.2 g/dl), the hematocrit had fallen (22%), the anemia was unresponsive and blood viscosity levels had risen (5.5 cps) (Table II). Severe epistaxis began two days following admission causing the hematocrit to fall to 14.7%. A decrease in serum protein levels occurred as well (10.9 g/dl) due to the blood loss (Table II). Plasmaphoresis was repeated with the dog under halothane anaesthesia. Eleven hundred and fifty ml of whole blood were removed and replaced by 650 ml of packed DEA negative red blood cells suspended in one liter of isotonic electrolyte solution. The hematocrit increased (30.4%) and total serum protein levels decreased (9.8 g/dl) although not as much as previously (Table II). Again epistaxis ceased. Maintenance therapy with systemic antibiotics,9 vitamin B complex with iron'0 and anabolic steroids" was reinstituted. Another bone marrow biopsy was examined
4Ampicillin, Ayerst Laboratories, Montreal, Quebec. 5Beminal with iron, Ayerst Laboratories, Montreal, Quebec. 6Winstrol, Winthrop Laboratories, Aurora, Ontario. 'Leukeran, Burroughs Wellcome Ltd., LaSalle, Quebec. XDeltacortisone, Upjohn Company of Canada, Orangeville, Ontario. 9Cephalexin monohydrate, Ceporex Glaxo Laboratories, Toronto, Ontario. '°Beminal with iron, Ayerst Laboratories, Montreal, Quebec. Winstrol, Winthrop Laboratories, Aurora, Ontario.
31
(Figure 5). The previously identified mature plasma cells were replaced by very immature plasmacytoid cells with a detectable maturation sequence. These cells now composed 60% of the marrow population. Normal leukocyte precursors were present but there was a relative depression of the red cell series. The hematocrit was 35%, leukocyte count 9.8 x 103/,ul and the serum protein 12 g/dl. L-phenylalanine mustard'2 therapy (0.25 mg/kg/day) was begun and continued for a total of seven doses (Figure 3). During this course of therapy the total serum protein level decreased and the blood leukocyte count fell to 2,000/,ul. Prednisone'3 therapy was initiated at the same time (2.2 mg/ kg/day) and continued after the withdrawal of the alkylating agent. Leukocyte levels returned to normal levels after discontinuation of alkeran therapy while the serum protein levels remained stable (Figure 2). The dog was discharged one week later with antibiotic therapy'4 to be continued at home and prednisone therapy to be gradually withdrawn. L-phenylalanine mustard therapy was to be repeated at six week intervals for a period of four days in an attempt to maintain remission. Two weeks following discharge the dog was presented in septic shock. A massive soft tissue swelling of the left hind limb was present. Escherichia coli was cultured from the peripheral blood. Euthanasia was performed because of the poor prognosis.
Gross and Histological Findings The dog was in good body condition with large amounts of both subcutaneous and intra-abdominal fat. The left hind limb was markedly swollen distal to the stifle joint with both subcutaneous and interfacial edema fluid. The spleen was congested and contained only limited amounts of follicular tissue. The previously identified rib lesions were easily located and consisted of areas of marrow cavity enlargement in the mid rib region. The bony cortex at these locations was thin, brittle and easily cut and marrow cells filled the cavity. The ratio of red to yellow marrow was within expected limits. The kidneys appeared to be normal. The heart appeared dilated due to the presence of a thickened and dilated left ventricle. On microscopic examination the edema fluid of the limb contained moderate numbers of mature neutrophils. The spleen contained a few karyorrhectic lymphocytes and a few differentiated plasma cells. This decreased number of plasma cells may be a reflection of previous chemotherapy. The liver histology was unremarkable with only periacinar lipidosis present. The bone marrow appeared hypercellular with the majority of the cells poorly differentiated medium-size mononuclear cells be-
longing to the lymphocytic series. The erythroid series was markedly reduced. Mature plasma cells were found in a few areas only. The medullary collecting ducts of the kidneys were found to contain proteinaceous casts. Many of the glomeruli had membranes thickened by eosinophilic material. Some of the Bowman's spaces contained proteinaceous material as well. Proteinuria was not evident in previous urinalysis. These kidney changes may have been due to secondary disease or directly related to the primary disease process.
Discussion Epistaxis may be associated with localized tissue destruction due to tumor masses, trauma, infection or primary blood coagulation defects. Macroglobulinemia or other dysproteinurias should be considered as a cause of epistaxis and be included in the differential diagnosis. Epistaxis was the presenting problem in this animal. The defective hemostasis is not believed to be due to one parameter derangement but to combined alterations in the clotting system. Precipitation of blood clotting factors (II, V, VII), interference of the thrombin-fibrinogen conversion reaction, inadequate platelet adhesiveness with altered platelet function due to platelet surface coating, Ca++ chelation, and vascular wall damage with the activation of the fibrinolytic system by the presence of the abnormal tumor product are believed to be responsible for the overall bleeding disorder in man (18). A routine total serum or plasma protein determination is important in the early recognition of dysproteinemias. A diagnosis of monoclonal gammopathy can only be reached by the evaluation of a serum electrophoretic pattern and the detection of a monoclonal protein peak (19). Immunoelectrophoresis with specific reagents and ultracentrifugation can be used to identify the protein involved and define its molecular weight. Identification of the specific paraprotein produced is important in human medicine for therapeutic and prognostic purposes (2). Only as this information is accumulated will the clinical importance be determined for animal species. Examination of bone marrow aspirate smears will often reveal the presence of an abnormal number or type of lymphocytic or plasmacytic cells. The cellular morphology and level of cellular maturity cannot alone be used to distinguish myeloma from macroglobulinemia. The course of the disease, the presence or absence of osteolysis, and the type of protein being produced do not correlate well with the morphological form of lymphocytic cell observed (24). Proteinuria, if present may be due to Bence Jones proteinuria or due to glomerular membrane
'2Alkeran, Burroughs Wellcome Ltd., LaSalle, Quebec. '3Deltacortisone, Upjohn Company of Canada, Orangeville, Ontario. '4Cephalexin monohydrate, Ceporex Glaxo Laboratories, Toronto, Ontario. 32
damage induced by circulating macroglobulins. In these cases impaired renal function is closely associated with greatly reduced patient survival time (2). Blood or serum viscosity measurements allow the identification of the hyperviscosity syndrome. Retinal vessel distention and sacculation, hematological defects and congestive heart failure due to the presence of an expanded blood volume may be present as a result of increased serum viscosity ( 14). Plasmaphoresis can serve as an important method of therapy not only in times of acute crisis during massive epistaxis, but also as a method of maintenance therapy during prolonged treatment of the disease (16). It is an effective method in reducing elevated serum protein levels and serum viscosity and halting continued bleeding episodes without the toxicity associated with chemotherapeutic agents. When used correctly and in conjunction with the alkylating agents, the patients' lifespan may be effectively lengthened.
Veterinary Therapy V. R.W. Kirk, Editor. pp. 372374. Philadelphia: W.B. Saunders Co. 1974. 11. KOK, D.A.. D.N. WHITMORE and R.W. AINWORTH. Four
cases of Waldenstrom's macroglobulinemia. J. clin. Path. 76: 351-361. 12. KYLE. R.A. Diagnosis of monoclonal gammopathies. In Manual of Clinical Immunology. N.R. Rose and H. Friedman, Editors. pp. 734-752. Washington, D.C.: American Society for Microbiology. 1976. 13. KYLE, R.A. and E.A. BAYRD. The Monoclonal Gammopathies. Springfield, Illinois: Charles C. Thomas. 1976. 14. MacKENZIE, M.R., E. BROWN, H.H. FUNDENBERG and L.
GOODENDAY. Waldenstrom's macroglobulinemia: Correlation between expanded plasma volume and increased serum viscosity. Blood 35: 394-408. 1970. 15. MacKENZIE, M.R. and H.H. FUNDEMBERG. Macroglobulinemia: An analysis for forty patients. Blood 39: 874-889. 1972. 16. ORENTREICH, N., S. ETTINGER, R.J. TASHJIAN, A.A.S.
STANISLOUISKI and R. HENKIN. Intensive chronic plasmaphoresis in dogs. Am. J. vet. Res. 29: 19291933. 1968. 17. OSBORNE, C.A., V. PERMAN, J.H. SAUTTER, J.B. STEVENS
Refi lelicel.S
and G.F. HANLON. Multiple myeloma in the dog. J. Am. vet. med. Ass. 153: 1300-1319. 1968.
1. ADNER, P.L., G. WALLENIUS and I. WERNER. Macro-
18. PERKINS, H.A., M.R. MacKENZIE and H.H. FUNDEMBERG.
globulinemia and myelomatosis. Acta med. scand. 168: 431-437. 1960. 2. ANONYMOUS. A comparative study by acute Leukemia Group B: Correlation of abnormal immunoglobulins with clinical features of myeloma. Archs intern. Med. 135: 46-52. 1975. 3. BLOCH, K.J. and D.G. MAKI. Hyperviscosity syndrome associated with immunoglobulin abnormalities. Semin. Hematol. 10: 113-124. 173. 4. DUTCHER, T.F. and J.F. FAHEY. The histopathology of the macroglobulinemia of Waldenstrom. J. natn. Cancer Inst. 22: 887-917. 1959.
Hemostatic defects in dysproteinemias. Blood 35: 695-707. 1970. 19. PUTMAN, F.W. Plasma-cell myeloma and macroglobulinemia. I. Physiochemical immunochemical and isotopic turnover studies of the abnormal serum and urinary proteins. New Engl. J. Med. 18: 902-908. 1959.
5. ENGLE. R.L. Jr. and L.A. WALLIS. Immunoglobulinopathies; Immunoglobulins, Immune Deficiency Syndromes, Multiple Myeloma and Related Disorders. Springfield, Illinois: C.C. Thomas. 1969. 6. FAHEY. T.L.. W.F. BARTH and A. SOLOMON. Serum
hyperviscosity syndrome. J. Am. med. Ass. 192:120133. 1965. 7. HARVEY, A. Mc., R.T. JOHNS, A.H. OWENS Jr. and R.S.
ROSS. Immunoglobulins and plasma cell dyscrasias. In The Principles and Practice of Medicine. 19th Edition. pp. 1324-1343. New York: Appelton Century Crofts. 1976. 8. HURVITZ, Al.., S.C. HASKINS and C.A. FISHER. Macro-
globulinemia with hyperviscosity syndrome in a dog. J. Am. vet. med. Ass. 157: 455-460. 1970. 9. HURVITZ, A.l., M. KEHOE, J.D. CAPRA and R. PRATA.
Bence Jones protein and proteinuria in a dog. J. Am. vet. med. Ass. 159: 1112-1116. 1971. 10. HURVITZ, A.l. Monoclonal Gammopathies in Current
20. SHEPARD, V.J., W.J. DODDS-LAFFIN and R.J. LAFFIN.
Gamma A myeloma in a dog with defective hemostasis. J. Am. vet. med. Ass. 1121-1127. 1972. 21. SMITH, E., S. KOCHWA and L.R. WASSERMAN. Aggregation of IgG globulin in vivo. I. The hyperviscosity syndrome in multiple myeloma. Am. J. Med. 39: 3548. 1965. 22. WALDENSTROM, J.C. Macroglobulinemia In Adv. Metab. Disord. 2: 115-153. New York: Academic Press. 1965. 23. WALDENSTROM, J.C. Monoclonal and Polyclonal Hypergammaglobulinemia. Clinical and Biological Significance. Nashville: Vanderbilt University Press. 1968. 24. WELTON, T., S.R. WALKER, G.C. SHARP, L.A. HERREMBERG, R. WISLAR and W.P. GREGORY. Macroglobu-
linemia with bone destruction. Am. J. Med. 44: 280288. 1968. 25. WINTROBE, M.M. Clinical Hematology, 7th Edition. pp. 1625-1626. Philadelphia: Lea and Febiger. 1974. 26. ZAWADZKI, Z.A. and G.A. EDWARDS. Nonmyelomatosis monoclonal immunoglobulinemia. In Progress in Clinical Immunology. R.S. Schwartz, Editor. pp. 105-156. New York: Grune and Stratton. 1972.
33
