Diagnostic Value of Biochemical Analysis of Pleural Effusions Carcinoembryonic Antigen and Beta2 Microglobulin ADRIAN O. VLADUTIU, M.D., RICHARD H. ADLER, M.D., AND F. WELLS BRASON, M.D.
PLEURAL EFFUSION is a common finding in medical practice; however, its etiology is sometimes obscure. The simultaneous presence of effusion and a disease that can produce pleural effusion does not establish a causal relationship between the disease and the effusion. A large proportion of pleural effusions is due to malignancy; therefore, differentiation between malignant and nonmalignant effusions is especially important. Accurate diagnosis of malignant effusions is often difficult, and biochemical analysis of pleural effusion has not proved helpful in this respect.29-30 For example, orosomucoid (alpha,-acid glycoprotein) was initially reported as increased in all pleural effusions due to malignant diseases, 26 but subsequently was found inadequate for differentiating malignant from nonmalignant effusions.1 We attempted to reassess the diagnostic value of biochemical analysis in pleural effusions obtained from patients admitted to the Buffalo General Hospital in Received November 7, 1977; received revised manuscript and accepted for publication December 27, 1977. Supported in part by grants from Margaret Duffy and Robert Cameron Troup Memorial Fund and the Junior Board of the Buffalo General Hospital. Address reprint requests to Dr. Vladutiu: Department of Immunopathology, The Buffalo General Hospital, 100 High Street, Buffalo, New York 14203.
Departments of Immunopathology, Surgery, and Pathology, The Buffalo General Hospital, and SUNY at Buffalo, School of Medicine, Buffalo, New York
1975-1976. The concentrations of orosomucoid, ceruloplasmin, alpha2 macroglobulin, carcinoembryonic antigen (CEA), beta 2 microglobulin, and the activity of lysozyme and hexosaminidase were measured. Protein electrophoresis, cytologic examination of effusions, and histologic examination of pleural biopsies were also performed. Materials and Methods One hundred and five consecutive patients, 40-85 years of age, admitted to the hospital were the subjects of the investigation. Sixty-seven patients (63%) had malignancies, usually cancer of the lung or breast, but some patients had pulmonary or pleural metastases, or both, from other tumors. The diagnosis was established by clinical and laboratory evaluation and patients with undetermined diagnosis were deleted from the study. An effusion was considered malignant when the clinical history and either the cytologic characteristics of the effusion or the histologic features of the pleural biopsy specimen were consistent with malignancy. Pleural effusion was collected in evacuated glass tubes,* with and without solid ethylenediaminetetraacetate, randomly during the day. The samples were immediately centrifuged for 15 min at 3,000 rpm in a Sorvall GLC-1 centrifuge and were stored at - 2 0 C for as long as two months before the assays. This storage had no effect on the concentrations of substances assayed. Carcinoembryonic antigen (CEA) was measured by radioimmunoassay with commercial reagents.t Pleural effusion was collected in tubes with ethylenediaminetetraacetate, and the method for CEA determination was similar to the plasma CEA assay. 11 Beta 2 microglobulin was measured by a solid-phase radioimmuno* Vacutainers; Becton and Dickinson, Rutherford, New Jersey. t Roche Diagnostics, Nutley, New Jersey.'
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Vladutiu, Adrian O., Adler, Richard H., and Brason, F. Wells: Diagnostic value of biochemical analysis of pleural effusions. Carcinoembryonic antigen and beta2 microglobulin. Am J Clin Pathol 71: 210-214,1979. Pleural effusions from 105 patients with malignant and nonmalignant diseases were examined for tumor cells, content of CEA, beta2 microglobulin, ceruloplasmin, alpha2 macroglobulin, orosomucoid, lysozyme, and hexosaminidase. Only CEA and beta2 microglobulin determinations were of diagnostic value. CEA concentrations greater than 11 ng/ml were found only in malignant effusions. Beta, microglobulin values were increased in pleural effusions due to lymphoma or immune diseases. Measurement of CEA and beta2 microglobulin in addition to the cytologic examination could increase the diagnostic significance of the analysis of pleural effusions. (Key words: Pleural effusion; Carcinoembryonic antigen; Beta2 microglobulin; Orosomucoid; Hexosaminidase; Ceruloplasmin; Alpha2 macroglobulin; Lysozyme.)
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assay.$ Orosomucoid, ceruloplasmin, and alpha2 macroglobulin were measured by radial immunodiffusion21 by use of commercial reagents.§ Lysozyme activity was measured with "Lysoplates"11 according to the method of Osserman and Lawlor.23 Hexosaminidase activity was measured according to the method of Leaback and Walker.18 Protein electrophoresis was carried out in 0.2% agarose gel in sodium phosphate buffer, pH 8.5, at 19 V/cm.** Cytologic examination of the effusions for malignant cells and histologic examination of pleural biopsy specimens were performed according to routine technics. Results
t Pharmacia Diagnostics, Piscataway, New Jersey. § Behring Diagnostics, Sommerville, New Jersey. ' Kallestad Laboratories, Chaska. Minnesota. ** Spectrophor I; Bausch and Lomb, Rochester, New York.
The highest beta2 microglobulin concentrations in pleural effusions were found for patients with lymphoma, connective-tissue diseases, and other putative immune diseases. The average value for 15 patients with lymphoma or myeloma was 10.3 ± 3.1 /xg/ml (SE) and that for eight patients with immune diseases (scleroderma, lupus erythematosus, rheumatoid arthritis, etc.) was 8.2 ± 1.1 /xg/ml. Pleural effusions of patients with other diseases had lower values, e.g. 3.86 ± 0.45 /xg/ml for 26 patients with pulmonary carcinoma and 3.16 ± 0.25 /xg/ml for 11 patients with mammary carcinoma (Table 1). Lysozyme activity had values below 6 /xg/ml in most of the specimens from patients with carcinoma (mammary or pulmonary), whereas patients with nonmalignant diseases had higher values, to 23 /xg/ml; the difference between the two groups was not statistically significant. Patients with empyema had very high lysozyme activity, but they were excluded from the study. Hexosaminidase activity (measured in only 49 samples) had a very wide range for patients with both malignant and nonmalignant diseases. The cytologic examination for malignant cells was positive in 57%, negative in 39%, and inconclusive in 4% of cases; four of nine patients with known malignancy and negative cytologic findings had histologic evidence of malignancy in pleural biopsy specimens. Pleural biopsy specimens also showed malignant changes in two of three patients with equivocal cytologic findings. In one case both pleural biopsy and cytologic findings were negative, while the pulmonary biopsy specimen was positive for malignant cells. Since CEA appeared to differentiate malignant from nonmalignant pleural effusions, the diagnostic value of CEA was assessed in effusions of patients with equivocal or negative cytologic findings but with histories of malignancy in whose cases the diagnosis had been made on the basis of histologic examination of pleural biopsy specimens or positive cytologicfindingsduring subsequent admissions to the hospital. For four patients with equivocal cytologic findings CEA concentrations in pleural effusions were 12.5,4.5, 5.2, and 19.9 ng/ml, respectively; pleural biopsy specimens from the first and third patients showed malignancy. Three patients with clinical evidence of malignancy but with negative cytologic examinations had CEA values of 10.1, 32.5, and 294 ng/ml in the effusions. At follow-up evaluations 3-12 months later, cytologic or histologic examinations of pleural biopsy specimens showed malignant cells. Discussion Various proteins have been measured in pleural effusions in order to find criteria for specific diagnosis or for discriminating between malignant and nonmalig-
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Protein electrophoresis could not differentiate malignant from nonmalignant effusions. Alpha2 macroglobulin showed values ranging from 0 to 332 mg/dl. The majority of patients had concentrations below 70 mg/dl; in specimens from nine patients no measurable alpha2 macroglobulin could be found. Patients with malignancies generally had higher concentrations of alpha2 macroglobulin in pleural effusions than patients without malignancy, but the difference between the two groups was not statistically significant. Ceruloplasmin values varied widely, from an unmeasurable concentration in a case of cirrhosis to the highest concentrations in patients with pulmonary carcinoma or lymphoma. Concentrations of orosomucoid showed a wide range (52-300 mg/dl) in patients with malignant effusions (Fig. 1). The lowest values (29 mg/dl) were found in two patients with hepatic cirrhosis. The average concentration for 53 patients with malignancies (including lymphoma) was 137.58 ± 6.96 mg/dl (SE) and that for 38 patients without malignancy was 104.97 ± 9.24 mg/dl. Carcinoembryonic antigen was undetectable in pleural effusions of 17 patients, most of whom had hepatic cirrhosis, congestive heart failure, or other nonmalignant diseases. CEA values greater than 10 ng/ml were found for only 57% of patients with malignancies; one , patient with bronchogenic carcinoma had a CEA value as high as 3,000 ng/ml. Only one patient without malignancy (diabetes and vasculitis) had a CEA value greater than 10 ng/ml, i.e., 11 ng/ml. The average CEA value for effusions from 48 cancer patients (excluding lymphoma) was 20.8 ± 0.5 ng/ml (SE), that for 15 patients with lymphoma or myeloma was 3.2 ± 0.5 ng/ml, and that for 32 patients with nonmalignant diseases was 2.1 ± 0.5 ng/ml (Fig. 2).
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with inflammatory diseases. However, in two subsequent studies 66 and 56 patients, respectively,1,4 orosomucoid concentration did not show a statistically significant difference between neoplastic and nonneoplastic inflammatory effusions. Very recently it was reported that all malignant effusions had orosomucoid values of more than 108 mg/dl and all nonmalignant effusions had values below 108 ng/ dl.8 In the present study we could not find a cutoff value for orosomucoid that could separate malignant from nonmalignant effusions. Therefore, new prospective studies should be performed to ascertain the significance of orosomucoid determination in pleural effusions.
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FIG. 1. Orosomucoid in pleural effusions.
nant effusions. Measurement of immunoglobulins, C3, albumin, prealbumin, alpha2Hs glycoprotein, transferrin, and beta2 glycoprotein in pleural effusions had no diagnostic value.1-4,8,25,27 Higher concentrations of fibrin/fibrinogen degradation products in pleural exudates from patients who had tumors compared with patients who had cardiac failure were reported,2 but high concentrations of these products were found in both benign and malignant exudates when compared with transudates.5 Zinc, copper, iron, calcium and phosphorus concentrations could not differentiate benign from malignant effusions.6,9 Ceruloplasmin was reported increased in pleural effusions of some patients with malignancies.8 However, our data and other data4 showed no diagnostic value for ceruloplasmin and alpha2 macroglobulin determination. Recently it was reported that the orosomucoid content of pleural effusions was higher in malignant than in nonmalignant diseases.26 Orosomucoid concentration was 130 ± 13 mg/dl (SD) for 58 patients with neoplastic diseases and 67 ± 17 mg/dl (SD) for 12 patients
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FIG. 2. CEA in pleural effusions.
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Although increased hexosaminidase activity was found in 32 of 35 malignant pleural effusions,20 in 49 patients we could not find a discriminative value for hexosaminidase assay. The present study confirmed preliminary data31 showing increased beta2 microglobulin concentrations in pleural effusions of patients with lymphoma and immune diseases. In one case with inconclusive cytologic findings the diagnosis of lymphoma was made only after pleural biopsy was prompted by a high value (9.1 /Lig/ml) of beta2 microglobulin in the effusion. Although we did not measure the concentration of beta2 microglobulin in serum or urine, it is conceivable that the high values in pleural effusions are due to the lymphoma itself and not to renal tubular damage, since our lymphoma patients did not have abnormal renal function. Beta2 microglobulin was increased in saliva of patients with Sjogren's syndrome22 and in spinal fluids of patients with bacterial meningitis.10 High concentrations of this globulin were also found in the sera of some patients with lymphoma, leukemia, and myeloma.13,28 The measurement of CEA in pleural effusions is of particular interest because CEA is one of the few wellknown tumor markers. This study showed that CEA or a CEA-like substance can be readily measured in pleural effusions with a method widely used for the measurement of CEA in plasma. With one exception, values of more than 10 ng/ml were found only for patients who had malignancies. Since CEA values were not increased in all malignant effusions, this test, despite a high specificity (96.6% for a cutoff value of 10 ng/ml), had a low sensitivity (57% for the same cutoff value) for detecting malignant effusions. When we take 20 mg/ml as a cutoff value for CEA, the specificity of the test is 100% but its sensitivity is only 40%.
Table I. Beta2 Microglobulin in Pleural Effusions* Lymphoma and myeloma Immune diseases Pulmonary carcinoma Mammary carcinoma Other diseases
10.3 8.2 3.8 3.1 2.9
± ± ± ± ±
3.1 (15)t 1.1 (8) 0.4 (20) 0.2(11) 0.2 (35)
* Expressed in /ig/ml. t Mean ± SE. Number of patients in parentheses.
Simultaneous measurements of CEA in blood and pleural effusion were not performed in this study, but it was reported that CEA values in pleural effusions were more consistently diagnostic than plasma values for showing malignancy.3 Recently, it was found that 70% of patients with various carcinomas had CEA values of more than 20 ng/ml in pleural effusions,3 but values as high as 23 ng/ml were found for patients with cirrhosis.12 The present study showed no specific pattern for any particular disease, and we could not find a biochemical test that consistently differentiated between malignant and nonmalignant effusions. The determination of CEA419 and beta2 microglobulin concentrations in pleural effusions promises to be of diagnostic help; however, a large number of patients should be investigated in order to obtain definitive information. Our study also showed that cytologic examination of pleural effusion is not sensitive enough for diagnosing all malignant effusions, confirming results of other investigations,7,17 which showed that cytologic examination for tumor cells was positive for no more than 60% of patients with malignancies. It is hoped that new biochemical assays, together with cytologic examination,15 will alter the view of pleural effusion as a "diagnostic dilemma."29 Acknowledgments. Dr. Georgirene D. Vladutiu performed the hexosaminidase determination. Mrs. Barbara M. Winiarski, Ms. Sharon Y. Clark and Mrs. Barbara Roach provided technical assistance.
References 1. Agostoni A, Marasini B: Orosomucoid contents of pleural and peritoneal effusions of various etiologies. Am J Clin Pathol 67:146-148, 1977 2. Astedt B, Adielsson G, Mattson W: Fibrin/fibrinogen degradation products in pleural exudate (letter). Lancet 2:414, 1976 3. Basta WR, Vandervoorde JP, Lyons HA: The use of pleural fluid CEA levels in the diagnosis of malignant pleural effusions (abstract). Am Rev Resp Dis 113:118, 1976 4. Booth SN, Lakin G, Dykes PW, et al: Cancer-associated proteins in effusion fluids. J Clin Pathol 30:537-540, 1977 5. Cohen NA, Lawton E: Fibrin/fibrinogen degradation products in effusions (letter). Lancet 2:909, 1976 6. Dines DE, Elveback LR, McCall JT: Zinc, copper and iron content of pleural fluid in benign and neoplastic disease. Thorax 27:368-370, 1972 7. Dines DE, Pierre RV, Franzen SJ: The value of cells in the
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The value of enzyme determinations in pleural effusions is also not clear. The measurement of total lactate dehydrogenase (LDH) activity can differentiate exudates from transudates, but it cannot distinguish malignant from benign exudates; no clear-cut difference in LDH isoenzyme patterns among various groups of benign exudative effusions was found.18 Leucine aminopeptidase and beta glucuronidase activities were higher in malignant than in nonmalignant pleural effusions, but there was a wide overlap between the two groups.24 Similar values for lysozyme concentrations were found in both malignant and nonmalignant effusions.32 However, high lysozyme activity was found in association with empyema and tuberculous pleurisy.14 In our study the highest lysozyme activity was also found in empyema; nevertheless, the diagnosis of empyema can be easily made without the need for lysozyme assay.
213
214 8. 9.
10.
11. 12. 13.
14.
16.
17. 18. 19.
pleural fluid in the differential diagnosis. Mayo Clin Proc 50: 571-572, 1975 Diret MF, Valdiguie P: Les glycoprot£ines des liquides pleuraux. Clin Chim Acta 77:219-225, 1977 Feldstein AM, Samacham J, Spencer H: Levels of calcium, phosphorus, alkaline phosphatase and protein in effusion fluid and serum in man. Am J Med 35:530-535, 1963 Gekle D, Kult J, Roth R: Lysozym und /32-Mikroglobulin in Liquor gesunder Kinder und bei Kindern mit Erkrankungerns des Zentral-nervensystems. Klin Wochenschr 55:189-191, 1977 Hansen HJ, Snyder JJ, Miller E, et al: Carcinoembryonic antigen (CEA) assay. Human Pathol 5:139-147, 1974 Kim YD, Hirata AA: Carcinoembryonic antigen-like substances in human cavity fluids. Immunol Commun 5:619-626, 1976 Kithier K, Cejka J, Belamaric J, et al: ft. microglobulin: Occurrence in fetal life and malignancy. Clin Chim Acta 67: 307-313, 1976 Klockars M, Petterson R, Riska H, et al: Pleural fluid lysozyme in tuberculous and non-tuberculous pleuresy. Br Med J 3: 1381, 1976 Krivinkova H, Ponten J, Blondal T: The diagnosis of cancer from body fluids. Acta Path Microbiol Scand Sect A, 84: 455-467, 1976 Leaback DH, Walker PG: Studies on glucosaminidase. 4. The fluorometric assay of N-acetyl-/3 glucosaminidase. Biochem J 78:151-156, 1961 Leuallen EC, Carr CY: Pleural effusion: A statistical study of 436 patients. New Engl J Med 252:79-83, 1955 Light RW, Ball WC: Lactate dehydrogenase isoenzymes in pleural effusions. Am Rev Resp Dis 108:660-664, 1973 Loewenstein ML, Rittgers RA, Kupchik HZ, et al: Improved detection of malignant ascites and pleural effusions by combined assay of fluid CEA and cytology (abstract). Clin Res 23:596A, 1975
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20. McCormack M: Diagnostic potential of lysosomal hydrolases in body cavity effusions. J Clin Pathol 29:124-129, 1976 21. Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-243, 1965 22. Michalski JP, Daniels TE, Talal N, et al: Beta 2 microglobulin and lymphocytic infiltration in Sjogren's syndrome. N Engl J Med 293:1228-1234, 1971 23. Osserman EF, Lawlor DP: Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med 124:921-951, 1966 24. Pineda EP, Goldbarg JA, Levitan R, et al: Leucine aminopeptidase and /3-glucuronidase activity in cancerous and non-cancerous effusions. Am J Dig Dis 7:797-803, 1962 25. Reabek E: Low density lipoproteins and immunoglobulins in human pleural effusions. Clin Chim Acta 76:363-364, 1977 26. Rudman D, Chawla RK, Del Rio AE, et al: Orosomucoid content of pleural and peritoneal effusions. J Clin Invest 54:147155, 1974 27. Shallenberg DW, Daniel TM: Quantitative determination of several pleural fluid proteins. Am Rev Resp Dis 106:121-122, 1972 28. Shuster J, Gold P, Poulik MD: /32 microglobulin levels in cancerous and other disease states. Clin Chim Acta 67:307-313, 1976 29. Storey DD, Dines DE, Coles DT: Pleural effusion. A diagnostic dilemma. JAMA 236:2186, 1976 30. Tinney WS, Olsen AM: The significance of fluid in the pleural space: A study of 274 cases. J. Thorac Surg 14:245-252, 1945 31. Vladutiu AO: Beta 2 microglobulin in pleural fluids (letter). N Engl J Med 294:903, 1976 32. Wu KK, Burns CP, Barret DA, II: Pleural fluids lysozyme in human disease. Proc Soc Exp Biol Med 152:132-134, 1976
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15.
VLADUTIU, ADLER, AND BRASON
PLEURAL EFFUSION is a common finding in medical practice; however, its etiology is sometimes obscure. The simultaneous presence of effusion and a disease that can produce pleural effusion does not establish a causal relationship between the disease and the effusion. A large proportion of pleural effusions is due to malignancy; therefore, differentiation between malignant and nonmalignant effusions is especially important. Accurate diagnosis of malignant effusions is often difficult, and biochemical analysis of pleural effusion has not proved helpful in this respect.29-30 For example, orosomucoid (alpha,-acid glycoprotein) was initially reported as increased in all pleural effusions due to malignant diseases, 26 but subsequently was found inadequate for differentiating malignant from nonmalignant effusions.1 We attempted to reassess the diagnostic value of biochemical analysis in pleural effusions obtained from patients admitted to the Buffalo General Hospital in Received November 7, 1977; received revised manuscript and accepted for publication December 27, 1977. Supported in part by grants from Margaret Duffy and Robert Cameron Troup Memorial Fund and the Junior Board of the Buffalo General Hospital. Address reprint requests to Dr. Vladutiu: Department of Immunopathology, The Buffalo General Hospital, 100 High Street, Buffalo, New York 14203.
Departments of Immunopathology, Surgery, and Pathology, The Buffalo General Hospital, and SUNY at Buffalo, School of Medicine, Buffalo, New York
1975-1976. The concentrations of orosomucoid, ceruloplasmin, alpha2 macroglobulin, carcinoembryonic antigen (CEA), beta 2 microglobulin, and the activity of lysozyme and hexosaminidase were measured. Protein electrophoresis, cytologic examination of effusions, and histologic examination of pleural biopsies were also performed. Materials and Methods One hundred and five consecutive patients, 40-85 years of age, admitted to the hospital were the subjects of the investigation. Sixty-seven patients (63%) had malignancies, usually cancer of the lung or breast, but some patients had pulmonary or pleural metastases, or both, from other tumors. The diagnosis was established by clinical and laboratory evaluation and patients with undetermined diagnosis were deleted from the study. An effusion was considered malignant when the clinical history and either the cytologic characteristics of the effusion or the histologic features of the pleural biopsy specimen were consistent with malignancy. Pleural effusion was collected in evacuated glass tubes,* with and without solid ethylenediaminetetraacetate, randomly during the day. The samples were immediately centrifuged for 15 min at 3,000 rpm in a Sorvall GLC-1 centrifuge and were stored at - 2 0 C for as long as two months before the assays. This storage had no effect on the concentrations of substances assayed. Carcinoembryonic antigen (CEA) was measured by radioimmunoassay with commercial reagents.t Pleural effusion was collected in tubes with ethylenediaminetetraacetate, and the method for CEA determination was similar to the plasma CEA assay. 11 Beta 2 microglobulin was measured by a solid-phase radioimmuno* Vacutainers; Becton and Dickinson, Rutherford, New Jersey. t Roche Diagnostics, Nutley, New Jersey.'
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Vladutiu, Adrian O., Adler, Richard H., and Brason, F. Wells: Diagnostic value of biochemical analysis of pleural effusions. Carcinoembryonic antigen and beta2 microglobulin. Am J Clin Pathol 71: 210-214,1979. Pleural effusions from 105 patients with malignant and nonmalignant diseases were examined for tumor cells, content of CEA, beta2 microglobulin, ceruloplasmin, alpha2 macroglobulin, orosomucoid, lysozyme, and hexosaminidase. Only CEA and beta2 microglobulin determinations were of diagnostic value. CEA concentrations greater than 11 ng/ml were found only in malignant effusions. Beta, microglobulin values were increased in pleural effusions due to lymphoma or immune diseases. Measurement of CEA and beta2 microglobulin in addition to the cytologic examination could increase the diagnostic significance of the analysis of pleural effusions. (Key words: Pleural effusion; Carcinoembryonic antigen; Beta2 microglobulin; Orosomucoid; Hexosaminidase; Ceruloplasmin; Alpha2 macroglobulin; Lysozyme.)
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assay.$ Orosomucoid, ceruloplasmin, and alpha2 macroglobulin were measured by radial immunodiffusion21 by use of commercial reagents.§ Lysozyme activity was measured with "Lysoplates"11 according to the method of Osserman and Lawlor.23 Hexosaminidase activity was measured according to the method of Leaback and Walker.18 Protein electrophoresis was carried out in 0.2% agarose gel in sodium phosphate buffer, pH 8.5, at 19 V/cm.** Cytologic examination of the effusions for malignant cells and histologic examination of pleural biopsy specimens were performed according to routine technics. Results
t Pharmacia Diagnostics, Piscataway, New Jersey. § Behring Diagnostics, Sommerville, New Jersey. ' Kallestad Laboratories, Chaska. Minnesota. ** Spectrophor I; Bausch and Lomb, Rochester, New York.
The highest beta2 microglobulin concentrations in pleural effusions were found for patients with lymphoma, connective-tissue diseases, and other putative immune diseases. The average value for 15 patients with lymphoma or myeloma was 10.3 ± 3.1 /xg/ml (SE) and that for eight patients with immune diseases (scleroderma, lupus erythematosus, rheumatoid arthritis, etc.) was 8.2 ± 1.1 /xg/ml. Pleural effusions of patients with other diseases had lower values, e.g. 3.86 ± 0.45 /xg/ml for 26 patients with pulmonary carcinoma and 3.16 ± 0.25 /xg/ml for 11 patients with mammary carcinoma (Table 1). Lysozyme activity had values below 6 /xg/ml in most of the specimens from patients with carcinoma (mammary or pulmonary), whereas patients with nonmalignant diseases had higher values, to 23 /xg/ml; the difference between the two groups was not statistically significant. Patients with empyema had very high lysozyme activity, but they were excluded from the study. Hexosaminidase activity (measured in only 49 samples) had a very wide range for patients with both malignant and nonmalignant diseases. The cytologic examination for malignant cells was positive in 57%, negative in 39%, and inconclusive in 4% of cases; four of nine patients with known malignancy and negative cytologic findings had histologic evidence of malignancy in pleural biopsy specimens. Pleural biopsy specimens also showed malignant changes in two of three patients with equivocal cytologic findings. In one case both pleural biopsy and cytologic findings were negative, while the pulmonary biopsy specimen was positive for malignant cells. Since CEA appeared to differentiate malignant from nonmalignant pleural effusions, the diagnostic value of CEA was assessed in effusions of patients with equivocal or negative cytologic findings but with histories of malignancy in whose cases the diagnosis had been made on the basis of histologic examination of pleural biopsy specimens or positive cytologicfindingsduring subsequent admissions to the hospital. For four patients with equivocal cytologic findings CEA concentrations in pleural effusions were 12.5,4.5, 5.2, and 19.9 ng/ml, respectively; pleural biopsy specimens from the first and third patients showed malignancy. Three patients with clinical evidence of malignancy but with negative cytologic examinations had CEA values of 10.1, 32.5, and 294 ng/ml in the effusions. At follow-up evaluations 3-12 months later, cytologic or histologic examinations of pleural biopsy specimens showed malignant cells. Discussion Various proteins have been measured in pleural effusions in order to find criteria for specific diagnosis or for discriminating between malignant and nonmalig-
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Protein electrophoresis could not differentiate malignant from nonmalignant effusions. Alpha2 macroglobulin showed values ranging from 0 to 332 mg/dl. The majority of patients had concentrations below 70 mg/dl; in specimens from nine patients no measurable alpha2 macroglobulin could be found. Patients with malignancies generally had higher concentrations of alpha2 macroglobulin in pleural effusions than patients without malignancy, but the difference between the two groups was not statistically significant. Ceruloplasmin values varied widely, from an unmeasurable concentration in a case of cirrhosis to the highest concentrations in patients with pulmonary carcinoma or lymphoma. Concentrations of orosomucoid showed a wide range (52-300 mg/dl) in patients with malignant effusions (Fig. 1). The lowest values (29 mg/dl) were found in two patients with hepatic cirrhosis. The average concentration for 53 patients with malignancies (including lymphoma) was 137.58 ± 6.96 mg/dl (SE) and that for 38 patients without malignancy was 104.97 ± 9.24 mg/dl. Carcinoembryonic antigen was undetectable in pleural effusions of 17 patients, most of whom had hepatic cirrhosis, congestive heart failure, or other nonmalignant diseases. CEA values greater than 10 ng/ml were found for only 57% of patients with malignancies; one , patient with bronchogenic carcinoma had a CEA value as high as 3,000 ng/ml. Only one patient without malignancy (diabetes and vasculitis) had a CEA value greater than 10 ng/ml, i.e., 11 ng/ml. The average CEA value for effusions from 48 cancer patients (excluding lymphoma) was 20.8 ± 0.5 ng/ml (SE), that for 15 patients with lymphoma or myeloma was 3.2 ± 0.5 ng/ml, and that for 32 patients with nonmalignant diseases was 2.1 ± 0.5 ng/ml (Fig. 2).
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with inflammatory diseases. However, in two subsequent studies 66 and 56 patients, respectively,1,4 orosomucoid concentration did not show a statistically significant difference between neoplastic and nonneoplastic inflammatory effusions. Very recently it was reported that all malignant effusions had orosomucoid values of more than 108 mg/dl and all nonmalignant effusions had values below 108 ng/ dl.8 In the present study we could not find a cutoff value for orosomucoid that could separate malignant from nonmalignant effusions. Therefore, new prospective studies should be performed to ascertain the significance of orosomucoid determination in pleural effusions.
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FIG. 1. Orosomucoid in pleural effusions.
nant effusions. Measurement of immunoglobulins, C3, albumin, prealbumin, alpha2Hs glycoprotein, transferrin, and beta2 glycoprotein in pleural effusions had no diagnostic value.1-4,8,25,27 Higher concentrations of fibrin/fibrinogen degradation products in pleural exudates from patients who had tumors compared with patients who had cardiac failure were reported,2 but high concentrations of these products were found in both benign and malignant exudates when compared with transudates.5 Zinc, copper, iron, calcium and phosphorus concentrations could not differentiate benign from malignant effusions.6,9 Ceruloplasmin was reported increased in pleural effusions of some patients with malignancies.8 However, our data and other data4 showed no diagnostic value for ceruloplasmin and alpha2 macroglobulin determination. Recently it was reported that the orosomucoid content of pleural effusions was higher in malignant than in nonmalignant diseases.26 Orosomucoid concentration was 130 ± 13 mg/dl (SD) for 58 patients with neoplastic diseases and 67 ± 17 mg/dl (SD) for 12 patients
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FIG. 2. CEA in pleural effusions.
Vol. 71 • No. 2
CEA AND BETA2 MICROGLOBULIN IN PLEURAL FLUIDS
Although increased hexosaminidase activity was found in 32 of 35 malignant pleural effusions,20 in 49 patients we could not find a discriminative value for hexosaminidase assay. The present study confirmed preliminary data31 showing increased beta2 microglobulin concentrations in pleural effusions of patients with lymphoma and immune diseases. In one case with inconclusive cytologic findings the diagnosis of lymphoma was made only after pleural biopsy was prompted by a high value (9.1 /Lig/ml) of beta2 microglobulin in the effusion. Although we did not measure the concentration of beta2 microglobulin in serum or urine, it is conceivable that the high values in pleural effusions are due to the lymphoma itself and not to renal tubular damage, since our lymphoma patients did not have abnormal renal function. Beta2 microglobulin was increased in saliva of patients with Sjogren's syndrome22 and in spinal fluids of patients with bacterial meningitis.10 High concentrations of this globulin were also found in the sera of some patients with lymphoma, leukemia, and myeloma.13,28 The measurement of CEA in pleural effusions is of particular interest because CEA is one of the few wellknown tumor markers. This study showed that CEA or a CEA-like substance can be readily measured in pleural effusions with a method widely used for the measurement of CEA in plasma. With one exception, values of more than 10 ng/ml were found only for patients who had malignancies. Since CEA values were not increased in all malignant effusions, this test, despite a high specificity (96.6% for a cutoff value of 10 ng/ml), had a low sensitivity (57% for the same cutoff value) for detecting malignant effusions. When we take 20 mg/ml as a cutoff value for CEA, the specificity of the test is 100% but its sensitivity is only 40%.
Table I. Beta2 Microglobulin in Pleural Effusions* Lymphoma and myeloma Immune diseases Pulmonary carcinoma Mammary carcinoma Other diseases
10.3 8.2 3.8 3.1 2.9
± ± ± ± ±
3.1 (15)t 1.1 (8) 0.4 (20) 0.2(11) 0.2 (35)
* Expressed in /ig/ml. t Mean ± SE. Number of patients in parentheses.
Simultaneous measurements of CEA in blood and pleural effusion were not performed in this study, but it was reported that CEA values in pleural effusions were more consistently diagnostic than plasma values for showing malignancy.3 Recently, it was found that 70% of patients with various carcinomas had CEA values of more than 20 ng/ml in pleural effusions,3 but values as high as 23 ng/ml were found for patients with cirrhosis.12 The present study showed no specific pattern for any particular disease, and we could not find a biochemical test that consistently differentiated between malignant and nonmalignant effusions. The determination of CEA419 and beta2 microglobulin concentrations in pleural effusions promises to be of diagnostic help; however, a large number of patients should be investigated in order to obtain definitive information. Our study also showed that cytologic examination of pleural effusion is not sensitive enough for diagnosing all malignant effusions, confirming results of other investigations,7,17 which showed that cytologic examination for tumor cells was positive for no more than 60% of patients with malignancies. It is hoped that new biochemical assays, together with cytologic examination,15 will alter the view of pleural effusion as a "diagnostic dilemma."29 Acknowledgments. Dr. Georgirene D. Vladutiu performed the hexosaminidase determination. Mrs. Barbara M. Winiarski, Ms. Sharon Y. Clark and Mrs. Barbara Roach provided technical assistance.
References 1. Agostoni A, Marasini B: Orosomucoid contents of pleural and peritoneal effusions of various etiologies. Am J Clin Pathol 67:146-148, 1977 2. Astedt B, Adielsson G, Mattson W: Fibrin/fibrinogen degradation products in pleural exudate (letter). Lancet 2:414, 1976 3. Basta WR, Vandervoorde JP, Lyons HA: The use of pleural fluid CEA levels in the diagnosis of malignant pleural effusions (abstract). Am Rev Resp Dis 113:118, 1976 4. Booth SN, Lakin G, Dykes PW, et al: Cancer-associated proteins in effusion fluids. J Clin Pathol 30:537-540, 1977 5. Cohen NA, Lawton E: Fibrin/fibrinogen degradation products in effusions (letter). Lancet 2:909, 1976 6. Dines DE, Elveback LR, McCall JT: Zinc, copper and iron content of pleural fluid in benign and neoplastic disease. Thorax 27:368-370, 1972 7. Dines DE, Pierre RV, Franzen SJ: The value of cells in the
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The value of enzyme determinations in pleural effusions is also not clear. The measurement of total lactate dehydrogenase (LDH) activity can differentiate exudates from transudates, but it cannot distinguish malignant from benign exudates; no clear-cut difference in LDH isoenzyme patterns among various groups of benign exudative effusions was found.18 Leucine aminopeptidase and beta glucuronidase activities were higher in malignant than in nonmalignant pleural effusions, but there was a wide overlap between the two groups.24 Similar values for lysozyme concentrations were found in both malignant and nonmalignant effusions.32 However, high lysozyme activity was found in association with empyema and tuberculous pleurisy.14 In our study the highest lysozyme activity was also found in empyema; nevertheless, the diagnosis of empyema can be easily made without the need for lysozyme assay.
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A.J.C.P. • February 1979
20. McCormack M: Diagnostic potential of lysosomal hydrolases in body cavity effusions. J Clin Pathol 29:124-129, 1976 21. Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-243, 1965 22. Michalski JP, Daniels TE, Talal N, et al: Beta 2 microglobulin and lymphocytic infiltration in Sjogren's syndrome. N Engl J Med 293:1228-1234, 1971 23. Osserman EF, Lawlor DP: Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med 124:921-951, 1966 24. Pineda EP, Goldbarg JA, Levitan R, et al: Leucine aminopeptidase and /3-glucuronidase activity in cancerous and non-cancerous effusions. Am J Dig Dis 7:797-803, 1962 25. Reabek E: Low density lipoproteins and immunoglobulins in human pleural effusions. Clin Chim Acta 76:363-364, 1977 26. Rudman D, Chawla RK, Del Rio AE, et al: Orosomucoid content of pleural and peritoneal effusions. J Clin Invest 54:147155, 1974 27. Shallenberg DW, Daniel TM: Quantitative determination of several pleural fluid proteins. Am Rev Resp Dis 106:121-122, 1972 28. Shuster J, Gold P, Poulik MD: /32 microglobulin levels in cancerous and other disease states. Clin Chim Acta 67:307-313, 1976 29. Storey DD, Dines DE, Coles DT: Pleural effusion. A diagnostic dilemma. JAMA 236:2186, 1976 30. Tinney WS, Olsen AM: The significance of fluid in the pleural space: A study of 274 cases. J. Thorac Surg 14:245-252, 1945 31. Vladutiu AO: Beta 2 microglobulin in pleural fluids (letter). N Engl J Med 294:903, 1976 32. Wu KK, Burns CP, Barret DA, II: Pleural fluids lysozyme in human disease. Proc Soc Exp Biol Med 152:132-134, 1976
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15.
VLADUTIU, ADLER, AND BRASON
