Cell counts of various biological fluids: Cerebrospinal fluid (CSF), Peritoneal fluid, Pleural fluid, and Pericardial fluid. HSBTE DMLT, IVth. Sem. Hematology.

Body fluids are liquids found within the human body. They are essential for various physiological functions. Examples of body fluids are blood, saliva, urine, cerebrospinal fluid, synovial fluid, lymph, Semen, Vaginal fluid, Gastric juice (stomach acid), Bile, Sweat, Interstitial fluid, Peritoneal fluid, Pleural fluid, Pericardial fluid, etc,

Cerebrospinal Fluid (CSF): Cerebrospinal fluid (CSF) is a clear, colorless fluid that surrounds the brain and spinal cord. It is present within the subarachnoid space and also within the ventricles of the brain. CSF provides protection and nutrient supply to these vital organs. The composition of CSF includes:

• 1. Water: approximately 99% of its volume.

• 2. Electrolytes: CSF contains various electrolytes such as sodium, potassium, chloride, calcium, and magnesium.

• 3. Proteins: CSF contains a small amount of proteins, including albumin and globulins.

• 4. Glucose: CSF contains glucose although at lower concentrations compared to blood glucose levels.

• 5. Waste products: CSF carries metabolic waste products away from the central nervous system.

• 6. White blood cells: Normally, CSF contains a few white blood cells (leukocytes).

• 7. Other substances: CSF may also contain hormones, neurotransmitters, vitamins, and other molecules.

Collection of the CSF:

The collection of cerebrospinal fluid (CSF) is typically performed through a procedure called lumbar puncture, also known as a spinal tap. The following steps are followed to collect sample of cerebrospinal fluid (CSF)

Purpose of CSF Collection: To diagnose various neurological conditions, including infections, inflammation, and hemorrhage.

• 1. Pre-procedure preparation: Ask the patient either to lie on their side with their knees drawn up to the chest or sit and lean forward with their head resting on a surface. Sanitize the lower back (lumbar region) to be punctured. Between the L3 and L4 or L4 and L5 vertebrae is chosen for the puncture. Apply local anesthetic.

• 2. Insert the needle: Insert the spindle needle between the vertebrae into the subarachnoid space, which contains CSF.

• 3. Collect the fluid: CSF flows into collection tubes due to the pressure gradient.

• 4. Monitoring: Monitor the patient's vital signs and assess for any signs of complications, such as headache, nausea, or neurological symptoms throughout procedure.

• 5. Remove the needle: Withdraw the needle slowly and apply pressure using sterile dressing to the puncture site to prevent CSF leakage and to minimize the risk of infection.

• 6. Post-procedure care: Instruct patient to be in clinic or hospital under observation for a few hours.

• 7. Labeling: Label the sample container with the name of the patient, date of birth, time of collection, and date of collection.

Preservation of synovial fluid: Refrigerate it immediately at 4 degrees C.

The procedure of cell count of CSF

The following are the steps of the CSF collection procedure:

• Preparation: Gather necessary equipment such as a hemocytometer (a specialized counting chamber), microscope, glass slides, pipettes, and appropriate stains (e.g., trypan blue).

• Dilution: CSF samples are too concentrated to count cells accurately. CSF is diluted using a diluting fluid, such as saline or formaldehyde solution, in a specified ratio. Common dilution ratios are 1:10 or 1:20.

• Loading the hemocytometer: Place a clean glass slide on a level surface. Place the hemocytometer on top of the glass slide. Use a pipette to load the diluted CSF into the counting chamber. Capillary action will draw the fluid into the chamber.

• Microscopic examination: Examine the hemocytometer under a microscope using low-power (10x) magnification. Focus on the grid lines of the counting chamber. Count the number of cells present in the specified areas of the grid (squares).

• Repeat the counting process and calculate the average number of cells per square.

• Calculation: Use the average number of cells per square and the dilution factor to calculate the total number of cells per microliter (μL) of CSF.

The formula for calculating the total cell count is:

Total cell count = (Average number of cells per square) × (Dilution factor) × (1/Depth factor) Depth factor is specific to the depth of the counting chamber (usually 0.1 mm).

Observation: Elevated cell counts may indicate conditions such as meningitis, encephalitis, or hemorrhage, while decreased counts may occur in certain neurological disorders.

Synovial fluid

Definition: Synovial fluid is a viscous, clear, and slightly yellowish fluid found within the synovial joints of the body. It lubricates the joint surfaces. It provides nutrients to the cartilage and acts as a shock absorber.

Composition: The composition of synovial fluid varies slightly between individuals and may change depending on factors such as age, health status, and joint activity. However, it generally consists of the following components:

• 1. Water: Synovial fluid is composed of water. 0.1 to 2.0 ml synovial fluid in a joint.

• 2. Hyaluronic Acid (Hyaluronan): It is a polysaccharide molecule. It contributes to the viscosity of synovial fluid. It lubricates the joint surfaces.

• 3. Lubricin (proteoglycan 4): lubricin is a glycoprotein. It reduces friction between the cartilage surfaces within the joint.

• 4. Proteins: Synovial fluid contains proteins such as albumin, globulins, and enzymes. These proteins maintain the fluid's viscosity. It provides nutrition to the cartilage. It adjusts inflammation and immune responses within the joint.

• 5. Lipids: Small amounts of cholesterol and fatty acids are present in synovial fluid. It contributes to its lubricating properties.

• 6. Glycosaminoglycans (GAGs): These are long unbranched polysaccharide chains. GAGs maintain synovial fluid structure and function.

• 7. Electrolytes: Synovial fluid contains ions such as sodium, potassium, chloride, and bicarbonate. It maintains the fluid's osmotic balance and pH.

• 8. Cells: Synovial fluid contains a small number of cells such as synoviocytes (cells of the synovial membrane), and leukocytes (white blood cells). WBCs number 20/cumm. Or 1-2.0 cells/cumm.

Contents per 100ml synovial fluid:

• 1. Lactate-10-20mg%

• 2. Uric acid-2-8mg%

• 3. Glucose-70-110

• 4. HYALURONATE-0.3-0.4gm%

• 5. Protein-1-3gm%

Synovial Fluid Collection:

Purpose of synovial fluid collection: To diagnose various conditions of joints such as arthritis, inflammation or infection, presence of WBC, bacteria, crystals, etc in synovial fluid,

The following steps are followed to collect a sample of synovial fluid

• 1. Pre-procedure preparation: Ask the patient to sit comfortably. Sanitize the area of the skin to be punctured. Apply local anesthetic.

• 2. Insert the needle: Insert the long thin sterile needle into the joints.

• 3. Collect the fluid: Use a sterile syringe to withdraw synovial fluid.

• 4. Remove the needle: Remove the needle after collecting a sufficient amount of synovial fluid sample. Apply a bandage at the puncture site to stop bleeding.

• 5. Post-procedure care: Instruct patient to be in clinic or hospital under observation for a few hours.

• 6. Labeling: Label the sample container with the name of the patient, date of birth, time of collection, and date of collection.

Preservation of synovial fluid: Refrigerate it immediately at 4 degrees C in a leak-proof container. Do not allow to freeze it. Ensure their transportation to laboratory in an ice bag to maintain a temperature of 4 degrees C. Assure analyze the sample within 24 hours of the collection..

Cell counts technique for synovial fluid

The following steps are followed for the synovial fluid cell count technique:

• 1. Sample Collection: Obtain synovial fluid from the joint using a procedure called arthrocentesis or joint aspiration.

• 2. Preparation of the Sample: place synovial fluid on a glass slide and spread it evenly. A thin layer of the fluid is then examined under a microscope.

• 3. Cell Counting: Count the number of cells microscopically present in a specific volume of synovial fluid. The total cell count is typically reported as the number of cells per cubic millimeter (cells/mm³) of synovial fluid.

• 4. Differentiation of Cell Types: The types of cells present in the synovial fluid are identified and differentiated after obtaining the total cell count. Lymphocytes, eosinophils, neutrophils, monocytes, red blood cells, etc may present in the synovial fluid

• 5. Interpretation of Results:.

  • · A normal cell count (<2000 cells/mm³) with predominantly mononuclear cells (lymphocytes and monocytes) may suggest a non-inflammatory condition or osteoarthritis.

  • · An elevated total cell count (>2000 cells/mm³) with neutrophils may indicate acute inflammation, infection, or crystal-induced arthritis.

  • · Chronic inflammatory conditions like rheumatoid arthritis may exhibit elevated total cell counts with a predominance of lymphocytes and macrophages.

  • · In cases of infection, the presence of bacteria or fungi in the synovial fluid, along with elevated neutrophil counts, may indicate septic arthritis.

BODY CAVITY FLUIDS (Serous Fluids)

The body cavities such as the pleural cavity, peritoneal cavity, and pericardial cavity (myocardial cavity) are lined by visceral and parietal layers. Pleural fluid (10 to 12c ml) is present between the visceral and parietal layers of the pleural cavity. Pleural fluids lubricate the lungs to reduce friction. Peritoneal fluid (10 to 50 ml) is present between the visceral and parietal layers of the peritoneal cavity. Peritoneal fluids lubricate abdominal cavities to reduce friction. Pericardial fluid (10 to 50 ml) is present in between the visceral and parietal layers of the pericardial cavity. The pericardial fluid lubricates the heart to reduce friction. These serous fluids are pale yellow, clear fluids. Normal serous fluid has 2 gm% protein, pH 7.0, glucose concentration similar to blood glucose, and a specific gravity of 1.015.

Mechanism of Serous fluid accumulation: Accumulation of fluid inside tissues develops diseases. Body fluid present inside tissues outside body cavities is called effusion. There are two types of effusion: Transudate and Exudate. The following are mechanisms of accumulation of fluids inside tissues.

1. Transudate: Increased venous pressure due to cardiac disturbance leads to the fluid exit from the vascular system. This fluid is called transudate. Trnsudate has low protein concentration, low specific gravity (less than 1.015), and low cell count.

2. Exudate: Toxic or inflammatory damage in pleural capillaries, peritoneal capillaries or pleural capillaries cause fluid accumulation. This fluid is called exudate. Exudate has high protein content, high specific gravity (more than 1.015), and cell concentration.

Comparison (Differentiation) of Trnsudate and Exudate

• 1. Appearance:

  • Transudate: Clear and watery.

  • Exudate: Cloudy and may be cloted

• 2. Specific gravity:

  • Transudate: Less than 1.015

  • Exudate: More than 1.015

• 3. Protein:

  • Transudate: Less than 2% Protein

  • Exudate: More than 2% protein

• 4. Fibrin:

  • Transudate: Poor

  • Exudate: Rich

• 5. Glucose:

  • Transudate: Higher than blood glucose

  • Exudate: Less than blood glucose

• 6. Cell count:

  • Transudate: Few

  • Exudate: Abundant.

Pleural fluid

Pleural Fluid collection: Pleural fluid is present inside the pleural cavity. Pleural fluid (10 to 12c ml) is present between the visceral and parietal layers of the pleural cavity.

Purpose to collect pleural fluid: To diagnose pleural effusion. Abnormal accumulation of the pleural fluid in pleural space is called pleural effusion.

Process to collect pleural fluid: The process to collect pleural fluid samples is called thoracentesis. The following steps are followed to collect pleural fluid samples:

• 1. Pre-procedure preparation: Instruct patient to sit upright, lean forward on the edge of bed or table, and hold breath during needle insertion.

• 2. Insert the needle: Sanitize the area of the skin to be punctured. Apply local anesthetic. Insert the thin hollow needle between the ribs and into the pleural space.

• 3. Collect the fluid: Collect pleural fluid.

• 4. Remove the needle: Remove the needle after collecting a sufficient amount of pleural fluid sample.

• 5. Post-procedure care: Instruct patient to be in clinic or hospital under observation for a few hours.

• 6. Labeling: Label the sample container with the name of the patient, date of birth, time of collection, and date of collection.

Pleural fluid sample preservation: Refrigerate it immediately at 4 degrees C in a leakproof container. Do not allow to freeze it. Ensure their transportation to the laboratory in an ice bag to maintain a temperature 4 degrees C. Assure to analyze the sample within 24 hours of the collection.

Pericardial Fluid

Pericardial Fluid collection: Pericardial fluid (10 to 50 ml) is present in between the visceral and parietal layers of the pericardial cavity. The process to collect pericardial fluid is called pericardiocentesis.

Purpose to collect pericardial fluid: Pericardial fluid is collected for both diagnostic and therapeutic purposes.

1. Diagnostic purposes: Pericarditis (Inflammation of pericardium),

Pericardial effusion and Cancer detection spread up to the pericardium.

2. Therapeutic purposes: Drainage of excess pericardial fluid.

Administration of medication into pericardial space.

Process to collect pericardial fluid: The following steps are followed to collect pericardial fluid.

• 1. Pre-procedure preparation: Ask the patient to lie down on his/her back. Sanitize the area of the skin to be punctured. Apply local anesthetic.

• 2. Insert the needle: Insert a thin hollow needle between the ribs and into the pericardium space.

• 3. Collect the fluid: Use a syringe to withdraw pericardial fluid.

• 4. Remove the needle: Remove the needle after collecting a sufficient amount of pericardial fluid sample. Apply bandage at the puncture site.

• 5. Post-procedure care: Instruct patient to be in clinic or hospital under observation for a few hours.

• 6. Labeling: Label the sample container with the name of the patient, date of birth, time of collection, and date of collection.

Preservation of pericardial fluid: Refrigerate it immediately at 4 degrees C in a leakproof container. Do not allow to freeze it. Ensure their transportation to the laboratory in an ice bag to maintain a temperature of 4 degrees C. Assure to analyze the sample within 24 hours of the collection.

Peritoneal Fluid

The peritoneum is a membrane to surrounds the abdominal cavity. Peritoneal fluid is present in the peritoneum. The process of collecting peritoneal fluid is called paracentesis. The following steps are followed to collect peritoneal fluid.

• 1. Pre-procedure preparation: Ask the patient to lie down on his/her back or sit. Sanitize the area of the skin to be punctured. Apply local anesthetic.

• 2. Insert the needle: Insert a long thin needle into the peritoneal cavity.

• 3. Collect the fluid: Use a sterile syringe to withdraw peritoneal fluid.

• 4. Remove the needle: Remove the needle after collecting a sufficient amount of peritoneal fluid sample. Apply bandage at the puncture site.

• 5. Post-procedure care: Instruct patient to be in clinic or hospital under observation for a few hours.

• 6. Labeling: Label the sample container with the name of the patient, date of birth, time of collection, and date of collection.

Preservation of peritoneal fluid: Refrigerate it immediately at 4 degrees C in a leakproof container. Do not allow to freeze it. Ensure their transportation to the laboratory in an ice bag to maintain a temperature of 4 degrees C. Assure to analyze the sample within 24 hours of the collection.

LABORATORY INVESTIGATION

Physical Examination:

• 1. Volume: Large volume of normal body fluid is due to congestive cardiac failure, chronic liver abscess, etc.

• 2. Appearance: Turbid or cloudy body fluid due to pus cells. It is due to inflammation. Milky white fluid indicates infectious disease.

• 3. Colour: Slight red indicates hemorrhage.

Microscopical examination:

• 1. Leucocytes: Centrifuge body fluid to concentrate leucocyte. Stain it with 1% methylene blue or Leishman stain.

  • Total cell count: Use Neubauer’s modified counting chamber. Both RBC (Erythrocytes) and WBC (Leucocytes) are counted.

  • Differential cell count: Use Leishman stained film to identify cell types. Polymorphonuclear leucocytes and mesothelial cells suggest cancer.

• 2. Mesothelial cells: Mesothelium produces mesothelial cells. Mesothelial cells are larger than normal and binucleated is abnormal. It is mainly checked in pleural fluid. 1% aqueous methylene blue is used to stain mesothelial cells. It is reported in the percentage of leucocytes.

• 3. Tumour cells: Tumour cell's size, shape, and numbers are very high The cells may be single, in sheets, or in papillary form. An increase in the nucleus and cytoplasmic ratio increases in tumor cells. These cells are identified by Papanicolaou’s staining.

Chemical Examination:

1. Protein: Protein contents less than 2 gm% indicate transudate. Protein contents of more than 2 gm% indicate exudates.

2. Glucose: Infective exudates contain low glucose content (less than blood glucose level).

Microbiological Examination

1. Smear Examination: Use a centrifuge deposit to prepare a smear. Stain with gram stain or Zeil Neelsen staining.

2. Culture: Routine cultures are used to identify bacteria. Use Petrof’s method to culture for tubercule bacilli.