SYNOVIAL FLUID

Physiology and Purpose of Examination

 Synovium

- Tissue lining synovial tendon sheaths, bursae, and diarthrodial joints except for the articular surface. - One to three cell layers: surface overlying fatty, fibrous, or periosteal joint tissue.
Synovial

Fluid

joints.

- Viscous liquid found in the cavities of the movable

- Imperfect ultrafiltrate blood plasma and hyaluronic acid (synoviocytes) - synoviocytes secrete the hyaluronic acid and a small amount of protein into the fluid - Lubrication and adhesion - Provides nutrients for the avascular articular cartilage

 Examination

- Arthritis - Acute or chronic inflammation of a joint

of Synovial Fluid

- Having diverse causes, such as: infection, crystal deposition or injury - Causes deformities much like the ones seen in the xray.
Arthrocentesis

- Often accompanied by pain and structural changes

- Needle aspiration of synovial fluid

Purpose

- Differentiate infectious from noninfectious arthritis - Classify pathological significance of joint disorders

of Examination

classifications
- Non inflammatory - Inflammatory - Septic - Hemorrhagic

 Specimen

Collection and Handling

Arthrocentesis Volume not always constant, should be recorded TUBES USED:

- Sterile heparin tube (Gram stain and culture)

- Heparin or EDTA tube (cell counts) - Nonanticoagulated tube (chemical and serological tests) - Sodium Fluoride tube (glucose analysis)
Powdered anticoagulants should not be used. They produce artifacts that may interfere with crystal analysis. NORMAL SYNOVIAL FLUID does not clot diseased joints may contain fibrinogen, which will clot Aspirate using syringe moistened with heparin

Color

Normal color: colorless to pale yellow Pale yellow diapedesis Straw to yellow xanthochromia

and Clarity

White blood cells escape from the vessels and enter the synovium
Indicative of synovial hemorrhage

Deeper yellow noninflammatory and/or inflammatory effusions Greenish tinge bacterial infection Red traumatic tap versus hemorrhage Note distribution of blood to distinguish
Traumatic tap uneven, receding distribution of blood in tubes Hemorrhage even, consistent distribution of blood across tubes

Red/brown, post-centrifuge pathologic hemarthrosis (bleeding into the joints)

 Synovial

comes from the Latin word for egg.

Normal consistency: resembles the viscosity of egg white Turbidity presence of WBCs, synovial cell debris and fibrin Milky presence of crystals Oily, shimmering cholesterol crystals

 Viscosity

From the polymerization of hyaluronic acid For proper lubrication of the joints
Note: Arthritis affects both the production of hyaluronate and its ability to polymerize (decreasing viscosity)

Methods:
String

method form a string with the tip of the syringe, 46cm is normal. or mucin clot test chemically measures the amount of hyaluronate polymerization

Ropes

Cell Counts

EXTREMELY viscous specimens: pre-treated with a pinch of hyaluronidase per 0.5mL of fluid or 1 drop of 0.05% hyaluronidase in phosphate buffer per mL of fluid and incubate at 37C for 5mins.

Total Leukocyte Count Perform ASAP or refrigerate to prevent cellular disintegration

Manual counts use Improved Neubauer Clear specimens are counted without dilution. Turbid or bloody specimens are first diluted prior to counting. Diluents: - Normal saline - 0.3% hypotonic saline or saline with saponin for RBC lysis (when necessary) - Methylene blue may be added to stain WBC nuclei (separation from RBC)

Count 5 secondary squares (4 corners + center)

Abnormal cells
Cell/Inclusion
LE cells

Description
Neutrophil containing a characteristic ingested round body Vacuolated macrophages with ingested neutrophils Neutrophils with small dark cytoplasmic granules that consist of precipitated rheumatoid factor Resemble polished rice macroscopically, show collagen and fibrin microscopically Large, multi-nucleated cell Refractile intra- and extracellular globules Inclusions within clusters of synovial cells

Significance
Lupus erythematosus

Reiter cells RA cells or ragocytes

Reiter syndrome Nonspecific inflammation Rheumatoid arthritis Immunologic inflammation

Rice bodies

TB, septic and rheumatoid arthritis Osteoarthritis Traumatic injury Chronic inflammation Villonodular synovitis

Cartilage cells Lipid/Fat droplets Hemosiderin granules

 Crystals

useful in the diagnosis and evaluation of cases of arthritis formation causes acute or chronic pain and affects both bone and cartilage. causes of crystal formation: Metabolic disorders Decreased renal excretion Medications that are injected into the joints

Crystal

Main

Chemical Tests: Glucose Determination -Most frequently requested chemical test Blood and synovial fluid samples obtained simultaneously (correlation)
sample

- Synovial fluid is handled similar to serum - Standard: Nelson-Somogyi method - Requires 8 hours fasting - NV: <10mL lower than blood glucose

 Total

Protein Determination - NV: <3 g/dL - Increased in inflammatory and hemorrhagic disorders - Methods same as serum protein determinations - Refractometers used to estimate the synovial fluid protein concentration - Measurements are nonspecific

 Uric

Acid Determination - Elevated in gout - Confirms status if uric acid crystals are not demonstrated microscopically
Suspected but unconfirmed gout

- Correlated with serum uric acid. - Little clinical value other than correlation.

 Mucin

Clot Test - Semi-quantitatively grades viscosity - Add acetic acid to specimen to precipitate hyaluronidase into a mucin clot - Fair to poor ratings reflect dilution and depolymerization of hyaluronic acid nonspecific finding in several inflammatory arthrites

 MICROBIOLOGIC TESTS 2 important tests: Gram stain and culture - Most frequent infection of the synovial joints is bacterial by origin. - Routine bacterial cultures use enriched medium such as chocolate agar, which supports the Haemophilus species and Neisseria gonorrheae. SEROLOGIC TESTS - Important in joint disorder diagnosis (as in serologic testing for rheumatoid factor, systemic lupus erythematosus, etc) - Majority of serologic tests are performed on serum. - Testing on synovial fluid is only done as a confirmatory measure due to the sensitivity of the arthrocentesis procedure. - Associated Autoimmune Diseases: RA and SLE Cause serious inflammation of the joints Autoantibodies are found in both serum and synovial fluid

SEROUS FLUID

 Closed

Cavities of body Pleural Pericardial Peritoneal cavities Parietal membrane in between (serous fluid) provides lubrication
Visceral

membrane lines the organ within cavity

Lubrication

to prevent fiction between 2 membranes

 Formation

Serous fluid ultra filtrates of plasma Production and reabsorption hydrostatic and colloidal (oncotic) pressure capillaries serves cavities and the capillary permeability Normal : same colloidal pressure from serum proteins in the capillaries on both sides ( hydrostatic pressure in the parietal and visceral capillaries to enter between the membranes) Filtration of plasma oncotic pressure (favors reabsorption of fluid back into the capillaries) Slight different amount of positive pressure in the parietal and visceral capillaries creates small excess of fluid that is reabsorbed by the lymphatic capillaries located in the membranes Effusion: disruption of the mechanism of serous fluid formation and reabsorption that causes in fluid between the membrane

Pathologic causes of effusion

1. Capillary hydrostatic pressure Congestive heart failure Salt and fluid retention

1. Oncotic pressure Hypoproteinemia

Nephrotic syndrome Hepatic cirrhosis Malnutrition Protein-losing enteropathy

1. Capillary permeability Inflammation and infection

Microbial; infection Membrane inflammations Malignancy

Malignant tumor, lymphomas Infection and inflammation Thoracic duct injury

1. Lymphatic obstruction

 Specimen

- Needle aspiration Thoracentesis (pleural) Pericardiocentesis (pericardial) Paracentesis (peritoneal) - >100 ml - EDTA tube cell counts and differential - Sterile heparinized evacuated tubes microbiology and cytology - Centrifugation (better recovery of microorganisms ) - Chemistry test clotted specimens ( plain tubes or heparinized tubes) - pH maintained anaerobically in ice - Chemical test compared with plasma chemical concentrations - Blood specimens also collected

Collection and Handling

General classification of the cause of an effusion separating the fluid into category of :
Transudates Systemic disorder disrupt balance in regulation of fluid filtration and reabsorption - Hydrostatic pressure congestive heart failure Hypoproteinemia nephritic syndrome Directly involved Infections Malignancies

Exudates

 Classification

: for initial diagnostic step (NO testing for transudate fluids) Laboratory tests: Appearance Total protein most reliable Lactic dehydrogenase Cell counts Spontaneous clotting

determination: fluid-toblood ratio

 General

Evaluation of the appearance and differentiation between transudate and exudates Effusion of exudates presence of microbiologic and cytologic abnormalities RBC and WBC counts not performed on serous fluid (little diagnosis) WBC <1000/mircoliter = Transudate > 1000/mircoliter =exudates Serous cell counts manually ( Neubauer counting chamber) Differential counts Wrights-stained, cytocentrifuged specimens Examine: WBC, normal and malignant cell tissue

Laboratory Procedures

PLEURAL FLUID

 PLEURAL

FLUID

- Obtained from the pleural cavity, located between parietal pleural membrane lining the chest wall and the visceral membrane covering the lungs - Pleural fluid cholesterol >60mg/dl exudates - Fluid:serum cholesterol >0.3 - Pleural fluid:serum total bilirubin ratio 0.6 exudates

Pleural Fluid Appearance and Disease

Appearance Clear, pale yellow Turbid white (related to presence of WBC) Bloody Disease Normal Microbial infection(tuberculosis) Immunologic disorders : Rheumatoid arthritis Hemothorax ( traumatic injury occurs in malignancy or traumatic aspiration) Traumatic tap : streaked and uneven Hemorrhagic effusion, pulmonary embolism, Tuberculosis malignancy Chylous material from thoracic duct leakage Pseudochylous material from chronic inflammation Rupture of amoebic liver abscess Aspergillous Malignant mesothelioma (increase hyaluronic acid)

Milky

Brown Black Viscous

Difference of Hemothorax and Hemorhagic Exudates

Hemothorax Hematocrit >50% of the whole blood hematocrit effusion occurs from the INPOURING of blood from the injury Hemorhagic exudates <50% Chronic membrane disease: contains both blood and increased pleural fluid.

Differentiation Between Chylous Effusion and Pseudochylous Effusion

Chylous Effusion Cause Appearance Leukocyte Cholesterol crystals Triglycerides Sudan III staining Thoracic duct leakage Milky/white Predominantly Lymphocytes Absent >110 mg/dl Strongly positive High cholesterol conc. Pseudochylous Effusion Chronic inflammation Milky/green tinge Mixed cells Present <50mg/dl Negative/weakly positive Cholesterol crystals

 HEMATOLOGY TEST Differential Count - Most significant hematology test performed. ( also seen in pericardial and peritoneal fluids)
Neutrophils
Lymphocytes
Pneumonia Pancreatitis Pulmonary infarction Tuberculosis Viral infection Autoimmune disorder ( RA and SLE) Malignancy Trauma ( presence of air or blood) Pneumothorax and hemothorax Allergic reactions Parasitic infections Increase: pneumonia and malignancy Decrease : mesothelial cells are associated with Tuberculosis Primary concern of examination

Eosinophils

Mesothelial cells Malignant cells Plasma cells

Tuberculosis

Significance of Chemical Testing of Pleural Fluid

Test Significance

Glucose
Parallel plasma levels with values <60 mg/dl considered Fluid values vs. plasma values Considered in addition to glucose level

in rheumatoid inflammation in tuberculosis in purulent infection in bacterial infection in chylous effusions in pneumonia not responding to antibiotics Marked with esophageal rupture <pH 6.0 Allowing influx of gastric fluid

Lactate Triglyceride
To confirm the presence e of chylous effusion than pH7.0 ( need for chest-tube drainage) In cases of acidosis : pleural fluid pH should be compared to blood pH. Pleural pH at least 0.30 or lower than blood pH : SIGNIFICANT

Ph

ADA(adenosine deaminase)
40U/L Elevated 1st in the pleural fluid Salivary amylase

in tuberculosis and malignancy in pancreatitis, esophageal rupture and malignancy

Amylase

 MICROBIOLOGIC

TESTS Staphylococcus aureus Enterobacteriaceae Anaerobes Mycobacterium tuberculosis

Tests:
Gram stains Cultures ( aerobic and anerobic) Acid-fast stains

 SEROLOGICAL

TESTS To differentiate effusions of immunologic origin from noninflammatory processes. Tests : Antinuclear antibody (ANA) Rheumatoid factore (RF) Detection of tumor markers ( diagnostic information for malignant origin): Carcinoembryonic antigen (CEA) CA 125 ( metastic uterine cancer) CA15.3 CA 549(breast cancer)

PERITONIAL FLUID

 PERITONEAL

FLUID

Characteristics - An ultrafiltrate of plasma - 50 mL of fluid is normally present in this mesothelial-lined space - Normal peritoneal fluid is clear and pale yellow
*Ascites

pathologic accumulation of excess fluid in the peritoneal cavity

 TRANSUDATES

Transudates - Increased hydrostatic pressure or deceased plasma oncotic pressure - Low specific gravity(<1.012), low protein content - Cirrhosis, CHF, nephrotic syndrome Exudates - Increased capillary permeability or decreased lymphatic resorption - High specific gravity(>1.020), high protein content - Bacterial infections (peritonitis) and malignancy

AND EXUDATES

 Serumascites

albumin gradient

- Widely considered as the most reliable method to differentiate peritoneal transudates from exudates. - The serum albumin concentration minus the ascitic fluid albumin concentration - A difference of 1.1 or greater suggests a transudate effusion of hepatic origin - Lower than 1.1 are associated with exudative effusions.

 SPECIMEN

COLLECTION Paracentesis Minimum of 30 mL is needed for complete evaluation Samples for cell counts should be placed in an EDTA-anticoagulated tube Culture specimens inoculated at the bedside with ascitic fluid (10 mL per culture bottle)

 MICROSCOPIC

EXAMINATION

Normal WBC counts are less than 350 cells/uL An absolute neutrophil count greater than 250 cells/uL or greater than 50% of the total WBC count is indicative of infection. Lymphocytes are the predominant cell in tuberculosis. Eosinophilia (>10%) is most commonly associated with the chronic inflammatory process associated with chronic peritoneal dialysis.

CHEMICAL ANALYSIS
Chemical examination of ascitic fluid consists primarily of glucose, amylase, and alkaline phosphatase determinations.
GLUCOSE Decreased below serum levels in bacterial and tubercular peritonitis and malignancy Low sensitivity and specificity AMYLASE To ascertain cases of pancreatitis, and it may be elevated in patients with gastrointestinal perforations ALKALINE PHOSPHATASE An elevated level is highly diagnostic of intestinal perforation.

PERICARDIAL FLUID

 PERICARDIAL

FLUID

- Normally, 10-50 mL is present in the pericardial space - Pericardial effusions may be due to viral, bacterial or fungal infections, autoimmune disorders, renal failure, myocardial infarction, drugs or may be idiopathic - HIV patients have asymptomatic pericardial effusions

Specimen Collection

Pericardiotomy the process wherein the pericardial fluid is obtained Pericardiocentesis sterile needle aspiration Normal pericardial fluid is clear and pale yellow. Turbid infection is present Clear and straw colored uremia is present Bloody hemorrhagic effusion or due to aspiration of blood from the heart
Cause may be differentiated by observing clots, hematocrit and blood gas analysis

Aspiration Hematocrit is similar to that of the peripheral blood Blood gas analysis similar to venous or arterial blood The blood forms clots Hemorrhagic effusions Hematocrit is lower than peripheral blood Blood gas pH and pO2 are lower, pcO2 is higher Does not clot Milky color chylous or pseudochylous The difference is similar to other serous fluids

Exudates and Transudates similar difference with other serous fluids Exudates are diagnosed by Lights criteria ( the most reliable diagnostic tool for identifying exudates and transudates)
Lights criteria Pleural fluid/serum LD ratio >0.6 Pleural fluid LD level >200 U/L

ROUTINE TESTING OF PERICARDIAL EFFUSIONS

Cell

count Glucose Total protein Lactate dehydrogenase (LD) Bacterial culture Cytology

CHEMICAL

1. Protein a value of > 3.0 g/dl has a sensitivity of 97% for exudates effusions but with only 22% specificity which limits its usefulness. Thus, no discriminating power in pericardial diagnosis.
2. Glucose < 60 mg/dl has a diagnostic accuracy of only 36% in identifying pericardial exudates <40 mg/dL is indicative of bacterial, tuberculous, rheumatic or malignant effusions.

3. pH Decreased pH (<7.10) rheumatic or purulent pericarditis Moderate decreases (7.20-7.30) malignancy, uremia or tuberculosis. Idiopathic disorders
4. Lipids Chylous vs. pseudochylous same difference as serous fluids. Effusions facilitated by triglyceride and cholesterol measurements.

5. Enzymes

Lactate dehydrogenase > 200 U/L (pericardial exudates cutoff) LD and CK measurement is important in postmortem death after 48 hours may be useful in establishing Acute myocardial infarction in cases wherein injury cannot be established by usual histologic methods. CK-MB, myoglobin and Troponin I in postmortem pericardial fluid is increased in myocardial infarction Adenosine deaminase is increased in tuberculosis pericarditis than pathologic effusions and is a better marker than acid fast stain. Cut off value 30 U/L = sensitivity is 94%, specificity is 97%. Cut off value 40 U/L = sensitivity is 93%, specificity is 97%.

6. Interferon gamma

High levels in tuberculosis serous effusions (1,000 pg/L). Higher than effusions from pathologic conditions. If cut off value is 200 pg/L, sensitivity and specificity is 100%

7. PCR

A more sensitive diagnosis in tuberculous pericarditis than Adenosine deaminase. Negative test in acid fast stain does not rule out tuberculosis.

IMMUNOLOGIC Negative ANA means a diagnosis of lupus serositis is unlikely. High ANA titers lack specificity in pericardial exudates. If there is a high ANA titer is unexplained, malignancy should be considred.
MICROBIOLOGICAL Gram stain sensitivity is similar to serous body fluids Important aerobic bacteria: S. aureus, S. pneumonia, S.pyogenes, Beta hemolytic group A streptococcus and Gram negative bacilli. Aerobic bacteria not often recognized due to inconsistent methods used for their isolation and identification. Viral pericarditis is difficult to diagnose because they are rarely isolated from pericardial fluid. Viral infection accounts for most idiopathic HIV associated pericardial exudates Sensitivity of culture and acid fast stain for tuberculous pericarditis is about 50%