ASCITIC & PLEURAL FLUID ANALYSIS

ASCITIC

FLUID ANALYSIS

BACK GROUND
THE WORD ASCITES IS OF GREEK ORIGIN ( ASKOS ) & MEANS A BAG OR SAC. ASCITES DESCRIBE THE CONDITION OF PATHOLOGIC ACCUMULATION WITH IN CAVITY. HEALTHY MEN HAVE LITTLE OR NO INTRA PERITONEAL FLUID. WOMEN MAY HAVE AS MUCH AS 20 ML DEPENDING UPON PHASE OF MENSTRUAL CYCLE.

PATHO PHYSIOLOGY
UNDER FILLING THEORY Inappropriate sequestration of fluid with in vascular bed. OVERFLOW THEORY Abnormal retention of sodium & water in absence of volume. PERIPHERAL ARTERIAL VASODILATION THEORY Combination of both theories.

CAUSES OF ASCITES
A) NORMAL PERITONEUM. 1) PORTAL HYPERTENSION (SAAG > 1.1 g/dl ) .

a) Hepatic congestion b) C.H.F. c) Constrictive pericarditis d) Budd chiari syndrome e) Cirrhosis f) Alcoholic hepatitis g) Fulminant hepatic failure h) Massive hepatic failure

CAUSES OF ASCITES
A)

NORMAL PERITONEUM

2) HYPO ALUBINEMIA (SAAG < 1.1 g /dl )

a) Nephrotic syndrome . b) Protein losing enteropathy . c) Severe malnutrition with anasarca .

CAUSES OF ASCITES

A) NORMAL PERITONEUM 3)MISCELLANEOUS (SAAG < 1.1 g/dl ) a) Chylous ascites. b) Pancreatic ascites . c) Bile ascites . d) Nephrogenic ascites . e) Urine ascites . f) ovarian disease.

CAUSES OF ASCITES
B) 1)

DISEASED PERITONEUM
INFECTIONS ( SAAG <1.1 g/dl )
a) Bacterial infections. b) Tubercular infections. c) Fungal infections . d) H.I.V. infections.

CAUSES OF ASCITES
B) DISEASED PERITONEUM 2) MALIGNANT CONDITIONS

a) Peritoneal carcinomatosis. b) Primary mesothelioma. c) Pseudo myxoma peritonei. d) Hepatic cellular carcinoma.

CAUSES OF ASCITES
B) DISEASED PERITONEUM 3) OTHER RARE CONDITIONS. (SAAG < 1.1 g/dl )

a) Familiar Mediterranean fever. b) Vasculitis. c) Granulomatous peritonei. d) Eosinophilic peritonei.

LAB. STUDIES/SPECIMEN COLLECTION

Peritoneal

fluid should be sent for cell count , total protein , sugar ,albumin level , culture , gram stain & cytology for newly onset ascites origin.

LAB STUDIES

A minimum of 30 ml is necessary for complete evaluation. Samples should be collected in heparin tubes to prevent clot formation. Samples for glucose should be adequately preserved with fluoride. Samples for ph should be collected an aerobically into heparinsed blood gas syringe . Blood stained samples are not suitable for analysis of total protein & LDH. All samples should be centrifuged prior to analysis. Ascitic fluid albumin should be determined along with blood sample for albumin. SAAG > 1.1 g/dl indicates presence of portal HTN. Very low serum albumin ( <12 g/l ) may make gradient less reliable.

LAB STUDIES

ADDITIONAL TESTS ARE REQUIRED IF A SPECIFIC QUESTION IS BEING ASKED.

a) Amylase suspected pancreatitis or gut perforation. b) Triglyceride suspected Chylous ascites. c) Bilirubin suspected biliary perforation . d) Differentiating spont. Bacterial peritonitis from secondary infection - Ascitic fluid glucose < or = 2.8 mmol / l . - LDH greater than upper limit of normal for serum. - total protein > 10 g /l Above findings are suggestive of secondary peritonitis.

PHYSICAL EXAMINATION

QUANTITY APPEARANCE clear , turbid or hemorrhagic. COLOR normal tinged yellow .A minimum of 10000 RBCs/ uL are required for Ascitic fluid to appear PINK & more than 20000 RBC s /uL are required to appear as blood tinged. This may attribute to traumatic tap or malignancy. Bloody fluid from traumatic tap is heterogeneous bloody & fluid will clot. Non traumatic bloody fluid is homogenously red & does not clot because it is already clotted & lysed. COAGULUM SEEN IN TUBERCULOSIS.

GROSS EXAMINATION
TURBID
-APPENDICITIS

AMBER
OBSTRUCTION - CIRRHOSIS -NEPHROTIC SYNDROME -C.C.F
- HEPATIC VEIN

-PANCREATITIS -BOWEL RUPTURE -INFECTED INTESTINE

GROSS EXAMINATION
GREENISH
INTESTINE -CHOLECYSTITIS -PERFORATED GALL BLADDER -APPENDICITIS
-PERFORATION

MILKY
-PARASITIC INFECTION -NEPHROTIC SYNDROME -CARCINOMA -LYMPHOMA

GROSS EXAMINATION
BLOODY

HEMORRHAGIC PANCREATITIS ORGAN RUPTURE

MICROSCOPIC EXAMINATION

A) CELL COUNT Normal Ascitic fluid contains fewer than 250 neutrophils / uL Approx. 90 % of patients with S.B.P will have leukocyte count > 500 / uL over 50 % of which are neutrophils. EOSINOPHILLIA (>10 % ) -chronic inflammatory processes asso with chronic peritoneal dialysis. -congestive heart failure. -Vasculitis -lymphoma. -ruptured hydatid cyst.

NEUBEAURS CHAMBER

DIAGRAM OF CHAMBER

MICROSCOPIC EXAMINATION
SPECIMEN 1) CLEAR SAMPLE DILUTION FACTOR CHAMBER CHARGED WITHOUT ANY DILUTION SAMPLE DILUTED WITH NORMAL SALINE IN 1: 20 RATIO

2) TURBID SAMPLE

CELL COUNT

1) UNDILUTED SPECIMENS WBC/cu mm = COUNTED CELLS/4 X 10.

. 2) DILUTED SPECIMENS WBC/ cu mm = COUNTED CELLS/4 X10 X 20

CHEMICAL EXAMINATION PROTEIN

1) 2) 3)

QUALITATIVE. RIVALTAS QUALITATIVE TEST.

PLACE 200 mL OF WATER IN A CYLINDER. ADD 2 DROPS OF GLACIAL ACETIC ACID AND MIX. ALLOW 2 OR 3 DROPS OF FLUID TO BE EXAMINED TO FALL INTO SOLUTION.

.INTERPRETATION : 1) IF FLUID IS AN EXUDATE , A DISTINCT CLOUD IS OBSERVED IN THE WAKE OF THE FALLING DROP. 2) IN CASE OF TRANSUDATE , NO SUCH TURBIDITY IS SEEN.

CHEMICAL EXAMINATION PROTEIN

NAME OF METHOD BIURET METHOD PRINCIPLE Protein reacts with cupric

ions in alkaline medium to form violet colored complex .The intensity of color produced is directly proportional to protein present in specimen which can be measured on a photometer at 530 nm.

REAGENTS

BIURET REAGENT PROTEIN STANDARD 6G/DL BOVINE ALBUMIN SAMPLE BLANK REAGENT

PROCEDURE
TEST
PROTEIN REAGENT SAMPLE

STANDARD

BLANK

5.0 ML 0.05 ML

5.0 ML -

5.0 ML -

PROTEIN STANDARD
DISTILLED WATER

0.05 ML
-

0.05 ML

PROCEDURE

MIX THOROUGHLY & KEEP AT ROOM TEMPERATURE FOR EXACTLY 10 MINUTES. CALCULATION
ASCITIC FLUID PROTEIN = O.D TEST /O.D STANDARD X 6

S.A.A.G

SERUM ALBUMIN ASCITIES GRADIENT SAAG is best single test for classifying ascites into portal HTN (SAAG >1.1G/DL) & non portal HTN (SAAG <1.1 G/DL ).

SAAG = SERUM ALBUMIN ASCITIC FLUID ALBUMIN

MEASUREMENT OF SAAG

NAME BROMOCRESOL GREEN METHOD PRINCIPLE Albumin present in serum binds

with bromocresol green at ph 4.1 to form green colored complex , intensity of which can be measured colorimetric by using 640 nm ( or a red filter ).

Normal serum albumin = 3.3 4.8 g/dl

REAGENTS

BROMOCRESOL REAGENT ALBUMIN STANDARD (4.0 G/DL ) SAMPLE BLANK REAGENT

PROCEDURE
TEST
BROMO CRESOL 5.0 ML

STANDARD
5.0 ML

BLANK
5.0 ML

SERUM/ FLUID
ALBUMIN STANDARD DISTILLED WATER

0.05 ML

0.05 ML -

0.05 ML

PROCEDURE

MEASURE INTENSITY OF TEST / STANDARD BY SETTING BLANK AT 100 % T USING 640 NM. CALCULATION
SERUM/ASCITIC FLUID ALBUMIN = O.D TEST/O.D STD X 4

CHEMICAL EXAMINATION GLUCOSE

ORTHO TOLUDINE METHOD

PRINCIPLE Glucose reacts with ortho toludine in

hot acidic medium to form green colored complex , intensity of which is measured by photometer at 620 660 nm

CHEMICAL EXAMINATIONGLUCOSE

GLUCOSE OXIDASE METHOD GLUCOSE + h2o +o2 = gluconic acid =h2o2

Aldehyde group of glucose is oxidized to form GLUCONIC ACID UNDER PEROXIDASE h2o2 = h2o + o2. o2 reacts with h aminophenazone in presence of phenol to form pink colored complex. Intensity read at 530 nm

CINICAL SIGNIFICANCE GLUCOSE

IN UNCOMPLICATED ASCITES , USUALLY SIMILAR TO SERUM. DECREASED IN MOST CASES OF T.B PERITONITIS & ABDOMINAL CARCINOMATOSIS IN LATER S.B.P (but not in early ) ASCITIC FLUID GLUCOSE LEVEL DROPS TO AS LOW AS ZERO mg / dl DUE TO BACTERIAL CONSUMPTION.

Ascitic & pleural fluid analysis

PLEURAL FLUID ANALYSIS

PLEURAL FLUID
The pleural cavity is potential space lined by mesothelium of visceral & parietal pleura. The normal pleural cavity contains approx 1 ml of fluid. This represents the balance between the hydrostatic/ oncotic forces in parietal pleural vessels & external lymphatic drainage. Pleural effusion results from disruption of this balance.

CAUSES OF PLEURAL EFFUSION

TRANSUDATES

EXUDATES
A)INFECTION -BACTERIAL PNEUMONIA -TUBERCULOSIS -HISTOPLASMOSIS -VIRAL PNEUMONIA -MYCOPLASMA PNEUMONIA B)NEOPLASMS C)NON INFECTIOUS INFLAMMATORY DISEASES

C.H.F HEPATIC CIRRHOSIS ATELECTASIS HYPOALBUMINEMIA NEPHROTIC SYNDROME PERITONEAL DIALYSIS MYXEDEMA CONSTRICTIVE PERICARDITIS

CAUSES OF PLEURAL EFFUSION

FLUID FROM EXTRA PERITONEAL SOURCES

A) Pancreatitis (increased amylase ) B) Ruptured esophagus (increased amylase + low ph ). C) Urinothorax (increased creatinine +low ph ).

PLEURAL FLUID EXUDATE

LIGHTS CRITERIA
PLEURAL PROTEIN/SERUM PROTEIN > OR = 0.5 PLEURAL LDH /SERUM LDH > OR = 0.6 PLEURAL FLUID LDH = > OR = 2/3 rd UPPER LIMIT OF NORMAL SERUM LDH.

OTHER CRITERIA
PLEURAL FLUID CHOLESTEROL = > 45 mg / Dl. PLEURAL CHOLESTEROL /SERUM CHOLESTEROL > OR = 0.3. SERUM PLEURAL FLUID ALBUMIN GRADIENT < OR = 1.2 g /dl. PLEURAL BILIRUBIN / SERUM BILIRUBIN > OR = 0.6

PLEURAL FLUID ANALYSIS

SPECIMEN COLLECTED IN THREE ASEPTIC TUBES

1)ONE WITH SODIUM FLOURIDE FOR SUGAR/ PROTEIN ESTIMATION.

2) PLAIN TUBE FOR OBSERVATION OF CLOT & BACTERIOLOGY.

3)EDTA TUBE FOR MICROSCOPIC EXAMINATION.

TRANSUDATE /EXUDATE
TRANSUDATE
1
2 3 4 5 6 7

EXUDATE
Inflammatory process
Clear or cloudy , serous , purulent , hemorrhagic, chylous or flocculent. Specific gravity > 1.018 Clots spontaneous. Protein > 3.5 g / dL Many neutrophils in acute & small lymphocytes in chronic inflammation. Bacteria usually present.

Non inflammatory process

Clear, serous ,light yellow Specific gravity < 1.018 Clot absent Protein < 2.5 g / dL Few lymphocytes & endothelial cells Bacteria usually absent

PHYSICAL EXAMINATION

NORMAL CLOUDY BLOODY FLUID

CHYLOUS

PSEUDOCHYLOUS

PALE/STRAW COLORED BACTERIAL/ VIRAL INFECTION TRAUMA MALIGNANCY TUBERCULOSIS PULMONARY INFARCT PANCREATITIS LYMPHOMA CARCINOMA LUNG TRAUMA TUBERCULOSIS RHEUMATIC ARTHRITIS MYXEDEMA

CHYLOUS/PSEUDOCHYLOUS
CHYLOUS 1)ONSET SUDDEN PSEUDOCHYLOUS GRADUAL

2)APPEARA MILKY WHITE / YELLOW or MILKY / GREENISH/ NCE BLOODY METALLIC SHINE 3)MICROSC LYMOHOCYTES OPIC 4)TRIGLYC ERIDES > OR = 110 MG / DL MIXED CELLULAR REACTION /CHOLESTEROL CRYSTALS < 50 MG / DL

5)LIPO PROTEINS

CHYLOMICRONS PRESENT

CHYLOMICRONS ABSENT

CLOT TEST
THE PLAIN TUBE IS OBSERVED FOR CLOT. NORMAL = DOES NOT CLOT.

INFLAMMATION/INCREASED FIBRINOGEN = CLOTS.

SPECIFIC GRAVITY

BY HAND REFRACTOMETER OR WEIGHT METHOD.

EXUDATE TRANSUDATE

> 1.018. < 1.018

MICROSCOPIC EXAMINATION
SPECIMEN 1) CLEAR SAMPLE DILUTION FACTOR CHAMBER CHARGED WITHOUT ANY DILUTION SAMPLE DILUTED WITH NORMAL SALINE IN 1: 20 RATIO

2) TURBID SAMPLE

CELL COUNT

1) UNDILUTED SPECIMENS WBC/cu mm = COUNTED CELLS/4 X 10.

. 2) DILUTED SPECIMENS WBC/ cu mm = COUNTED CELLS/4 X10 X 20

CELLULAR DIFFERENTIAL OF PLEURAL EFFUSION

NEUTROPHILLIA
1)BACTERIAL PNEUMONIA 2)PULMONARY INFARCTION 3)PANCREATITIS 4)SUBPHRENIC ABSCESS 5)EARLY T.B. 6)TRANSUDATE.

CELLULAR DIFFERENTIAL OF PLEURAL EFFUSION

LYMPHOCYTOSIS ( > 50 % )
1) T.B (MESOTHELIAL CELLS ARE RARE ). 2)VIRAL INFECTION. 3)MLIGNANCY. 4)TRUE CHYLOTHORAX. 5)RHEUMATOID PLEURITIS. 6)S.L.E. 7)UREMIC EFFUSION. 8)TRANSUDATES (APPROX. 50 % )

CELLULAR DIFFERENTIAL OF PLEURAL EFFUSION

EOSINOPHILLIA.( > 10 % )
1)PNEUMOTHORAX. 2)TRAUMA. 3)PULMONARY INFARCTION. 4)C.H.F. 5)INFECTION ( PARASITIC , FUNGI ). 6)HYPERSENSITIVITY SYNDROME. 7)DRUG REACTION. 8)RHEUMATOLOGIC DISEASES. 9)HODGKINS DISEASE. 10)IDIOPATHIC

CHEMICAL EXAMINATION
PROTEIN RIVALTAS QUALITATIVE METHOD. BIURET METHOD

PLEURAL PROTEIN = O.D. TEST/O.D.STD X 4. PROTEIN >3 G/DL = EXUDATE. PROTEIN <3 G/DL = TRANSUDATE.

CHEMICAL EXAMINATION

GLUCOSE.
Normally equal to serum level.

Determined by orthotoludine method.

CHEMICAL EXAMINATION

DECREASED PLEURAL GLUCOSE : 1)RHEUMATOD PLEURITIS. 2)PURULENT PARAPNEUMONIC EXUDATE. 3)MALIGNANCY. 4)T.B. 5)NON PURULENT BACTERIAL INFECTION. 6)LUPUS PLEURITIS. 7)ESOPHAGEAL RUPTURE.

CHEMICAL EXAMINATION

LACTATE : -INCREASED IN

1)BACTERIAL PLEURAL INFLAMMATION. 2)T.B. PLEURAL INFECTION.

3)MALIGNANT EFFUSION.

CHEMICAL EXAMINATION

AMYLASE : - elevation above serum

levels usually 1.5 2.0 times indicate : 1) PANCREATITIS. 2) ESOPHAGEAL RUPTURE. 3) MALIGNANT EFFUSION. NOTE: Elevated amylase from last two causes is of SALIVARY ISOFORM.

MICROBIOLOGIC EXAMINATION

DONE BY PREPARING TWO SMEARS DRIED IN AIR , HEAD FIXED & STAINED WITH GRAM STAIN & Z.N. STAIN. CONFIRMED REPORT ONLY AFTER CULTURE. BACTERIA MOST COMMONLY ASSO WITH PARAPNEUMONIC EFFUSION .

1) S.AUREUS 2) S.PNEUMONIA 3) BETA HEMOLYTIC GROUP A STREPTOCOCCI. 4) GAMMA STREPTOCOCCI 5) SOME GRAM NEGATIVE BACILLI.

REFERENCES
HENRYS CLINICAL DIAGNOSIS & MANAGEMENT BY LAB. METHODS. GODKARS T.B. OF MEDICAL LAB. TECHNOLOGY. MILLERS CLINICAL PATHOLOGY. VARIOUS WEB SITES.

THANKS
BY : DR RAVI JAIN