Serum cholinesterase and pleural cholinesterase levels and its usage to differentiate pleural transudates and exudates.
A primary diagnostic step is the identification of pleural effusion as either transudate or an exudate. Transudates are secondary to disease elevating hydrostatic pressures in the systemic or pulmonary circulation, or disease decreasing plasma colloid oncotic pressure. (3) Exudates are due to disease which alters the permeability of the pleural surface or due to impaired lymphatic drainage.
Levels of pleural fluid protein and lactic acid dehydrogenase (LDH) are commonly analyzed to classify an effusion as an exudate or transudate. But the results have not always been satisfying. Lights criteria for pleural effusion has been widely used to differentiate between transudates and exudates.
However several reports 2-7 have shown that the Light et al criteria misclassify a large number of effusion, even up to 20 to 30% of transudates as exudates. (4) Therefore several alternative parameters have been proposed e.g., pleural fluid cholesterol level and pleural fluid/serum cholesterol ration pleural fluid/serum bilirubin ratio, serum-pleural effusion albumin gradient, alkaline phosphatase vale and many others. But their superiority with respect to the criteria of Light et al remains doubtful (5) on 1978. Eduardo et al2 and recently, in 2000, Janmeja et al (6) have estimate pleural fluid cholinesterase and pleural fluid/serum cholinesterase ratio and found these to be more efficient for separating pleural transudates from exudates.
* To evaluate the usefulness of estimating the cholinesterase level in pleural fluid and pleural fluid to serum cholinesterase ratio for differentiating pleural transudates from exudates.
* To compare the diagnostic efficacy of these parameters with that of lights criteria.
METHODOLOGY: The study was conducted in Department of Medicine, Basaveshwara Teaching and general hospital, attached to Mahadevappa Rampure Medical College, Gulbarga. 50 patients with pleural effusion were selected for study between December 2006 to April 2008.
* Tubercular effusion was diagnosed by X-ray, pleural fluid and sputum AFB.
* Malignant effusion--malignant cells in the pleural fluid with or without histological evidence.
* CHF effusion as diagnosed by cardiomegaly on roentgenogram and echocardiography, presence of pulmonary congestion and absence of other lesions in the chest X-ray.
* Nephrotic syndrome as diagnosed by establishing protenuria of > 3 gm/24 hours, oedema, hypoalbuminemia and hypercholesterolemia.
* Pancreatitis as diagnosed by history, serum amylase > 1000 u/ml and ultrasound abdomen.
* Pleural effusion due to other well determined cause.
* Effusions of undetermined origin.
* Effusions with more than are possible cause.
* Persons with history of exposure to organophosphorus compounds.
All the patients selected for the study were evaluated in detail, comprising of detailed history, clinical examination and relevant investigations.
Patients with clinical evidence of pleural effusion were first send for chest X -ray PA and lateral view and ultrasound thorax if required.
Following investigations were done on all the patients in the study Group:
1. Partial haemogram including ESR.
2. Urine albumin, sugar.
4. Blood urea/creatinine.
5. Liver function tests.
6. Serum lactic dehydrogenase.
7. Serum cholinesterase.
Then the diagnostic thoracocentesis was performed taking great care not to let the fluid mix with blood.
Pleural fluid was immediately sent for following Investigations:
1. Pleural fluid cytology including malignant cells.
2. Proteins, sugar.
3. Lactic dehydrogenase.
Effusions were individually classified as transudates or exudates after careful evaluation of all clinical data and investigation results. The criteria are analyzed for separation of transudative and exudative pleural effusions are as follows:
1. The criteria of Light et al, is in based on three parameters:
a. Pleural fluid to serum proteins >0.5
b. Pleural fluid to serum LDH > 0.6
c. Pleural fluid LDH > 200 IU
Exudative pleural effusion meets at least one of the following criteria, whereas transudative pleural effusion meets none.
2. Pleural fluid cholinesterase values > 2000 U/L are taken as exudates and those < 200 U/L are taken as transudate.
3. Pleural fluid to serum cholinesterase ratio: Ratio > 0.5 is taken as exudates and those < 0.5 are taken as transudate.
ESTIMATION OF CHOLINESTERASE: PRINCIPLE: REACTIONS INVOLVED IN THE CHOLINESTERASE ASSAY ARE AS FOLLOWS: Cholinesterase hydrolyses Butyryl thiocholine to from thiocholine which reacts with 5.5- dithiobis-2-nitrobenzoic acid (DTNB) to yield coloured 5MNBA which is measured spectrophotometric ally at 410nm. Therefore, the rate of change in absorbance at 410nm is directly proportional to cholinesterase activity.
REAGENT COMPOSITION: CHOLINESTERASE R1.
BUFFER SOLUTION: Phosphate buffer. (pH 7.7) 50mmol/L, CHOLINESTERASE R2
5, 5 DTNB 0.25mmol/L.
Butyrylthocholine 7mmol/ L.
NORMAL RANGE: It is recommended that each laboratory establish its own reference vale.
SERUM: 4659--14443 U/L.
REAGEMT PREPATATION: Dissolve one tablet R2 in one vial of Reagent R1. Wait for 10 Minutes for complete dissolution.
REAGENT STORAGE AND STABILITY: Dry regent is stored at 2-8[degrees] C, and is stable till the expiry date or reagent. Reconstituted regent is stable for 2 hours at 2-8[degrees] C
* 20[micro] of sample (10[micro]l of serum/pleural fluid + 10^l of saline solution) is added to 3000^l of the working reagent. Test is performed on semi-automated biochemistry analyzer.
* This cholinesterase assay is a kinetic test with a delay time of 60 sec, followed by two readings of absorbance again at 60 sec interval each. Average difference in.
Absorbent/min (AOD/ min) is obtained and multiplied by the factor 22710 to obtain the results. This whole process was programmed in to the analyzer and result was automatically obtained. Test was performed at 37[degrees] C.
STATISTICAL ANALYSIS: Different formulas used for result analysis are:
1. Student unpaired 't' test was used to compare various parameters used in the study. This test in a parametric test described by W.S. Gossett whose pen name was 'Student'. It is used to comparison of small number of samples is less than 30.
Sensitivity = TP TP+FN/TP Sensitivity = TP+FP/TP Positive predictive value = TP+FP/TP Negative predictive value = TP+FP/TP+TN Efficacy = TP+FN+TN+FP TP-True positive, TN-True Negative, FP-False Positive, FN-False Negative
Cut off values of pleural fluid proteins, PF/S protein ratio, pleural fluid LDH, PF/S LDH ratio are fixed according to the Light's Criteria. Pleural fluid cholinesterase value of 2000 IU and PF/S cholinesterase ratio 0.50 are based on mean and standard deviation (Mean-2SD) with clinical features and positivity of the test.
The misclassification of transudates and exudates were less pleural fluid to serum cholinesterase ration, compared to all other parameters and Light's criteria. But even pleural fluid to serum cholinesterase ratio misclassified one each case of transudate and exudate.
Light's criteria misclassified 3 transudates, while PF/S cholinesterase 1 transudates.
Light's criteria misclassified 2 exudates PF/S cholinesterase ratio misclassified only 1 exudate and pleural fluid cholinesterase misclassified none.
DISCUSSION: Pleural effusion occurs in a large variety of conditions, but correct diagnosis of underlying diseases is essential for rationale management of pleural effusion. Early evidence of the transudative or exudative nature of a pleural effusion may be of considerable clinical value and is often used as a basis for further diagnostic procedures and therapeutic considerations. Many tests we used to differentiate transudate from exudates by specific gravity, cell count and presence or absence of clotting of fluids. But no single chemical tests or series of tests has yet proved to be completely reliable.
In their study in 1972, Light et al used pleural and serum levels of proteins and LDH to establish criteria for segregation transudates from exudates with a sensitivity and specificity of 99% and 98% respectively. Recently however, several prospective studies 2-7 were unable to reproduce the excellent results obtained by light et al.
In most of those studies sensitivity for exudates remained high, about 95% but specificity did not surpass 78%. Therefore several alternative parameters have been proposed e.g. plural fluid/ serum cholesterol ratio 2. pleural fluid/ serum bilirubin ratio 3, serum pleural effusion albumin gradient4, alkaline phosphatese5 and many others.
In 1978, Carbrer et al analyzed the cholinesterase activity in pleural effusions of diverse causes and found a significant difference in the average level of cholinesterase. Between transudates and exudates. This fact later became the basis for using cholinesterase levels for differentiating transudative from exudative plural effusions.
In the present study, by using pleural to serum cholinesterase ratio, we could accurately classily 96% of pleural effusion cases. Out of the 50 cases evaluated, 1 each case of transudate and exudates were misclassified. By using pleural fluid choliesterease. Level 2 cases of transudates were misclassified. By using Light's criteria 10 % of cases were misclassified.
Eduardo et al in 1996, conducted study on 153 patients with pleural effusion to differentiate transudate and exudates. They compared Lights criteria with pleural fluid cholesterol, pleural fluid to serum cholesterol, pleural fluid cholinesterase and pleural fluid to serum cholinesterease ratio.
RESULTS WERE AS FOLLOWING:
* Misclassification rates by Lights criteria was 7.8 %
* Misclassification rates by pleural fluid cholesterol level with cut off value of mg/dl was 7.8 %
* Misclassification rates by Pleural fluid to serum cholesterol with cut off value 0.30 was 6.5 %
* Misclassification rates by pleural fluid cholinesterease with cut off values of 1140 for Group A and Group B was 8.5%.
Misclassification rates by pleural fluid to serum cholinesterase ratio with cut of value 0.23% was only 1.38%. These criteria correctly classified 98.77% of 153 pleural effusion patients.
In the present study, the misclassifications using PF to serum cholinesterase ratio is higher than the study by Eduardo et al. But both the studies shows that compared to Light et al criteria, pleural fluid to serum cholinesterase ratio is a better parameter to separate transudate from exudates. Jasnmeja et al in 2000 studied 50 cases of pleural effusion with different etiology using PF/S cholinesterase as criteria.
THEY MADE TWO GROUPS IN STUDY POPULATION:
Group A: Compromise 10 case each of PTB, malignancy and para pneumonic plural effusion total of 30 cases.
Group B: Compromised 10 pleural effusions due to CHF and 10 cases due to nephritic syndrome total of 20 cases.
By using 0.5 as the cutoff value for PF/S cholinesterase ratio they were able to classify 98% of effusion accurately, by taking 1600 IU of pleural fluid cholinesterase as cutoff value they misclassified 3 cases of effusion, and by using Light's criteria 5 cases were misclassified.
In the present study the misclassifications using PF to serum cholinesterase is higher than the study of Janmeja et al. Misclassifications using Light's criteria is also high with the present study. But both studies show that PF to serum cholinesterase is a better parameter to separate transudates from exudates.
The present study and the study by Eduardo et al shows that number of misclassification is higher using only pleural fluid cholinesterase, compared to pleural fluid to serum cholinesterase ratio. While present study, using PF cholinesterase misclassified 6% of pleural effusions, Eduardo et al and Janmeja et al misclassified 8.5% and 6% cases each respectively. This could be attributed to the fact that, since the cholinesterase is synthesized in the liver, the levels can be influenced by different disorders like hepatitis, cirrhosis, acute infections, pulmonary embolism chronic renal failure and after surgical procedures.
In the present study the cut of value of pleural fluid cholinesterase and pleural fluid to serum cholinesterase ratio is 2000IU and 0.5 respectively. Pleural fluid cholinesterase >2000 and Pleural fluid to cholinesterase ration> 0.5 is taken as exudates and the value <2000 a ratio <0.5 is taken as transudates. This cut of value is based on Mean and standard deviation (Mean-2 SD) with clinical features and positively of the test. For transudate the value is 1111 [+ or -] 362 and for exudates the value is 5433[+ or -] 1587. The difference between transudates and exudates are statistically highly significant (p<0.001).
The present study and various other studies, brings to the light the fact that Light's et al criteria. Misclassify transudate more than the exudates. In the present study using Light's criteria the misclassification ratio were 6% for transudates 4% for exudates.
In the present study the sensitivity and specificity using pleural fluid to serum cholinesterase ratio is 97 % and 95% respectively while the corresponding values using Light's Criteria is 93% and 85% respectively. This shows that sensitivity and specificity of pleural fluid to serum cholinesterase, to separate transudate from exudates is higher than that of Light's criteria. Also the efficiency of pleural fluid to serum cholinesterase ratio to separate transudate is 96% while that of Light's criteria is only 90%.
The most analyzed parameters in the recent past to separate transudative from exudative effusions have been pleural fluid cholesterol level and pleural fluid to serum cholesterol ratio. In the study of Valdes et al the misclassification rates were 6.8 by pleural fluid cholesterol, while pleural fluid to serum cholesterol ratio misclassified 6.6% cases. Meisel et al in his study using pleural fluid to serum bilirubin ratio as parameters to separate transudate and exudates misclassified 19% Pleural effusion. Roth et al analyzed the serum-albumin gradient in their study of 59 patients correctly classified all the transudates and 3% of the 41 exudates but other authors could not reproduce similar satisfactory results. Tahaoglou et al found the level of alkaline phosphate lower in transudates compared to exudates but misclassification rate was 6.5%.
The results of our study compared with the parameters used by others suggest that the pleural fluid to serum cholinesterase ratio criterion is the accurate parameter for differentiation transudative and exudative pleural effusions.
* Misclassification is present with all parameters used.
* Number of misclassifications using PF/S cholinesterase ratio and pleural fluid cholinesterase level are less compared to other parameter used.
* Sensitivity and specificity of PF/S cholinesterase ratio and pleural fluid cholinesterase level as diagnostic parameter to differentiate transudates and exudates are higher than all other parameters used in the study.
* The numbers of misclassification are more with the Light's criteria than PF/S cholinesterase ratio.
* Hence, the pleural fluid to serum cholinesterase ratio and pleural fluid cholinesterase level are the reliable method in separating pleural transudates from exudates. If further studies with larger study group confirm our results, cholinesterase ratio and pleural fluid cholinesterase level could be used as the first step in diagnostic study of pleural effusion.
* Fifty patients with pleural effusion were studied over a period of 17 months from December 2006 to April 2008 in Basaveshwara Teaching and General Hospital, Gulbarga.
* Out of 50 cases, 33 were males and 17 females.
* Study group comprised of mainly two types of pleural effusion.
Group A: transudative pleural effusion -20 cases.
Group B: Exudative pleural effusion--30 cases.
* Pleural fluid cholinesterase value of >2000 IU and PF/S cholinesterase ratio of > 0.50 was taken as exudates and corresponding values of <2000 IU and <0.50 was taking as transudate.
* Misclassifications are present with all parameters used.
* The number of misclassifications are more with the Light's criteria than the pleural fluid cholinesterase and PF/S cholinesterase ration, and pleural fluid cholinesterase level as diagnostic parameter to separate transudative from exudative pleural effusion are higher than the Light's criteria.
LIST OF ABBREVIATIONS USED: PF Pleural Fluid PF/S Pleural Fluid to Serum SChe Serum Cholinesterase PChe Pleural Fluid Cholinesterase Che Cholinesterase LDH Lactate Dehydrogenase TB Tuberculosis AFB Acid Fast Bacilli ADA Adenosine Deaminase CT Computed Tomography
(1.) Bartter T, Santarelli R, Askers SM et al., (1994). The evaluation of pleural effusion. Chest 106 1209. Cabrer B, Bofill D and Grau A (1978). Valore de la colinesrasa en liquido plerual para su diagnostico etiolgico. Revista Clinica Espanola 150 183-4.
(2.) Eduardo GP, Isabel PN, Jose FS, Benedicto J and Juan C (1996). Pleural fluid to serum cholinesterase ratio for separation of transudates and exudates. Chest 110 97.
(3.) Garquez I, Porcel JM, Vives M, Rubio M and Rivas MC (1998). Comparative analysis of Light's criteria and other biochemical parameters for distinguishing transudates and exudates. Respiratory Medicine 92(5) 762.
(4.) Gupta KB, Tandon S, Singh GP, Dhania OP and Janmeja AK (1999). Pleural fluid cholesterol and serum cholesterol ratio as a parameter to differentiate between pleural transudates and exudate. Indian Journal of Tuberculosis 46 255.
(5.) Hamm H, Brohan VB, Bohmer R et al., (1987). Cholesterol in pleural effusion: a diagnostic aid. Chest 92 296.
(6.) Janmeja AK, Goyal KM and Manju Sharma (2000). Pleural fluid/serum cholinesterase ratio for differentiation between transudative and exudative pleural effusions. The Indian Journal of Tuberculosis 47 83-86
Suresh S. Harsoor , Shoukath A. R 
[1.] Suresh S. Harsoor
[2.] Shoukath A. R.
PARTICULARS OF CONTRIBUTORS:
[1.] Professor, Department of Medicine, MRMC, GLB, Rajiv Gandhi University of Health Science, Bangalore.
[2.] Associate Professor, Department of Medicine, MRMC, GLB, Rajiv Gandhi University of Health Science, Bangalore.
NAME ADDRESS EMAIL ID OF THE CORRESPONDING AUTHOR:
Dr. Shoukath A. R, # 6-41/1A, Mijgori Road, Mominpura, Gulbarga.
Date of Submission: 28/10/2014.
Date of Peer Review: 05/11/2014.
Date of Acceptance: 21/11/2014.
Date of Publishing: 26/11/2014.
Table No.--1 Showing Sex Distribution Yes No. of Cases Percentage Males 33 66 Females 17 34 Total 50 cases were included in this study, 33 were males and 17 females. Table No. 2 Table showing Age Distribution Age Male Female 11-20 04 03 21-30 05 03 31-40 01 02 41-50 04 00 51-60 10 04 61-70 06 05 > 70 03 00 Table 3: Showing Types of Effusion and their Percentage Type No. of Cases Percentage TRANSUDATES 20 40 a) CCF 18 36 b) Nephrotic syndrome 01 02 c) Cirrhosis 01 02 EXUDATE 30 60 a) Malignant 04 08 b) Non Malignant 26 52 * Tubercular 17 34 * Pneumonia 07 14 * CRF 01 02 * Post CABG 01 02 Table 4: Showing Cutoff Values of Different Parameters of Differentiate between Transudate and Exudate Investigation Transudate Exudate Pleural <3gm >3gm PF/S * Protein ratio <0.5 >0.5 Pleural LDH <200 IU >200 IU PF/S * LDH ratio <0.6 >0.6 Pleural fluid cholinesterase <2000 IU >2000 IU Table 5: Showing Comparison of Different Parameters between Transudates and Exudates Investigation Transudate (n=20) Exudate (n=30) Protein Pleural 2.27 [+ or -] 0.77 4.38 [+ or -] 0.98 fluid PF/S * 0.26 [+ or -] 0.17 0.66 [+ or -] 0.12 ratio LDH Pleural 346.2 [+ or -] 234.8 1247.43 [+ or -] 994 fluid PF/S 0.72 [+ or -] 0.68 1.92 [+ or -] 1.42 ratio Cholinesterase Pleural 1111 [+ or -] 362 5433 [+ or -] 1587 fluid PF/S 0.18 [+ or -] 0.11 0.64 [+ or -] 0.06 ratio Investigation t-value * p-value ** Protein Pleural 8.51 <0.001 fluid PF/S * 9.30 <0.001 ratio LDH Pleural 4.78 <0.001 fluid PF/S 4.10 <0.001 ratio Cholinesterase Pleural 14.36 <0.001 fluid PF/S 16.15 <0.001 ratio * t value--Student t-test. PF/S- Pleural fluid to serum ratio. ** P-value <0.5 significant <0.001 Highly significant Table 6: Showing Number and Percentage of Misclassification Transudates and Exudates by using different Parameters Transudates Exudate No. of No. of Cases Cases Investigations (n=30) Percentage (n=30) Percentage Light's 03 06 02 04 criteria Pleural fluid 02 04 02 00 Cholinesterase PF/S 01 02 01 02 cholinesterase ratio Table 7: Number and Percentage of Misclassified Transudates CCF Nephrotic Cirrhosis Criteria's (n=18) Syndrome (n=1) (m=01) Light's Criteria 03 00 00 Pleural fluid 02 00 00 Cholinesterase PF/S 01 00 0 cholinesterase ratio Total Criteria's (n=20) Percentage Light's Criteria 03 06 Pleural fluid 02 04 Cholinesterase PF/S 01 02 cholinesterase ratio Table 8: Number and Percentage of Misclassified Exudates Criteria's PTB Pneu. CRF Post CABG Light's 2 0 0 0 Criteria Pleural fluid 0 0 0 0 Cholinesterase PF/S 0 1 0 0 cholinesterase ratio Criteria's Malig. Others Total Percentage Light's 0 0 2 4 Criteria Pleural fluid 0 0 0 0 Cholinesterase PF/S 0 0 1 2 cholinesterase ratio Table 9: Showing Diagnostic Validity of various Parameters Investigation Sensitivity Specificity PPV % NPV % Efficiency Light's 93 85 90 89 90 Criteria PF 100 90 94 100 97 Cholinesterase PF/S 97 95 97 95 96 Cholinesterase Table 10: Showing Comparison of Misclassification of Pleural Effusion by Eduardo et al study and Present Study using Various Parameters Study of Eduardo et al Present Study Criteria Used % of Misclassification % of Misclassification Light's criteria 7.8 10 PF Cholinesterase 8.5 4 PF/S Cholinesterase 1.38 4 Table 11: Showing Comparison of Misclassification of Pleural Effusion by Janmeja et al and Present Study using Various Parameters Study of Eduardo et al Present Study Criteria Used % of Misclassification % of Misclassification Light's 10 10 criteria PF 06 04 Cholinesterase PF/S 02 04 Cholinesterase Table 12: Showing Difference Between Misclassification to Transudates and Exudates by Various Studies using Light's Criteria Study Transudates % Exudates Eduardo et al 25 2.5 Janmeja et al 15 6.6 Bernard J. Roth et al 8 0 Present study 6 4
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Harsoor, Suresh S.; Shoukath, A.R.|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Nov 27, 2014|
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