Dr Piyush Tailor

PURPOSE OF EXAMINATION:

For the diagnosis of certain diseases like

1. Infection- bacterial pneumonia, Tuberculosis, meningitis, encephalitis.
2. Inflammatory diseases- Rheumatoid arthritis, SLE, multiple sclerosis, Guillain Barre syndrome.
3. Malignancy( primary or metastatic in lung, mesothelioma, lymphoma, CNS neoplasm)
4. To differentiate between transudates and exudates for diseases like Congestive cardiac failure, cirrhosis of liver, hypoproteinemia.
5. CSF examination to diagnose subarachnoid haemorrhage.

PRINCIPLE OF EXAMINATION:

Physical Examination-various physical characteristics of fluid like Volume, color, Appearance are examined.

Microscopic Examination-

The microscopic elements present in fluid are examined as wet preparation and then after dilution with WBC diluting fluid, cell count is done with the help of neubauer chamber. Differential leukocyte count is done after giemsa stain of smear prepared from sediment after centrifugation at 2500 RPM for 5 min.

Performance specification:Not applicable

Sample type required:freshly collected

Pericardial Fluid

Peritoneal Fluid,

Pleural Fluid

Cerebrospinal fluid

Preservative needed:not required

Requirements:

1. Physical Examination- not applicable
2. Microscopic Examination-

Centrifuge machine

Glass slides and coverslips

Test tubes

Neubauer chamber

Micropipette

WBC diluting fluid

giemsa stain

Calibration method: not applicable

Detailed work bench instruction:

Physical Examination- see for the following physical characteristics

Volume

Color

Appearance

Chemical Examination

Chemical examination is carried out in biochemistry dept.

Microscopic Examination

1. Wet preparation of properly mixed sample. Mix the fluid well. Prepare direct examination of fluid by putting one drop over a slide and lay a cover slip over it.
2. Cell count- it should be made soon after collection of the fluid. Leukocyte cell count is done with the help of Neubauer's chamber and micropipette. Since leukocytes are present in much smaller number than R.B.C the dilution required is much less.

Method

1. Take 100 micro liter of fluid into one test tube and mix it with 100 micro liter of WBC diluting fluid*. Mix it and keep for 10-15 minutes.
2. Mixed well and Neubauers chamber is charged as for WBC count.
3. The rulings are bought into focus by using the low power objective. The cells in four larger corner squares of the Neubauer chamber are counted.
4. Perform microscopic examination as follows

*WBC DILUTING FLUID:

The diluting fluid used should stain the leukocytes distinctly and lyses the Red Cells. Most commonly used is

10% Glacial acetic acid…….…2.0 ml

1 %(w/v)gentian violet……….. 1.0ml

Distilled Water…… 97 ml

CALCULATION:

Total leukocyte count: N x D N: Average no. of leukocyte

(V) Counted in one square

D: Dilution

: N x 2 V: Volume of 1 large square

(1 x 1x 0.1)

: N x 20/cu. mm

Differential count of cells: Sample is centrifuged at 2500 rpm for 5 minutes. Examine color of supernatant and smear is prepared from the centrifuged deposit, dried and stained by Giemsa stain.Differential leukocyte count is done by counting 500 cells manually on giemsa stain sediment smear. The smear is cross checked by senior doctor simultaneously.

QUALITY CONTROL

Internal quality control:

Monthly one body fluid sample is assessed by one resident (R2/R3) and two faculties.

Results are entered in record register CP: C/Records/File/9/Internal Quality Control records-E.

External quality control:

Yearly four body fluid samples sent to one NABL accredited lab, results are compared and records are kept in record file CP: C/Records/file/2/Results of EQA and interlaboratory comparison.

Interference:contaminated containers or centrifuge tubes if used it may interfere with the results

CONCLUSION OF RESULTS AND UNCERTAINTY:

Total leukocyte count: >1000 cells /cu mm (suggest exudates)

<1000 cells /cu mm (suggest transudate)

BIOLOGICAL REFERENCE INTERVAL:

Physical Examination-

Volume: The pleural cavity normally contains a small amount of fluid that facilitates movement of the two membranes against each other. This fluid is a plasma filtrate derived from capillaries of the parietal pleura.

Color: Transudates are typically clear, pale yellow to straw

Turbidity: Exudative processes may look like transudates but most show variable
degrees of cloudiness or turbidity and often clot if not heparinized. Normally pleural fluid turbidity is clear.

Total Cell Count: A leukocyte count of 1000 cells/cmm has been used as a cut-off pointbetween transudates and exudates.

Differential Count:Mesothelial cells are common in pleural fluids and should be differentiated from inflammatory process.

Pleural fluid

Neutrophils predominate in pleural fluid from patient with inflammation of the pleura. Over 10% of transudates also show a predominance of neutrophil but this has no clinical significance.

Lymphocyte predominates in the disorders summarized in most are small but medium, large and reactive variant may be seen. Nucleoli and nuclear cleaving are more prominent in effusions than in the peripheral blood. Plasma cells may also be observed. Lymphocytosis associated with transudates is of no clinical significance.

An eosinophilic effusion is defined as an effusion that has more than 10% eosinophils. The most common cause is related to the presence of air or blood in the pleural cavity. A small number of mast cells or basophils often accompany the eosinophils. Eosinophil-derived Charcot-leyden crystal may be seen.

Mesothelial cells are common in pleural fluids inflammatory processes. Carcinoma cells may closely mimic mesothelial cell.

Pericardial Fluid

Normal leukocyte differential adds little diagnostic information but a stained smear should always be examined. Cytological identification of malignant cells is usually not difficult. Metastatic carcinoma of the lung and breast are most frequently observed in malignant pericardial effusion and are virtually never the initial presentation.

Peritoneal Fluid

Fluid absolute neutrophil counts have become the preferred method for the diagnosis of spontaneous bacterial peritonitis (SBP) Cut off values of 250-500 neutrophils /ul has used. Diagnostic accuracy is about 94% for 500 cells/ul and about 90% for 250 cells/ul.

Eosinophil (>10%) is most commonly associated with chronic peritoneal dialysis but has also been reported with congestive heart failure, vasculitis, lymphoma and ruptured hydatid cyst.

REPORTABLE INTERVAL FOR EXAMINATION RESULTS:

Physical Examination-

Volume: 10-50 ml

Color: pale yellow, dark yellow, red, white turbid,green

Appearance: clear, turbid, milky

CRITICAL VALUES:

• Presence of large numbers of granulocytes( in CSF >10/cmm)
• Presence of blast cells or malignant cells.

INTERPRETATION BY THE LABORATORY

Physical Examination-

Color:

normal: pale yellow or straw color with no clot on standing

Dark yellow: Jaundice

Reddish: traumatic tap, pulmonary infarction or malignancy

White- chyluria, pus

Brown: In CSF it indicates meningeal metastatic melanoma

Green: In ascetic fluid it indicates bile stain fluid may be due to perforation of biliary tract.

Appearance:

Straw colored transudative fluid: occurs in congestive cardiac failure, pulmonary embolism and cirrhosis of liver

Turbid: due to increased leukocytes, proteins or malignant cells.

Hemorrhagic fluid: indicates traumatic tap, recent surgery, pulmonary infarction, malignancy

Milky: results from obstruction of lymphatic duct due to inflammation or malignancy (lymphoma, carcinomatosis) or from abdominal injury

POTENTIAL SOURCES OF VARIABILITY:

Appearance:

Turbid CSF may be due to aspiration of epidural fat during lumber puncture.

Hemorrhagic appearance may be due to traumatic tap.

Thick viscous CSF may be due to needle injury to the intervertebral disk.

Bacteria: after storage at room temperature bacterial growth may take place

LIMITATIONS:

• The sample should not be degenerated.
• The Sample should be sent in aseptic conditions.
• Red cell counts of fluid have limited diagnostic value but may allow a useful approximation of the true fluid Leukocyte counts or a total protein in the presence of traumatic tap in which these levels could be elevated.
• Differential performed in a counting chamber without centrifugation is unsatisfactory because the low cell numbers have poor precision and identifying granulocytes and mono nuclear is difficult in a wet preparation.
• Direct smears of the centrifuged sediments can cause mild cell distortion and fragmentation.

Retention Period of Samples

Samples Retention Period Storage

Body Fluid 24hrs 2-80C

Morphology Slide 24hrs Room Temp.

SAFETY PROCEDURE

The sample is received in a sterile leak proof container. Contaminated / infected sample should not be examined. The Technicians should wear proper gloves and whitecoat then only start doing the test procedure. Do not refrigerate specimen until after microscopy .The specimen should then be refrigerated till it is discarded. After the sample has been examined the technician/s should properly wash their hands with detergent / liquid soap.

WASTE DISPOSAL GUIDANCE

Fluid samples are disposed in a chemical disaffection bin (CDB) with 1-% hypocholoride.
All other safety and waste disposable guidelines / outlines by the laboratory followed.