CSF & Synovial Fluid

EspirituGeronimoGuittapManuelRivera

Cerebrospinal Fluid is a clear, colorless, sterile, extravascular fluid

produced by the brain serves as a protective fluid cushioning and

lubricating the brain and the spinal column potentially infectious and it must always be

collected and handled with Standard precautions.

CSF differs from serous and synovial fluids because of the selective permeability of the membranes and adjacent tissues containing CSF which referred to as the blood-brain barrier.

four main functions 1. Mechanical buffer which prevents trauma 2. Regulator of the volume of intercranial

contents 3. Nutrient medium for the CNS 4. Acts as excretory channel for metabolic

products of the CNS

CSF evaluation performing a lumbar puncture

lumbar puncture done only if serious concerns

exist because it involves potential harm to the patient

should not be done if infection or inflammation exists over the puncture site

Indications include diagnosis of meningitis, hemorrhage, neurological disease, and suspected malignancies.

Laboratory examination includes: gross examination chemical examination microscopic examination cell counts differential counts, and Immunology or Serology studies.

Specimen Collection Lumbar Puncture (Lumbar TAP)

Contraindication to performing this puncture in the presence of infection at the puncture site

Most common site used for lumbar puncture is the intervertebral space between L3 and L4.

For small children and infants, intervertebral space between L4 and L5.

10 – 20 mL of CSF is slowly removed into three or four sterile tubes that are numbered sequentially

CSF specimen must be processed immediately Lumbar puncture through an area of infection

may cause the spread of infection into the meninges

A common protocol is the performance of:1. Tube 1: microbiology 2. Tube 2: Hematology, Flow cytometry, Cytology;3. Tube 3: Chemistry4. Tube 4: Hematology , Molecular

Must be processed within an Hour. In transporting CSF, 22-25ºC is needed for

room temperature. In storing CSF, incubate for 6 hrs allocating 37C

if it has viruses 3 days maximum are allotted at 4 C

Laboratory Examination Normal CSF is clear and colorless and demonstrates a

viscosity similar to water Abnormal turbidity is observed if blood cells,

microorganism, or flecks of protein are present. Normal specific gravity is 1.006 to 1.008.

Pleocytosis -varying degrees of CSF cloudiness due to the

presence of cells.

Microscopic Examination CSF cell count must be performed as soon as possible,

because cell deterioration begins within 2 hours of collection.

Cell counts are performed manually rather than using automation because of the low level of cells present.

1. Cell Count Cell counts are performed on well-mixed,

undiluted specimens unless the CSF is grossly bloody

Normally, no red cells are present Dilution with HCl eliminates RBCs and

enhances nuclei Normal CSF contains 0-5 WBCs er µL Children can exhibit higher CSF WBC counts

2. Differential Count Usually performed on centrifuged preparations

stained with Wright stain. Lymphocytes and monocytes are predominant Neutrophils are rarely seen

Pleocytosis Increased amount of WBCs in a body

fluid. Varying degrees of CSF cloudiness due

to the presence of cells.

Neutrophilic Pleocytosis Present in bacterial meningitis and in

the early stages of other forms of meningitis

Cerebral abscess Subdural empyema CNS hemorraghe Intrathecal treatments Postseizure

Lymphocytic Pleocytosis Later stages of viral, tubercular,

fungal, or syphilitic meningitis. Guillian-Barré Syndrome

Plasmacytes Not normally found in normal CSF Appear in the same disorders in which there is

lymphocytic pleocytosis Seen in multiple sclerosis Eosinophils Rare finding in normal CSF Eosinophilic Pleocytosis: Eosinophils >10% of

cells in CSF Increased in parasitic and fungal infections of

the CSF or allergic reactions to malfunctioning intracranial shunts, radiographic contrast media, and drugs.

Monocytes Monocytic pleocytosis is a rare finding Increased number of monocytes occurs with

increased number of other cells, mixed pleocytosis

Mixed Pleocytosis Present in chronic bacterial meningitis Tubercular or fungal meningitis Rupture of a cerebral abscess

Macrophages Originate from monocytes Not a normal finding in CSF Present after CNS hemorrhage Seen with phagocytized RBC, digested RBC, and

hemosiderin. Increased in tuberculotic and mycotic

meningitis, acute intracranial bleeding, contrast media

Others Cells Ependymal Cells

Normally increased in neonates Increased in children with

hydrocephalus Choroidal Cells

Increased after traumatic brain injury, pneumoencephalography, surgery, myelography, ischemic infarction of the brain, ventricular shunts, and intrathecal injections

CSF Protein Derived from plasma proteins that are transported

across endothelial cells of the choroid plexus and meninges and from intrathecal synthesis

Normal Values: 15mg/dL - 45mg/dL Neonates and Adults >40 years old (exhibit higher

protein concentrations) Protein levels collected from lumbar puncture are

higher. Protein levels collected from ventricles are lower. Contains small amounts of IgG Methods for Determining CSF Proteins: Most are performed in Chemistry sections

A. Dye-binding B. Immunochemistry C. Modified Biuret Method D. Turbidimetric Method

Increased in: Endocrine disorders Hemorrhage Multiple Sclerosis Obstruction Traumatic Tap Toxic conditions Meningitis

Decreased in: Hyperthyroidism Loss of fluid from a dural tear Sudden loss of CSF volume

Increase absorption by arachnoid villi Leakage of fluid from CNS

CSF Albumin One of the specific proteins evaluated in CSF Derived from transport across blood-brain barrier

CSF IgG Can be increased due to increased plasma

protein transport IgG that are specifically increased in intrathecal

synthesis can be identified using albumin. Normal Ratio = 1:369 Normal Index = 3-8

Decrease index: compromised blood-brain barrier

Increased index: multiple sclerosis inflammatory neurologic disorders increased intrathecal production

Chemical Analyses Lab tests commonly performed on CSF include:

Protein Electrophoresis Glucose and Lactate

Protein Electrophoresis Performed on concentrated CSF Identify and evaluate CSF protein fractions Detect oligoclonal bands:

Guillian-Barre syndrome HIV type 1 Acute Necrotizing Encephalitis Bacterial, Viral, and Cryptococcal

Meningitis Neurosyphilis Subacute Sclerosing Panencephalitis

CSF Protein Bands: Transthyretin or Prealbumin Albumin Beta Gamma Transferrin Alpha-1-Antitrypsin Tau Transferrin

*Otorrhea and Rhinorrhea - having tau transferrin band in the fluid from the ear or nose

Myelin Basic Protein (MBP) Myelin sheath

provides proper nerve function MBP

present in CSF in demyelinating diseases

Normal Values: <4ng/mL Increased in:

Multiple sclerosis Acute exacerbations of multiple sclerosis Trauma Hypoxia Myelopathy Chemotherapy

CSF Glucose CSF glucose level accounts for 60% of the

serum glucose value, 80% in newborns Reference range: 40-45 mg/100ml

it is rare to find values below 45mg/100ml

Takes 2 ½ hours for maximum change in CSF values after a change in serum values

Only decreased in CSF glucose is given more clinical significance

Decreased CSF Glucose: cause d by acute bacterial meningitis. The real major problem is the effect of

blood glucose levels on CSF glucose levels.

Decrease in CSF values is masked by the elevation of blood glucose levels

Determination of blood glucose level must be done at the same time the CSF specimen was obtained.

Low CSF glucose may be due to peripheral blood hypoglycaemia

Extensive involvement of meninges by metastatic carcinoma

Patterns of CSF Abnormality: Cell Type & Glucose Level

Polymorphonuclear:1. Low Glucose

Acute bacterial meningitis 2. Low or Normal Glucose

Some cases of early phase acute bacterial meningitis

Primary amebic (Naegleria species) meningoencephalitis

Early phase Lepstospira meningitis

3. Normal Glucose Brain abscess Early phase coxsackievirus and

echovirus meningitis CNS syphilis (some patients) Acute bacterial meningitis with IV

glucose therapy Listeria (about 20% of cases) Lymphocytic:

4. Low Glucose Tuberculosis meningitis Cryptococcal (Torula) meningitis Mumps meningoencephalitis

(some cases) Meningeal carcinomatosis (some

cases) Meningeal sarcoidosis (some

cases) Listeria (about 15% of cases)

2. Normal Glucose Viral meningitis Viral encephalitis Postinfectious encephalitis Lead encelopathy CNS syphilis (majority of patients) Brain tumor (occassionally) Leptospira meningitis (after the early phase) Leptospira (about 15% of cases)

CSF Lactate neonates 0-2 days old had mean values nearly

60% higher than those obtained after 10 days of life

neonates aged 2-10 days old only had 25% higher levels than after 10 days

Normal persons and those with viral (“aseptic”) meningitis do not have elevations

Increased CSF Lactate Acute bacterial, tuberculous, and fungal

meningitis In viral meningitis, the elevation is less than

twice the reference upper limits Lactate levels remain elevated for 2-3 days CNS tissue destruction from various causes

(including brain tumor, head trauma, CVAs, and intracerebral hemorrhage, cerebral hypoxia, and seizures) may also produce elevated CSF levels

CSF Lactate Assay Sensitivity range: 66-100% Test for acute bacterial meningitis: 93-95%

sensitive

Microbiological Procedures Detection of meningitis is among the most

serious diagnosis made on CSF Stains Cultures Immunologic tests

Stains CSF is concentrated using standard centrifugation

or cytocentrifugation. Cytocentrifugation results in a higher yield of

microorganisms. Gram stain

-demonstrate 60 – 90% sensitivity Wright stain Ziehl-Neelsen and fluorescent rhodamine

stain -used to stain M. tuberculosis

India Ink or Nigrosin stain -best detects C. neoformans (25 – 50% sensitivity)

Cultures Sediment of centrifuged CSF is inoculated into

thioglycolate broth, BAP, CAP and MacConkey agar.

Strips of X-V are applied to BAP if Haemophilus is suspected

Sabouraud dextrose agar : if fungal meningitis is

suspected Middlebrook broth and agar:

if Mycobacteria is suspected

Microorganisms most commonly responsible for causing meningitis:

Haemophilus influenza Neisseria meningitidis Streptococcus pneumonia Klebsiella sp. Less frequently caused by: Staphylococci M. tuberculosis Other Streptococci C. Neoformans Listeria monocytogenes Leptospira Coliform bacteria Anaerobic bacteria Amoebae Parasites

Immunologic Tests provide a rapid method for detection of

meningitis-causing agents Sensitivity and specificity vary among assays. Immunologic tests do not replace microbiologic

stains and cultures as standard procedure

Methods used in immunologic testing: Coagulation Counterimmunoelectrophoresis Enzyme-linked immunosorbence Fluorescent treponemal antibody test Latex agglutination RIA Venereal Disease Research Laboratory (VDRL)

Molecular techniques (e.g. PCR) - used for identification of infectious agents in body fluids.

Synovial Fluid

Joint fluid

- “synovial” – because of its resemblance to egg white- Viscous, mucinous substance- Lubricates most joints- Analysis is important in the diagnosis of joint disease- Aspiration of joint fluid = joint effusion or inflamed joints *helpful for patients with gout and pseudogout

Physiology and Compostion

- Synovium: all joints are lined with a tissue except those that are weight bearing : produces synovia (synovial fluid)

- This fluid capsules cushions diarthrotic joints allowing them to freely articulate- Dense connective tissue layer of collagen surrounds the synovial capsule- Synovial fluid: ultrafiltrate or dialysate of plasma : contains levels of glucose and uric acid (equivalent to plasma levels)

: protein = synovial < plasma (about 1/3 less then that in plasma)

- Plasma constituents must cross a double-barrier membrane to enter joint fluid *Endothelial lining of capillaries

Matrix surrounding the synovial cells

- Ultrafiltrate is combined with a mucopolysaccharide: hyaluronate *synthesized by the synovium

Specimen Collection

- (+) bugle test

Arthocentesis: aspiration of effected joint

: two-step process

1. first puncture is made through the skin2. second puncture into the synovial capsule

:Tubes: Heparinized – cell counts

Sterile tubes – microbiology

Plain tubes – chemistry and immunology testing

- specimens should be handled like STAT specimens - delivered immediately to avoid: alteration of chemical constituents, cell lysis, problems in microorganism detection and identification- glucose test = fasting for at least 6 hours prior to collection *necessary to establish an equilibrium between

plasma and joint glucose levels

Laboratory Testing- Volume : usually very small amount

: knee joint = up to 4mL of fluid- Color and Clarity : colorless and clear :other appearances may indicate various disease Yellow/Clear – non-inflammatory processes Yellow/Cloudy – inflammatory processes

White/Cloudy – may contain crystals Red, brown or xanthrohromic – hemorrhage : Inclusions Rice bodies – free-floating aggregates

- rheumatoid arthritis- degenerated synovium (enriched with fibrin)

Ochronotic shards – debris from metal and plastic joint prosthesis

- look like ground pepper- Viscosity : may be very viscous = high concentration of polymerized hyaluronate : evaluated by a string test

- Remove the needle from syringe Expressed into a test tube one drop at a time

Normally: Synovial fluid will form a “string” approximately 5 cm long before breaking. Fluid may cling to side of tube. *poor viscosity = shorter strings <3cm; like water = indicative of inflammatory response

- Clotting : clotting can result if fibrinogen is present : fibrinogen may enter the synovial capsule during damage to the membrane or traumatic tap : interfere with performance of cell counts : heparin to avoid clotting- Mucin clot : mucin clot test also known as Rope’s test

- Estimation of integrity of the hyaluronic acid-protein complex (mucin)- Normal: formation of a tight ropy clot upon addition of acetic acid- A good mucin clot indicates good integrity of hyaluoronate- Poor mucin clot (breaks up easily) = associated with destruction or dilution of hyaluronate

Chemical Examination

- Protein : contains all proteins found in plasma Exception: various high molecular weight proteins: fibrinogen, beta 2 microglobulin, alpha 2 macroglobulin (may be present or

absent in small amounts)

: serum protein procedures can be used for synovial fluid

NV: 1-3 g/dL

Increased: ankylosing spondylitis, arthritis, arthropathies that accompany Crohn disease, gout, psoriasis, Reiter syndrome and ulcerative colitis

- Glucose : interpreted using serum glucose levels : fasting specimen should be used (or at least 6-8 hours postprandially)

NV: less then 10 mg/dL lower then serum levels

Decreased: infectious joint disorders (20-100mg/dL less then serum levels), other joint disorders (0-20mg/dL less then serum)

- Uric Acid : diagnosis of gout : crystal identification is use for determination

NV: 6-8 mg/dL

- Lactic Acid : rarely measured : diagnosis of septic arthritis

NV: less then 25 mg/dL

Increased: septic arthritis (as high as 1000 mg/dL)

- Lactate Dehydrogenase : can be elevated in synovial fluid but normal in serum levels

: neutrophils are increased in acute phase of these disorders

Increased: RA, increased arthritis, gout

- Rheumatoid factor : antibody to immunoglobulins : present in the serum of patients with RA

: may only be produced by joint tissue, synovial fluid RF may be positive while serum RF is negative

: false-positive RF: chronic inflammatory disease

Microscopic Examination of Synovial Fluid

Cell Counts

- Should be performed within 1 hour of collection- Hemacytometer and manual differentials are usually performed- Saline may be used as a diluent for synovial flyids with a high number of cells- Diluents used when RBCs are present: Hypotonic saline, weak acid, commercial diluents- Instruments are available to automate counts- Cytocentrifugation for Wright staining

Differential

- Normal: contains small amounts of lymphocytes and only a few neutrophils- WBC NV: 0-150 cells per microliter- Mean distribution of these nucleated cells Neutrophils 7% vacuolated, contain bacteria, crystals

Lymphocytes 24%

Monocytes 48%

Macrophages 10%

Synovial lining cells 4% not clinically significant

- Cells may exhibit pyknotic nuclei or karyohexxis- Other cells may be seen: plasma cells, eosinophils, LE cells

- may indicate presence of joint disease- eosinophils: allergic disease with arthritis, hemorrhagic joint effusions, Lyme disease, parsitic arthritis, rheumatoid diseases and

tubercular arthritis- Increase in neutrophils: septic arthritis : later stages of RA (With inclusions containing immune complexes such as IgG, IgM, complement and RF. They appear

to have dark cytoplasmic granules and are called RA cells or ragocytes

Increase in lymphocytes: early stages of RA

Increase in monocytes: arthritis associated with serum sickness, viral infections and crystal induced arthritis

- LE cells : in 10% of patients with SLE and some in RA : large neutorphils that have engulfed a nucleus of a lymphocyte that has been altered by antinuclear antibody

- Tart cells : monocytes that have engulfed nuclear material : may be confused with LE cells

- Reiter cells: neutrophil-laded macrophage : seen in Reiter syndrome but not specific for it

- Lipophages : result of lipids released from bone marrow after injury to the boneCrystals

- Routine test for most laboratories- Used to diagnose gout = presence of monosodium urate crystals

- Thin, needle like crystals

- Polarize light and negatively birefringent *crystals aligned with compensator filter = yellow Lying perpendicular to filter = blue

- Calcium pyrophosphate dehydrate : pseudogout : smaller and rhomboid or rodlike : polarize light and positively birefringent *aligned with compensator filter = blue Lying perpendicular to the filter = yellow- Corticosteroid crystals : needle-shaped : intra-articular effusion: osteoarthritis or RA

- Apatite crystals: small chunky rods : calcific periarthritis, osteoarthritis and inflammatory arthritis

Microbiologic Examination

- Bacteria (most common), fungi, Mycobacteria and viruses can enter synovial fluid - Microorganims enter the synovial capsule through:

1. bloodstream2. deep penetrating wounds3. rupture of osteomyelitis into the join4. arthroscopy, intra-articular steroid injections, prothetic joint surgery [Bacteria]

- Gram stain = smears prepared by centrifugation or cytocentrifuation- Diluting with saline helps separate cells - Aerobic and anaerobic cultures should be performed- Gram stains are positive in only 50% of those with joint sepsis

Classification of Synovial Fluids

Group Category Visual Viscosity Mucin Clot

Cell Count Glucose Blood:SF

Others

Normal Colorless – straw clear

High Good <150 WBCs <25% neutrophils

0-10

I Noninflammatory Yellow, Slightly cloudy

Decreased Fair <-1000 WBCs <30% neutrophils

0-10

II Inflammatory White, gray, yellow, Cloudy, turbid

Absent Poor <100,000 WBCs >50% neutrophils

0-4

III Septic White, gray, yellow or green, cloudy, purulent

Absent Poor 50,000 – 200,00 WBCs >90% neutrophils

20-100 Positive Cultures

IV Crystal Induced White, Cloudy, turbid, opaque, milky

Absent Poor 500 – 200,000 WBCs <90% neutrophils

0-80 Crystals present

V Hemorrhagic Sanguinous, xanthochromic, red or brown, cloudy

Absent Poor 50-10,000 WBCs <50% neutriphils

0-20 RBCs present