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Urinalysis & Body Fluids

Microscopic Analysis

We have progressed through the physical and chemical analysis of CSF, gathering crucial clues about the state of the central nervous system. Now, we arrive at the microscopic examination - the part of our analysis that provides the most direct and visually compelling evidence

This is not just about counting cells. It’s about identifying the specific type of inflammatory response, searching for malignant invaders, and sometimes, seeing the causative organism itself on a Gram stain. This examination, performed on Tube #3 to minimize contamination, often provides the definitive diagnosis that all other tests have been pointing towards. The stakes are incredibly high; accuracy and speed are paramount

Cell Count: Quantifying the Response

The first step is to determine the number of cells present. This is a quantitative measure of the degree of inflammation or bleeding

Methodology: The Hemacytometer

  • Instrument: Neubauer counting chamber (hemacytometer)
  • Procedure
    1. The CSF specimen must be well-mixed
    2. If the fluid is clear, it can be loaded undiluted. This is called a “neat” count
    3. If the fluid is cloudy or bloody, it must be diluted with saline to allow for an accurate count
    4. To perform a WBC count in a bloody specimen, RBCs must be lysed using a weak acid (e.g., 3% acetic acid) mixed with methylene blue stain to aid in visualizing the WBC nuclei
  • Calculation
    • (Cells Counted × Dilution Factor) / (Area Counted (mm²) × Depth (0.1 mm)) = Cells/µL
    • For a full Neubauer grid (9 large squares = 9 mm²): (Cells Counted × Dilution Factor) / (9 × 0.1) = Cells/µL

Total Cell Count

  • This is a count of all nucleated cells before any lysing agent is added. It’s a quick screen for pleocytosis (increased cell count)

Red Blood Cell (RBC) Count

  • Normal: 0 RBCs/µL
  • Significance: The presence of RBCs indicates bleeding. The key is differentiating a traumatic tap from a subarachnoid hemorrhage (SAH), which we discussed in the handling lecture (uneven vs. even distribution, xanthochromia, etc.)

White Blood Cell (WBC) Count

  • Reference Range
    • Adults: 0 - 5 WBCs/µL
    • Neonates (up to 1 month): 0 - 30 WBCs/µL (Their BBB is more permeable)
  • Clinical Significance: A WBC count greater than 5 in an adult is always abnormal and is referred to as pleocytosis. It is a cardinal sign of CNS inflammation or infection. The degree of elevation provides a major clue:
    • Mild Pleocytosis (5-100 WBCs/µL): Often seen in viral or fungal meningitis, multiple sclerosis
    • Moderate to Marked Pleocytosis (hundreds of WBCs/µL): Can be seen in more severe viral cases or early bacterial meningitis
    • Severe Pleocytosis (thousands to tens of thousands of WBCs/µL): Hallmark of acute bacterial meningitis.

Cell Differential: Identifying the Responders

A total WBC count tells us if there’s inflammation. The differential tells us what kind of inflammation it is. This is arguably the most diagnostically powerful piece of the entire CSF workup

Methodology: The Cytospin

  • Standard slide smears are useless for CSF because the cell concentration is too low. We would be looking at an empty slide
  • Cytocentrifugation (Cytospin): This is the required method
    1. A small volume of CSF (a few drops) is placed in a special conical chamber with a filter card and a glass slide
    2. The apparatus is spun at high speed
    3. The fluid is forced out and absorbed by the filter paper, while the cells are gently concentrated into a small, monolayer button on the slide
    4. The slide is then stained with Wright-Giemsa stain: and is ready for microscopic review

Interpreting the Cellular Players

(To do: add images of each cell type)

  1. Neutrophils (Polymorphonuclear cells, PMNs)
    • Appearance: Multi-lobed nucleus, granular cytoplasm
    • Significance: The “first responders” to acute bacterial infection
    • Predominance (>80%) is the classic finding in BACTERIAL MENINGITIS.
    • Can also be seen in early viral or fungal infections, but rarely at such high percentages
    • May contain phagocytized bacteria
  2. Lymphocytes
    • Appearance: Small cells with a large, round nucleus and a scant rim of blue cytoplasm
    • Significance: Associated with the adaptive immune response
    • Predominance is the classic finding in VIRAL MENINGITIS.
    • Also seen in fungal, tuberculous meningitis, and in inflammatory conditions like Multiple Sclerosis
  3. Monocytes / Macrophages
    • Appearance: Larger cells, indented or kidney-bean shaped nucleus, abundant gray-blue cytoplasm, may have vacuoles
    • Significance: The “clean-up crew” of the CNS
    • Seen in conjunction with lymphocytes in viral, fungal, and especially tuberculous meningitis
    • Macrophages in SAH: After a hemorrhage, macrophages will enter the CSF to clear out the debris. We may see:
      • Erythrophages: Macrophages that have engulfed intact RBCs
      • Hemosiderin-laden macrophages: Macrophages containing golden-brown, granular hemosiderin pigment from the breakdown of hemoglobin. This is definitive proof of a bleed that occurred at least 12-24 hours prior
  4. Eosinophils
    • Appearance: Bilobed nucleus, large, bright red/pink granules
    • Significance: Their presence is unusual but highly significant
    • Associated with parasitic infections: (e.g., Angiostrongylus cantonensis), fungal infections (especially Coccidioidomycosis), and allergic reactions (e.g., to shunts or medications)
  5. Malignant Cells
    • Appearance: These are the “abnormal” cells we look for. They exhibit classic signs of malignancy: large size, high nuclear-to-cytoplasmic ratio, irregular nuclear contours, prominent nucleoli, and often occur in clumps or clusters
    • Significance
      • Leukemia/Lymphoma: Blasts or lymphoma cells that have infiltrated the CNS
      • Metastatic Carcinoma: Often from lung, breast, or melanoma. Finding these cells is crucial for staging cancer and determining treatment

Search for Microorganisms: Finding the Cause

While the differential tells us about the host response, microbiology stains can identify the pathogen directly. This is performed on Tube #2

Gram Stain

  • The most critical STAT test in the microbiology lab for CSF.: A positive result is a medical emergency
  • A cytospin smear is prepared and stained
  • Allows for the rapid, presumptive identification of bacteria based on their morphology and cell wall characteristics
    • Gram-positive cocci in pairs: Streptococcus pneumoniae
    • Gram-negative diplococci: Neisseria meningitidis
    • Gram-positive cocci in clusters: Staphylococcus aureus
    • Small, pleomorphic gram-negative coccobacilli: Haemophilus influenzae
    • Gram-positive rods: Listeria monocytogenes

India Ink Stain

  • A special stain used to identify the encapsulated yeast Cryptococcus neoformans
  • The ink provides a dark background, making the large, clear polysaccharide capsule of the yeast visible as a distinct halo around the budding yeast cell
  • This is a common and dangerous opportunistic infection in immunocompromised patients (e.g., HIV/AIDS)

Acid-Fast Stain

  • Used for the detection of Mycobacterium tuberculosis in cases of suspected tuberculous meningitis
  • This is a low-yield procedure, as the number of organisms in the CSF is often very small. A negative result does not rule out TB meningitis. Culture or molecular methods are more sensitive

Conclusion: Synthesizing the Complete Picture

Let’s revisit our diagnostic table, now with the microscopic findings integrated. This is the complete puzzle

  • CSF Profile: Bacterial Meningitis
    • Appearance: Turbid / Purulent
    • WBC Count: >1,000 /µL (Often in the thousands)
    • Differential: >80% Neutrophils
    • Protein: Markedly Elevated
    • Glucose: Markedly Decreased
    • Microbiology: Positive Gram Stain/Culture
  • CSF Profile: Viral Meningitis
    • Appearance: Clear to slightly hazy
    • WBC Count: 50-1,000 /µL
    • Differential: Predominantly Lymphocytes
    • Protein: Moderately Elevated
    • Glucose: Normal
    • Microbiology: Negative
  • CSF Profile: Tuberculous Meningitis
    • Appearance: Hazy / Viscous (may form a web-like clot)
    • WBC Count: 100-500 /µL
    • Differential: Lymphocytes & Monocytes
    • Protein: Markedly Elevated
    • Glucose: Decreased
    • Microbiology: Positive Acid-Fast Stain/Culture

The microscopic analysis is the pinnacle of our CSF investigation. It moves beyond chemical shadows to provide direct visualization of the conflict between the host’s immune system and the invading pathogen. A well-prepared, well-stained cytospin slide, interpreted by a skilled laboratory scientist, is one of the most powerful diagnostic tools in modern medicine