AUBF - CSF and Synovial Fluid PDF

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Summary

PHYSIOLOGY● Meninges (3 layers) Dura 2. Arachnoid 3. Pia “hard” – “spiderweb” – “gentle” outer – inner; filamentous – thin skull, – brain, vertebral canal spinal cord ● CSF ✓ Supply nutrients to nervous tissue ✓ Remove metabolic waste ✓ Mechanical barrierVolume: Adults: 90 – 50 mL Neonates: 10 – 60 ...

Description

AUBF | CSF and Synovial Fluid Compiled by: Vienna Cari-cari

Appearance Cause

Appearance

Major significance

Crystal clear

PHYSIOLOGY ●

Meninges (3 layers) 1. Dura 2. Arachnoid – “hard” – outer – skull, vertebral canal

●

Hazy, Turbid, milky, cloudy

3. Pia

– “spiderweb” – inner; filamentous

– “gentle” – thin – brain, spinal cord

CSF ✓ Supply nutrients to nervous tissue ✓ Remove metabolic waste ✓ Mechanical barrier Volume:

Normal Meningitis

Microorganisms

Meningitis

Protein

BBB disorders IgG production within CNS

Oily

Radiographic contrast media

Bloody

RBCs

Hemorrhage Traumatic tap

Hgb

Old hemorrhage Lysed cells from traumatic tap

Melanin

RBC degradation Elevated serum bilirubin level Increased serum levels Protein Meningeal melanosarcoma

Protein

BBB disorders

Protein

BBB disorders

Clotting factors

Traumatic tap

Protein

BBB disorders

Clotting factors

Tubercular meningitis

Bilirubin Xanthochromic Carotene

Adults: 90–50 mL Neonates:10–60 mL Clotted

Production – Choroid plexuses of 2 lumbar ventricles and 3rd & 4th ventricles – (every hour) adult: 20mL approx. – flows through subarachnoid space – reabsorbed into capillaries in arachnoid granulations

WBCs

Pellicle

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Xanthochromia – pink/orange/yellow CSF supernatant Pink = very slight oxyhemoglobin Orange = heavy hemolysis Yellow = conv. of oxyhemoglobin to unconjugated bilirubin

→ to maintain volume → reabsorption rate = production rate

→ CAUSES:

(main) RBC degradation Elevated serum bilirubin and carotene ● Increased protein conc. ● Melanoma ●

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Choroid plexus – forms CSF from plasma CSF is NOT an ultrafiltrate ✓ Selective filtration ✓ Hydrostatic pressure ✓ Active transport secretion

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Blood-Brain Barrier – tight fitting structure of endothelial cells in the choroid plexus – meningitis Disruption of BBB – multiple sclerosis

Traumatic collection (TAP) ●

3 visual exams to know if bloody CSF is due to hemorrhage or traumatic tap:

1. Uneven blood distribution

Specimen collection & handling ●

Lumbar puncture between 3rd and 4th/5th lumbar vertebra Precautions: – measurement of intracranial pressure – prevent infx or neural tissue damage

FACTORS: ● ●

Volume removed: based on volume available (adult vs. neonate) Opening pressure of CSF → Measured when needle first enters subarachnoid space → ↑ = slow withdrawal of fluid

– Cerebral hemorrhage = even – Traumatic tap = heaviest blood conc. in tube 1 > 2 > 3 2. Clot Formation – Traumatic tap = clots (due to plasma fibrinogen) – C. Hemorrhage = not enough fibrinogen to clot – Damaged BBB = clots; but no bloody CSF – Tubercular meningitis = classic web-like pellicle seen after refrigeration overnight 3. Xanthochromic supernatant

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3 sterile tubes (labeled 1,2,3 in order of draw) 1

– Chemical & serologic tests – Least affected by blood/bacteria

– Centrifuge in microhematocrit tube; Then examine against white background

2

– Microbiology

– Additional tests: D-dimer test and Microscopic tests

3

– Cell count – Least likely to contain cells

4

– Optional – Microbiology (to better exclude skin contamination) – Additional serologic tests

– Excess fluid is NOT discarded; Frozen until no further use – If only 1 tube can be collected, test first in Microbiology ●

STAT; but if not possible, – Hematology: Refrigerate

C. Hemorrhage = macrophages; erythrophagocytosis = hemosiderin granules = D-dimer by latex agglutination immunoassay Fibrin degradation product

Cell count – Routine: Leukocyte count – Perform immediately; (if can’t, refrigerate) → Lysis begins within 1 hr

– Microbiology: Room temp – Chemical & Serologic: Frozen

→ 40% of WBCs disintegrate after 2hrs

RBC count = Total cell count – WBC count

AUBF | CSF and Synovial Fluid Compiled by: Vienna Cari-cari

METHODOLOGY Normal: ● adult : 0–5 WBCs/μL ● children : higher ● newborns : 30 mononuclear cells/ μL

CYTOCENTRIFUGATION Page 186 in Strasinger book (p.200 in pdf)

– 200 WBCs or 400 RBCs /μL = clear – Neubaeur Counting Chamber Cells /μL =

cells counted x dilution cells counted x V of 1 square *for both diluted and undiluted

TOTAL CELL COUNT ●

Clear spx can be counted undiluted

DILUTION:

normal saline mixed by inversion, loaded into hemocytometer using Pasteur pipette Cells /μL = cells counted x dilution

WBC COUNT ●

Lyse RBC first before performing

CSF CELLULAR CONSTITUENTS ●

Primary:

DILUTION:

– 3% glacial acetic acid – to lyse RBC – Methylene blue – to stain *note: same procedure w/ Total cell count NO DILUTION:

– 4 drops of spx in tube – Rinse Pasteur pipette w/ 3% glacial acetic acid – 4 drops of spx into pipette – Let sit for 1 min; then mix – Discard 1st drop – Load the hemocytometer

✓ Lymphocytes – more dominant in adults ✓ Monocytes – more dominant in children ✓ Neutrophils – occasional

→ Pleocytosis – abnormal increase in #s of these normal cells ●

Abnormal/ ✗ Immature leukocytes not found: ✗ Eosinophils

✗ Increased tissue cells

✗ Malignant cells ✗ Plasma cells ✗ Macrophages

● High

CSF WBC (majority: Neutrophils) = Bacterial meningitis = Early (1– 2 days) Viral, Fungal, Tubercular, Parasitic meningitis

● Moderate

QC OF CSF AND OTHER BODY FLUID CELL COUNTS – Liquid commercial controls – In-house controls – All diluents: Check biweekly - Use counting chamber at 400x magnification - Contaminated = discard; prepare new – Cytocentrifuge: Check monthly - Speed: Tachometer - Timing: Stopwatch – Nondisposable counting chambers - Soak in bactericidal sol’n (at least 15mins) - Rinse w/ water - Clean w/ isopropyl alcohol after each use

Differential count on csf ●

Stained smears; not from cells in the counting chamber → Reason: may overlook abnormal cells Conc. spx before preparing smear → Methods:

1. Sedimentation 2. Filtration 3. Cytocentrifuge 4. Centrifugation - if no cytocentrifuge - 5-10mins centrifuge; save supernatant for other tests - Stain: Wright

increase CSF WBC (majority: Lymphocytes, Monocytes) = Viral, Fungal, Tubercular, Parasitic meningitis SUMMARY OF CSF CELLULAR CONSTITUENTS

Cell Type

Clinical Significance Normal

Lymphocytes

Microscopic Findings All stages of development

Viral, tubercular, and fungal meningitis Multiple sclerosis

Neutrophils

Bacterial meningitis (Early) Viral, tubercular, and fungal meningitis Cerebral hemorrhage Normal

Monocytes

Less prominent granules Cells disintegrate rapidly

Mixed with lymphocytes

Viral, tubercular, and fungal meningitis Multiple sclerosis

Macrophages

RBCs in spinal fluid

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

Blast forms

Acute leukemia

Lymphoblast, myeloblast, or monoblast

Lymphoma cells

Disseminated lymphomas

Resemble lymphocytes with cleft nuclei

Multiple sclerosis

Traditional & classic forms

Lymphocyte reactions

Reactive lymphs

Diagnostic procedures

Clusters Distinct nuclei & cell walls

Metastatic carcinomas

Clusters Fused cell borders & nuclei

Plasma cells Ependymal, Choroidal, and Spindle-shaped Malignant

Primary central nervous

- Differential count: 100 cells * Report: percentage * If low (35 mg/dL:

8 – 12 mg/dL >35 mg/dL: Disturbance of consciousness

MICROBIOLOGY TESTS ● Purpose:

identify causative agent of meningitis

Positive: microorganism recovered from spx by growing it on appropriate culture medium → bacterial = 24 hrs → tubercular = 6 weeks

CSF culture – confirmatory

CSF LACTATE Importance: • diagnosing and managing meningitis → >25 mg/dL = bacterial, fungal, tubercular → >35 mg/dL = frequently in bacterial → 35 mg/dL

CSF GLUTAMINE – Produced from ammonia and α -ketoglutarate by brain cells → Process serves to remove ammonia from CNS

Positive: ✓ Gram stain ✓ Bacterial antigen tests

VIRAL

TUBERCULAR

FUNGAL

Elevated

Elevated

Elevated

Lymphocytes

Lymphocytes & monocytes

Lymphocytes & monocytes

Moderate

Moderate to marked

Normal

Decreased

Moderate to marked Normal to decreased

Normal

>25 mg/dL

>25 mg/dL

Cryptococcus neoformans Pellicle formation

Positive: ✓ India ink ✓ Immunologic test

• Normal conc.: 8 to 18 mg/dL → Elevated = liver disorders Increased blood and CSF ammonia

= 75% of children w/ Reye syndrome determining CSF glutamine = indirect test for the presence of excess ammonia in the CSF; because excess ammonia in CNS increases glutamine synthesis

• Methods of assaying glutamine: → based on measurement of ammonia liberated from

glutamine preferred over direct measurement of CSF ammonia bec. glutamine conc. is more stable than volatile ammonia conc.

GRAM STAIN Routine from all suspected meningitis ● Concentrated spx ● 10% chance negative; so, perform blood cultures ●

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Centrifuge: 1500g for 15mins → Slides and culture – prepared from sediment

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Cytocentrifuge = highly concentrated spx

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One of the most difficult slides to interpret because: - Few organisms - Easily overlooked - False-positive from precipitated stain/debris

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Frequently encountered: ✓ Streptococcus pneumoniae (gram-positive cocci) ✓ Haemophilus influenzae (pleomorphic gram-negative rods) ✓ Escherichia coli (gram-negative rods) ✓ Neisseria meningitidis (gram-negative cocci)

• As CSF ammonia conc. increases, α -ketoglutarate supply

becomes depleted → glutamine can no longer be produced to remove the toxic ammonia, and coma ensues • >35 mg/dL = disturbance of consciousness CSF glutamine test is frequently requested for patients with coma of unknown origin

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May be in newborns: ✓ Streptococcus agalactiae (gram-positive cocci) ✓ Listeria monocytogenes (gram-positive rods)...