copyright © 2014. f.a. davis company chapter 9 cerebrospinal fluid

Copyright © 2014. F.A. Davis Company

CHAPTER 9CHAPTER 9

CEREBROSPINAL FLUIDCEREBROSPINAL FLUID


Upon completing this chapter, the reader will be able to1.State the three major functions of cerebrospinal fluid (CSF).2.Distribute CSF specimen tubes numbered 1, 2, 3, and possibly 4 to their appropriate laboratory sections and correctly preserve them. 3.Describe the appearance of normal CSF and the causes of abnormally appearing CSF.4.Define xanthochromia and state its significance.5.Differentiate between CSF specimens caused by intracranial hemorrhage and a traumatic tap.

Learning ObjectivesLearning Objectives


6. Calculate CSF total, white blood cell (WBC), and red blood cell (RBC) counts when given the number of cells seen, amount of specimen dilution, and the squares counted in the Neubauer chamber.

7. Describe the leukocyte content of the CSF in bacterial, viral, tubercular, and fungal meningitis.

8. Describe and state the significance of macrophages in the CSF.

9. Differentiate between the appearance of normal choroidal cells and malignant cells.

Learning Objectives Learning Objectives (cont’d)(cont’d)


10.State the reference values for CSF total protein and name three pathologic conditions that produce an elevated CSF protein.

11.Determine whether increased CSF albumin or immunoglobulin is the result of damage to the blood-brain barrier or central nervous system production.

12.Discuss the significance of CSF electrophoresis, immunophoresis, and isoelectric focusing findings in multiple sclerosis and the identification of CSF.

13.State the reference values for CSF glucose and name the possible pathologic significance of a decreased CSF glucose.



14.Discuss the diagnostic value of CSF lactate and glutamine determinations.

15.Name the microorganism associated with a positive India ink preparation.

16.Discuss the diagnostic value of the bacterial and cryptococcal antigen tests.

17.Determine whether a suspected case of meningitis is of bacterial, viral, fungal, or tubercular origin, when presented with pertinent laboratory data.

18.Describe the role of the Venereal Disease Research Laboratories test and fluorescent treponemal antibody-absorption test for syphilis in CSF testing.



• Brain and spinal cord lined by meninges• Three layers of meninges

– Dura mater: outer– Arachnoid: middle– Pia mater: surfaces of brain and spinal cord

• Cerebrospinal fluid (CSF) produced in choroid plexuses of the four ventricles– 20 mL/min produced in adults– Volume adults 90 to 150 mL, neonates 10 to 60 mL

Formation and PhysiologyFormation and Physiology


The MeningesThe Meninges


• CSF flows through subarachnoid space between arachnoid and pia mater

• Reabsorbed into blood in arachnoid granulations/villae (one-way valves)

• Formation by selective filtration– Hydrostatic pressure and active transport– Not an ultrafiltrate– Very tight-fitting endothelial cells, prevent filtration of

large molecules—called the blood-brain barrier



Flow of Spinal Fluid Flow of Spinal Fluid Through the BrainThrough the Brain


• Blood-brain barrier– Essential to protect brain– Chemicals and harmful substances do not pass– Antibodies and medications are excluded– CSF composition differs from plasma– Meningitis, multiple sclerosis disrupt membrane– Test for substances that pass through: cells, protein,

bacteria, immunoglobulins



• CSF collected between third to fifth lumbar vertebrae

• Three sterile tubes in this order1. Chemistry/serology2. Microbiology (avoid skin contamination)3. Hematology (avoid cells from tap)– Save leftover fluid/fourth tube for additional tests– Volume removed based on patient volume and

opening pressure

Specimen Collection Specimen Collection & Handling& Handling


CSF Specimen Collection TubesCSF Specimen Collection Tubes


• Usually STAT requests• Handle carefully to avoid repeat taps• Preservation

– Hematology– Refrigerate– Microbiology– Room temperature– Chemistry/serology– Frozen

Specimen CollectionSpecimen Collection& Handling & Handling


• Crystal clear, cloudy/turbid, milky, xanthochromic, hemolyzed/bloody

• Cloudy = infection; milky = lipid or protein• Xanthochromic

– Pink, orange, yellow – RBC degradation products– Also jaundice, ↑ ↑ protein, carotene– Pathologic = cerebral hemorrhage

AppearanceAppearance


Tubes of CSFTubes of CSF


• Blood vessel punctured during tap• Differentiate from cerebral hemorrhage• Uneven blood distribution in tubes with

traumatic tap– Erythrophagocytosis, hemosiderin granules– Hemorrhage = even distribution in all tubes– Traumatic tap = decreasing tubes 1 through 3

Traumatic TapTraumatic Tap


• Clot formation– Clots present = traumatic tap (plasma)– Hemorrhage does not have enough fibrinogen– Other causes of clot formation

• Nonbloody CSF = damage to blood-brain barrier• TB meningitis: web-like pellicle after refrigeration

• Xanthochromia– Not present in a recent traumatic tap– Indicates older hemorrhage– D-dimer test for hemorrhage

Traumatic Tap Traumatic Tap (cont’d)(cont’d)


• White blood cell (WBC) and total cell count• Red blood cell (RBC) count seldom done• Granulocytes lyse within 1 hour; STAT

– Normal adult 0 to 5 WBCs/µL– Neonates up to 30 mononuclear cells/µL

• Neubauer counting chamber– Automated cell counters can be used

• Body fluid specific automation is available

Cell CountCell Count


Neubauer Counting Chamber Neubauer Counting Chamber


• Standard Neubauer calculation formula (cells/µL)

Number of cells counted × dilutionNumber of cells counted × volume of 1 square

= cells/µL• Can be used for diluted and undiluted samples

Calculating CSF Cell CountCalculating CSF Cell Count


• Example# cells counted × dilution × 1 μL = cells/µL

1 μL (0.1 × 10) (volume counted) </exeq)

Calculating CSF Cell Count Calculating CSF Cell Count (cont’d)(cont’d)


• Total cell count– Clear specimens count undiluted unless overlapping

cells are seen– Load with transfer pipette– Dilute with normal saline if necessary

• WBC count– Dilute with 3% acetic acid; methylene blue helps to

see cells; undiluted rinse transfer pipette with acetic acid, gently rotate pipette

Calculating CSF Cell Count Calculating CSF Cell Count (cont’d)(cont’d)


• Commercial cell controls are available• Check diluents for contamination biweekly• Monthly check on cytocentrifuge speed and

timing• Soak nondisposable chambers in bactericidal

solution for 15 minutes; rinse; clean with isopropyl alcohol

Quality ControlQuality Control


• Valuable diagnostic aid• Stained smear only• Must concentrate specimen

– Sedimentation, filtration, centrifugation, and cytocentrifugation

• 100 cells should be counted, classified, and reported in terms of percentage

Differential CountDifferential Count


• Cytocentrifuge– Forces cells onto a slide in a monolayer– Filter paper absorbs moisture– 0.1 mL CSF to 1 drop 30% albumin

• Albumin increases the cell yield and decreases the cellular distortion

– Positively charged slides to attract cells– Daily control of 0.2 mL saline and two drops of

albumin stained for bacterial contamination

CytocentrifugationCytocentrifugation


• Normal lymphocytes and monocytes• Adults: normal lymphocytes:monocytes = 70:30• Children’s ratio is reversed• Occasional neutrophils are normal• Pleocytosis: increased amounts of normal cells• Pleocytosis of normal cells is valuable in determining the

cause of meningitis– Neutrophils = bacterial– Lymphocytes = viral, tubercular, fungal, parasitic

Cellular ConstituentsCellular Constituents


Prominent Cells Seen in CSFProminent Cells Seen in CSF

Type of Cell Major Clinical Significance Microscopic FindingsLymphocytes Normal All stages of development may be found Viral, tubercular, and fungal

meningitis

Multiple sclerosis Neutrophils Bacterial meningitis Granules may be less prominent than in blood Early cases of viral, tubercular,

and fungal meningitisCells disintegrate rapidly

Cerebral hemorrhage Monocytes Normal Found mixed with lymphocytes Viral, tubercular, and fungal

meningitis

Multiple sclerosis


Prominent Cells Seen in CSF Prominent Cells Seen in CSF (cont’d)(cont’d)

Type of Cell Major Clinical Significance

Microscopic Findings

Macrophages RBCs in spinal fluid from hemorrhageContrast media

May contain phagocytized RBCs appearing as empty vacuoles or ghost cells, hemosiderin granules, and hematoidin crystals

Blast forms Acute leukemia Lymphoblasts, myeloblasts, or monoblastsLymphoma cells Disseminated lymphomas Resemble lymphocytes with cleft nucleiPlasma cells Multiple sclerosis Traditional and classic forms seen Lymphocyte reactions Reactive lymphsEpendymal, choroidal, and spindle-shaped cells

Diagnostic procedures Seen in clusters with distinct nuclei and distinct cell walls

Malignant cells Metastatic carcinomas Primary central nervous system carcinoma

Seen in clusters with fusing of cell borders and nuclei


NeutrophilsNeutrophils

• Primarily in bacterial meningitis

• Often contain phagocytized bacteria

• Increased early viral, fungal, tubercular, parasitic

• Vacuoles may be present


Nucleated RBCs (NRBC)Nucleated RBCs (NRBC)

• Seen with bone marrow contamination from tap in 1% of specimens

• Neutrophils with pyknotic nucleii may resemble NRBCs

• Capillary structures and epithelial cells from traumatic taps


Lymphocytes and MonocytesLymphocytes and Monocytes

• Lymphs and monos in viral, fungal, tubercular

• Reactive lymphocytes with viral

• Multiple sclerosis has 50 or fewer lymphocytes/μL, both normal and reactive

• Seen in HIV and AIDS


EosinophilsEosinophils

• Parasitic and fungal infections– (primarily Coccidioides

immitis)

• Medications and shunts into the central nervous system


MacrophagesMacrophages

• Purpose is to remove cellular and other debris

• May be seen after repeated taps

• Hemorrhage: enter CSF within 2 hours to phagocytize RBCs

• RBCs degraded to hematoidin crystals representing unconjugated bilirubin


Macrophages Macrophages (cont’d)(cont’d)


Nonclinically Significant CellsNonclinically Significant Cells

• Seen after diagnostic procedures• Choroidal cells• Epithelial lining of choroid

plexus, singular and in clumps, uniform cells

• Ependymal cells lining ventricles and neural canal; less defined cell membranes in clumps

• Spindle cells lining arachnoid seen in clumps


• Leukemias– Lymphoblasts, monoblasts, and myeloblasts– Nucleoli may be more prominent than in blood

• Lymphomas– Dissemination from lymph organs– Cleaved nucleii and prominent nucleoli

Malignant Cells of Malignant Cells of Hematologic OriginHematologic Origin


Malignant Cells of Malignant Cells of Hematologic Origin (cont'd)Hematologic Origin (cont'd)


• Metastatic carcinoma cells– Lung, breast, renal,

gastrointestinal, and melanoma

– Fused cell walls, nuclear irregularities, and hyperchromatic nucleoli

• Primary tumors– Astrocytomas,

retinoblastomas, medulloblastomas

Malignant Cells of Malignant Cells of Nonhematologic OriginNonhematologic Origin


• CSF formed by plasma filtration• Normal values differ from plasma because of

selectivity of blood-brain barrier• Abnormal values result

– Alterations in the permeability of the blood-brain barrier

– Increased production or metabolism by the neural cells in response to a pathologic condition

Chemistry TestsChemistry Tests


• Total protein is the most common test– Normal 15 to 45 mg/dL (mg, not grams)

• Method dependent• Increased in infants and persons >40

– Albumin is predominant, prealbumin is second– Alpha globulins-haptoglobin and ceruloplasmin– Transferrin is major beta globulin– TAU, carbohydrate-deficient transferrin seen in CSF, not

in blood; used to identify CSF– IgG major gamma globulin

Cerebrospinal ProteinCerebrospinal Protein


• Decreased protein levels = fluid leakage• Elevated levels = damage to blood-brain barrier,

IG production within CNS, decreased clearance, degeneration of neural tissue

• Meningitis/hemorrhage most common causes of increased damage to blood-brain barrier

• Find abnormal results on clear fluid with low cell counts from neurologic disorders

Clinical SignificanceClinical Significance


Elevated Results• Meningitis• Hemorrhage• Primary CNS tumors• Multiple sclerosis• Guillain-Barré syndrome• Neurosyphilis• Polyneuritis• Myxedema• Cushing disease• Connective tissue disease• Polyneuritis• Diabetes• UremiaDecreased results• CSF leakage/trauma• Recent puncture• Rapid CSF production• Water intoxication* Reference values for protein are usually 15 to 45 mg/dL, but are method dependent, and higher values are found in infants and people older than 40 years.

Clinical Significance of Elevated Clinical Significance of Elevated Protein ValuesProtein Values


• Turbidity • Automated instrumentation available

– Nephelometry

MethodologyMethodology


• Comparisons between serum and CSF levels of albumin and IgG

• CSF/serum albumin index– Blood-brain barrier integrity

• CSF IgG index• Comparison of the CSF/serum albumin index with

the CSF/serum IgG index• Values for CSF albumin and globulin adapted for

automated instruments

Protein FractionsProtein Fractions


IgG index = CSF IgG (mg/dL)/serum IgG (g/dL)

CSF albumin (mg/dL)/serum albumin (g/dL)

• Values >0.70 indicate IgG production within the CNS

Protein Fractions Protein Fractions (cont’d)(cont’d)


• Detection of oligoclonal bands– Represent inflammation within the CNS– Located in the gamma region of the protein

electrophoresis– Simultaneous serum electrophoresis must be

performed

Electrophoresis and Electrophoresis and Immunophoretic TechniquesImmunophoretic Techniques


• Multiple sclerosis (MS) = no bands in serum, bands in CSF

• Leukemia, lymphoma, viral, HIV: bands in both• Primary purpose for MS, compare also to IgG

index• Encephalitis, neurosyphilis, Guillain-Barré, and

neoplasms may give same pattern • Consider symptoms

Electrophoresis and Immunophoretic Electrophoresis and Immunophoretic Techniques (cont'd)Techniques (cont'd)


• Presence in CSF indicates demyelination of myelin sheath around axons of neurons

• Monitors the course of multiple sclerosis• Effectiveness of treatment• Immunoassay procedures available

Myelin Basic Protein Myelin Basic Protein


• Selective transport across blood-brain barrier• Approximately 60% to 70% plasma glucose

– Plasma = 100 mg/dL; CSF = 65 mg/dL– Draw blood 2 hours before spinal tap

• Significance– Values that are decreased relative to plasma values– Elevated CSF glucose values are always a result of

plasma elevations

CSF GlucoseCSF Glucose


• Markedly decreased with increased neutrophils in bacterial meningitis

• Tubercular meningitis decreased with increased lymphocytes

• Viral/fungal meningitis, normal glucose and increased lymphocytes

CSF GlucoseCSF GlucoseClinical SignificanceClinical Significance


• Diagnosis and management of meningitis– Bacterial, TB and fungal levels >25 mg/dL – Viral <25 mg/dL

• More reliable than CSF glucose• Levels remain elevated until treatment becomes

effective, then fall rapidly• Can result from any condition that decreases

oxygen flow to the tissues– Monitor severe head injuries

CSF LactateCSF Lactate


• Produced by brain cells from ammonia and α-ketoglutarate to remove toxic ammonia

• Indirect test for the presence of excess ammonia in the CSF

• Normal: 8 to 18 mg/dL– Elevated in liver disease– Elevated in children with Reye syndrome– Disturbance of consciousness when glutamine levels are more

than 35 mg/dL • More reliable than direct CSF ammonia

CSF GlutamineCSF Glutamine


• Gram stain and cultures must be performed on sediment from centrifuged CSF; cytocentrifuge helps Gram stains

• Blood cultures also must be drawn• Difficult to interpret Gram stains, few organisms and

often debris• Organisms: S. pneumocystis<<AU: Should this be

Streptococcus pneumocystis or pneumoniae?>>, Haemophilus influenza, Escherichia coli, Neisseria meningitidis, Listeria monocytogenes, S. agalactiae

Microbiology TestsMicrobiology Tests


• Cultures also plated on chocolate agar• TB smears very important because of growth time delay• Latex agglutination tests are available for

– Group B streptococcus, Haemophilus influenzae, S. pneumocystis<<AU: Should this be Streptococcus pneumocystis or pneumoniae?>>, Neisseria meningitidis, and Escherichia coli

• Gram stain is the best for detection• Compare with hematology and chemistry results

Microbiology Tests Microbiology Tests (cont’d)(cont’d)


• Naegleria fowleri– Found in ponds, lakes, and

some pools– Enters nasal passages and

migrates to the brain– Motile amoeba seen in wet

preps, nonmotile in cytospin preps

– Elongated with tapered posterior

ParasitesParasites


• Primary test is for neurosyphilis, third stage• Performed less now that people have been treated

early with penicillin• Detect active cases within the CNS• The Venereal Disease Research Laboratories (VDRL)

produces the recommended test for specificity• Should be accompanied by a positive serum

Fluorescent Treponemal Antibody Absorption (FTA-ABS)<<AU: defined FTA-ABS: ok?>>

Serologic TestingSerologic Testing

copyright © 2014. f.a. davis company chapter 9 cerebrospinal fluid

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