- Email: [email protected]
Hemostatic Abnormalities Associated With Monoclonal Gammopathies
Hemostatic Abnormalities Associated With Monoclonal Gammopathies FRANCISCO ROBERT, MD,* MARLY MIGNUCCI, MT, MS,* SHIRLEY A. McCURDY, MT,t NORMAN MALDONADO, MO,t
ABSTRACT: To confirm and expand previous observations about the association of monoclonal gammopathies with hemostatic defects, a prospective evaluation was made in 42 patients with lymphoplasmacytic disorders. The incidence of bleeding complications was low, despite the diversity of abnormal hemostatic tests observed in these patients. Patients with myeloma frequently had abnormal coagulation tests, including thrombin time (64%), fibrin degradation products (32%), platelet aggregation tests with different agonist (30% to 55%), and bleeding time (22%). The lack of platelet response to ristocetin and normal ristocetin cofactor activity in four patients with myeloma may suggest a BernardSoulier-type defect. Serum viscosity was negatively correlated with platelet aggregation with collagen, ristocetin, and adenosine diphosphate. In patients with immunoglobulin myeloma, there was a positive correlation between an increased viscosity and a prolonged thrombin time. This study demonstrates the wide variety of coagulation abnormalities in lymphoplasmacytic disorders, usually without significant clinical implications. KEY INDEXING TERMS: Plasma cell dyscrasias; Monoclonal gammopathies; Dysproteinemias; Coagulation. [Am J Med Sci 1993;306(6):359-366.]
A
variety of lymphoplasmacytic disorders characterized by the presence of monoclonal-circulating immunoglobulins have been associated with From the *Medical Service, Hematology-Oncology Section, VA Medical Center, San Juan, Puerto Rico, the t Laboratory Service, VA Medical Center, San Juan, Puerto Rico, the +Department of Medicine, University of Puerto Rico, San Juan, Puerto Rico, and the §Biostatistics Unit, Comprehensive Cancer Center, University of Alabama at Birmingham. Correspondence: Francisco Robert, MD, Department of Medicine, Hematology-Oncology Section, Comprehensive Cancer Center, University of Alabama at Birmingham, VA Medical Center, 700 South 19th Street, Birmingham, AL 35233. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
JEANNETTE Y. LEE, PHD§
multiple coagulation abnormalities. 1- 3 These acquired hemostatic defects are features of a number of dysproteinemic disorders, including multiple myeloma, Waldenstrom's macroglobulinemia, systemic amyloidosis, and lymphoma. In general, these alterations of hemostasis may be expressed as either hemorrhage or thrombosis, or a combination of the two. Hemorrhagic manifestations are more common with the actual incidence, varying depending on the particular dysproteinemia. Bleeding diatheses 3 are observed more often in macroglobulinemia (36%) or immunoglobulin A (IgA) myeloma (33%) than immunoglobulin G (IgG) myeloma (15%). The pathophysiology of these hemostatic defects are complex and only partially understood. Most of the abnormalities may be explained on the basis of four mechanisms: (1) circulating paraproteins (monoclonal immunoglobulins) that bind to and inhibit components involved in hemostasis, (2) circulating paraproteins that bind to coagulation factors, promoting an enhanced clearance of the complex, (3) monoclonal cellsurface immunoglobulins that bind and clear circulating coagulation proteins, and (4) abnormal proteins deposited into the extracellular space of certain tissues that bind to normal coagulation proteins. 4 In addition, bleeding diatheses may be associated with conditions that complicate the primary disorder, such as uremia, hypersplenism, bone marrow failure, liver disease, sepsis, or chemotherapy. To confirm and expand these earlier observations about the relationship of dysproteinemias with acquired hemostatic abnormalities, we report the results of a prospective laboratory evaluation of coagulation in 42 patients with lymphoplasmacytic disorders. In addition, the coagulation profile of 12 cases with essential or secondary monoclonal gammopathies were examined. Results were correlated with the amount of circulating monoclonal protein, type of immunoglobulin or light chain, and serum viscosity. Patients and Methods Patients. Fifty-four patients with the appearance of
a homogenous monoclonal protein in the serum or urine were studied from August 1984 to March 1990. Through 359
Hemostatic Defects in Monoclonal Gammopathies
the duration of the study (6 years), each patient was observed for possible hemorrhagic or thrombotic events. Bleeding time determinations and blood sample collections were done with informed consent from the patients. Serum and urine found to contain monoclonal protein components by zonal electrophoresis were studied by immuno-electrophoresis to identify the class of heavy chain and the type of light chain of the protein. Diagnosis of the various causes of monoclonal gammopathy was based on a thorough history, physical examination, and pertinent laboratory examination. Laboratory studies performed included a complete blood cell count, serum chemistry, special hematologic studies for the diagnosis of lymphoplasmacytic disorders, and other procedures to search for underlying diseases. Serum viscosity was determined as previously described. 5 Based on these studies and using standard diagnostic criteria,6-8 the patients were classified as follows: Group 1. Multiple myeloma: Thirty-four patients with active disease were studied; most before chemotherapy. Of the 34,19 had IgG myeloma, 9 had IgA myeloma, and 6 had light chain disease. There were 31 men and three women, ranging in age from 44 to 85 years (median, 62 years). Group 2. Diseases associated with monoclonal (IgM) macroglobulinemia: Four patients were included in this category; three had Waldenstrom's macroglobulinemia and one had an extramedullary lymphoplasmacytic neoplasm. There were three men and one woman, ranging in age from 56 to 62 years (median, 60 years). Group 3. Diseases associated with monoclonal IgG gammopathy: Four male patients ranging in age from 41 to 70 years (median 63 years) were studied; one had primary amyloidosis, one had a solitary osseous plasmacytoma, and two had a lymphocytic neoplasm. Group 4. Essential and secondary monoclonal gammopathies: Twelve male patients with a serum monoclonal protein either without an associated disease or with a non-Iymphoplasmacytic disorder were evaluated. They ranged in age from 58 to 79 years (median, 71 years). Methods. The patients had not taken aspirin or other drugs interfering with platelet function for at least 7 days before testing. Fasting blood samples were obtained for all coagulation studies. The activated partial thromboplastin time, prothrombin time, thrombin time, and fibrinogen levels were performed by standard methods. 9 The reptilase clotting time of plasma was determined according to the Sigma Diagnostics procedures (Atroxin Test; Sigma Chemical Co, St Louis, MO). Fibrin degradation products were measured with the use of the Thrombo-W ellcotest assay (Wellcome Diagnostics, Dartford, England). More recently, the fragment D-dimer determination was included in the
360
coagulation profile of these patients (Fibrinosticon; Organon Teknika, Durham, NC). Circulating anticoagulant screening tests (mixing studies) were performed as previously described. 9 A Lupus-type inhibitor was detected in one of the patients by methods previously described;9.10 the patient's plasma was treated with an anti-immunoglobulin M (IgM) anti-serum (Sigma Chemical Co, St Louis, MO) in an attempt to confirm the nature of the anticoagulant. Prolonged thrombin times were evaluated for heparin-like anticoagulants using in vitro protamine sulfate (Sigma Chemical Co, St Louis, MO) neutralization of the patient's plasma. Platelet function was evaluated with a bleeding time (General Diagnostics Simplate, Morris Plains, New Jersey), and platelet aggregation studies were performed as previously described. n - 13 Bleeding time was not performed if the platelet count was below 100,000/ J.LL. The concentrations of inducing agents were 5 J.LM and 10 J.LM adenosine diphosphate (Sigma Chemical, St Louis, MO); 0.2 mg/mL collagen (Worthington Biochemical, Freehold, NJ); 5 J.LM and 10 J.LM epinephrine (Parke Davis, Detroit, MI); and 1.5 mg/mL ristocetin (Abbott Laboratories, North Chicago). The extent of platelet aggregation was expressed as the percent of increase in light transmission using patient's autologous platelet-poor plasma as 100% standard. The von Willebrand factor (Ristocetin Cofactor) assay (Bio/Data Co, Horsham, PA) was completed on patients with an abnormal aggregation induced by ristocetin. Statistical Methods. Statistical analyses were carried out using the Statistical Analysis System software package (SAS Institute, Cary, NC). Group comparisons of continuous data were made using the Wilcoxon rank sum test. 14 Correlations between variables were assessed using the Spearman rank correlation coefficient. All statistical tests were carried out at the two-tailed significance level. Results
Fifty-four patients with a monoclonal protein (Mprotein) in the serum or urine were evaluated. Table 1 outlines the laboratory features of the M-proteins in each group of patients. The number of patients in the categories will occasionally be smaller because of incomplete information in some cases. The predominant immunoglobulin was IgG: 56% in the lymphoplasmacytic disorders and 67% in group 4 (essential/secondary gammopathies) . The various hemostatic parameters in each group of patients are shown in Table 2. There was a small number of patients evaluated in certain groups, and the available number of results for each group is different. For these reasons, calculation of the probable significance of differences using a one-way analysis of variance procedure was limited between groups 1 and 4. There was a trend for prolonged thrombin times and December 1993 Volume 306 Number 6
Robert et 01
Table 1. Laboratory Evaluation of M-Proteins in Patients With Lymphoplasmacytic Disorders and Essential (or Secondary) Monoclonal Gammopathies*
Group
No. of Patients
Serum Monoclonal Spike (gm/L)
1. Multiple myeloma IgG IgA Light chain disease 2. IgM disorders 3. IgG disorders 4. Essential/secondary gammopathies§
34 19 9 6 4 4 12
46.2 ± 19.2:j: 43.2 ± 18.1 52.4 ± 21.0
Serum Viscosityt 2.46 2.49 3.03 1.51 6.45 1.73 1.73
25.9 ± 20.4 13.8 ± 6.05 23.5 ± 10.2
± 1.05 ± 1.08 ± 0.96 ± 0.16 ± 5.8 ± 0.25 ± 0.42
Light Chain K K K K K K K
(21), L (13) (11), L (8) (7), L (2) (3), L (3) (3), L (1) (2), L (2) (8), L (4)
* Results were expressed as mean ± standard deviation. .
.
..
. Flow Time of Serum the ratw o f . (normal range 1.4-1.8). Flow Time of Water :j: Cases of light chain disease are not included. § IgG (7), IgA (5).
t The relatwe VISCOSity
IS
reduced platelet aggregation with adenosine diphosphate (ADP), collagen, and ristocetin in patients with multiple myeloma as compared with patients in group 4, but these differences are not statistically significant. Patients with multiple myeloma had significantly higher titers of fibrin degradation products as compared with patients in group 4 (p = 0.03, by analysis of variance, unpublished data). Hemostatic abnormalities were fewer and less severe in group 4. One patient with chronic myelomonocytic leukemia (IgGk) had a bleeding time over 900 seconds and slight elevation of the fibrin degradation products. Another patient with squamous cell carcinoma of the penis (IgGk) had a slight reduction of the platelet aggregation with ADP (50%), collagen (60%), and ristocetin (47%), but had a normal bleeding time and ristocetin cofactor assay. Two additional cases in group 4 had a slight reduction of the platelet aggregation with
ristocetin, but normal bleeding times. None of these patients had a history of previous hemorrhagic complications, and their family history was unremarkable. In this series of patients, the incidence of bleeding complications was low, despite the diversity of abnormal hemostatic tests observed in groups 1 and 2. Two major hemorrhagic events were noted in two patients: one patient (JO) in group 1 developed significant bleeding after a dental extraction during his initial evaluation; the other patient (JM), in group 2, had an upper gastrointestinal bleeding. The clinical and laboratory data of these cases are summarized in Table 3. In Case JO, the bleeding time was prolonged and the abnormal prothrombin time was not corrected with a mixture of equal parts of normal plasma and patient plasma. Treatment with chemotherapy produced a good response; a follow-up examination of his hemostatic parameters 8 months later revealed a normal
Table 2. Summary of Coagulation Assays and Platelet Function Studies*
Test Bleeding time Platelet count Platelet aggregation Adenosine diphosphate Collagen Epinephrine Ristocetin Ristocetin cofactor assay Prothrombin time Partial thromboplastin time Thrombin time Fibrinogen
Multiple Myeloma (Group 1) N =' 34 488
± 37 (22) 226 (15)
64.3 ± 75 ± 60.8 ± 62.4± 143 ± 12.3 ± 27.6 ± 13.8 ± 3.3 ±
4.8 (38) 4.4 (30) 8 (31) 6 (55) 20.9 (1/13) 0.3 (11) 0.7 (6) 1.1 (64) 0.2.(8)
IgM Disorders (Group 2) N =' 4 414
± 50 (O)t 214 (I;")
68.5 ± 10.9 (2/4) 65.5 ± 11 (2/4) 43 (1/1) 70.8 ± 12 (2/4) 77 ± 28 (1/3) 14.9 ± 2.5 (1/4) 40.9 ± 12.5 (1/4) 9.6 ± 0.2 (0) 3.7 ± 1.1 (0)
IgG Disorders (Group 3) N = 4 450 ± 151 (';..) 218 ('II) 75.9 ± 5 (0) 3.5 (0) 81 ± 70 (0/1) 2.7 (0) 83 ± 0.4 (0) 11.2 ± 2.1 (0) 27 ± 8.3 (0) 0.4 (0) 3.2 ±
Essential/Secondary Gammopathies (Group 4) N = 12 422
± 53 (8) 265 (0)
74.7 ± 80 ±
3 3
(8) (8)
± 3.5 (25) 78.5 (0/2) 12.1 ± 0.3 (8) 26 ± 0.9 (0) 9.9 (0) 3.1 ± 0.3 (0)
78
Normal Range 180-540 sec 150-400 X 109/L 68.8%-91.6% 63.8%-91% 67%-97% 76%-99% 58%-166% 10.9-13.3 sec 22.7-36.7 sec 5.9-10.7 sec 1.55-3.51 gm/L
* Results were expressed as mean ± standard error of the mean. t Numbers in parenthesis indicate the percentage or fraction (if few cases were tested) of patients with an abnormal value. THE AMEIlICAN JOUIlNAL OF THE MEDICAL SCIENCES
361
Hemostatic Defects in Monoclonal Gammopathies
Table 3. Clinical and Laboratory Characteristics of Patients With Hemorrhagic Events* Patients
Diagnosis Serum M-spike (gm/L) Serum viscosityt Platelet count (X 109 /L) Bleeding time Platelet aggregation Adenosine diphosphate Collagen Ristocetin Ristocetin cofactor assay Prothrombin time Partial thromboplastin time Fibrinogen (gm/ L)
* The normal range
JO
JM
IgGk myeloma 82.1 4.8 233 900 sec
IgMk macroglobulinemia 38.3 12.9 302 510 sec
67.5 % 67.5% 0% 154% 17 sec:j: 26.8 sec 2.75
50% 45% 50% 29% 12.6 34.3 sec 2.75
~f
values is given in Table 2. 1.4-1.8). :j: The abnormality was not corrected with 50:50 mixture of normal plasma and patient plasma.
t Relative viscosity (normal range
bleeding time (510 seconds) and prothrombin time (13.2 seconds). The abnormal prothrombin time was interpreted as indicating the presence of an inhibitor (probably to Factor VII), but a specific inhibitor assay or a Russell's viper venom (Stypven) time was not performed. There was a lack of platelet aggregation in response to ristocetin, but this inhibitory activity of the patient plasma was not observed in the ristocetin cofactor assay. In Case JM, the bleeding time was within normal range despite some suppression of the platelet aggregation to different agonists and marked inhibitory activity in the ristocetin cofactor assay. Cryoglobulin was absent. There was no history of previous hemorrhagic complications, and his family history was negative for significant bleeding. Viscosity was elevated in both patients, but it was markedly abnormal in Case JM, suggesting a possible explanation for the bleeding diathesis in this patient. He was treated with plasmapheresis and chemotherapy with a resolution of the bleeding diathesis; a follow-up examination of his hemostatic parameters was not performed. None of the patients studied had thrombotic complications. The association of lupus inhibitor and Waldenstrom's macroglobulinemia was observed in one of the cases in group 2. Case EB was a patient with an IgM lambda monoclonal gammopathy who had skin lesions and marked prolongation of the partial thromboplastin time. She had no history of thrombotic or hemorrhagic complications, and did not have a bleeding tendency during diagnosis or throughout her illness. The results of the screening coagulation tests and specialized laboratory studies are shown in Table 4. The activated partial thromboplastin time was markedly prolonged
362
on several occasions and did not decrease significantly with a splenectomy or chemotherapy. The prothrombin time was always prolonged, but to a lesser degree than the partial thromboplastin time. The mixing studies showed that the defect in coagulation was not corrected with normal plasma at different time intervals. The tissue thromboplastin inhibition studies were prolonged at the 1:200 and the 1:1000 dilution with a ratio of 2.9 to 3.2, respectively. The platelet neutralization test showed a dramatic correction when a suspension of washed normal platelets were incubated with the mixture of the activated partial thromboplastin test. The control showed no change. The mixture of patient plasma with an anti-IgM antiserum showed a shortening of the partial thromboplastin time, but especially of the prothrombin time. The frequency of abnormal coagulation tests in the myeloma subtypes is listed in Table 5. The frequency of abnormal values was high among patients with myeloma for bleeding time (22%), platelet aggregation tests with different agonists (30% to 55 %), thrombin time (64 %) and the fibrin degradation product assay (32%). A significant difference was detected between IgA myeloma, IgG myeloma, and light chain disease with respect to fibrinogen levels (p = 0.034). Among the patients with IgG myeloma, thrombin time was significantly prolonged in IgG myeloma containing lambda chains as compared with those with kappa chain (p = 0.04), with mean values of27.3 seconds and 10.6 seconds, respectively. For all patients with myeloma combined, the mean value of platelet aggregation with collagen of 85 % for myelomas with kappa chains (n = 20) was significantly higher than the 53% with those containing lambda chains (n = 13; p < 0.01).
Table 4. Results of Coagulation Studies in a Patient (EB) with Waldenstrom's Macroglobulinemia Coagulation Assays Prothrombin time (PT) Partial thromboplastin time (PTT) Thrombin time Circulating anticoagulant Screening PT (50:50) PTT (50:50) Tissue thromboplastin Inhibitor (dilution/ratio) Platelet neutralization procedure (PNP) Control saline PNP Bleeding time Platelet aggregation (ADP)
Results
(Normal Range)
22.3 sec
(10.9- 13.3 sec)
71.5 sec 9.8 sec
(22.7-36.7 sec) (5.9-10.7 sec)
20.5 sec* 67.5 sec* 1:100/2.9 1:1000/3.2
«1.3) «1.3)
74.8 sec 43.8 sec 300 sec 95 %
(180-540 sec) (68.8%-91.6% )
* The mixing studies showed that the defect in coagulation occurred immediately, with no significant potentiation with incubation for 1 hour. December 1993 Volume 306 Number 6
Robert et 01
Table 5. Abnormal Tests of Hemostatic Function in Multiple Myeloma* Test
All Cases
IgG
IgA
Light Chain Disease
Bleeding time >540 sec Platelet count <150 X 109/L Platelet aggregation Adenosine diphosphate < 68% Collagen < 63% Epinephrine < 67% Ristocetin < 76% Ristocetin cofactor assay < 58% Prothrombin time> 13.3 sec Partial thromboplastin time> 36.7 sec Thrombin time> 10.7 sec Fibrinogen < 1.5 giL Fibrin degradation products:j:
22% (32) 15% (34)t
29% (17) 16% (19)
11% (9) 11% (9)
16% (6) 17% (6)
38% 30% 31% 55% 8% 11% 6% 64% 8% 32%
32% 16% 33% 58% 10% 11 % 0 70% 0 38%
33% 38% 0 50% 0% 22% 11% 33% 22% 14%
(34) (33) (13) (33) (13) (34) (34) (14) (34) (28)
(19) (19) (9) (19) (10) (19) (19) (10) (19) (16)
(9) (8) (3) (8) (2) (9) (9) (3) (9) (7)
50% 0 100% 50% 0 0 16% 100% 0 40%
(6) (6) (1) (6) (1) (6) (6) (1) (6) (5)
* Percent abnormal followed by number tested. t Mean platelet count of 226 X 1(fJ/L (range 81-538 X 1(fJ/L) :j: Fibrin degradation products (> 10 Ilg/mL) or cross-linked fibrin derivatives containing the D-dimer domain (>500 ng/mL).
Abnormal bleeding times were more common in patients with IgG myeloma (29%). There was no correlation between bleeding time and the results of other platelet function tests, except for platelet aggregation with ADP (Spearman rank correlation coefficient -0.378, p = 0.01). Defective platelet aggregation with ristocetin was observed in 18 patients with multiple myeloma (mean ± standard error of the mean 62.4% ± 6%; range, 0 to 71 %). None of these patients had abnormal partial thromboplastin times. Four of these cases had prolonged bleeding times, and only one had a low ristocetin cofactor activity «25%). This patient had IgGk myeloma with a normal bleeding time and a normal platelet aggregation response to ADP and collagen. He had no history of hemorrhagic complications, and did not have a bleeding tendency during diagnosis. Two patients with IgM macroglobulinemia (Group 2) also had decreased platelet aggregation with ristocetin, but only one had a significant reduction of the ristocetin cofactor assay (Case JM, Table 3). Characterization ofthe von Willebrand factor multimers was not done on these cases. The thrombin clotting time of plasma was prolonged in 9 (64%) of the 14 patients with myeloma tested. This abnormality was correlated with increased serum viscosity and circulating monoclonal proteins (Table 6); six out of nine cases had mild to moderate increased levels of fibrin degradation products. Only one of the patients (IgAk) evaluated with Atroxin (reptilase test) had a normal clotting time. Protamine sulfate neutralized the anticoagulant activity detected in his plasma in vitro. Another patient with IgGk myeloma and significant cryoglobulinemia (16%) had prolonged plasma clotting times with both thrombin!Atroxin reagents, and complete in vitro neutralization with protTHE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
amine sulfate. None of these cases had significant bleeding complications. Abnormal prothrombin times and partial thromboplastin times were observed in only a few patients with myeloma (Table 5). Mixing studies in two cases with isolated prolonged prothrombin time and partial thromboplastin time demonstrated that a deficiency was responsible for these abnormalities. One patient discussed previously (Case JO, Table 3) had a prolonged prothrombin time not corrected with normal plasma, and most likely an inhibitor is the basic mechanism. Table 6. Relationship of Significant Hemostatic Variables With Serum Viscosity and Monoclonal Spike M-Spike
Viscosity
IgG /IgA myeloma Platelet aggregation Collagen Ristocetin Thrombin time IgG myeloma Platelet aggregation Ristocetin Thrombin time All patients* Platelet aggregation Adenosine diphosphate Collagen Ristocetin Thrombin time
p
Correlation Coefficient
p
Correlation Coefficient
0.01 <0.001 0.001
-0.448 -0.667 0.813
NS <0.01 NS
NS -0.498 NS
<0.001 <0.001
-0.800 0.916
0.005 0.02
-0.614 0.705
0.05 <0.01 <0.01 0.03
-0.287 -0.443 -0.439 0.568
0.04 0.05 <0.001 0.03
-0.301 -0.290 -0.586 0.558
* All patients included in Table 1, except those cases with light chain disease. NS = not significant.
363
Hemostatic Defects in Monoclonal Gammopathies
The presence of elevated levels of serum fibrin degradation products (>10 JLg/mL) or cross-linked fibrin derivatives containing D-dimer domain (>500 ng/mL) was demonstrated in nine patients (32%) in group 1. Another patient with IgM macroglobulinemia (Case JM, Table 3) had elevated fibrin degradation products but also had high titers of rheumatoid factor. Most of these cases had slight to moderate increased levels of fibrin degradation products, and there was no clinical evidence of thromboembolic disorders during diagnosis. None of these patients had hypofibrinogenemia, thrombocytopenia, or prolongation of the prothrombin time and partial thromboplastin time. Correlation analysis of multiple hemostatic tests was used to identify relationships with serum viscosity or circulating monoclonal proteins. A subgroup of patients with light chain disease was excluded from this analysis because of the low concentration of abnormal proteins in the serum and normal viscosity (mean ± standard deviation 1.5 ± 0.16). The hemostatic variables with the most significant correlation coefficients are shown in Table 6. Viscosity was negatively correlated with platelet aggregation with collagen, ristocetin, and ADP. Prolonged thrombin time was proportional to the increase in serum viscosity with a highly significant correlation coefficient. Similar relationships were apparent with the serum monoclonal proteins, but to a lesser degree of significance. There was no correlation between serum viscosity and monoclonal spikes and the results of the bleeding time, platelet aggregation with epinephrine, and fibrin degradation products. Discussion
A multiplicity of hemostatic defects have been described in patients with lymphoplasmacytic disorders associated with abnormal monoclonal proteins. 1- 4,15 The current comprehensive evaluation of 54 patients was designed to update the clinical implications of monoclonal immunoglobulins with acquired hemostatic disorders. We found a wide variety of hemostatic abnormalities without any correlation between the magnitude of the defect(s) and the presence of a clinical bleeding syndrome. One of the most important mechanisms in the pathophysiology of these hemostatic disorders is the presence of circulating inhibitors of blood coagulation.4 Endogenous inhibitors of hemostasis have been characterized previously as immunoglobulins that bind specifically to either individual coagulation factors or phospholipids, or that interfere nonspecifically with fibrin polymerization or platelet aggregation. 16- 19 The most commonly described paraprotein inhibitors of coagulation are directed against fibrin monomer polymerization. The laboratory features of this abnormality include a prolonged clotting time with both thrombin/Atroxin reagents and a gelatinous friable clot that exhibits poor clot retraction partially corrected by the addition of calcium. 4,18 Coleman and co-workers 18
364
have demonstrated that the inhibition of fibrin polymerization resides on the Fab fragment of the IgG and IgA immunoglobulins, but not on the Fc fragment or isolated heavy and light chains. The thrombin time was prolonged in 64% of our patients with multiple myeloma and was significantly correlated with the presence of lambda light chains, as in Coleman's study. However, it is apparent that both types of light chains may be associated with abnormal fibrin formation. 2o A significant correlation between thrombin time and serum viscosity was demonstrated in patients with IgG/ IgA myeloma (Table 6). A possible explanation for this correlation (IgG-myelomas) is that an increased amount of IgG paraproteins may accentuate this inhibition of the fibrin polymerization. Serum viscosity was significantly correlated (p = 0.001) with the IgGM spike of these patients (unpublished data). Several cases of heparin-like anticoagulant in association with plasma cell dyscrasias have been reported. 21- 24 In contrast to previous reports of circulating paraprotein inhibitors, these anticoagulants are not directly related to the myeloma protein. These endogenous heparin-like inhibitors are circulating proteoglycans with electrophoretic mobility similar to heparan or chondroitin sulfates. Although no clear evidence as to the source of these inhibitors has been found, Kaufman and collaborators21 were able to demonstrate the production of a similar anticoagulant by the neoplastic plasma cells of a patient with plasma cell leukemia. The characteristic laboratory features of these inhibitors are a prolonged thrombin time with a normal reptilase time assay as well as neutralization of the anticoagulant activity with protamine sulfate, platelet factor 4, or toluidine blue. Unlike inhibitors that interfere with fibrin polymerization, these heparinlike anticoagulants are associated with a bleeding diathesis. We identified two patients with myeloma who had a prolonged thrombin time corrected by the addition (in vitro) of protamine sulfate. One of these patients also had an abnormal reptilase assay, though neither experienced bleeding complications. Circulating immunoglobulins may also have properties characteristic of the lupus anticoag-ulant. 4 In 1980, Thiagarajan and collaborators 17 reported a patient with macroglobulinemia and a lupus-type anticoagulant whose purified monoclonal IgMX immunoglobulin possessed the inhibitory activity. This patient had a prolongation of all phospholipid-dependent coagulation tests with no bleeding manifestations. The patient's lambda paraprotein reacted with anionic phospholipids, but not with neutral phospholipids. We have studied a similar patient with IgMX (Table 4) macroglobulinemia and prolongation of the phospholipid-dependent coagulation tests. Our patient is the second reported patient, to our knowledge, with a plasma cell disorder ofthe Waldenstrom's macroglobulinemia type to have a lupus anticoagulant. Like most patients, she had no bleeding tendencies and underwent December 1993 Volume 306 Number 6
Robert et al
a splenectomy without bleeding problems. The IgM macroglobulin was shown to be present in the neoplastic cells by immunoperoxidase staining and flow cytometry (data not shown). The inhibitory activity of the lupus anticoagulant was neutralized with an antiIgM antiserum, confirming the IgM nature of the antibody. Monoclonal immunoglobulins are known to interfere with a number of coagulation proteins, including von Willebrand factor, fibrinogen, prothrombin, and factors V, VII, VIII, and X. l ,3,4,25 One mechanism of this rare phenomenon is the binding of circulating paraproteins with selective coagulation factors, which inhibit their function. 4 A conceivable explanation for the prolonged prothrombin time in Case JO (Table 3) may be related to this paraprotein-coagulation protein interaction. The mixing studies and a normal partial thromboplastin time are compatible with the presence of an inhibitor, probably affecting factor. VII activity. Another mechanism that could possibly cause low levels of one or more clotting factors might be the binding of the abnormal paraprotein to various coagulation factors, resulting in accelerated clearance of them. 4 These interactions may occur in the plasma, or at the surface of the neoplastic B cells. 3 ,26-28 Accelerated clearance is an attractive hypothesis to explain the isolated clotting time abnormalities (prothrombin and partial thromboplastin times) and low ristocetin cofactor activity occasionally observed in the plasma of our patients. The present study is in agreement with other reports with respect to the diversity of the platelet function test abnormalities in these patients. l ,3 There is a disagreement in the literature as to whether abnormalities in primary hemostasis are related to the clinical tendency for hemorrhage. Data from our study show a poor correlation between platelet function abnormalities and the development of clinically significant bleeding. We found abnormal bleeding times more common in patients with IgG myeloma. However, there was no correlation between the bleeding time and the results of the platelet aggregation tests, except for platelet aggregation with ADP (p = 0.01). The mechanism of the effect of the monoclonal immunoglobulins in the impairment of platelet function is poorly understood. It has been stated that these abnormalities occur as a consequence of nonspecific adherence of the paraprotein to the platelet surface. l ,3,4,19 In agreement with this theory is the negative correlation observed in this study between platelet aggregation with several agonists and serum viscosity or the paraprotein concentration (Table 6). DiMinno 29 and coworkers have demonstrated specific interaction of a monoclonal immunoglobulin with the platelet glycoprotein IlIa of a patient with IgGk myeloma having impaired platelet aggregation. Their data support the concept that most monoclonal immunoglobulins elaborated in multiple myeloma and related plasma cell THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
dyscrasias are functional antibodies for specific antigens. 30 The lack of response to ristocetin and normal ristocetin cofactor activity observed in four patients with myeloma (including Case JO) may suggest another specific interaction of monoclonal proteins with platelet glycoproteins, resulting in a Bernard-Soulier type defect. Three of these patients also had prolonged bleeding time. Multiple myeloma has also been associated with chronic low-grade disseminated intravascular coagulation, although this appears to be uncommon. 25 ,31 The mechanism by which this occurs is unclear. Nine patients with myeloma in this study had elevated fibrin degradation products or abnormal d-dimer assays, even though there was no clinical manifestations of bleeding or thrombotic complications. The presence of fibrin degradation products in these patients may also be due to other causes of reactive fibrinolysis, or represent false positive results. Rheumatoid factor may interfere with these assays by triggering false positive values. Although most monoclonal rheumatoid factors are IgMk (as in Case JM, Table 3), there are reports of monoclonal IgG and IgA rheumatoid factors. 3o Therefore, it is important to check for the presence of rheumatoid factor in unexpected positive samples. In summary, the interaction between monoclonal immunoglobulins with platelets and coagulation factors results in a panorama of abnormal hemostatic tests, usually without significant clinical implications. Our results indicate that laboratory evaluation of coagulation in patients with dysproteinemias does not offer information of predictive value regarding bleeding or thrombotic complications. Acknowledgments
The authors thank Dr. Pradip K. Rustagi for his critical comments and Lee Ann Grimmett for her preparation of the manuscript. References 1. Lackner H: Hemostatic abnormalities associated with dyspro-
teinemias. Semin HematollO:125-133, 1973. 2. Sanchez-Avalo J, Soong BCF, Miller SP: Coagulation disorders in cancer. 2. Multiple myeloma. Cancer 23:1388-1398, 1969. 3. Perkins HA, Mackenzie MR, Fundenburg HH: Hemostatic defects in dysproteinemias. Blood 35:695-707, 1970. 4. Furie B: Acquired coagulation disorders and dysproteinemias, in Colman RW, Hirsh J, Marder VJ, Salzman EW (eds): Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 2nd edition, Philadelphia, JB Lippincott, 1987, pp 841-845. 5. Wright DJ, Jenkins DE Jr: Simplified method for estimation of serum and plasma viscosity in multiple myeloma and related disorders. Blood 36:516-522, 1970. 6. Chronic Leukemia-Myeloma Task Force, National Cancer Institute. Proposed guidelines for protocol studies. 2. Plasma cell myeloma. Cancer Chemotherapy Reports 4:145-158, 1973. . 7. Kyle RA: Monoclonal gammopathy of undetermined significance: Natural history in 241 cases. Am J Med 64:814-826, 1978. 8. Stein RS, Ellman L, Bloch KJ: The clinical correlates of IgMM components: An analysis of thirty-four patients. Am J Med Sci 269:209-216, 1975.
365
Hemostatic Defects in Monoclonal Gammopathies
9. Triplett DA, Harms CS: Procedures for the coagulation laboratory. Chicago, American Society of Clinical Pathologist, 1st edition, 1981, pp 6-80. 10. Triplett DA, Brandt JT, Kaczor D: Laboratory diagnosis of lupus inhibitors: A comparison of the tissue thromboplastin inhibition procedure with a new platelet neutralization procedure. Am J Clin Pathol 79:678-682, 1983. 11. Born G VR: Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194:927-929, 1962. 12. Harms CS: Laboratory evaluation of platelet function, in Triplett DA, Harms CS, Newhouse P, Clark C (eds): Platelet Function, Laboratory Evaluation and Clinical Application, 1st edition, Chicago, ASCP, 1978, pp 39-43. 13. Newhouse P, Clark CS: The variability of platelet aggregation, in Triplett DA, Harms CS, Newhouse P, Clark C (eds): Platelet Function, Laboratory Evaluation and Clinical Application, 1st edition, Chicago, ASCP, 1978, pp 63-109. 14. Snedecor GW, Cochran WG: Statistical methods. Iowa State University Press, 1957. 15. Farhangi M, Merlini G: The clinical implications of monoclonal immunoglobulins. Semin OncoI13:366-379, 1986. 16. Shapiro SS, Hultin M: Acquired inhibitors to the blood coagulation factors. Semin Thromb Hemost 1:336-385, 1975. 17. Thiagarajan P, Shapiro SS, DeMarco L: Monoclonal immunoglobulin MA coagulation inhibitor with phospholipid specificity: Mechanism of a lupus anticoagulant. J Clin Invest 66:397-405, 1980. 18. Coleman M, Vigliano EM, Weksler ME, Nachman RL: Inhibition of fibrin monomer polymerization by lambda myeloma globulins. Blood 39:210-223, 1972. 19. Penny R, Castaldi P A, Whitsed HM: Inflammation and hemostasis in paraproteinemias. Br J Haematol 20:35-44, 1971. 20. Glueck HI, MacKenzie MR, Glueck CJ: Crystalline IgG protein in multiple myeloma: Identification effects on coagulation and on lipoprotein metabolism. J Lab Clin Med 79:731-744, 1972.
366
21. Kaufman PA, Gockerman JP, Greenberg CS: Production of a novel anticoagulant by neoplastic plasma cells: Report of a case and review of the literature. Am J Med 86:612-616, 1989. 22. Palmer RN, Rick ME, Rick PD, Zeller JA, Gralnick HR: Circulating heparan sulfate anticoagulant in a patient with a fatal bleeding disorder. N Engl J Med 310:1696-1699, 1984. 23. Khoory MS, Nesheim ME, Bowie EJW, Mann KG: Circulating heparan sulfate proteoglycan anticoagulant from a patient with a plasma cell disorder. J Clin Invest 65:666-675, 1980. 24. Tefferi A, Nichols WL, Walter Bowie EJ: Circulating heparinlike anticoagulants: Reports of five consecutive cases and a review. Am J Med 88:184-188,1990. 25. Bick RL: Alterations of hemostasis associated with malignancy: Etiology, pathophysiology, diagnosis and management. Semin Thromb Hemost 6:18-21, 1978. 26. Brody JI, Haidar ME, Rossman RE: A hemorrhagic syndrome in Wa!denstrom's macroglobulinemia secondary to immunoabsorption of factor VIII: Recovery after splenectomy. N Engl J Med 300:408-410, 1979. 27. Mant MJ, Hirsh J, Gauldie J, Bienenstock J, Pineo GP, Luke KH: von Willebrand's syndrome presenting as an acquired bleeding disorder in association with a monoclonal gammopathy. Blood 42:429-435, 1973. 28. Zettervall 0, Nilsoon 1M: Acquired von Willebrand's disease caused by monoclonal antibody. Acta Medica Scandinavia 204: 521-528, 1978. 29. DiMinno G, Coraggio F, Cerbone AM, Capitanio AN, Manzo C, Spina M, Scarpato P, Datolli GMR, Mattioli PL, Mancini M: A myeloma paraprotein with specificity for platelet glycoprotein IlIa in a patient with a fatal bleeding disorder. J Clin Invest 77: 157-164, 1986. 30. Merlini G, Farhangi M, Osserman EF: Monoclonal immunoglobulins with antibody activity in myeloma, macroglobulinemia and related plasma cell dyscrasias. Semin Oncol13:350-365, 1986. 31. Bick RL: Disseminated intravascular coagulation and related syndromes: A clinical review. Semin Thromb Hemost 14:299338,1988.
December 1993 Volume 306 Number 6
ABSTRACT: To confirm and expand previous observations about the association of monoclonal gammopathies with hemostatic defects, a prospective evaluation was made in 42 patients with lymphoplasmacytic disorders. The incidence of bleeding complications was low, despite the diversity of abnormal hemostatic tests observed in these patients. Patients with myeloma frequently had abnormal coagulation tests, including thrombin time (64%), fibrin degradation products (32%), platelet aggregation tests with different agonist (30% to 55%), and bleeding time (22%). The lack of platelet response to ristocetin and normal ristocetin cofactor activity in four patients with myeloma may suggest a BernardSoulier-type defect. Serum viscosity was negatively correlated with platelet aggregation with collagen, ristocetin, and adenosine diphosphate. In patients with immunoglobulin myeloma, there was a positive correlation between an increased viscosity and a prolonged thrombin time. This study demonstrates the wide variety of coagulation abnormalities in lymphoplasmacytic disorders, usually without significant clinical implications. KEY INDEXING TERMS: Plasma cell dyscrasias; Monoclonal gammopathies; Dysproteinemias; Coagulation. [Am J Med Sci 1993;306(6):359-366.]
A
variety of lymphoplasmacytic disorders characterized by the presence of monoclonal-circulating immunoglobulins have been associated with From the *Medical Service, Hematology-Oncology Section, VA Medical Center, San Juan, Puerto Rico, the t Laboratory Service, VA Medical Center, San Juan, Puerto Rico, the +Department of Medicine, University of Puerto Rico, San Juan, Puerto Rico, and the §Biostatistics Unit, Comprehensive Cancer Center, University of Alabama at Birmingham. Correspondence: Francisco Robert, MD, Department of Medicine, Hematology-Oncology Section, Comprehensive Cancer Center, University of Alabama at Birmingham, VA Medical Center, 700 South 19th Street, Birmingham, AL 35233. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
JEANNETTE Y. LEE, PHD§
multiple coagulation abnormalities. 1- 3 These acquired hemostatic defects are features of a number of dysproteinemic disorders, including multiple myeloma, Waldenstrom's macroglobulinemia, systemic amyloidosis, and lymphoma. In general, these alterations of hemostasis may be expressed as either hemorrhage or thrombosis, or a combination of the two. Hemorrhagic manifestations are more common with the actual incidence, varying depending on the particular dysproteinemia. Bleeding diatheses 3 are observed more often in macroglobulinemia (36%) or immunoglobulin A (IgA) myeloma (33%) than immunoglobulin G (IgG) myeloma (15%). The pathophysiology of these hemostatic defects are complex and only partially understood. Most of the abnormalities may be explained on the basis of four mechanisms: (1) circulating paraproteins (monoclonal immunoglobulins) that bind to and inhibit components involved in hemostasis, (2) circulating paraproteins that bind to coagulation factors, promoting an enhanced clearance of the complex, (3) monoclonal cellsurface immunoglobulins that bind and clear circulating coagulation proteins, and (4) abnormal proteins deposited into the extracellular space of certain tissues that bind to normal coagulation proteins. 4 In addition, bleeding diatheses may be associated with conditions that complicate the primary disorder, such as uremia, hypersplenism, bone marrow failure, liver disease, sepsis, or chemotherapy. To confirm and expand these earlier observations about the relationship of dysproteinemias with acquired hemostatic abnormalities, we report the results of a prospective laboratory evaluation of coagulation in 42 patients with lymphoplasmacytic disorders. In addition, the coagulation profile of 12 cases with essential or secondary monoclonal gammopathies were examined. Results were correlated with the amount of circulating monoclonal protein, type of immunoglobulin or light chain, and serum viscosity. Patients and Methods Patients. Fifty-four patients with the appearance of
a homogenous monoclonal protein in the serum or urine were studied from August 1984 to March 1990. Through 359
Hemostatic Defects in Monoclonal Gammopathies
the duration of the study (6 years), each patient was observed for possible hemorrhagic or thrombotic events. Bleeding time determinations and blood sample collections were done with informed consent from the patients. Serum and urine found to contain monoclonal protein components by zonal electrophoresis were studied by immuno-electrophoresis to identify the class of heavy chain and the type of light chain of the protein. Diagnosis of the various causes of monoclonal gammopathy was based on a thorough history, physical examination, and pertinent laboratory examination. Laboratory studies performed included a complete blood cell count, serum chemistry, special hematologic studies for the diagnosis of lymphoplasmacytic disorders, and other procedures to search for underlying diseases. Serum viscosity was determined as previously described. 5 Based on these studies and using standard diagnostic criteria,6-8 the patients were classified as follows: Group 1. Multiple myeloma: Thirty-four patients with active disease were studied; most before chemotherapy. Of the 34,19 had IgG myeloma, 9 had IgA myeloma, and 6 had light chain disease. There were 31 men and three women, ranging in age from 44 to 85 years (median, 62 years). Group 2. Diseases associated with monoclonal (IgM) macroglobulinemia: Four patients were included in this category; three had Waldenstrom's macroglobulinemia and one had an extramedullary lymphoplasmacytic neoplasm. There were three men and one woman, ranging in age from 56 to 62 years (median, 60 years). Group 3. Diseases associated with monoclonal IgG gammopathy: Four male patients ranging in age from 41 to 70 years (median 63 years) were studied; one had primary amyloidosis, one had a solitary osseous plasmacytoma, and two had a lymphocytic neoplasm. Group 4. Essential and secondary monoclonal gammopathies: Twelve male patients with a serum monoclonal protein either without an associated disease or with a non-Iymphoplasmacytic disorder were evaluated. They ranged in age from 58 to 79 years (median, 71 years). Methods. The patients had not taken aspirin or other drugs interfering with platelet function for at least 7 days before testing. Fasting blood samples were obtained for all coagulation studies. The activated partial thromboplastin time, prothrombin time, thrombin time, and fibrinogen levels were performed by standard methods. 9 The reptilase clotting time of plasma was determined according to the Sigma Diagnostics procedures (Atroxin Test; Sigma Chemical Co, St Louis, MO). Fibrin degradation products were measured with the use of the Thrombo-W ellcotest assay (Wellcome Diagnostics, Dartford, England). More recently, the fragment D-dimer determination was included in the
360
coagulation profile of these patients (Fibrinosticon; Organon Teknika, Durham, NC). Circulating anticoagulant screening tests (mixing studies) were performed as previously described. 9 A Lupus-type inhibitor was detected in one of the patients by methods previously described;9.10 the patient's plasma was treated with an anti-immunoglobulin M (IgM) anti-serum (Sigma Chemical Co, St Louis, MO) in an attempt to confirm the nature of the anticoagulant. Prolonged thrombin times were evaluated for heparin-like anticoagulants using in vitro protamine sulfate (Sigma Chemical Co, St Louis, MO) neutralization of the patient's plasma. Platelet function was evaluated with a bleeding time (General Diagnostics Simplate, Morris Plains, New Jersey), and platelet aggregation studies were performed as previously described. n - 13 Bleeding time was not performed if the platelet count was below 100,000/ J.LL. The concentrations of inducing agents were 5 J.LM and 10 J.LM adenosine diphosphate (Sigma Chemical, St Louis, MO); 0.2 mg/mL collagen (Worthington Biochemical, Freehold, NJ); 5 J.LM and 10 J.LM epinephrine (Parke Davis, Detroit, MI); and 1.5 mg/mL ristocetin (Abbott Laboratories, North Chicago). The extent of platelet aggregation was expressed as the percent of increase in light transmission using patient's autologous platelet-poor plasma as 100% standard. The von Willebrand factor (Ristocetin Cofactor) assay (Bio/Data Co, Horsham, PA) was completed on patients with an abnormal aggregation induced by ristocetin. Statistical Methods. Statistical analyses were carried out using the Statistical Analysis System software package (SAS Institute, Cary, NC). Group comparisons of continuous data were made using the Wilcoxon rank sum test. 14 Correlations between variables were assessed using the Spearman rank correlation coefficient. All statistical tests were carried out at the two-tailed significance level. Results
Fifty-four patients with a monoclonal protein (Mprotein) in the serum or urine were evaluated. Table 1 outlines the laboratory features of the M-proteins in each group of patients. The number of patients in the categories will occasionally be smaller because of incomplete information in some cases. The predominant immunoglobulin was IgG: 56% in the lymphoplasmacytic disorders and 67% in group 4 (essential/secondary gammopathies) . The various hemostatic parameters in each group of patients are shown in Table 2. There was a small number of patients evaluated in certain groups, and the available number of results for each group is different. For these reasons, calculation of the probable significance of differences using a one-way analysis of variance procedure was limited between groups 1 and 4. There was a trend for prolonged thrombin times and December 1993 Volume 306 Number 6
Robert et 01
Table 1. Laboratory Evaluation of M-Proteins in Patients With Lymphoplasmacytic Disorders and Essential (or Secondary) Monoclonal Gammopathies*
Group
No. of Patients
Serum Monoclonal Spike (gm/L)
1. Multiple myeloma IgG IgA Light chain disease 2. IgM disorders 3. IgG disorders 4. Essential/secondary gammopathies§
34 19 9 6 4 4 12
46.2 ± 19.2:j: 43.2 ± 18.1 52.4 ± 21.0
Serum Viscosityt 2.46 2.49 3.03 1.51 6.45 1.73 1.73
25.9 ± 20.4 13.8 ± 6.05 23.5 ± 10.2
± 1.05 ± 1.08 ± 0.96 ± 0.16 ± 5.8 ± 0.25 ± 0.42
Light Chain K K K K K K K
(21), L (13) (11), L (8) (7), L (2) (3), L (3) (3), L (1) (2), L (2) (8), L (4)
* Results were expressed as mean ± standard deviation. .
.
..
. Flow Time of Serum the ratw o f . (normal range 1.4-1.8). Flow Time of Water :j: Cases of light chain disease are not included. § IgG (7), IgA (5).
t The relatwe VISCOSity
IS
reduced platelet aggregation with adenosine diphosphate (ADP), collagen, and ristocetin in patients with multiple myeloma as compared with patients in group 4, but these differences are not statistically significant. Patients with multiple myeloma had significantly higher titers of fibrin degradation products as compared with patients in group 4 (p = 0.03, by analysis of variance, unpublished data). Hemostatic abnormalities were fewer and less severe in group 4. One patient with chronic myelomonocytic leukemia (IgGk) had a bleeding time over 900 seconds and slight elevation of the fibrin degradation products. Another patient with squamous cell carcinoma of the penis (IgGk) had a slight reduction of the platelet aggregation with ADP (50%), collagen (60%), and ristocetin (47%), but had a normal bleeding time and ristocetin cofactor assay. Two additional cases in group 4 had a slight reduction of the platelet aggregation with
ristocetin, but normal bleeding times. None of these patients had a history of previous hemorrhagic complications, and their family history was unremarkable. In this series of patients, the incidence of bleeding complications was low, despite the diversity of abnormal hemostatic tests observed in groups 1 and 2. Two major hemorrhagic events were noted in two patients: one patient (JO) in group 1 developed significant bleeding after a dental extraction during his initial evaluation; the other patient (JM), in group 2, had an upper gastrointestinal bleeding. The clinical and laboratory data of these cases are summarized in Table 3. In Case JO, the bleeding time was prolonged and the abnormal prothrombin time was not corrected with a mixture of equal parts of normal plasma and patient plasma. Treatment with chemotherapy produced a good response; a follow-up examination of his hemostatic parameters 8 months later revealed a normal
Table 2. Summary of Coagulation Assays and Platelet Function Studies*
Test Bleeding time Platelet count Platelet aggregation Adenosine diphosphate Collagen Epinephrine Ristocetin Ristocetin cofactor assay Prothrombin time Partial thromboplastin time Thrombin time Fibrinogen
Multiple Myeloma (Group 1) N =' 34 488
± 37 (22) 226 (15)
64.3 ± 75 ± 60.8 ± 62.4± 143 ± 12.3 ± 27.6 ± 13.8 ± 3.3 ±
4.8 (38) 4.4 (30) 8 (31) 6 (55) 20.9 (1/13) 0.3 (11) 0.7 (6) 1.1 (64) 0.2.(8)
IgM Disorders (Group 2) N =' 4 414
± 50 (O)t 214 (I;")
68.5 ± 10.9 (2/4) 65.5 ± 11 (2/4) 43 (1/1) 70.8 ± 12 (2/4) 77 ± 28 (1/3) 14.9 ± 2.5 (1/4) 40.9 ± 12.5 (1/4) 9.6 ± 0.2 (0) 3.7 ± 1.1 (0)
IgG Disorders (Group 3) N = 4 450 ± 151 (';..) 218 ('II) 75.9 ± 5 (0) 3.5 (0) 81 ± 70 (0/1) 2.7 (0) 83 ± 0.4 (0) 11.2 ± 2.1 (0) 27 ± 8.3 (0) 0.4 (0) 3.2 ±
Essential/Secondary Gammopathies (Group 4) N = 12 422
± 53 (8) 265 (0)
74.7 ± 80 ±
3 3
(8) (8)
± 3.5 (25) 78.5 (0/2) 12.1 ± 0.3 (8) 26 ± 0.9 (0) 9.9 (0) 3.1 ± 0.3 (0)
78
Normal Range 180-540 sec 150-400 X 109/L 68.8%-91.6% 63.8%-91% 67%-97% 76%-99% 58%-166% 10.9-13.3 sec 22.7-36.7 sec 5.9-10.7 sec 1.55-3.51 gm/L
* Results were expressed as mean ± standard error of the mean. t Numbers in parenthesis indicate the percentage or fraction (if few cases were tested) of patients with an abnormal value. THE AMEIlICAN JOUIlNAL OF THE MEDICAL SCIENCES
361
Hemostatic Defects in Monoclonal Gammopathies
Table 3. Clinical and Laboratory Characteristics of Patients With Hemorrhagic Events* Patients
Diagnosis Serum M-spike (gm/L) Serum viscosityt Platelet count (X 109 /L) Bleeding time Platelet aggregation Adenosine diphosphate Collagen Ristocetin Ristocetin cofactor assay Prothrombin time Partial thromboplastin time Fibrinogen (gm/ L)
* The normal range
JO
JM
IgGk myeloma 82.1 4.8 233 900 sec
IgMk macroglobulinemia 38.3 12.9 302 510 sec
67.5 % 67.5% 0% 154% 17 sec:j: 26.8 sec 2.75
50% 45% 50% 29% 12.6 34.3 sec 2.75
~f
values is given in Table 2. 1.4-1.8). :j: The abnormality was not corrected with 50:50 mixture of normal plasma and patient plasma.
t Relative viscosity (normal range
bleeding time (510 seconds) and prothrombin time (13.2 seconds). The abnormal prothrombin time was interpreted as indicating the presence of an inhibitor (probably to Factor VII), but a specific inhibitor assay or a Russell's viper venom (Stypven) time was not performed. There was a lack of platelet aggregation in response to ristocetin, but this inhibitory activity of the patient plasma was not observed in the ristocetin cofactor assay. In Case JM, the bleeding time was within normal range despite some suppression of the platelet aggregation to different agonists and marked inhibitory activity in the ristocetin cofactor assay. Cryoglobulin was absent. There was no history of previous hemorrhagic complications, and his family history was negative for significant bleeding. Viscosity was elevated in both patients, but it was markedly abnormal in Case JM, suggesting a possible explanation for the bleeding diathesis in this patient. He was treated with plasmapheresis and chemotherapy with a resolution of the bleeding diathesis; a follow-up examination of his hemostatic parameters was not performed. None of the patients studied had thrombotic complications. The association of lupus inhibitor and Waldenstrom's macroglobulinemia was observed in one of the cases in group 2. Case EB was a patient with an IgM lambda monoclonal gammopathy who had skin lesions and marked prolongation of the partial thromboplastin time. She had no history of thrombotic or hemorrhagic complications, and did not have a bleeding tendency during diagnosis or throughout her illness. The results of the screening coagulation tests and specialized laboratory studies are shown in Table 4. The activated partial thromboplastin time was markedly prolonged
362
on several occasions and did not decrease significantly with a splenectomy or chemotherapy. The prothrombin time was always prolonged, but to a lesser degree than the partial thromboplastin time. The mixing studies showed that the defect in coagulation was not corrected with normal plasma at different time intervals. The tissue thromboplastin inhibition studies were prolonged at the 1:200 and the 1:1000 dilution with a ratio of 2.9 to 3.2, respectively. The platelet neutralization test showed a dramatic correction when a suspension of washed normal platelets were incubated with the mixture of the activated partial thromboplastin test. The control showed no change. The mixture of patient plasma with an anti-IgM antiserum showed a shortening of the partial thromboplastin time, but especially of the prothrombin time. The frequency of abnormal coagulation tests in the myeloma subtypes is listed in Table 5. The frequency of abnormal values was high among patients with myeloma for bleeding time (22%), platelet aggregation tests with different agonists (30% to 55 %), thrombin time (64 %) and the fibrin degradation product assay (32%). A significant difference was detected between IgA myeloma, IgG myeloma, and light chain disease with respect to fibrinogen levels (p = 0.034). Among the patients with IgG myeloma, thrombin time was significantly prolonged in IgG myeloma containing lambda chains as compared with those with kappa chain (p = 0.04), with mean values of27.3 seconds and 10.6 seconds, respectively. For all patients with myeloma combined, the mean value of platelet aggregation with collagen of 85 % for myelomas with kappa chains (n = 20) was significantly higher than the 53% with those containing lambda chains (n = 13; p < 0.01).
Table 4. Results of Coagulation Studies in a Patient (EB) with Waldenstrom's Macroglobulinemia Coagulation Assays Prothrombin time (PT) Partial thromboplastin time (PTT) Thrombin time Circulating anticoagulant Screening PT (50:50) PTT (50:50) Tissue thromboplastin Inhibitor (dilution/ratio) Platelet neutralization procedure (PNP) Control saline PNP Bleeding time Platelet aggregation (ADP)
Results
(Normal Range)
22.3 sec
(10.9- 13.3 sec)
71.5 sec 9.8 sec
(22.7-36.7 sec) (5.9-10.7 sec)
20.5 sec* 67.5 sec* 1:100/2.9 1:1000/3.2
«1.3) «1.3)
74.8 sec 43.8 sec 300 sec 95 %
(180-540 sec) (68.8%-91.6% )
* The mixing studies showed that the defect in coagulation occurred immediately, with no significant potentiation with incubation for 1 hour. December 1993 Volume 306 Number 6
Robert et 01
Table 5. Abnormal Tests of Hemostatic Function in Multiple Myeloma* Test
All Cases
IgG
IgA
Light Chain Disease
Bleeding time >540 sec Platelet count <150 X 109/L Platelet aggregation Adenosine diphosphate < 68% Collagen < 63% Epinephrine < 67% Ristocetin < 76% Ristocetin cofactor assay < 58% Prothrombin time> 13.3 sec Partial thromboplastin time> 36.7 sec Thrombin time> 10.7 sec Fibrinogen < 1.5 giL Fibrin degradation products:j:
22% (32) 15% (34)t
29% (17) 16% (19)
11% (9) 11% (9)
16% (6) 17% (6)
38% 30% 31% 55% 8% 11% 6% 64% 8% 32%
32% 16% 33% 58% 10% 11 % 0 70% 0 38%
33% 38% 0 50% 0% 22% 11% 33% 22% 14%
(34) (33) (13) (33) (13) (34) (34) (14) (34) (28)
(19) (19) (9) (19) (10) (19) (19) (10) (19) (16)
(9) (8) (3) (8) (2) (9) (9) (3) (9) (7)
50% 0 100% 50% 0 0 16% 100% 0 40%
(6) (6) (1) (6) (1) (6) (6) (1) (6) (5)
* Percent abnormal followed by number tested. t Mean platelet count of 226 X 1(fJ/L (range 81-538 X 1(fJ/L) :j: Fibrin degradation products (> 10 Ilg/mL) or cross-linked fibrin derivatives containing the D-dimer domain (>500 ng/mL).
Abnormal bleeding times were more common in patients with IgG myeloma (29%). There was no correlation between bleeding time and the results of other platelet function tests, except for platelet aggregation with ADP (Spearman rank correlation coefficient -0.378, p = 0.01). Defective platelet aggregation with ristocetin was observed in 18 patients with multiple myeloma (mean ± standard error of the mean 62.4% ± 6%; range, 0 to 71 %). None of these patients had abnormal partial thromboplastin times. Four of these cases had prolonged bleeding times, and only one had a low ristocetin cofactor activity «25%). This patient had IgGk myeloma with a normal bleeding time and a normal platelet aggregation response to ADP and collagen. He had no history of hemorrhagic complications, and did not have a bleeding tendency during diagnosis. Two patients with IgM macroglobulinemia (Group 2) also had decreased platelet aggregation with ristocetin, but only one had a significant reduction of the ristocetin cofactor assay (Case JM, Table 3). Characterization ofthe von Willebrand factor multimers was not done on these cases. The thrombin clotting time of plasma was prolonged in 9 (64%) of the 14 patients with myeloma tested. This abnormality was correlated with increased serum viscosity and circulating monoclonal proteins (Table 6); six out of nine cases had mild to moderate increased levels of fibrin degradation products. Only one of the patients (IgAk) evaluated with Atroxin (reptilase test) had a normal clotting time. Protamine sulfate neutralized the anticoagulant activity detected in his plasma in vitro. Another patient with IgGk myeloma and significant cryoglobulinemia (16%) had prolonged plasma clotting times with both thrombin!Atroxin reagents, and complete in vitro neutralization with protTHE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
amine sulfate. None of these cases had significant bleeding complications. Abnormal prothrombin times and partial thromboplastin times were observed in only a few patients with myeloma (Table 5). Mixing studies in two cases with isolated prolonged prothrombin time and partial thromboplastin time demonstrated that a deficiency was responsible for these abnormalities. One patient discussed previously (Case JO, Table 3) had a prolonged prothrombin time not corrected with normal plasma, and most likely an inhibitor is the basic mechanism. Table 6. Relationship of Significant Hemostatic Variables With Serum Viscosity and Monoclonal Spike M-Spike
Viscosity
IgG /IgA myeloma Platelet aggregation Collagen Ristocetin Thrombin time IgG myeloma Platelet aggregation Ristocetin Thrombin time All patients* Platelet aggregation Adenosine diphosphate Collagen Ristocetin Thrombin time
p
Correlation Coefficient
p
Correlation Coefficient
0.01 <0.001 0.001
-0.448 -0.667 0.813
NS <0.01 NS
NS -0.498 NS
<0.001 <0.001
-0.800 0.916
0.005 0.02
-0.614 0.705
0.05 <0.01 <0.01 0.03
-0.287 -0.443 -0.439 0.568
0.04 0.05 <0.001 0.03
-0.301 -0.290 -0.586 0.558
* All patients included in Table 1, except those cases with light chain disease. NS = not significant.
363
Hemostatic Defects in Monoclonal Gammopathies
The presence of elevated levels of serum fibrin degradation products (>10 JLg/mL) or cross-linked fibrin derivatives containing D-dimer domain (>500 ng/mL) was demonstrated in nine patients (32%) in group 1. Another patient with IgM macroglobulinemia (Case JM, Table 3) had elevated fibrin degradation products but also had high titers of rheumatoid factor. Most of these cases had slight to moderate increased levels of fibrin degradation products, and there was no clinical evidence of thromboembolic disorders during diagnosis. None of these patients had hypofibrinogenemia, thrombocytopenia, or prolongation of the prothrombin time and partial thromboplastin time. Correlation analysis of multiple hemostatic tests was used to identify relationships with serum viscosity or circulating monoclonal proteins. A subgroup of patients with light chain disease was excluded from this analysis because of the low concentration of abnormal proteins in the serum and normal viscosity (mean ± standard deviation 1.5 ± 0.16). The hemostatic variables with the most significant correlation coefficients are shown in Table 6. Viscosity was negatively correlated with platelet aggregation with collagen, ristocetin, and ADP. Prolonged thrombin time was proportional to the increase in serum viscosity with a highly significant correlation coefficient. Similar relationships were apparent with the serum monoclonal proteins, but to a lesser degree of significance. There was no correlation between serum viscosity and monoclonal spikes and the results of the bleeding time, platelet aggregation with epinephrine, and fibrin degradation products. Discussion
A multiplicity of hemostatic defects have been described in patients with lymphoplasmacytic disorders associated with abnormal monoclonal proteins. 1- 4,15 The current comprehensive evaluation of 54 patients was designed to update the clinical implications of monoclonal immunoglobulins with acquired hemostatic disorders. We found a wide variety of hemostatic abnormalities without any correlation between the magnitude of the defect(s) and the presence of a clinical bleeding syndrome. One of the most important mechanisms in the pathophysiology of these hemostatic disorders is the presence of circulating inhibitors of blood coagulation.4 Endogenous inhibitors of hemostasis have been characterized previously as immunoglobulins that bind specifically to either individual coagulation factors or phospholipids, or that interfere nonspecifically with fibrin polymerization or platelet aggregation. 16- 19 The most commonly described paraprotein inhibitors of coagulation are directed against fibrin monomer polymerization. The laboratory features of this abnormality include a prolonged clotting time with both thrombin/Atroxin reagents and a gelatinous friable clot that exhibits poor clot retraction partially corrected by the addition of calcium. 4,18 Coleman and co-workers 18
364
have demonstrated that the inhibition of fibrin polymerization resides on the Fab fragment of the IgG and IgA immunoglobulins, but not on the Fc fragment or isolated heavy and light chains. The thrombin time was prolonged in 64% of our patients with multiple myeloma and was significantly correlated with the presence of lambda light chains, as in Coleman's study. However, it is apparent that both types of light chains may be associated with abnormal fibrin formation. 2o A significant correlation between thrombin time and serum viscosity was demonstrated in patients with IgG/ IgA myeloma (Table 6). A possible explanation for this correlation (IgG-myelomas) is that an increased amount of IgG paraproteins may accentuate this inhibition of the fibrin polymerization. Serum viscosity was significantly correlated (p = 0.001) with the IgGM spike of these patients (unpublished data). Several cases of heparin-like anticoagulant in association with plasma cell dyscrasias have been reported. 21- 24 In contrast to previous reports of circulating paraprotein inhibitors, these anticoagulants are not directly related to the myeloma protein. These endogenous heparin-like inhibitors are circulating proteoglycans with electrophoretic mobility similar to heparan or chondroitin sulfates. Although no clear evidence as to the source of these inhibitors has been found, Kaufman and collaborators21 were able to demonstrate the production of a similar anticoagulant by the neoplastic plasma cells of a patient with plasma cell leukemia. The characteristic laboratory features of these inhibitors are a prolonged thrombin time with a normal reptilase time assay as well as neutralization of the anticoagulant activity with protamine sulfate, platelet factor 4, or toluidine blue. Unlike inhibitors that interfere with fibrin polymerization, these heparinlike anticoagulants are associated with a bleeding diathesis. We identified two patients with myeloma who had a prolonged thrombin time corrected by the addition (in vitro) of protamine sulfate. One of these patients also had an abnormal reptilase assay, though neither experienced bleeding complications. Circulating immunoglobulins may also have properties characteristic of the lupus anticoag-ulant. 4 In 1980, Thiagarajan and collaborators 17 reported a patient with macroglobulinemia and a lupus-type anticoagulant whose purified monoclonal IgMX immunoglobulin possessed the inhibitory activity. This patient had a prolongation of all phospholipid-dependent coagulation tests with no bleeding manifestations. The patient's lambda paraprotein reacted with anionic phospholipids, but not with neutral phospholipids. We have studied a similar patient with IgMX (Table 4) macroglobulinemia and prolongation of the phospholipid-dependent coagulation tests. Our patient is the second reported patient, to our knowledge, with a plasma cell disorder ofthe Waldenstrom's macroglobulinemia type to have a lupus anticoagulant. Like most patients, she had no bleeding tendencies and underwent December 1993 Volume 306 Number 6
Robert et al
a splenectomy without bleeding problems. The IgM macroglobulin was shown to be present in the neoplastic cells by immunoperoxidase staining and flow cytometry (data not shown). The inhibitory activity of the lupus anticoagulant was neutralized with an antiIgM antiserum, confirming the IgM nature of the antibody. Monoclonal immunoglobulins are known to interfere with a number of coagulation proteins, including von Willebrand factor, fibrinogen, prothrombin, and factors V, VII, VIII, and X. l ,3,4,25 One mechanism of this rare phenomenon is the binding of circulating paraproteins with selective coagulation factors, which inhibit their function. 4 A conceivable explanation for the prolonged prothrombin time in Case JO (Table 3) may be related to this paraprotein-coagulation protein interaction. The mixing studies and a normal partial thromboplastin time are compatible with the presence of an inhibitor, probably affecting factor. VII activity. Another mechanism that could possibly cause low levels of one or more clotting factors might be the binding of the abnormal paraprotein to various coagulation factors, resulting in accelerated clearance of them. 4 These interactions may occur in the plasma, or at the surface of the neoplastic B cells. 3 ,26-28 Accelerated clearance is an attractive hypothesis to explain the isolated clotting time abnormalities (prothrombin and partial thromboplastin times) and low ristocetin cofactor activity occasionally observed in the plasma of our patients. The present study is in agreement with other reports with respect to the diversity of the platelet function test abnormalities in these patients. l ,3 There is a disagreement in the literature as to whether abnormalities in primary hemostasis are related to the clinical tendency for hemorrhage. Data from our study show a poor correlation between platelet function abnormalities and the development of clinically significant bleeding. We found abnormal bleeding times more common in patients with IgG myeloma. However, there was no correlation between the bleeding time and the results of the platelet aggregation tests, except for platelet aggregation with ADP (p = 0.01). The mechanism of the effect of the monoclonal immunoglobulins in the impairment of platelet function is poorly understood. It has been stated that these abnormalities occur as a consequence of nonspecific adherence of the paraprotein to the platelet surface. l ,3,4,19 In agreement with this theory is the negative correlation observed in this study between platelet aggregation with several agonists and serum viscosity or the paraprotein concentration (Table 6). DiMinno 29 and coworkers have demonstrated specific interaction of a monoclonal immunoglobulin with the platelet glycoprotein IlIa of a patient with IgGk myeloma having impaired platelet aggregation. Their data support the concept that most monoclonal immunoglobulins elaborated in multiple myeloma and related plasma cell THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
dyscrasias are functional antibodies for specific antigens. 30 The lack of response to ristocetin and normal ristocetin cofactor activity observed in four patients with myeloma (including Case JO) may suggest another specific interaction of monoclonal proteins with platelet glycoproteins, resulting in a Bernard-Soulier type defect. Three of these patients also had prolonged bleeding time. Multiple myeloma has also been associated with chronic low-grade disseminated intravascular coagulation, although this appears to be uncommon. 25 ,31 The mechanism by which this occurs is unclear. Nine patients with myeloma in this study had elevated fibrin degradation products or abnormal d-dimer assays, even though there was no clinical manifestations of bleeding or thrombotic complications. The presence of fibrin degradation products in these patients may also be due to other causes of reactive fibrinolysis, or represent false positive results. Rheumatoid factor may interfere with these assays by triggering false positive values. Although most monoclonal rheumatoid factors are IgMk (as in Case JM, Table 3), there are reports of monoclonal IgG and IgA rheumatoid factors. 3o Therefore, it is important to check for the presence of rheumatoid factor in unexpected positive samples. In summary, the interaction between monoclonal immunoglobulins with platelets and coagulation factors results in a panorama of abnormal hemostatic tests, usually without significant clinical implications. Our results indicate that laboratory evaluation of coagulation in patients with dysproteinemias does not offer information of predictive value regarding bleeding or thrombotic complications. Acknowledgments
The authors thank Dr. Pradip K. Rustagi for his critical comments and Lee Ann Grimmett for her preparation of the manuscript. References 1. Lackner H: Hemostatic abnormalities associated with dyspro-
teinemias. Semin HematollO:125-133, 1973. 2. Sanchez-Avalo J, Soong BCF, Miller SP: Coagulation disorders in cancer. 2. Multiple myeloma. Cancer 23:1388-1398, 1969. 3. Perkins HA, Mackenzie MR, Fundenburg HH: Hemostatic defects in dysproteinemias. Blood 35:695-707, 1970. 4. Furie B: Acquired coagulation disorders and dysproteinemias, in Colman RW, Hirsh J, Marder VJ, Salzman EW (eds): Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 2nd edition, Philadelphia, JB Lippincott, 1987, pp 841-845. 5. Wright DJ, Jenkins DE Jr: Simplified method for estimation of serum and plasma viscosity in multiple myeloma and related disorders. Blood 36:516-522, 1970. 6. Chronic Leukemia-Myeloma Task Force, National Cancer Institute. Proposed guidelines for protocol studies. 2. Plasma cell myeloma. Cancer Chemotherapy Reports 4:145-158, 1973. . 7. Kyle RA: Monoclonal gammopathy of undetermined significance: Natural history in 241 cases. Am J Med 64:814-826, 1978. 8. Stein RS, Ellman L, Bloch KJ: The clinical correlates of IgMM components: An analysis of thirty-four patients. Am J Med Sci 269:209-216, 1975.
365
Hemostatic Defects in Monoclonal Gammopathies
9. Triplett DA, Harms CS: Procedures for the coagulation laboratory. Chicago, American Society of Clinical Pathologist, 1st edition, 1981, pp 6-80. 10. Triplett DA, Brandt JT, Kaczor D: Laboratory diagnosis of lupus inhibitors: A comparison of the tissue thromboplastin inhibition procedure with a new platelet neutralization procedure. Am J Clin Pathol 79:678-682, 1983. 11. Born G VR: Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194:927-929, 1962. 12. Harms CS: Laboratory evaluation of platelet function, in Triplett DA, Harms CS, Newhouse P, Clark C (eds): Platelet Function, Laboratory Evaluation and Clinical Application, 1st edition, Chicago, ASCP, 1978, pp 39-43. 13. Newhouse P, Clark CS: The variability of platelet aggregation, in Triplett DA, Harms CS, Newhouse P, Clark C (eds): Platelet Function, Laboratory Evaluation and Clinical Application, 1st edition, Chicago, ASCP, 1978, pp 63-109. 14. Snedecor GW, Cochran WG: Statistical methods. Iowa State University Press, 1957. 15. Farhangi M, Merlini G: The clinical implications of monoclonal immunoglobulins. Semin OncoI13:366-379, 1986. 16. Shapiro SS, Hultin M: Acquired inhibitors to the blood coagulation factors. Semin Thromb Hemost 1:336-385, 1975. 17. Thiagarajan P, Shapiro SS, DeMarco L: Monoclonal immunoglobulin MA coagulation inhibitor with phospholipid specificity: Mechanism of a lupus anticoagulant. J Clin Invest 66:397-405, 1980. 18. Coleman M, Vigliano EM, Weksler ME, Nachman RL: Inhibition of fibrin monomer polymerization by lambda myeloma globulins. Blood 39:210-223, 1972. 19. Penny R, Castaldi P A, Whitsed HM: Inflammation and hemostasis in paraproteinemias. Br J Haematol 20:35-44, 1971. 20. Glueck HI, MacKenzie MR, Glueck CJ: Crystalline IgG protein in multiple myeloma: Identification effects on coagulation and on lipoprotein metabolism. J Lab Clin Med 79:731-744, 1972.
366
21. Kaufman PA, Gockerman JP, Greenberg CS: Production of a novel anticoagulant by neoplastic plasma cells: Report of a case and review of the literature. Am J Med 86:612-616, 1989. 22. Palmer RN, Rick ME, Rick PD, Zeller JA, Gralnick HR: Circulating heparan sulfate anticoagulant in a patient with a fatal bleeding disorder. N Engl J Med 310:1696-1699, 1984. 23. Khoory MS, Nesheim ME, Bowie EJW, Mann KG: Circulating heparan sulfate proteoglycan anticoagulant from a patient with a plasma cell disorder. J Clin Invest 65:666-675, 1980. 24. Tefferi A, Nichols WL, Walter Bowie EJ: Circulating heparinlike anticoagulants: Reports of five consecutive cases and a review. Am J Med 88:184-188,1990. 25. Bick RL: Alterations of hemostasis associated with malignancy: Etiology, pathophysiology, diagnosis and management. Semin Thromb Hemost 6:18-21, 1978. 26. Brody JI, Haidar ME, Rossman RE: A hemorrhagic syndrome in Wa!denstrom's macroglobulinemia secondary to immunoabsorption of factor VIII: Recovery after splenectomy. N Engl J Med 300:408-410, 1979. 27. Mant MJ, Hirsh J, Gauldie J, Bienenstock J, Pineo GP, Luke KH: von Willebrand's syndrome presenting as an acquired bleeding disorder in association with a monoclonal gammopathy. Blood 42:429-435, 1973. 28. Zettervall 0, Nilsoon 1M: Acquired von Willebrand's disease caused by monoclonal antibody. Acta Medica Scandinavia 204: 521-528, 1978. 29. DiMinno G, Coraggio F, Cerbone AM, Capitanio AN, Manzo C, Spina M, Scarpato P, Datolli GMR, Mattioli PL, Mancini M: A myeloma paraprotein with specificity for platelet glycoprotein IlIa in a patient with a fatal bleeding disorder. J Clin Invest 77: 157-164, 1986. 30. Merlini G, Farhangi M, Osserman EF: Monoclonal immunoglobulins with antibody activity in myeloma, macroglobulinemia and related plasma cell dyscrasias. Semin Oncol13:350-365, 1986. 31. Bick RL: Disseminated intravascular coagulation and related syndromes: A clinical review. Semin Thromb Hemost 14:299338,1988.
December 1993 Volume 306 Number 6