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					CEREBROSPINAL FLUID
     DR.P.G.KONAPUR
  VMKV MEDICAL COLLEGE
          SALEM
•   Introduction
•   Specimen collection: LP Technique
•   Complications of LP
•   Routine examination of CSF.
•   Physical examination
•   Chemical examination
•   Cytological examination
•   Microbiological examination

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• found in the subarachnoid space
  surrounding the brain and spinal cord..
• an ultrafiltrate of plasma
• protects the central nervous system from
  injury




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• Spinal needle - 22 gauge:
• AGE& Length of needle:
      Less than 1 year--3.75 cm (1.5 inch)
      1 year to middle childhood--6.25cm (2.5 inch)
      Older children to adolescents--8.75 cm (3.5
    inch)
•   Povidone-iodine solution.
•   1% Lidocaine and 25 gauge needle for local
    anesthesia.
•   Sterile 4 x 4 gauze.
•   3-4 sterile specimen tubes.
•   For viral cultures: an additional tube
•   CSF manometers and 3 way stopcock.
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Use Sitting Position:
Patients with pulmonary disorders.
Young infants




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INDICATIONS
• Meningitis and encephilitis--viral, bacterial,
  fungal, or parasitic infections.
• metastatic tumors (e.g., leukemia) and
  central nervous system tumors that shed
  cells into the CSF
• Syphilis
• bleeding (hemorrhaging) in the brain and
  spinal cord
• Guillain-Barré-- a demyelinating disease
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COMPLICATIONS
•   Post-tap headaches.
•   Vomiting.
•   Paralysis (low risk)
•   Subarachnoid epidermal cyst.
•   Epidural hematomas.
•   Subdural or subarachnoid hemorrhage.
•   Spinal cord bleeding.
•   Acute neurologic or respiratory deterioration.
•   Hypoxemia or apnea
•   Cerebral herniation.
•   Introduction of infection with resultant bacterial
    meningitis, epidural abscess, diskitis or osteomyelitis.
    (low risk)
•   Ocular muscle palsy. (transient)
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ROUTINE EXAMINATION
Physical examination:

• Normal CSF is clear and colourless,

• specific gravity is 1.0032.

• Colour – Red colour is seen due to trauma occurring
    during L.P
•    yellow colour called xanthochromia
      is due to previous hemorrhage with lysis of RBC’S in
    the CSF and due to tumour.
•   Turbidity – or cloudiness is seen when

              increase in number of cells in CSF ( ie 400 –
    500/ul)
                           or
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              numerous bacteria
• Coagulum: protein content is increased.

• tuberculous meningitis (cobweb coagulum
 is seen)




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• Chemical examination:
     Glucose
           two-thirds of the fasting plasma glucose.
           A glucose level below 40 mg/dL is significant
                bacterial and fungal meningitis and in malignancy..
    Protein
        High levels -------
•                     bacterial
•                     fungal meningitis,
•                     tumors,
•                     subarachnoid hemorrhage,
•                     traumatic tap.
    Lactate
               bacterial and fungal meningitis V/S viral meningitis
               bacterial and fungal meningitis------ increased lactate,
               viral meningitis------------------------NORMAL
    Lactate Dehydrogenase
                elevated in
                             bacterial and fungal meningitis,
                             malignancy,
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                             subarachnoid hemorrhage.
Cytological Examination

Centrifuge
  smears from deposit
  stain -romanowasky
Cell Count

immediately
(pus cells stick to each other)
Method

count all 9 squares

Normal--- 0 – 5 lymphocytes per cubic mm.
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Neutrophils – increased
                      acute pyogenic meningitis.
 Lymphocytes-----increased
                      viral meningitis.,
                      syphilitic meningitis.,
                      tubercular meningitis.
                      fungal meningitis.

 RBCs : subarachnoid hemorrhage,
           stroke,
           traumatic tap
  Malignant cells:
        50 percent of--- metastatic cancers
        10 percent of------CNS tumors( shed cells
  into the CSF).
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Micobiological Examination:
Gram stain :on a sediment
Positive in---
               60 percent of cases of bacterial
                meningitis.
Culture: aerobic and anaerobic bacteria.
Other stains:
The Z-N for Mycobacterium tuberculosis,
Fungal culture:


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feature           normal        Ac.pyo.meni       Ac.viral    Chr.tb.meningit
                                ngitis            meningiti   is

Naked eye         Clear         Cloudy,frank Clear/slightt    Clear/slightturb
                  &colorless    ly purulent  urbid            id,cobweb
pressure          60-150        >180              >250        >300

Cell count,type   0-4lympho     10-               10-         100-
                                10,000neutr       100lympho   1000lympho
                                o
protein           15-45mg/dl    raised            raised      raised


glucose           50-80         reduced           normal      reduced


bacteria          sterile       +                 sterile     Tb bacilli+


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Serological examination:
Syphilis serology --neurosyphilis.
The fluorescent treponemal antibody-absorption
(FTA-ABS) test: positive
               !.with active and treated syphilis.
               !.used in conjunction with the VDRL
                  (for nontreponemal antibodies)

                 is positive-- in active syphilis,
                    negative in treated cases.




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PLEURAL FLUID ANALYSIS
•   Specimen collection Procedures
•    Diagnostic thoracentesis
•    Therapeutic thoracentesis
•   Tube thoracostomy  
•   Causes of pleural effusion
•   Difference between transudate and exudate
•   Routine examination of Pleural fluid.
•             Physical examination
•             Chemical examination
•               Immunological examination
•                Cytological examination
•   Algorythym for pleural effusion.
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• Diagnostic thoracentesis:
 @if the etiology of the effusion is unclear
 @if the presumed cause of the effusion does not
    respond to therapy as expected.
 @Pleural effusions do not require thoracentesis
 –
   underlying congestive heart failure(bilateral
 effusions)
 @by recent thoracic or abdominal surgery.
 @Relative contraindications:
            bleeding diathesis
            systemic anticoagulation,
            mechanical ventilation,
            cutaneous disease over site.
            Mechanical ventilation
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Complications:
• pain at the puncture site,
• cutaneous or internal bleeding,
• pneumothorax,
• empyema,
• spleen/liver puncture
• Pneumothorax -12-30% of
  thoracenteses( requires treatment with a
  chest tube in less than 5% of cases)
• Use of needles larger than 20 gauge
  increases the risk of a pneumothorax
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Therapeutic thoracentesis

• to remove larger amounts of pleural fluid




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• DIFFERENCES BETWEEN A
  TRANSUDATE AND A EXUDATE
• CHARACTERISTICS TRANSUDATE
• TRANSUDATE CLEAR,
• STRAW – YELLOW
• Sp gr:< 1.018
• PROTEIN :< 2G/DL

• INFLAMMATORY CELLS :LOW COUNT

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• EXUDATE
• Appearance:CLOUDY MAY BE CLOTTED
• Colour: YELLOW TO RED
• Sp gr:> 1.018
• Protein:> 2G/DL
• INFLAMMATORY CELLS: HIGH COUNT

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• Physical examination:
• 1. Volume: Measure and record the
  volume of fluid received.
• Appearance, colour, clot formation: Note
  colour whether clear or cloudy, whether
  clot is formed on standing



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• Chemical examination:
• Protein estimation:
• Glucose estimation:




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• Immunological studies:
• ANA titres are useful in diagnosing
 effusion due to SLE,and rheumatoid factor
 is commonly present in pleureal effusion
 associated with sero positive rheumatoid
 arthritis



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• Immunological studies:
• ANA titres are useful in diagnosing
  effusion due to SLE,
• rheumatoid factor is commonly present in
  pleureal effusion associated with sero
  positive rheumatoid arthritis



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NEUBAUER COUNTING
CHAMBER




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Count in the four corners




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Count in the four corners




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Cells in one corner square




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MESOTHELIAL CELLS




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BENIGN MESOTHELIAL CELLS




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FOAMY MACROPHAGES




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INFLAMMATORY PLEURAL FLUID




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ACID FAST BACILLI




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CANDIDA IN PLEURAL FLUID




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Abnormal mitosis




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SMALL CELL CA




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METASTIC CA FROM BREAST




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ATYPICAL PLASMA CELLS




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ASCITIC FLUID ANALYSIS
• Specimen collection: Procedure Abdominal
  paracentesis fluid.
• Causes of Ascitis
• Routine examination of Ascitic fluid.
• Physical examination
• Chemical examination
• Cytological examination
• Microbiological examination
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SPECIMEN COLLECTION
• Abdominal paracentesis:
• The removal of 5 L of fluid is considered large-
    volume paracentesis.
•    Total paracentesis, ie, removal of all ascites
    (even >20 L),
•   Recent studies demonstrate that supplementing
    5 g of albumin per each liter over 5 L decreases
    complications of paracentesis, such as
    electrolyte imbalances, and increases in serum
    creatinine secondary to large shifts of
    intravascular volume

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CAUSES FOR ASCITIS
•   alcoholic liver disease.

• Obesity,                       steatosis        cirrhosis

• hypercholesterolemia         steatosis          cirrhosis

• type 2 diabetes mellitus       steatosis         cirrhosis


• cancer, (especially gastrointestinal cancer)      malignant
    ascites.




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•Portal hypertension (serum-ascites albumin gradient
[SAAG] >1.1 g/dL)
   •Hepatic congestion,
   •congestive heart failure,
   •constrictive pericarditis,
   • tricuspid insufficiency,
   •Budd-Chiari syndrome
   •Liver disease,
            cirrhosis,
            alcoholic hepatitis,
            fulminant hepatic failure,
            massive hepatic metastases
         Hypoalbuminemia (SAAG <1.1 g/dL)
            Nephrotic syndrome
            Protein-losing enteropathy
            Severe malnutrition with anasarca
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     Miscellaneous conditions (SAAG <1.1 g/dL)
        • Chylous ascites
        • Pancreatic ascites
        • Bile ascites
        • Nephrogenic ascites
        • Urine ascites
        • Ovarian disease
•   Diseased peritoneum (SAAG <1.1 g/dL)
•      Infections
       • Bacterial peritonitis
       • Tuberculous peritonitis
       • Fungal peritonitis
       • HIV-associated peritonitis



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  Malignant conditions :

  •Peritoneal carcinomatosis
  •Primary mesothelioma
  •Pseudomyxoma peritonei
  •Hepatocellular carcinoma

Other rare conditions:

  •Familial Mediterranean fever
  •Vasculitis
  •Granulomatous peritonitis
  •Eosinophilic peritonitis.

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Routine examination

PHYSICAL EXAMINATION:
•     transparent and tinged yellow.
•     A minimum of 10,000 red blood cells/µL is required
  for ascitic fluid to appear pink,
•    more than 20,000 red blood cells/µL is considered
  distinctly blood tinged.
 a traumatic tap or malignancy.
         Bloody fluid from a traumatic tap is
  heterogeneously bloody, and the fluid will clot.
        Nontraumatic bloody fluid is homogeneously red
  and does not clot because it has already clotted and
  lysed.
        Neutrophil counts of more than 50,000 cells/µL
  have a purulent cloudy consistency and indicate
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  infection.
Chemical examination:
  SERUM-ASCITES ALBUMIN GRADIENT (SAAG):
  The SAAG ascites into portal hypertensive (SAAG >1.1
      g/dL) and non–portal hypertensive (SAAG <1.1
      g/dL) causes.
  Calculated by subtracting the ascitic fluid albumin value
      from the serum albumin value, it correlates directly
      with portal pressure.
  TOTAL PROTEIN:
  In the past, ascitic fluid ---an exudate (if the protein
      level is greater than or equal to 2.5 g/dL). However,
      the accuracy is only approximately 56% for
      detecting exudative causes.
  The total protein level +SAAG.
  An elevated SAAG and a high protein           ascites due to
      hepatic congestion.
  Those patients with malignant ascites            have a low
      SAAG and a high protein level.
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Cytological examination:
 Cytology:
 58-75% sensitive           Mal cells
sediment is smeared on slides.

Papanicolaou stain and Leishman stains

A cytospin preparation can be used for clear fluid.

A cell block may also be prepared if adequate
 sediment is available.

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• Cell count:
• Normal <500 leukocytes/µL
         < 250 polymorphonuclear
 leukocytes/µL.

 A neutrophil count > 250 cells/µL - highly
 suggestive of bacterial peritonitis.

 In tuberculous peritonitis
           &
  peritoneal carcinomatosis ______ a
 predominance of lymphocytes usually occurs.


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Microbiological examination:


CULTURE/GRAM STAIN:

 The sensitivity with bedside inoculation of
 blood culture bottles with ascites results in
 92% detection of bacterial growth in
 neutrocytic ascites.

AFB stain may be done if required.

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MESOTHELIAL CELLS




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MALIGNANCY IN ASCITIC FLUID




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PERICARDIAL FLUID
EXAMINATION

• Causes of pericardial fluid accumulation.

• Routine examination of pericardial fluid.
• Physical examination
• Chemical examination
• Cytological examination
• Microbiological examination
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Pathophysiology of pericardial
effusion:
• The pericardial space normally contains 15-50 mL of
    fluid,
•   Lubrication------ for the visceral and parietal layers
•   originate from the visceral pericardium
      an ultrafiltrate of plasma.
      Total protein levels are generally low
•   The cause of abnormal fluid production
    ---------underlying etiology
      secondary to------ pericarditis.
    1.Transudative ------obstruction of drainage(lymphatics)

    2. Exudative --------- inflammatory
                           infectious
                           malignant
                           autoimmune processes within the
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• CAUSES OF PERICARDIAL FLUID
    ACCUMULATION
•   Infectious
    – Viral (coxsackievirus A and B, hepatitis, HIV)
    – Pyogenic (pneumococci, streptococci, staphylococci,
      Neisseria, Legionella species)
    – Tuberculous
    – Fungal (histoplasmosis, coccidioidomycosis, Candida)
    – Other infections (syphilitic, protozoal, parasitic)
• Noninfectious
    –   Acute idiopathic
    –   Uremia
    –   Neoplasia
         • Primary tumors (benign or malignant, mesothelioma)
         • Tumors metastatic to pericardium (lung and breast cancer,
           lymphoma, leukemia)
    – Myxedema
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  –   Acute myocardial infarction
  –   Postirradiation
  –   Aortic dissection (with leakage into pericardial sac)
  –   Trauma
  –   Cholesterol
  –   Chylopericardium
  –   Familial Mediterranean fever
  –   Whipple disease
  –   Sarcoidosis
• Hypersensitivity or autoimmunity related :
  – Rheumatic fever
  – Collagen vascular disease (systemic lupus
    erythematosus, rheumatoid arthritis, ankylosing
    spondylitis, scleroderma, acute rheumatic fever,
    Wegener granulomatosis)
  – Drug-induced (eg, procainamide, hydralazine,
    isoniazid, minoxidil, phenytoin, anticoagulants,
    methysergide)
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ROUTINE EXAMINATION

• Physical examination:
• Colour. Clot formation. Specific gravity:
 Altered colour is seen in Bacterial
 pericarditis,Tuberculosis, SLE, Rheumatoid
 pleuritis, Lymphoma, carcinoma.




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Chemical examination:


• Includes test for glucose and proteins




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Cytological examination:

• Includes WBC count
• RBC count,
• Differential count
• malignant cells.




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Microbiological examination

• Gram”s stain

• AFB stains

• Pericardial fluid culture


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SUMMARY
• Normally -10-50 ml
• excess fluid -----pericardial effusion.
• Fluid is obtained by using a sterile needle
  under aseptic precaution called as
  pericardiocantisis.
• Physical examination
• chemical examination
• Microbiological examination
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SYNOVIAL FLUID ANALYSIS

• Specimen collection: Procedure

• Causes of Synovial fluid accumulation

• Routine examination of Synovial fluid


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SIGNIFICANCE
• Synovial fluid is found around the joint.
• Chemical composition: is similar to that of other
  body fluids except it has hyaluronic acid.
• Hyaluronic acid ----mucodysacchride that
  acts as a binding and protective agent for
  connective tissue.
CLINICAL SIGNIFICANCE:
• Diagnosis of – Arthritis
                 Gout
                 Infection (septic arthritis)
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SPECIMEN COLLECTION


Obtained by aspiration of a joint

Anticoagulant (EDTA) :-----cell counting

Fluoride:---------------------- glucose analysis


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ROUTINE EXAMINATION
 PHYSICAL EXAMINATION:
• APPEARANCE – Normal synovial fluid –
  straw coloured celar and viscous
• TURBIDITY – Increase in case of
  inflammatory and infected conditions.
• Grossly Purulent fluid with an increased
  leucocyte count is typical of acute Septic
  arthritis.
• XANTHOCHROMIA – Supernatent
  synovial fluid indicates – Tumours,
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• VISCOSITY – Synovial fluid is viscous ----
 hyaluronic acid.

• INFLAMMATORY DISORDERS of the
 joint rendor an enzymatic (hyaluronidase)

   Breakdown of hyaluronic acid

   Loss of viscosity of synovial fluid


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TESTS
• STRING TEST –
 Hold a drop of specimen between thumb
 and index finger.
 A drop of normal synovial fluid will form a
 string.
 4 –6cm in length_______normal
 <3cm__________viscosity is lower than
 normal

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• MUCIN CLOT TEST-
  Clots in the presence of acetic acid. If
 there is breakdown of hyaluronic acid does
 not allow the formation of firm clot.
 PROCEDURE – Synovial fluid is added
 drop by drop in a dilute solution of acetic
 acid.
  firm clot---------- Normal and non
 inflammatory conditions
  poor clot------ inflammatory conditions
 (Hyaluronic acid content decreases)

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CHEMICAL EXAMINATION:
• GLUCOSE – Synovial fluid for glucose
 ANALYSIS - Must be taken from a fasting patient (6 –
 12 hrs) and treated with fluoride
 Samples of the patient synovial fluid and blood specimen
 must be obtained at the same time for a comparison of
 two values.

 In case of non inflammatory arthritis, the difference of
 blood glucose and synovial fluid glucose is only 10mg/dl

 Increase to 25 – 50mg/dl in case of infectious septic
 arthritis

 In mild inflammatory conditions (gout pseudogout
 Rheumatoid arthritis)

 Glucose content of synovial fluid is close to normal.
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MICROSCOPIC EXAMINATION
• Total leucocyte count
• Differential count is important for
  diagnosis of joint related disorders.
• Leucocyte count of normal synovial fluid is
  very low (50 cells/cu mm). If specimen
  turbid saline containing methylene blue
  are diluent
• If specimen bloody, haemolyse the
  erythrocytes by diluting with O 1N Hcl or
  1% saporin in saline. Smear the slide.
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NORMAL SYNOVIAL FLUID

• NORMAL SYNOVIAL FLUID – Has a
  few mononuclear white cells
• Increased neutrophil count (>70 %) is
  suggestive of bacterial arthritis
• In inflammatory disorders white cell count
  is moderately high (>10m000/cu mm)



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MICROSCOPIC EXAMINATION
OF CRYSTALS

• Clear a slide and a coverslip with alcohol and acetone.
• Place a few drops of synovial fluid on the slide just
    sufficient enough to reach the periphery of the cover
    slip.
•   Needle shaped intracellular urate crystals (sodium and
    urate) - Gouty arthritis
•   Rhomboid calcium pyrophosphate crystals in
    pseudogiant
•   Rheumatoid arthritis – Cholesterol crystals

• Recognized by their flat, clear rhombic appearance with
    one corner punched out
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• MICROBIOLOGY EXAMINATION:
 Gram staining and acid fast staining.




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Lab Test N                Non          Inflammatory                   Infection
                          inflam
                          matory
APEARANC   Clear Yellow   Clear        Mild            Severe         Turbid to
E                         Yellow                                      purulent



VISCOSIT   High           High         TurbidDecreas   turbid         Decreased
                                       ed              Decreased
Y
LEUCOCY    0 – 200        0- 5,000     0- 10,000       500 – 50,000   500-20,000
TE /cu
mm
NEUTROP    0 – 25         0- 25        0- 50           0 – 90         40-100
HILS %

GLUCOSE    0 – 10         0- 10        0- 20           0 – 40         20-100
(mg/dl)
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