Aspirates and Exudates

Synovial Fluid

This is an imperfect ultra-filtrate of blood plasma combined with hyaluronic acid produced by synovial cells

Ions and molecules such as Na+, K+, glucose, urea pass into joint space hence similar to that of plasma.

Resorption of synovial fluid is by the lymphatics

It is found in large joints such as Knee, wrist, ankle, hip, elbow and shoulder

About 1 ml of the fluid is normally present.

This fluid is commonly sterile and therefore should be collected in sterile containers.

The fluid should be sent for microbiological examination, for serological and hematological test.

EDTA or heparanized tubes are used to prevent clotting.

Viscosity of synovial fluid

Normal fluid forms a tenacious strand about 4-6cm long if allowed to drop from the end of needle

A strand of less than 3cm indicates reduced viscosity.

Viscosity is evaluated in the laboratory by mucin clot test

Mucin Clot Test (Rope’s test)

Principle: Test is based on polymerization of hyaluronidase
Purpose: This measures how well-polymerized the hyaluronic acid is.
Method: Add synovial fluid drop by drop to about 20ml of 5% acetic acid in a beaker, swirled for a minute.
The resulting "clot" is rated:
Results:
o Good: a solid/firm clot in a clear solution
o Fair: a soft clot in a slightly turbid solution
o Poor: a friable (crumbly) clot in a turbid solution (clot fragments easily)
o Very poor: no clot/ only flakes in a cloudy fluid

Inflammation in the joint space damages the hyaluronic polymers, shortening them.
Thus, the "mucin clot" is:
o GOOD in osteoarthritis, trauma, and hemophilic arthritis
o FAIR in sub-acute and chronic inflammatory diseases such as lupus or rheumatoid arthritis
o POOR TO VERY POOR in septic and acute crystalline arthritis.

Synovial Fluid String Test ("Viscosity Test")

This test (not to be confused with Ropes, which is a name) measures the polymerization of hyaluronic acid
plus how much it has been diluted by the rapid entry of extracellular fluid into an inflamed joint.

All the different ways of doing this test involve stringing a drop of the fluid out (from the end of a syringe,
etc., but not between your fingers because of the HIV risk). If you can pull the drop into a string longer than
3 cm, the test is normal.

Interpretation is the same as for the mucin clot test.

NB
We analyzed the relationship between the mucin clot test and the synovial fluid (SF) leukocyte count in
osteoarthritis, rheumatoid arthritis (RA), gout, and calcium pyrophosphate dihydrate crystal deposition
disease.
Microscopic examination of synovial fluid
• Total and differential WBC count is performed as for csf
• Crystals such as monosodium urates (MSU), which are needle like/rod shaped may be present
• Calcium pyrophosphate dehydrate (CPPD) crystals which are rhomboid/rod shaped may be present.
• MSU crystals may be present in cases gout joints while CPPD in pseudogout.

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Pleural fluid

Normal pleural fluid is straw colored

Fluid present between the pleurae (two thin sheet of tissues that covers the lungs)

The inner sheet is known as the visceral pleura while the outer is parietal pleural

This fluid facilitates movement of the two membranes against each other.

Accumulation of this fluid is known as pleural effusion

This results from imbalance in production and re-absorption

Collection of this fluid is known as thoracentensis.

Normal pleural cavity contains 1-10ml of fluid

Volume may increase in case of pneumonia, TB, lung cancer etc

Blood stained fluid may appear due to haemorrage or trauma.

Cloudy/ turbid/milky fluid is due to large number of pus cell seen in TB, inflammatory or RA.

Microscopic examination
• Wbc count of over 50% polymorphs in differential count is indicative of septic inflammation.
• A predominance of lymphocyte (over 75%) is suggestive of TB, lymphoma or carcinoma.
• Rheumatoid Arthritis (RA) cell seen in rheumatoid pleural effusion.

Biochemical and microbiological examination

• Do gram stain and culture to exclude TB
• Estimate glucose and protein levels. In bacterial infection, glucose in pleural is less than that of
blood.

Pericardial fluid

Fluid present between pericardial space of the heart.

Normal pericardial fluid is pale yellow and clear

About 10-50ml of this fluid is present in this cavity

Accumulation of this fluid in the pericardial space is known as pericardial effusion

This fluid is collected by a process known as pericardiotomy/pericardiocentensis.

Causes of pericardial effusion are: viral infection (enterovirus) and malignancy.

Peritoneal fluid

Fluid present within the peritoneal cavity

Peritoneal membrane is a serous membrane that forms the lining of the abdominal cavity.

Normal peritoneal fluid is clear and pale/straw colored

Cloudy/turbid may indicate inflammatory reaction

Blood stained specimen may be due to various diseases or traumatic tap.

About 100ml of this fluid is present in this cavity

Pathological accumulation of this fluid in the peritoneal cavity is known as ascites

Collection of this fluid is known as paracentensis

Collected in sterile containers containing 3.8% sodium citrate or heparin to prevent clotting.

Peritoneal fluid is examined under the following conditions:
 Possible ruptured abdominal organs
 Post-operative complications
 Acute abdominal pain of unknown origin
 Ascitis of known and unknown aetiology.

Increased in wbc with predominant neutrolphils indicates acute peritonitis

High lymphocyte count indicates TB peritonitis

Papanicoleau stained smears of exfoliated cells may rule out neoplasm.

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Semen Analysis
 Seminal fluid (semen) consists of a combination of products of various male reproductive organs:
testes and epididymis, seminal vesicles, prostate and bulbourethral and urethral glands.
 Each product or fraction varies in its individual composition, each contributing to the whole
specimen. During ejaculation, the products are mixed in order to produce the normal viscous semen
specimen or ejaculate.
 Semen analysis is done for several reasons.
 These include assessment of fertility or infertility, forensic purposes, determination of the
effectiveness of vasectomy, and determination of the suitability of semen for artificial insemination
procedures.
Collection of semen specimen
 Give the person a clean, dry, leak-proof container, and request him to collect a specimen of semen at
home following 3-7 days of sexual abstinence.
 When a condom is sued to collect the fluid, this must be well- washed to remove the powder which
coats the rubber.
 It must be dried completely before being used.
 Coitus interruptus method of collection should not be used because the first portion of the ejaculate
(often containing the highest concentration of spermatozoa) may be lost.
 Also the acid pH of vaginal fluid can affect sperm motility and the semen may become contaminated
with cells and bacteria.
 During transit to the laboratory, the fluid should be kept as near as possible to body temperature.
 This is best achieved by placing the container inside a plastic bag and transporting it in a pocket in
the person’s clothing.
Laboratory assays
 The sample should be handled with car because it may contain infectious pathogens, e.g. HIV,
hepatitis, viruses, herpes viruses.
 When investigating infertility, the basic analysis of semen (seminal fluid) usually includes:
• Measurement of volume
• Measurement of pH
• Examination of a wet preparation to estimate the percentage of motile spermatozoa and viable
forms and to look for cells and bacteria
• Sperm count
• Examination of a stained preparation to estimate the percentage of spermatozoa with normal
morphology
Measurement of volume
 Normal semen is thick and viscous when ejaculated.
 It becomes liquefied usually within 60 minutes due to a fibrinolysin in the fluid.
 When liquefied, measure the volume of fluid in milliliters using a small graduated cylinder.
 A normal specimen is usually 2ml or more.

Measurement of pH
 Using a narrow range pH paper, e.g. pH 6.4-8.0, spread a drop of liquefied semen on the paper.
 After 30 second, record the pH.
 pH of normal semen: Should be pH 7.2 or more within 1 hour of ejaculation.
 When the pH is over 7.8 this may be due to infection.
 When the pH is below 7.0 and the semen is found to contain no sperm, this may indicate dysgenesis
(failure to develop) of the vas deferens, seminal vesicles or epididymis.

Estimate the percentage of motile and viable spermatozoa

Motility:
a) Place 1 drop (one drop falling from a 21g needle is equivalent to a volume of 10-15µl) of well- mixed
liquefied semen on a slide and cover with a 20x20mm or 22x22mm cover glass.

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 b) Focus the specimen using the low power objective.
c) Close the condenser iris sufficiently to give good contrast.
d) Ensure the spermatozoa are evenly distributed (if not, re-mix the semen and examine a new
preparation). Using the high power objective, examine several fields to assess motility, i.e. whether
excellent (rapid and progressive) or weak (slow and non-progressive).
e) Count a total of 100 spermatozoa, and note out of the hundred how many are motile.
f) Record the percentages that are motile and non-motile.

Normal motility:
 Over 50% of spermatozoa are motile within 60 minutes of ejaculation.
 The spermatozoa remain motile for several hours.
 When more than 60% of spermatozoa are non-motile, examine an eosin preparation to assess
whether the spermatozoa are viable or non-viable.
 Report when more than a few leucocytes (pus cells) or red cells are present.
 When pus cells are seen, examine a Gram stained smear for bacteria.

Viability:
 Mix one drop (10-15µl) of semen with 1 drop of 0.5% eosin solution on a slide.
 After 2 minutes examine the preparation microscopically.
 Use the low power objective to focus the specimen and the high power objective to count the
percentage of viable and non-viable spermatozoa.
 Viable spermatozoa remain unstained, non-viable spermatozoa stain red.
Normal viability:
o 75% or more of spermatozoa should be viable (unstained).
o A large proportion of non-motile but viable spermatozoa may indicate a structural defect in the
flagellum.

Perform a sperm count

 Using a graduated tube or small cylinder, dilute the semen 1 in 20 with a staining solution (sodium
bicarbonate, formalin, and a stain of trypan blue or saturated aqueous gentian violet is one diluent
that can be used).
 Using a Pasteur pipette, fill an Improved Neubauer ruled chamber with well-mixed diluted semen.
 Wait 3-5 minutes for the spermatozoa to settle.
 Using the low power objective with the condenser iris closed sufficiently to give good contrast, count
the number of spermatozoa in an area of 2 sq mm, i.e. 2 large squares.
 Calculate the number of spermatozoa in 1ml of fluid by multiplying the number counted by 100000.
 Normal count: 20x106 spermatozoa/ml or more. Counts less than 20x106/ml are associated with
male sterility.
Estimate the percentage of spermatozoa with normal morphology in a stained preparation
 Make a thin smear of the liquefied well-mixed semen on a slide.
 While still wet, fix the smear with 95% v/v ethanol for 5-10 minutes, and allow to air-dry.
 Wash the smear with sodium bicarbonate-formalin solution to remove any mucus which may be
present. Rinse the smear with several changes of water.
 Cover the smear with dilute (1 in 20) carbon fuchsin and allow to stain for 3 minutes.
 Wash off the stain with water.
 Counterstain, by covering the smear with dilute (1 in 20) Loeffler’s methylene blue for 2 minutes.
 Wash off the stain with water.
 Drain, and allow the smear to air-dry.
 Other staining techniques used to stain spermatozoa include Giemsa and Papanicolaou.
 Examine the preparation for normal and abnormal spermatozoa using the high power objective.
 Use the 100x objective to confirm abnormalities.
 Count 100 spermatozoa and estimate the percentage showing normal morphology and the percentage
that appear abnormal.

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  Abnormal semen findings should be checked by examining a further specimen, particularly when the
sperm count is low and the spermatozoa appear non- viable and abnormal.
 When the abnormalities are present in the second semen, further tests are indicated in a specialist
center.

Normal spermatozoa:
 Measures 50-70µm in length.
 Each consists of an oval-shaped head (with acrosomal cap) which measures 3-5 x 2-3µm, a short
middle piece, and a long thin tail (at least 45µm in length).
 In normal semen, at least 50% of spermatozoa should show normal morphology.
 Most specimens contain no more than 20% abnormal forms.
 Staining feature: Nucleus of head-dark blue; cytoplasm of head-pale blue; Middle piece and tail-pink-
red.

Abnormal spermatozoa:
The following abnormalities may be seen:
Head:
Greatly increased or decreased in size; abnormal shape and tapering head (pyriform); acrosomal cap
absent or abnormally large; Nucleus contains vacuoles or chromatin in unevenly distributed; two
heads; additional residual body, i.e. cytoplasmic droplet.
Middle piece:
Absent or markedly increase in size; appears divided (bifurcated); angled where it meets tail.
Tail:
Absent or markedly reduced in length; double tail; bent or coiled tail.

Reference ranges for semen analysis

Test parameter Reference range

1) Volume 2.0ml or more
2) pH 7.2-8.0
3) Sperm concentration >20 x 106 spermatozoa/ml
4) Total sperm count >40 x 106 spermatozoa per ejaculate
5) Morphology >30% with normal forms
6) Vitality/viability >75% live forms
7) White blood cells <1 x 106/ml
8) Red blood cells None

Review Questions
1) What are the different types of body fluids
2) Explain the analysis of cerebrospinal fluid
3) Describe the analysis of serous fluid, synovial fluid and semen

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