C-reactive protein, sialic acid and adenosine deaminase levels in serum and pleural fluid from patients with pleural effusion.

INTRODUCTION

Laboratory analysis of the pleural fluid is essential for the diagnosis of underlying pleural diseases. In the past, differentiation between transudate and exudate was dependent on their specific gravities, protein and glucose concentration, lactate dehydrogenase (LDH) levels and differential cell counts; but recently pleural fluid to serum ratio of the protein and lactate dehydrogenase concentration, and total lactate dehydrogenase levels of the pleural fluids are more often used.[1)] However, in exudates, underlying diseases are not easily differentiated by such a pleural fluid analysis, and cytologic examinations of the pleural fluids or pleural biopsy is usually specific but not sensitive enough for definitive diagnosis.[2)]

In Korea, incidences of pulmonary tuberculosis are high and malignant diseases such as bronchogenic cancer are increasing in incidence.

Therefore, other diagnostic methods which easily differentiate tuberculous pleural effusion from malignant effusions are recommended.

Thus, the author and co-workers studied c-reactive protein(CRP),[3)] an acute phase reactant, and sialic acid which can be elevated in various cancers and inflammatory diseases[4,5)] by quantitative measurement, and measured adenosine deaminase activities, which are known to be elevated in tuberculosis,[6)] typhoid fever and liver cirrhosis,[7)] in order to evaluate the differential diagnostic significance of these tests on pleural effusion.

MATERIALS AND METHODS

Materials

We tested 73 pleural effusion and 43 serum specimens obtained from patients with pleural effusions who had been admitted to Chunnam Medical School Hospital and St. Colomban’s Hospital. Diseases were classified as malignancy, pulmonary tuberculosis, pyothorax and transudate groups by cytologic, histologic, and bacteriologic culture examinations in conjunction with clinical findings. The normal control group was 12 adult men and women who had no abnormal findings on their biochemical serum examinations (Table 1)

Table 1.

Diseases and Number of Specimens

Diseases	No. of specimens
	pleural fluid	serum
Malignancy[*]	22	11
Tuberculous	29	10
Pyothorax	9	6
Other diseases[**]	13	10
Control	0	12
Total	73	49

includes squamous cell carcinoma (7), adenocarcinoma (10), mucoepidermoid carcinoma (1), lymphoma (2), oat cell carcinoma (1) and osteogenic sarcoma (1).

includes trauma (3), hepatoma (5), chronic renal failure (2) and cirrhosis (3).

METHODS

We tested the pleural fluid and serum specimens within 3 days after sampling. All speciments were refrigerated at −20°C.

C-RP measurement: C-RP level was determined quantitatively with a turbidimetric immunoassay kit.[8)]

Sialic acid measurement: Sialic acid levels was measured by an enzymatic method employing neuraminidase.[9)]

Adenosine deaminase (ADA) activity measurement: ADA was measured by the colorimetric assay of Guisti.[10)]

RESULTS

C-RP, Sialic Acid Levels and ADA Activities in Transudates and Exudates

We differentiated the transudates by the standard methods, namely pleural fluid to serum ratio of total protein and LDH levels, total LDH levels of the pleural fluids, and total leukocyte counts of the pleural fluids (in exudates: protein ratio >0.5, LDH ratio >0.6, total LDH level >200 IU/L, and total leukocyte counts >1000/cu mm). The C-RP, Sialic acid and ADA levels in the transudates (mean ± SD) were 0.18 ± 0.21 mg%, 29.60 ± 32.43 mg% and 20.68 ± 11.30 IU/L respectively, but in exudates, the C-RP level was 4.86 ± 5.96 mg%, the Sialic acid level was 72.40 ± 29.83 mg%, and ADA activity was 75.89 ± 59.22 IU/L, indicating a significant increase in the exudates (p<0.05, <0.01). (Table 2)

Table 2.

Camparison of C-Reactive Protein (CRP), Sialic acid (SA), and Adenosine Deaminase (ADA) Activities Between Transudate and Exudate of Pleural Fluid (mean ± SD)

	No.	CRP (mg/dl)	SA (mg/dl)	ADA (IU/L)
Transudate	4	0.18 ± 0.21	29.60 ± 32.43	20.68 ± 11.30
Exudate	50	4.86 ± 5.96	72.40 ± 29.83	75.79 ± 59.22
P-value		<0.01	<0.05	<0.01

C-RP Levels in the Pleural Fluids and Serums

C-RP levels of the sera of patients with pleural effusion were higher compared to the normal control group (0.21 ± 0.33mg%), and were significantly increased in the exudates (p<0.01). On the other hand, C-RP levels of the pleural fluids and sera were especially high in the pyothorax among patients with pleural effusion, with results of 9.17 ± 7.32mg% in the pleural fluid and 12.38 ± 5.51 mg% in the serum, and were more useful for the differentiation of malignancy from tuberculosis (p<0.05). (Table 3)

Table 3.

Comparison of Pleural Fluid and Serum Levels (mean ± SD) of C-RP (mg/dl) in Various Diseases

Group (N1, N2)	Malignancy	Tuberculosis	Pyothorax	Other disease	Control
Sample	(22, 11)	(28, 10)	(9, 6)	(13, 10)	(0, 12)
pleural fluid	3.75 ± 4.39	3.83 ± 5.84	9.17 ± 7.32[a,c]	2.56 ± 2.86
Serum	6.41 ± 7.23[h]	5.07 ± 3.58[h]	12.38 ± 5.51[a,h]	8.46 ± 14.56	0.21 ± 0.33
Ratio pleural fluid serum	0.58 ± 0.48	1.99 ± 2.68	0.94 ± 0.72	0.83 ± 0.66

Number of tested pleural fluid

Number of tested serum samples

Significant difference from malignancy group, p<0.05

Significant difference from malignancy group, p<0.01

Significant difference from tuberculosis group, p<0.05

Significant difference from tuberculosis group, p<0.01

Significant difference from pyothroax group, p<0.05

Significant difference from pyothroax group, p<0.05

Significant difference from serum control, p<0.05

Significant difference from serum control, p<0.01

Sialic Acid Levels in the Pleural Fluids and Serums

Sialic acid levels were significantly higher in the exudates secondary to malignancy and tuberculosis than in the transudates (p<0.05). Sialic acid levels in malignancies were 65.20 ± −26.29 mg% in the pleural fluids and 127.79 + 70.28 mg% in the sera which were comparatively higher than the serum levels in tuberculosis (86.90 ± −36.15 mg%) with a significant difference in their pleural fluid to serum ratios (p<0.05). (Table 4)

Table 4.

Comparison of Pleural Fluid and Serum Levels (mean ± SD) of Sialic Acid (mg/dl) in Various Diseases

Group (N1, N2)	Malignancy	Tuberculosis	Pyothorax	Other disease	Control
Sample	(22, 11)	(29, 10)	(9, 6)	(13, 10)	(0, 12)
Pleural fluid	65.20 ± 26.29	82.98 ± 67.44	72.97 ± 52.53	35.46[b,d,e] ± 27.99
Serum	127.79[h] ± 70.28	86.90[a,g] ± 36.15	113.82[h] ± 29.59	55.57[b,e,f] ± 23.18	57.96 ± 5.09
Radio pleural fluid serum	0.60 ± 0.33	1.13[a] ± 0.85	0.68 ± 0.77	0.73 ± 0.47

Abbreviations are the same as in Table 3

ADA Activities in the Pleural Fluids and Sera

ADA activities in the pleural fluids and sera were not significantly increased in malignancies compared to the normal control value (22.40 ± −8.40 IU/L), but ADA activities were high in the sera and pleural fluids, especially in tuberculosis (p<0.01, 0.05) (Table 5).

Table 5.

Comparison of Pleural Fluid and Serum Levels (mean ± SD) of Adenosine Deaminase (IU/L) in Various Diseases

Group (N1, N2)	Malignancy	Tuberculosis	Pyothorax	Other disease	Control
Sample	(22, 11)	(28, 10)	(9, 6)	(13, 10)
Pleural fluid	35.18 ± 54.57	94.74[b] ± 46.3	87.59[a] ± 54.31	30.04[d,e] ± 20.97
Serum	28.40 ± 19.18	43.36[g] ± 34.17	45.47[g] ± 25.42	43.37[a,h] ± 18.05	22.40 ± 8.40
Radio pleural fluid serum	1.21 ± 1.33	3.68[a] ± 2.73	1.47[c] ± 0.54	0.84[d,e] ± 0.48

Abbreviations are the same as in Table 3

Presumptive sensitivity of ADA Assay in the Pleural Fluids Between Malignancy and Tuberculosis

Using an ADA value of pleural fluids in malignancies of 41.01 IU/L, determined by taking the mean (24.12) + 1 SD (16.89) as the presumptive differential value, 19 of 22 malignant effusions were below this value and 25 of 28 tuberculosis effusions were above it. Thus, the calculated sensitivity was 86.4%, specificity was 89.3% and efficiency was 88.0%. Therefore, we found that these values were very efficient in differentiating between malignant and tuberculous effusions (Table 6).

Table 6.

Presumptive Sensitivity of Adenosine deaminase (ADA) Assay in Pleural fluid Between Malignancy and Tuberculosis

	No. of cases		Total number of cases
	less than 41.01 IU/L[*]	More than 41.01 IU/L
Malignancy	19	3**	22
Tuberculosis	3	25	28
Total	22	28	50

ADA value of pleural fluid in malignant disease, determined by mean (24.12) + 1SD (16.89)

Includes malignant lymphoma (2) and metastatic adenocarcinoma (1)

Sensitivity: 86.4% (19/22)

Specificity: 89.3 (25/28)

(+) Predictive value: 86.4% (19.22)

(−) Predictive value: 89.3% (25/28)

Efficiency 88.0% (44/50)

Correlation of C-RP, Sialic Acid, Adenosine Deaminase Levels and Other Parameters of Pleural Fluid Examination (Correlation Efficient)

The C-RP levels of the pleural fluids showed positive correlation to serum C-RP levels and LDH levels of the pleural fluids. The sialic acid and ADA levels didn’t show a correlation to the pleural fluids and sera, but showed positive correlation to the protein levels of the pleural fluids only. (Table 7)

Table 7.

Correlation of C-RP, Sialic Acid (SA), Adenosine Deaminase (ADA) Levels and Other Parameters of Pleural fluid Examination (Correlation Coefficient)

Sample	Pleural fluid			Cell		Serum
#	Protein	Sugar	LDH	Neut.	Lymph	C-RP	SA	ADA
Test	(N = 57)	(N = 57)	(N = 54)	(N = 57)		(N = 31)	(N = 32)	(N = 32)
C-RP	0.2139	−0.2017	0.4171***	0.0545	−0.0421	0.4631	0.0104	0.1559
SA	0.3359*	−0.0988	0.1369	−0.1588	−0.0147	−0.0713	0.1855	0.2339
ADA	0.4459**	−0.1208	0.1226	0.0917	0.1512	0.0334	−0.1421	0.2327

level of pleural fluid

p<0.05

p<0.01

DISCUSSION

Since discovery of the C-RP by Tillet and Francis,[11)] it has been used as an adjunctive diagnostic method for the various inflammat diseases, tissue trauma and malignant diseases, or as a useful means of clinical course observation.[3)] However, it was usually measured semiquantitatively by the latex agglutination method and studies using it were largely restricted to its sera level analysis, and those using body fluids such as pleural effusion were rare. In this study, we tested pleural effusions and sera of patients with pleural effusion by quantitative measurement of the C-RP levels using a turbidimetric immunoassay which is more rapid and accurate.

C-RP levels of the pleural fluids and sera were higher in exudative pleural diseases than in transudative pleural diseases, and especially higher in diseases causing pyothorax than in malignancies and tuberculous diseases. This result was correlated to that of Cho and his coworkers’ study[13)] in which sera levels of C-RP were higher in non-specific inflammatory diseases than in tuberculous peritonitis.

Sialic acid is present as a component of cellular membrane glycoproteins, glycolipids,[15)] and sera glycoproteins. Several studies[4,5,16,17)] showed that sialic acid was increased in malignant and inflammatory diseases, shock, trauma, myocardial infraction, diabetes mellitus and liver diseases. But these studies were done by measurement of sera sialic acid levels only and pleural fluid study[13)] on sialic acid was rare.

In this study, sialic acid measurement in the pleural fluids and sera was useful for differentiating exudate from transudate, and was helpful in distinguishing tuberculous from malignant effusion, but not in differentiating nonspecific pyothorax. These findings were not correlated to the results of Cho and his co-workers’ study[13)] showing that sialic acid measurement in ascites was helpful in distinguishing exudate from transudate, but not in differentiating similar ascitic fluids of various diseases.

ADA is an enzyme which participates in the catabolism of nucleic acids and nucleoproteins. It is distributed widely in tissues and blood of mammals and in the reticuloendothelial system, spleen and digestive tract tissue (duodenum, stomach, small intestine, cecum) of man.[18)]

Giblett et al[19)] reported a deficiency of ADA in patients with cellular immune deficiency. Meuwissen and Pollara[20)] reported thymus regression, lymphocyte disappearance and T-lymphocyte functional deficiency associated with ADA deficiency, and they suggested that ADA activity was closely related to immune function and differential proliferation of lymphocytes. Kim et al[21)] reported decreased ADA activities in lepromatous leprosy patients with cellular immune disturbances.

ADA activities in sera have been known to be increased in typhoid fever,[22)] infectious mononucleosis,[23)] viral hepatitis, liver cirrhosis and hepatoma.[7,24,25)] On the other hand, Blake and Berman,[26)] Martinez-Vazquez,[27)] Piras et al,[28)] Cha et al[25)] have reported higher ADA activities in tuberculous ascites, pleural effusions and CSF than in other diseases, as in Cho’s report[13)] on ascites and Cho’s report[23)] on CSF.

In this study, ADA activities in pleural fluids were higher in exudates than in transudates, as were C-RP and sialic acid levels. Cha et al[25)] suggested that an increase in ADA activity might not be characteristic of exudates because of non-correlation of ADA activities to protein and LDH levels measured in exudative ascites. But in this study, ADA activities, C-RP and sialic acid levels measured in the pleural fluids were higher in exudates compared to transudates, and sialic acid levels and ADA activities of the pleural fluids were positively correlated to the protein concentrations of the pleural fluids. Thus, ADA activities were thought to be useful in evaluating exudates.

In this study, ADA activities were increased in tuberculosis rather than malignancy, which was helpful in differentiating malignancy from tuberculosis, but not in differentiating tuberculosis from nonspecific pyothorax. This fact causes problems to arise in the explanation that ADA activities are increased by increases in lymphocyte numbers in serum or subpopulations of lymphocytes in the pleural fluid.[13,20,25)]

When we used a value of 41.01 IU/L, the mean of ADA activities + 1 SD, in malignancy as a differential value for the differentiation of tuberculosis from malignancy in pleural fluids, 19 of 22 malignant specimens were below this value and 25 of 28 tuberculous ones were above it, and thus the calculated sensitivity, specificity, and efficiency are all above 85%.

In the correlation of C-RP, sialic acid, ADA levels and other parameters of pleural fluid examinations, C-RP levels of pleural fluids showed positive correlation to serum C-RP and LDH levels of pleural fluids. Alternatively, sialic acid and ADA levels showed only correlation to protein concentrations of the pleural fluids, which suggested some correlation to the characteristics of exudates. The fact that sialic acid levels and ADA activities in the pleural fluids and sera have no correlation with each other suggested that sialic acid and C-RP levels in pleural fluids might not have merely entered from the serum, but they seemed to have some correlation to lymphocytes infiltration caused by pleural disease itself or mesothelial reactions.