CSF & Synovial Fluid

Espiritu Geronimo Guittap Manuel Rivera

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

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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: Chemistry 4. 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 4C

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
   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. 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

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

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Specimen Collection
 Lumbar Puncture (Lumbar TAP)  Contraindication to performing this puncture in the presence of infection at the puncture site

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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 

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Increase absorption by arachnoid villi Leakage of fluid from CNS

Myelin Basic Protein (MBP)

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

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CSF IgG
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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

Chemical Analyses
 Lab tests commonly performed on CSF include:  Protein  Electrophoresis  Glucose and  Lactate

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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

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)

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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)

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

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

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Specimen Collection
(+) bugle test Arthocentesis: aspiration of effected joint : two-step process 1. first puncture is made through the skin 2. 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

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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 Monocytes Macrophages 24% 48% 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

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Lipophages : result of lipids released from bone marrow after injury to the bone Crystals Routine test for most laboratories Used to diagnose gout = presence of monosodium urate crystals - Thin, needle like crystals

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- 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

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Microbiologic Examination Bacteria (most common), fungi, Mycobacteria and viruses can enter synovial fluid Microorganims enter the synovial capsule through: 1. bloodstream 2. deep penetrating wounds 3. rupture of osteomyelitis into the join 4. 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 Normal I II Noninflammatory Inflammatory Visual Colorless – straw clear Yellow, Slightly cloudy White, gray, yellow, Cloudy, turbid White, gray, yellow or green, cloudy, purulent White, Cloudy, turbid, opaque, milky Sanguinous, xanthochromic, red or brown, cloudy Viscosity High Decreased Absent Mucin Clot Good Fair Poor Cell Count <150 WBCs <25% neutrophils <-1000 WBCs <30% neutrophils <100,000 WBCs >50% neutrophils 50,000 – 200,00 WBCs >90% neutrophils 500 – 200,000 WBCs <90% neutrophils 50-10,000 WBCs <50% neutriphils Glucose Blood:SF 0-10 0-10 0-4 Others

III

Septic

Absent

Poor

20-100

Positive Cultures

IV

Crystal Induced

Absent

Poor

0-80

Crystals present RBCs present

V

Hemorrhagic

Absent

Poor

0-20