Cerebrospinal Fluid Lecture Notes Flashcards

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Course Information

• Continuing Education Credits:
  • P.A.C.E. Contact Hours: $1.50$ hour(s)
  • Course Number: $578-110-24$
  • Florida Board of Clinical Laboratory Science CE - General (Hematology): $1.50$ hour(s)
  • Course Number: $1223432$
• Level of Instruction: Intermediate
• Intended Audience: Medical laboratory scientists, medical laboratory technicians, medical laboratory science students, and pathology residents.
• Course Description: Cerebrospinal fluid analysis including: specimen collection and handling, macroscopic characteristics, cell counting techniques, and cell identification with images and interactive questions.

Introduction to Cerebrospinal Fluid (CSF)

• CSF is a clear fluid circulating outside the brain, within brain ventricles, and around the spinal cord.
• Most CSF originates in the choroid plexus (network of tiny blood vessels in the third, lateral, and fourth ventricles).
• About 30% of CSF is formed in other sites (subarachnoid space, ependymal lining of ventricles).
• Volume of CSF:
  • Adults: Approximately $90–150$ mL
  • Babies (up to four weeks): Average volume of $10–60$ mL

Three Main Functions of CSF

• Protect the brain and spinal cord from trauma.
• Supply nutrients to nervous system tissue.
• Remove waste products from cerebral metabolism.

Chemical Substances Present in CSF

• CSF composition is based on selective secretions from plasma.
• CSF has higher concentrations of some solutes and lower concentrations of others compared to plasma.
• Normal CSF has dramatically lower protein levels than plasma.
• Chemical Substances and Reference Intervals:
  • Chloride: $120–132$ mmol/L
  • Total Protein: $15–45$ mg/dL
  • Glucose: $40–70$ mg/dL
• Other chemicals present: Albumin, Calcium, Cholesterol, Creatinine, Immunoglobulins, Lactic acid dehydrogenase (LDH), Magnesium, Potassium, Phosphorus, Urea, Uric acid

Cells Present in Normal CSF

• Adults: $0–5$ WBC/µL is considered normal.
• Children: Slightly higher cell counts; newborns up to $30$ WBC/µL within normal limits.
• Lymphocytes account for $40–80\%$ of these cells.

CSF Evaluation and Diagnosis

• Examination of CSF provides vital information for diagnosing several disorders:
  • Benign disorders: Meningitis, Encephalitis, Brain abscess, Subarachnoid hemorrhage, Multiple sclerosis, Guillain-Barrè syndrome
  • Malignant disorders: Leukemia with CNS involvement, Malignant tumors of the brain or spinal cord, Metastasis of malignant tumors

CSF Specimen Collection and Processing

CSF Specimen Collection Process

• Obtained by a physician via lumbar puncture, usually in the L3-L4 region.
• Sterile technique is essential to reduce infection risk.
• Care must be taken to avoid injury to neural tissue.
• A syringe is used to remove $6–20$ mL of spinal fluid in adults.
• If opening pressure is elevated (greater than $200$ mm), no more than $1–2$ mL of CSF should be withdrawn.
• Less fluid is removed from babies and small children.
• CSF sample divided into 3–4 tubes (2–4 mL in each).
• Glass tubes should not be used due to cell adhesion.
• Tubes are numbered in order of collection.

Collection Tubes

• Sterile tubes are pre-numbered 1 through 3 or 4.
• Tube 1: Chemistry and immunology testing.
• Tube 2: Microbiology testing.
• Tube 3: Hematology testing.
• Tube 4: Reserved for non-routine studies.
• The first tube should not be used for microbiology or hematology due to possible contamination from the lumbar puncture and to ensure peripheral blood contamination from the puncture has cleared.

Specimen Labeling and Transport

• CSF sample should be properly labeled with tube number, patient's name, and hospital number/unique identifier.
• Samples should be transported to the laboratory immediately.

Specimen Handling and Storage

• Stability of CSF sample varies depending on ordered procedures.
• Hematologic analysis should be performed within one hour of fluid aspiration.
• RBCs and WBCs have limited stability in CSF due to its hypotonic nature, causing rapid lysis.
• Timing is critical for WBCs in diagnosing meningitis and detecting CNS leukemic involvement.
• CSF samples for hematologic testing should be maintained at room temperature prior to testing.
• Refrigeration is not recommended for culture specimens as fastidious organisms like Haemophilus influenzae and Neisseria meningitidis may not survive.

Initial Specimen Examination

• The medical laboratory scientist/technician is responsible for examining CSF samples upon receipt.
• Testing could be affected if any of the following conditions are present:
  • Tubes are not labeled or contain errors in patient identification.
  • Tubes are not numbered.
  • Specimen contains a blood clot.
  • Specimen contains less than $0.5$ mL CSF.
  • Delay in testing from the time of collection.
• Unlabeled or incorrectly labeled specimens should usually be rejected; however, because CSF is difficult to recollect, the laboratory should have a policy that permits the correction of patient identification information.

Safety Precautions

• CSF is a potentially infectious material; standard precautions must be followed.
  • Use appropriate personal protective equipment.
  • Semi-automatic micropipettes and disposable plastic counting chambers are the safest option for manual CSF counts.
  • If disposable materials are not used, soak contaminated reusable pipettes, hemocytometer, and coverslip in 70% alcohol or other appropriate disinfectant.
  • All disposable items must be placed in a biohazard container for appropriate disposal.
  • Wash hands thoroughly after analysis.
  • Spinal fluid samples that will be discarded must be placed in biohazard containers.

Macroscopic Characteristics

Abnormalities

• Normal CSF is clear and colorless.
• Abnormalities that may be observed:
  • Turbidity
  • Fibrinogen clot/pellicle
  • Blood
  • Xanthochromia

Turbidity

• Spinal fluid samples can be either clear or turbid.
• Quantifying CSF Turbidity:
  • 0: Clear fluid
  • 1+: Faintly cloudy, smoky, or hazy
  • 2+: Turbidity evident but newsprint read easily through the tube
  • 3+: Turbid with newsprint not easily read through the tube
  • 4+: Turbid with newsprint not able to be seen through the tube
• Turbidity may be caused by WBCs, RBCs, fungi, bacteria, amoebae, contrast media, or epidural fat aspiration.
• $200$ WBCs/mm³ cause slight turbidity (1+); increased numbers of WBCs cause increased turbidity.
• At least $400$ erythrocytes/mm³ are needed to produce 1+ turbidity.
• CSF may have an oily appearance due to substances remaining after radiologic procedures.

Clot/Pellicle

• Clot formation is always abnormal, often due to increased protein levels, especially fibrinogen.
• Clot formation is likely when the protein level is $1000$ mg/dL, but may occur at lower levels.
• Fine clots may appear as a thin membrane or "scum" on the surface of the CSF specimen (pellicle), composed of fibrinogen and WBCs.
• CSF Specimen Clots:
  • Bacterial meningitis: Pellicle forms quickly; large clot follows.
  • TB meningitis: Web-like clot (pellicle) after $12–24$ hours (enhanced by refrigeration).
  • Paresis: Incomplete clot.
  • Blockage of CSF circulation: Completely clotted due to high protein levels.

Bloody Specimen

• When blood is present, determine if it's due to a traumatic puncture or a pathologic condition.
• Differentiating Traumatic Taps and Subarachnoid Hemorrhages:
  • Traumatic Tap: Blood in Tube 1 is greater than in tubes 2, 3, 4; Supernatant is clear after centrifugation; Blood clots on standing.
  • Subarachnoid Hemorrhage: Blood is evenly distributed in all tubes; Supernatant is pink or yellow; Blood does not clot on standing.

Detecting Subarachnoid Hemorrhage

• RBC count may be requested on the first and last tubes to differentiate SAH from a traumatic tap.
• Similar RBC counts in the first and last tubes suggest SAH.
• Significantly lower RBC count in the last tube suggests a traumatic tap.
• However, a traumatic tap can mask an SAH.
• If RBC count is high in the last tube (but less than in the first tube), the physician must use additional clinical information.

Xanthochromia

• CSF samples from patients with SAH may have a pink- to yellow-tinged supernatant when centrifuged within one hour of collection.
• Xanthochromia is due to RBC breakdown products from the SAH.
• Breakdown of RBCs and xanthochromia begin $2–3$ hours after the SAH and may persist for weeks.
• High serum bilirubin can also cause xanthochromia, as can plasma from a grossly bloody traumatic tap.
• RBCs introduced by a traumatic tap haven’t been in the fluid long enough to begin the breakdown process if the CSF is examined within one hour.

Other Causes of Xanthochromia

• Other sources of pigment:
  • Methemoglobin
  • Increased CSF protein (>$150$ mg/dL)
  • Contamination with skin antiseptic (iodine or merthiolate)
• Xanthochromia in premature infants due to:
  • Elevated bilirubin in the blood
  • Immaturity of the blood-brain barrier
  • Elevated protein in CSF

Important Aspects of Xanthochromia

• Xanthochromia is the appearance of color in the supernatant of a fresh, centrifuged CSF sample.
• There are a variety of reasons for xanthochromia.
• Xanthochromia must be reported; the physician is responsible for determining the reason for its presence.

Cell Counting Techniques

Normal Cell Counts

• Normal adult CSF may have up to $5$ WBCs/µL.
• Children have slightly higher counts; newborns up to $30$ WBCs/µL are within normal limits.
• CSF containing up to $200$ WBCs/mm³ or $400$ RBCs/mm³ may appear clear or only slightly hazy, so all specimens must be examined microscopically.

Examining CSF Using the Hemocytometer

• Manual cell counts are performed using a standard hemocytometer, most commonly the improved Neubauer counting chamber.
• The Neubauer chamber contains 2 ruled areas, each composed of 9 large squares of equal size, further divided for counting larger numbers of cells.
• A disposable chamber is recommended for CSF samples (especially from suspected cases of meningitis and Creutzfeldt-Jakob disease).
• Example of a disposable chamber: C-Chip (one-piece improved Neubauer hemocytometer with an integrated coverslip).
• Due to limited precision, both sides of the hemocytometer should be charged with fluid and counted.
• Counts from each side must be within acceptable limits of agreement as established by your laboratory’s procedure.
• Normally, nucleated cell counts on CSF are quite low, but they can be dramatically elevated in conditions such as meningitis.
• Clear specimens may be counted undiluted if cells do not overlap.
• Identification of nucleated cells can be improved by rinsing the pipette with new methylene blue or crystal violet stain or by using phase microscopy.
• Cloudy fluids should be briefly examined microscopically to approximate necessary dilutions.
• The laboratory should have clear guidelines concerning the area of the hemocytometer to count and when to make a new dilution.

Examining CSF Using the Hemocytometer Continued

• White cells are less refractile than red cells and appear somewhat granular; in general, white cells will be larger than red cells.
• Procedures beyond unstained brightfield may be needed to distinguish RBCs from other cells.
• If cells are counted in the four corner squares and the center square on both sides of the hemocytometer, the number of cells counted equals the number of cells/mm³, which equals cells/µL.
• Standard Neubauer Counting Chamber Formula:
  • $\frac{\text{Number of cells counted on each side of the hemacytometer} \cdot \text{Dilution factor}}{\text{Area} \cdot 0.1}$
  • Area is the number of larger squares that were used to count.
  • 0.1 is the standard chamber depth, this number does not change.

Counting Nucleated Cells in a Bloody CSF Specimen

• Increased numbers of RBCs can make it difficult to count nucleated cells.
• Making excessive dilutions to dilute out RBCs can also dilute out nucleated cells.
• A better strategy is to perform the total cell count and then use commercially available Spinal Diluting Fluid to minimally dilute the CSF.
• Spinal Diluting Fluid: A mixture of acetic acid and crystal violet.
  • Acetic acid lyses RBCs.
  • Crystal violet slightly stains nucleated cells for more accurate counting.
• Historically, calculations were used to correct the nucleated cell count for the presence of red blood cells.
• Current recommendations are to generally assume there are 1 to 2 WBCs present for every 1000 RBCs because using a standard correction factor over-corrects the nucleated cell count.

Cell Counting Guidelines

• If cells are numerous and overlapping, a dilution must be made.
• When the macroscopic appearance is turbid or milky, significant dilution is usually necessary.
• Calibrated automatic pipettes should be used for specimens that require a dilution.
• Automated cell counters may not be the best choice due to variation in background counts, which could cause a false increase in normal or slightly elevated counts.
• The dilution required is based on the appearance of the sample.
• Cloudy fluids should be briefly examined microscopically to approximate necessary dilutions.

CSF Cell Count Guidelines Table 5: Undiluted Fluid

• Nucleated Cells / Square Area to Count on Both Chambers
  • <10: Count all 9 squares
  • 10–100: Count 4 corner squares
  • >100: Dilute for nucleated cell count

Suggested Dilutions Table 6:

• Appearance / Dilution Ratio / Volume of Sample / Volume of Diluent
  • Slightly hazy / 1:10 / 30 µL / 270 µL
  • Hazy / 1:20 / 30 µL / 570 µL
  • Slightly cloudy / 1:100 / 30 µL / 2970 µL
  • Cloudy / 1:200 / 30 µL / 5970 µL

CSF Cell Count Guidelines Table 7: Diluted Fluid

• Nucleated Cells / Square Area to Count on Both Chambers
  • <10: Make a smaller dilution
  • 10–100: Count 4 corner squares
  • >100: Make a larger dilution

Cell Identification

Stained Cytospin Preparations of CSF

• Identifying the types of white blood cells present in CSF provides important information for accurate diagnosis and treatment.
• A well-prepared CSF slide is crucial.
• The slide should be made from the same tube as the tube used for the cell count.
• The slide should be clearly labeled to ensure positive patient identification.
• Slides for microscopic examination of CSF should be made using a cytocentrifugation technique (cytospin).
• Alternate methods of slide preparation (traditional centrifugation and manual smear methods) are not recommended.
• Stain the slides with Wright or Wright-Giemsa stain and perform differential counts.

Cytocentrifuge Technique

• The cytocentrifuge technique concentrates cells on a slide in a uniform monolayer approximately 6 mm in diameter.
• The monolayer distribution enhances the morphological appearance of the cells present.
• Follow manufacturer guidelines for drops of fluid to use based on nucleated cell count.
• Add a drop of 11–22% albumin to the volume of fluid used for slide preparation before cytocentrifugation to help preserve the integrity and morphology of cells in the hypotonic CSF.
• Allow slides to air dry for several minutes, then stain with Wright or Wright-Giemsa stain (automated stainer or manually).

Nucleated Cells That May Be Present in CSF

• The nucleated cells seen in normal adult CSF are predominantly lymphocytes and monocytes/macrophages. A rare neutrophil may be seen.
• An increased number of lymphocytes, monocytes, or neutrophils in CSF is termed pleocytosis.
• Morphologically normal cells can be seen in abnormal numbers in meningitis and inflammation.
• The monocyte/macrophage appears when clean-up of the CSF is necessary because of degenerating cells and debris, often due to a subarachnoid hemorrhage (SAH) or meningitis.
• Cells that may be seen in cerebrospinal fluid can be divided into four categories:
  • Mature peripheral blood cells
  • Immature hematopoietic cells
  • Tissue cells
  • Malignant cells
• It is important to recognize tissue cells so that they are differentiated from tumor and blast cells.
• Pathologists must review any slides with presumptive malignant cells, unidentified cells, or immature stages of cells, such as blasts.

Nucleated Cells That May Be Present in CSF Table 8:

• Lymphocytes: Low number is normal. Increased in viral, tubercular, fungal, syphilitic meningitis; multiple sclerosis; drug abuse; lymphoma; leukemia; Guillain-Barré syndrome; chronic alcoholism; polyneuritis.
• Monocytes/Macrophages: Low number is normal. Increased in previous subarachnoid hemorrhage; response to foreign material in the CSF; meningitis; inflammation; tumors.
• Neutrophils: Low number is normal following cytocentrifugation. Increased in prominent pleocytosis in bacterial meningitis; early tuberculosis and fungal infections; hemorrhage; cerebral abscess; tumors.
• Eosinophils: Increased in response to foreign CSF material including allergic reaction medications, shunts, dyes, parasites, fungal meningitis, leukemia, CSF contamination with blood.
• Choroidal and Ependymal cells: Cells of the epithelial lining of the CNS; not clinically significant. May be seen in CSF following neurosurgical procedures. Often seen in clumps with no nuclear or cytoplasmic irregularities.
• Malignant cells: Metastatic carcinoma.
• Blast cells: Leukemia, Lymphoma.

Neutrophils

• Bacterial meningitis is associated with prominent neutrophilic pleocytosis.
• Bacteria may also be observed on the stained smear.

Monocyte/Macrophage

• Some laboratory professionals may attempt to make a distinction between monocytes and macrophages.
• Macrophages are the phagocytes that originate from monocytes.
• However, it is not necessary to morphologically distinguish these cells.
• The transformed monocyte/macrophage appears when clean-up of the CSF is necessary because of degenerating cells and debris, often due to a subarachnoid hemorrhage (SAH) or meningitis.

Macrophages As Indicators of Previous Subarachnoid Hemorrhage (SAH)

• Macrophages that have phagocytized red blood cells (erythrophages) or hemosiderin (siderophages) are indications that an SAH had occurred sometime before the collection of the CSF sample.
• Macrophages begin to appear in CSF approximately two hours after bleeding into the central nervous system occurs.
• Macrophages ingest RBCs and erythrophages may be observed on the stained CSF smear.
• As the RBCs degenerate further, the breakdown products are seen in the phagocytic cells as dark, granular, iron-laden hemosiderin deposits or yellow crystalline iron-free hematoidin crystals.
• The formation of hemosiderin deposits and hematoidin crystals occurs approximately 18 hours following a subarachnoid hemorrhage.
• The hemosiderin deposits, hematoidin crystals, and siderophages may be present in the CSF for several months.

Normal Peripheral Blood Cells

• CSF contaminated with peripheral blood from a traumatic tap or an SAH may have all the nucleated cell types seen in the patient's peripheral blood.
• Therefore, if abnormal or immature cells are in the peripheral blood, they can also be observed in the CSF.

Bone Marrow Contamination of CSF Sample

• Bone marrow contamination of the CSF can occur if a vertebral process that is part of the spinal column vertebra is nicked or pierced when performing the lumbar puncture.
• In this case, both immature myeloid cells and erythroid cells may be seen but are not a reflection of the patient's peripheral blood picture.

Blast Cells

• Blast cells may be seen in the spinal fluid when cell proliferation in acute leukemia or lymphoma spreads to the central nervous system.
• Notice the smooth chromatin pattern in the nucleus and nucleoli.

More Blast Cells

• Notice the smooth chromatin pattern, prominent nucleoli, high nuclear-to-cytoplasm ratio (N/C ratio), and irregularly shaped nuclei.

Malignant Cells

• Malignant cells that have broken away from a tumor within the brain or meninges may also be present in spinal fluid.
• Central nervous system (CNS) involvement is most common with melanoma, breast carcinoma, and lung carcinoma.
• Dramatic vacuolation is one characteristic of malignant cells in CSF specimens.

Malignant Cells Continued

• Malignant cells are often found in clumps, and cells may have more than one nucleus due to their erratic mitotic patterns.
• All of the following characteristics of malignant cells can be observed:
  • Giant cells
  • Multinucleation
  • Cellular crowding
  • Irregular nuclear shapes
  • High nuclear-to-cytoplasmic ratio (N/C ratio)
  • Vacuolation (sometimes dramatic) that may be seen in both the cytoplasm and the nucleus

Malignant Cells Continued Again

• Malignant cells often have nucleoli.
• They can also be phagocytic and phagocytize (cannibalize) other malignant cells.
• The provided characteristics do not apply to all malignant cells that may be present in a CSF sample.
• For this reason, CSF smears containing abnormal or suspicious cells must be reviewed by the pathologist, and presumptive malignant cells correlated with cytology findings.

References

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• Cytospin Protocol, Figure 2. "Cytospin preparation technique." EuroMAbNet. Available at: https://www.euromabnet.com/protocols/cytospin.php.
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