The document provides an extensive overview of body fluid analysis, particularly focusing on amniotic fluid, semen, and other bodily fluids. It discusses collection procedures, testing methodologies, and the clinical significance of different fluid analyses for diagnoses such as genetic disorders, fetal lung maturity, and male infertility. Key concepts include the roles and characteristics of various fluids, physiological impacts of imbalances, and laboratory procedures for testing.

1. Body fluid analysis
(excluding body cavity fluids & urine)
Feb 5th, 2013
By Dr. Niraj Gupta,
MD (Pathology)

2. Contents
Introduction
Amniotic fluid
Semen analysis
Synovial fluid
Gastric fluid
Other body fluids: Sweat,saliva,tear

3. FLUID
• A fluid is a non solid substance such as liquid or
gas that tends to flow or conform to the shape of
the container. Includes urine,fluids in the body
cavity, joint fluid,sweat, semen etc.
• They are generally involved with the process of
transport ,excretion and lubrication. They allow
the distribution of oxygen and nutrients to the
tissues and organs and the transport of waste
products from the tissues, enabling their
elemination from body.

4. Total body
water
Extracellular
fluid(33%)
Intracellular
fluid(66%)
Interstitial fluid
(25%)
Plasma
(7%)
Transcellular
fluid(~1%)
50-70%

5. • Transcellular fluid is a small compartment
that represents all those body fluids which are
formed from the transport activities of cells. It is
contained within the epithelial lined space.
• It includes CSF, synovial fluid, GIT fluids ,
aqueous humor and serous fluid of bodycavity
like pleural, peritoneal and pericardial fluid.
• It is important because of specialized function
involved. It is the smallest component of
extracellular fluid.

6. ACCUMULATION OF EXCESS
BODY FLUIDS
• Body fluids are necessary for lubrication of the body
cavity/organ interface during movement.
• A delicate equilibrium is maintained by the
capillaries and the lymphatic vessels. Any
obstruction or altered pressure in these vessels can
affect the amount of fluid and its constituents.
• Several forces, within and outside of the capillaries,
work together to maintain fluid equilibrium.

7. • The tissue’s colloidal osmotic pressure (interstitial fluid
pressure), along with the capillary’s hydrostatic pressure
(filtration pressure),regulates the outward flow of fluid from
the capillary.
• The colloidal osmotic pressure of the capillary and the
tissue’s hydrostatic pressure regulate the inward flow of
fluid into the capillary from the tissue.
• However, an imbalance in pressures causes excess egress
of fluid into tissue spaces and can lead to accumulation of
fluid.

8. AMNIOTIC FLUID
• Amniotic fluid is found around the developing
fetus,inside a membranous sac, called the amnion.
• This fluid serves to cushion and protect the
developing fetus and also serves a key role in the
exchange of water and molecules between the fetus
and the maternal circulation.
• The fluid has a composition similar to that of the
maternal plasma and contains a small amount of
sloughed fetal cells.
• These cells provide the basis for cytogenetic
analysis.

9. • The volume of amniotic fluid increases steadily
throughout the pregnancy up to a maximum of
1000–1200 mL at 36 weeks of gestation.
• Measurement of amniotic fluid creatinine has
been used to determine fetal age.
• Prior to 36 weeks’ gestation,the amniotic fluid
creatinine level ranges between 1.5 and 2.0
mg/dL.
• After 36 weeks rises to >2.0 mg/dL.

10. • The laboratory performs several crucial tests on
amniotic fluid to assess the status of the fetus.
• These tests can be divided into these groups:
(a) tests to diagnose genetic and congenital
disorders before birth,
(b) tests to detect fetal distress from hemolytic
disease of the newborn (HDN) or from infection,
(c) tests to assess fetal lung maturity

11. Specimen Collection
• Amniotic fluid is obtained by needle aspiration into the amniotic
sac, usually transabdominally with simultaneous use of
ultrasound,a procedure called amniocentesis.
• Amniocentesis is generally performed between 15 and 18
weeks of gestation for genetic studies,whereas tests for fetal
distress and maturity are performed later in the third trimester.
•A maximum of 30 mL of
amniotic fluid is collected in
sterile syringes.
•The first 2 or 3 mL collected
can be contaminated by
maternal blood, tissue fluid,
and cells and are discarded.

12. • In case of possible premature membrane rupture
or maternal bladder puncture or rupture, it may be
necessary to differentiate amniotic fluid from
maternal urine.
• To differentiate these two fluids,chemical levels of
creatinine, urea, glucose, and protein can be of
assistance.
• Levels of creatinine and urea are much higher
in urine than in amniotic fluid.
• Glucose and protein levels tend to be higher in
amniotic fluid than in urine.

13. • Normal amniotic fluid is colorless to pale
yellow and slightly cloudy.

14. TESTING AMNIOTIC FLUID FOR GENETIC
AND CONGENITAL DISORDERS
• Valuable cytogenetic information related to the sex of
the fetus and to genetic abnormalities can be obtained
via amniocentesis.
• Amniocentesis is often performed to detect Down
syndrome and anencephaly prior to birth.
• Fetal neural tube defects such as anencephaly and
spina bifida cause elevated alpha fetoprotein (AFP)
in amniotic fluid and in the maternal circulation.
• In normal fetal development, AFP peaks at about
16 weeks of gestation and then declines gradually to
term.

15. • Measurement of amniotic fluid AFP levels
is indicated when maternal serum levels
are elevated or a family history of previous
neural tube defects exists.
• Both serum and amniotic fluid AFP levels
are reported in terms of multiples of the
median (MoM).
• The median is the laboratory’s reference
level for a given week of gestation.

16. A value two times the median value is
considered abnormal (>2 MoM) for both maternal
serum and amniotic fluid.
• Elevated amniotic fluid AFP levels are followed
by measurement of amniotic
acetylcholinesterase (AChE).
• The test is more specific for neural tube
disorders than AFP, provided it is not
performed on a bloody specimen, because
blood contains AChE.
Amniotic fluid AchE = > 5 U/L in NTD

17. Fetal karyotype:
• Retrieval of fetal somatic cells can be used to identify
genetic constitution of fetus.
• The problem of karyotyping is that it take 3 or more
weeks.
• Nowadays ,numerical abnormalities in the
chromosomes 21,18,13,X & Y can be detected more
rapidly by using chromosome specific DNA probes
tagged with fluorescent dyes,known as FISH.(90%
specificity & gives results in 2days.)

18. FETAL LUNG MATURITY
• Respiratory distress is the most frequent complication of early
delivery.
• Therefore, laboratory tests must be performed to determine the
maturity of the fetal lungs.
• Lecithin:Sphingomyelin Ratio and Phosphatidylglycerol-
• Fetal lung surfactants include these three phospholipids:lecithin
(also known as phosphatidylcholine), sphingomyelin, and
phosphatidyl glycerol. It is produced by type II pneumocytes in
the form of lamellar bodies.
• The ratio of lecithin to sphingomyelin is used to assess fetal
lung maturity. Up until the 33rd week of gestation,the levels of
these two phospholipids are relatively equal.

19. • After 34 weeks of gestation,the level of
sphingomyelin decreases, whereas the level of lecithin
increases significantly.
L/S ratio ≥ 2.0 usually indicate maturity, and
ratio <1.5 indicate immaturity.
• This is done by centrifugation and acetone
precipitation.
• Amniotic fluid surfactant lipids are chromatographed
on thin-layer silica .
• The phospholipids are made visible by heat charring
or staining.
• Densitometric quantification determines the L/S ratio.

20. • Many laboratories have replaced the L/S ratio with
the more cost-effective phosphatidyl glycerol
immunoassays, fluorescence polarization, and
lamellar body density procedures.
• Development of an immunologic agglutination test
for phosphatidyl glycerol has provided a more rapid
method for assessment of fetal maturity that does
not require a laboratory to be equipped to perform
thin-layer chromatography.
• The Aminostat-FLM (Irving Scientific, Santa
Ana,CA) uses antisera specific for phosphatidyl
glycerol and is not affected by specimen
contamination with blood and
meconium.

21. Phosphatidylglycerol measurement in AF
• Thin-layer chromatography
– Absent: Fetal lung immaturity
– Present: Fetal lung maturity
• Slide-agglutination test
– Negative: <0.5 mg/L (immature)
– Positive : > 0.5 mg/L (mature)
• Enzymatic assay: 19-83 mg/L
< 19 = Immature lung
> 83 = Mature lung

22. Fluorescence Polarization Assay (Microviscosity)
• This assay provides a fluorescence polarization (P)
surfactant:albumin ratio.
• The microviscosity of lipid aggregates in the amniotic fluid may
be assayed by mixing the fluid with a specific fluorescent dye
that becomes incorporated in the lipids of surfactant.
• The intensity of fluorescence is measured by polarized light.
• Dye bound to surfactant has a longer fluorescence lifetime and
exhibits a low polarization.
P value is high with low levels of surfactant ,
P value is low with high levels of surfactant.
• The degree of fluorescence polarization is inversely
proportional to the quantity of pulmonary surfactant present.

23. FOAM STABILITY (SHAKE TEST)
1. Mix equal parts of amniotic fluid with 95% ethanol.
2. Vigorously shake for 15 seconds.
3. Allow to sit undisturbed for 15 minutes.
4. Observe for the presence of a continuous line of bubbles
around the outside edge.
• The presence of bubbles indicates that a sufficient amount of
phospholipid is available to reduce the surface tension.
bubbles present -- POSITIVE
bubbles absent -- NEGATIVE
• The highest concentration of 95% ethanol that is able to
support a ring of foam is known as the foam stability index.
Foam stability index= ≥0.47 indicate fetal lung maturity

24. Lamellar Bodies
• Fetal lung surfactants are produced by fetal type II
pneumocytes of the fetal lung and are stored as
lamellar bodies after about 20 weeks of gestation.
• Lamellar bodies are about the size of small platelets.
• Lamellar bodies are storage forms of lung
phospholipids and they enter the fetal lungs and the
amniotic fluid at about 20–24 weeks of gestation.
• Lamellar bodies affect the optical density of amniotic
fluid and a measurement of the optical density of
0.150 at 650 nm has been shown to correlate with an
L/S ratio of 2.0 and to correlate with the presence of
phosphatidyl glycerol.

25. • Lamellar body counts provide a reliable estimate of fetal
• lung maturity. Lamellar body counts can be performed
• easily with many hematology analyzers using the platelet
• count channel.
• Lamellar body counts of approximately 35,000 per microliter
correspond to adequate fetal lung surfactant levels.
Fig: Lamellar bodies in
amniotic fluid on
hematology counters

26. HEMOLYTIC DISEASE OF THE NEWBORN
• HDN, also known as erythroblastosis fetalis, is caused when
mother develops antibodies to an antigen on the fetal
erythrocytes and these maternal antibodies cross the placenta
to destroy many fetal red blood cells (RBCs).
• Most frequently,HDN is caused by the sensitization of an Rh-negative
mother to fetal Rh antigen.
• The destruction of these fetal RBCs results in the appearance
of elevated level of bilirubin in the amniotic fluid.
• The measurement of amniotic fluid bilirubin is performed by
spectrophotometric analysis.
• the optical density (OD) of the fluid is measured in intervals
between 365 nm and 550 nm and the readings plotted on
semilogarithmic graph paper.

27. • When bilirubin is present, a rise in OD will be seen at
450 nm because this is the wavelength of maximum
bilirubin absorption.
• The amount that the curve deviates from a straight
line at 450 nm (the ΔA450) is directly proportional to
the amount of bilirubin in the amniotic fluid.
Fig:
Spectrophotometric
bilirubin scan showing
bilirubin and
oxyhemoglobin peaks.

28. • This difference in OD, referred to as the absorbance difference
at 450 nm(A450), is then plotted on a Liley graph to determine
the severity of the hemolytic disease.
• The Liley graph plots the A450 against gestational age and is
divided into three zones that represent the degree of hemolytic
severity.

29. Infection
• Evidence is mounting of the importance of
microorganisms in the amniotic fluid contributing
to the incidence of preterm delivery and
spontaneous abortion.
• Herpes,candidiasis, trichomoniasis and even
bacterial vaginosis have been linked to preterm
birth.
• Gram stain, wet mount, culture, and molecular
tests may be used on amniotic fluid to look for
potential infectious agents.

30. Semen Analysis
• The semen is a fluid that is ejaculated
at the time of orgasm,contains sperms
and secretions of the seminal
vesicle,prostate, cowper’s gland and
bulbourethral glands.

31. indications for performing a semen
analysis
• Male infertility
• Determining the effectiveness of a vasectomy,
• Forensic cases, eg. rape-case
• Paternity cases
• Sperm donor evaluation for IVF

32. Specimen Collection
• The preferred method of semen collection is by masturbation.
• Specimens are collected following a period of sexual
abstinence of at least 3 days and not longer than 5 days.
• Two or three samples are usually tested at 2-week intervals
with two abnormal samples considered significant.
• Specimen collection containers should be clean glass or plastic
and have a wide opening.
• specimen should be delivered to the laboratory within 1 hour of
collection.
• Shortly after ejaculation, the semen coagulates because of the
action of a clotting enzyme, formed in the prostate, on a
fibrinogenlike precursor substance that is produced by the
seminal vesicles. Fresh sample should liquefy within 30 to 60
minutes after collection.
Failure to liquefy indicate inadequate prostate secretion.

33. MACROSCOPIC EXAMINATION
APPEARANCE:
• Normal semen - Gray-white color, translucent
• white turbidity - Presence of white blood cells
(infection)
• Red coloration - Red blood cells(infection,cancer)
• Yellow coloration - Urine contamination, specimen
collection following prolonged
abstinence, and
medications.

34. • Viscosity:
Slightly viscous and easily drawn into a pipette.
Incompletely liquefied specimens will be clumped and
highly viscous. Increased viscosity and incomplete
liquefaction will impede sperm motility.
Fators affects semen viscosity (liqification time):
Dehydration,Infection of prostate and seminal vesicle and drugs
• pH:
Normal pH:7.2 to 8.0
Increased pH -- infection within the reproductive tract.
decreased pH – increased prostatic fluid.

35. • Volume: N= 2-5 ml
• Decreased volume is more frequently associated with
infertility and may indicate improper functioning of one
of the semen-producing organs.
• Low volume ejaculates are caused by 3 disorders:.
• Ejaculatory disorders: Neurological
diseases,Diabetes,Prior surgeries & Medications; can
cause retrograde ejaculation, aspermia or both.
• Genital tract obstructions :Prostate
cysts,inflammatory disorders,Infections,Scars from
prior surgeries.
• Congenital anomalies : Seminal vesical aplasia
(absence),congenital absence of the vas deferens

36. MICROSCOPIC EXAMINATION
1. Sperm count -- ≥20 million/ml(20-160million/ml)
2. Total sperm count
per ejaculate -- ≥ 40 million
3. Motility -- > 60% motile
class A ≥ 25% rapidly progressive
class A & B ≥ 50% progressive
3. Morphology -- > 30% normal morphology
4. Viability -- > 75% live
5. WBCs -- ≤ 1 million/ml

37. SPERM CONCENTRATION/COUNT
• In the clinical laboratory, sperm concentration is
usually performed using the Neubauer counting
chamber,by diluting the specimen and counting the
cells in the Neubauer chamber.
• The amount of the dilution and the number of squares
counted vary among laboratories.
• Method : Semen is diluted 1:20 with sodium
bicaronate-formalin diluting fluid (1ml liquified semen
in graduated tube and fill with diluting fluid to 20ml
mark & mix well).
• Place a coverslip over Neubauer counting chamber
and filled with well mixed diluted semen.
• Spematozoa is counted in 4 large corner squares.

38. Fig; Inclusion criteria of counting cells.
Count cells (sperm heads, not tails) that
touch the upper and left boarders of the
counting grid.
Do not count cells (sperm heads, not tails)
that touch the lower and
right boarders of the counting grid. Count
only complete sperm.

39. • Sperm count per ml is calculated as follows
Sperms counted (N) X correction factor for dilution (D)
Sperm conct(C) = ---------------------------------------------------------------------------
X 1000
No. of squares counted X volume of 1 square
Sperms counted(N) X 20
= --------------------------------------- X 1000
4 X 0.1
= sperms counted (N) X 50,000
eg. 500 X 50,000 = 25,000,000/ml

40. • Aspermia : is the complete lack of semen
• Oligospermia : is a sperm count less than
10 million/ml
• Azoospermia : is the complete absence of
sperm.
• Oligozoospermia : is a sperm count less
than 20 million/ml

41. • Causes of Aspermia:
• Retrograde ejaculation due to prostate surgery ,excessive drug
use(tamsulosin & antidepressants),or benign prostatic
hyperplasia
• Ejaculatory duct obstruction
• Lesion of presacral sympathetic nerve fibers
• Causes of Oligospermia :
• Pre-testicular causes:
• Hypogonadism
• Drugs, alcohol, smoking
• Strenuous riding (bicycle riding,horseback riding)
• Medications, including androgens.

42. • Testicular factors:
• Age
• Genetic defects on the Y chromosome
Y chromosome microdeletions
• Abnormal set of chromosomes
Klinefelter syndrome
• Neoplasm, e.g. seminoma
• Cryptorchidism
• Varicocele
• Trauma
• Hydrocele
• Mumps
• Radiation
• Post-testicular causes:
• Vas deferens obstruction
• Infection, e.g. prostatitis
• Ejaculatory duct obstruction

43. Causes of Azoospermia
• Pretesticular azoospermia
• Hypopituitarism
• Hyperprolactinemia,
• Testosterone
• Testicular azoospermia:
• Klinefelter syndrome
• Cryptorchidism
• Sertoli cell-only syndrome
Infection (mumps orchitis),
• Surgery/trauma,
• Radiation
• Posttesticular azoospermia;
• Vasectomy
• Agenesis of the vas deferen
• Obstructive azoospermia

44. MOTILITY
• The presence of sperm capable of forward, progressive
movement is critical for fertility, because once presented to the
cervix, the sperm must propel themselves through the cervical
mucosa to the uterus, fallopian tubes, and ovum.
• Motility should be evaluated within 1 hour of specimen
collection.
• To evaluate sperm motility, place a small drop of liquefied
semen on a prewarmed slide and coverslipped.
• Atleast 200 spermatazoa counted (under 40X).
• Normally ≥ 60% sperm should be motile.
• “Asthenospermia" or Asthenozoospermia is the medical term
for reduced sperm motility.
• The movement of sperm is evaluated and may be subjectively
estimated or counted into four categories

45. A. Rapidly progressive spermatazoa: Moving fast forward in a
straight line.
B. Slowly progressive spermatazoa: Slow linear or nonlinear,
eg. Crooked/curved movement.
C. Non progressive spermatazoa: Movement of tails,but with
no forward progress.
D. Immotile spermatazoa: No movement at all.

46. Causes of Asthenospermia
• Infections: like mumps, tuberculosis, brucellosis,
gonorrhea and Chlamydia, typhoid, influenza,
smallpox, and syphilis.
• Epididymitis
• Prostatitis
• Nutrition:
• Vitamin deficiencies (vitamin C,Vit B12 & E, selenium,
zinc and folate, specifically) can cause low motility.
• Anabolic steroids ,cigarettes, alcohol and marijuana
also have a negative impact on motility.

47. SPERM MORPHOLOGY
• A smear is prepared by spreading a drop of seminal
fluid on a glass slide,stained and percentage of
normal & abnormal spermatozoa are counted.
• 200 spermatozoa should be counted under oil
immersion.
• Normal spermatozoa consists of : head,neck & tail.
•
Principal
piece
End piece
• A normal spermatozoa has
a flattened oval head and an
elongated tailpiece

48. • Teratospermia or teratozoospermia is a condition characterised by the
presence of sperm with abnormal morphology that affects fertility in males.
Excessive
cytoplasmic
membrane

49. Causes of abnormal sperm morphology
• Infections
• High fever
• Organic solvents causes coiled tails
• Usage of illegal drugs (cannabis, heroin, cocaine and
LSD)
• Alcohol consumption
• Congenital testicular abnormalities
• Varicocele
• Cigarette smoke

50. SPERM VIABILITY
• Decreased sperm viability may be suspected when a specimen
has a normal sperm concentration with markedly decreased
motility.
• Viability is evaluated by mixing the specimen with an eosin-nigrosin
stain, preparing a smear, and counting the number of
dead cells in 100 sperm. Living cells are not infiltrated by the
dye and remain a bluishwhite color, whereas dead cells stain
red against the purple background.
• Normal viability requires 75% living cells and should correspond
to the previously evaluated motility.

51. ANTISPERM ANTIBODIES
• Antisperm antibodies can be present in both men
and women. They may be detected in semen,
cervical mucosa,or serum and are considered a
possible cause of infertility.
• Two frequently used tests to detect the presence of
antibody- coated sperm are
• Mixed agglutination reaction(MAR) test and
• Immunobead test.

52. Mixed agglutination reaction(MAR) test
• The MAR test is a screening procedure used primarily to detect
the presence of IgG antibodies.
• The semen sample containing motile sperm is incubated with
IgG antihuman globulin (AHG) and a suspension of IgG coated-latex
particles.
• The bivalent AHG will bind simultaneously to both the antibody
on the sperm and the antibody on the latex particles, forming
microscopically visible clumps of sperm and particles.
• Less than 10 percent of the motile sperm attached to the
particles is considered normal.

53. The immunobead test
• It is a more specific procedure
• It can be used to detect the presence of IgG,
IgM, and IgA antibodies and will demonstrate
what area of the sperm (head, neck, or tail) the
autoantibodies are affecting.
• Sperm are mixed with polyacrylamide beads
known to be coated with either anti-IgG, anti-
IgM, or anti-IgA.
• Microscopic examination of the sperm will
show the beads attached to sperm at particular
areas.
• Depending on the type of beads used, the test
could be reported as “IgM tail antibodies,” “IgG
head antibodies,” and so forth.
• The presence of beads on less than 20 percent
of the sperm is considered normal.

54. Biochemical analysis
Table :

55. SEMINAL FLUID FRUCTOSE
• Specimens for fructose levels should be tested within
2 hours or frozen to prevent fructolysis.
• Procedure:
1. Prepare reagent.(50 mg resorcinol dissolved in 33 mL
concentrated HCl; diluted upto 100 mL with distilled
water).
2. Mix 1 mL semen with 9 mL reagent.
3. Boil.
4. Observe for orange-red color.
• Absence of fructose indicates obstructed or
absent vas deferens.

56. Sims-Huhner test (Postcoital test)
• A Sims-Huhner test is done when a couple is
experiencing difficulty conceiving, and is usually done
after the man has had a normal semen analysis.
• Consistency of cervical mucus as well as the ability of
spermatozooa to penetrate the mucus and maintain
activity are two useful indicators.
• The PCT is scheduled close to ovulation(12-13th day)
when mucus is abundant.
• Following 2-3 hours after intercourse,vaginal and
cervical mucus is removed and examined to
determine how well the sperm are able to move
through the mucus.

57. Interpretation of postcoital test

58. Hamster oocyte sperm penetration test
• The test is done for unexplained infertility with normal
semen.
• Hamster oocytes are enzymatically treated to remove
the outer layers(zona pellucida),hence zona-free.
• Oocytes are incubated with sperms and observed for
penetration rate compared with control.
• Reported as
a)No.of eggs penetrated
penetration rate < 15% - low fertility
b)No. of sperm penetration/egg
Normal = ≥ 5
• Test has high incidence of false negative results

59. Microbiology of semen specimens
• Microorganisms can be detected in semen, including
Enterobacteriaceae ,
• Gardnerella vaginalis ,
• Chlamydia trachomatis ,
• Mycoplasma genitalium , and
• Ureaplasma urealyticum .
• Semen quality was neither related to occurrence of
microorganisms nor pyospermia.
• However, pyospermia is associated with simultaneous
growth of Gardnerella vaginalis and Ureaplasma
urealyticum.
• Culture can be done.

60. SYNOVIAL FLUID
• Joint fluid is called as synovial fluid because of its
resemblance to egg white.It is viscous, mucinous substance
that lubricates most joints. It is an imperfect ultrafiltrate of
plasma combined with hyaluronic acid produced by synovial
cells.
• It acts as a lubricant and adhesive and provides nutrients for
avascular articular cartilage.
• Specimen collection – Arthrocentesis is performed by
physicians and the affected joint aspirated.Two step process in
which 1st thrust made in skin and next into synovial capsule.
specimen should be separated into 3 parts –
1)5-10ml into sterile heparanized tube for microbiological studies.
2) 2-5ml in an anticoagulant tube for microscopic examination.
3)5ml fluid in plain tube and allowed to clot (normal SF doesn’t
clot)

62. Laboratory testing
Normal Synovial Fluid Values
•Volume <3.5 mL
•Color Pale yellow ,transparent
•Clarity Clear
•Viscosity Able to form a string 4–6 cm long
•Erythrocyte count None
•Leukocyte count <0-150/ml
•Neutrophils <20% of the differential
•Lymphocytes <15% of the differential
•Monocytes and 65% of the differential
macrophages
•Crystals None present
•Glucose <10 mg/dL lower than the blood glucose
•Lactate <250 mg/dL
•Total protein <3 g/dL
•Uric acid Equal to blood value

63.  Volume –recorded at bed side. usually less, knee joint has upto
4ml
 Colour- normal colorless and clear.
• Yellow/clear-non inflammatory effusions
• Yellow/cloudy-inflammatory
• White/cloudy-crystals
• Red/brown/xanthochromatic-hemorrhage.
 Inclusions-
1)free floating aggregates of tissue
Rice bodies-degenerated synovium with fibrin. Seen in RA.
2) Ochronotic shards- debris from metal and plastic prosthesis
(ground pepper appearance).
 Viscosity – normal- high viscosity.
String test- normally – app. 5 cm string formed or cling to the
sides of tube. Abnormal – when <3cm/or fall to bottom.
• ↓ viscosity in inflammatory conditions

64. Synovial fluid inclusions.
A. “Ground pepper” ochronotic
shards.
B. “Rice bodies” fibrin-enriched
synovium fragments
String test showing normal
synovial fluid viscosity

65.  Clotting – Clotting of synovial fluid can result when fibrinogen is
present as a result of a traumatic tap.
• Depositing part of the specimen into a heparin tube avoid
clotting of synovial fluid.
 Mucin clot test /Ropes TEST – indicates integrity of hyaluronic
acid-protein complex (mucin).
• .
•Normally SF forms tight ropy
clot on addition of acetic acid.
•A good mucin clot indicates
good integrity of the hyaluronate.
•A poor mucin clot--breaks up
easily, is associated with
destruction or dilution of
hyaluronate
Fig.: Mucin clot test of
normal synovial fluid.

66. Chemical examination
 Proteins – normally 1-3 g/dL. All proteins present in plasma except high
molecular weight.
• increased protein level in Inflammatory contitios like ankylosing spondylitis
and arthritis.
 Glucose- normally synovial fluid glucose level is 10mg/dL lower than
serum levels.
• Infectious joint disorders demonstrate large decreases in synovial fluid
glucose and can be as much as 20–100 mg/dL less than serum levels.
 Uric acid- SF uric acid level = 6-8mg/dL .
• Useful for diagnosing gout. Crystal identification is used for this
determination,
 Lactic acid- Normal level = < 25mg/dL .
• In septic arthritis level = >1000mg/dL
 Lactate dehydrogenase- levels increased in RA,infectious arthritis and
gout. This is due to increased neutrophils.
 Rheumatoid factor -RF is an antibody to immunoglobulin.
• Found in serum and SF of patients of RA.

67. Synovial Fluid Findings by Disease Category

68. Microscopic examination
• Synovial fluid cell counts,should be performed within 1 hour of
collection.
• It is done by hemacytometer and automated cell counter.
• Normal WBC count = < 200 cells/μL (0-150 cells/μL
 DIFFERENTIAL CELL COUNT-
• NEUTROPHILS = 7%
• LYMPHOCYTES = 24%
• MONOCYTES = 48%
• MACROPHAGE = 10%
• SYNOVIAL LINING CELL = 4%
• EOSINOPHILS = > 2%
( ↑ed eosinophils in allergic diseases with arthritis and parasitic
arthritis)

69. • Septic arthritis - high no. of neutrophil. In later stages have
inclusions like IgG, IgM and RF. They appear to have dark
cytoplasmic granules called as RA cells or RAGOCYTES.
• LE cells -are also seen in patients with
SLE and in some cases of RA.
These are neutrophils that have engulfed
nucleus of lymphocyte that has been
altered by AntiNuclearAntibody(ANA).
• Reiter cell- Macrophage that has
phagocytosed 1 or more neutrophils.
Not specific for Reiter’s Syndrome.
LE cell

70. • Tart cell: a macrophage containing
a phagocytized nucleus that retains
some nuclear detail
(Macrophage with engulfed nuclear
material).
• Lipophage :Lipids may be released from bone marrow after
injury to the bone.
Lipid-laden macrophage may be
present in synovial fluid.

71. Crystals examination
MATERIAL SHAPE/SIZE BIREFRINGENE CAUSE MONOSODIUM
URATE
Needle/rod.
Strongly -ve GOUT 8-20 microm
Calcium
pyrophosphate
dihydrate
Rhomboid/rod/square
Weakly +ve PSEUDOGOUT <10 micro m
CALCIUM OXALATE bipyramidal strong Long term renal
dialysis
hydroxypatite Aggregates
only/small round
weak Acute/chronic
arthritis
CHOLESTROL Flat/plate corner
notch,<100microm
variable TB arthritis/RA
CHARCOT LEYDEN spindle variable Eosinophilic
synnovitis
Crystalline
corticosteroids
Blunt, jagged edges +ve Intraarticular
injection
Lipid crystal Maltese cross app +ve Acute arthritis

72. Monosodium urate crystals. The needle-shaped
crystals demonstrate negative
birefringence, because they are yellow when
aligned with the compensator.
MSU
Calcium pyrophosphate dihydrate crystals.
The rhomboidal crystal demonstrates positive
birefringence, because it is blue when
aligned with the compensator filter.
CPPD
Cholesterol crystals in synovial fluid.
Polarized light

73. Microbiologic Examination
• Infectious agents can enter the synovial fluid,between them
bacteria being the most common.
• Bacteria and other microorganisms enter the synovial capsule
through the bloodstream,deep penetrating wounds, and rupture
of osteomyelitis into the joint.
• Bacteria may be introduced during procedures such as
arthroscopy, intra-articular steroid injections, and prosthetic
joint surgery.
• Gram stain is performed on synovial fluid smears prepared by
centrifugation or cytocentrifugation.
• Synovial fluid Gram stains are positive in only 50% of cases
with joint sepsis
• So, if Gram staining does not suggest the presence of
infectious agents, both aerobic and anaerobic cultures should
be performed.

74. Gastric fluid analysis
• Gastric analysis consists of a series of tests used to analyze
the contents of the stomach for acidity, appearance, and
volume.
• Gastric juice can be aspirated through an oral or nasogastric
tube(Ryle’s tube) or during endoscopy.
• In some diseases of Stomach alterations in gastric secretion
occurs, thereby chemical examination of gastric contents has
limited but specific value in diagnosis & assessment of
disorders of upper GIT.
• Hence to get complete data of gastric function, the contents of
stomach should be examined -
During resting period
During digestion after meals
After stimulation
• Normal Gastric acid secretion = 1500-2000ml/24hr

75. Chief constituents of Gastric juice
Renin / Chymosin:
A milk clotting
factor found in the
stomach of young
animals,
but is probably
absent in humans.

76. Abnormal constituents
a) Blood :bleeding ulcer,malignancy
b) Food remnants: after 6 hrs of eating(delayed
emptying)
c) Large quantity of mucus or bile
d) Pyogenic bacteria,yeast cell,lactobacillus: mostly
found in Achlorhydria
e) Parasites(round worm), amoeba(cyst form)
f) Organic acid,lactic acid :found in absence of Hcl
g) Tubercular bacilli: through swallowed sputum
h) Tissue fragments,large no. of epithelial cells from
exfoliation
i) Malignant cells: by exfoliation

77. Indications of Gastric Function Tests
• To diagnose Gastric Ulcers
• To exclude the diagnosis of Pernicious Anaemia & Peptic ulcer
• For presumptive diagnosis of Zollinger Ellison Syndrome
• To determine the completeness of Surgical Vagotomy.
Contraindication
• Esohageal strictures or varices
• Active nasopharyngeal disease
• Diverticula
• Recent H/o severe gastric hemorrhage
• Aortic aneurysm
• Congestive cardiac failure
• Cardiac arrythmias
• Pyloric stenosis

78. • Basic acid output(BAO) : is the amount of Hcl
secreted by the stomachin the absence of any
external stimuli (visual/olfactory/auditory).
• Maximum acid output(MAO) : is the amount of Hcl
secreted by the stomach following stimulation by
pentagastrin.
• Peak aci output(PAO) : is calculated from the 2
highest consecutive 15-minute samples following
stimulationand multiplied by 2.It indicates greatest
possible acid secretory capacity.
• Acidity is estimated by titration

79. Estimation of BAO
• Sample is collected in the morning after 12 hr overnight fast.
• Gastric secretion that has accumulated overnight is aspirated and
discarded.Followed by
• Aspiration of gastric secretions at 15-minute interval for 1hr.
• Total 4 consecutive samples are collected.
• Each sample is titrated to pH 3.5 using 0.1N sodium hydroxide.
• The acid output in 4 samples is totaled and result is expressed as
concentration of acid in mEq/hr or mmol/hr.
• Normal BAO=upto 5mEq/hr
 MAO & PAO estimation : After determination of BAO pt is given a s/c or
I/M injection of pentagastrin(6μg/kg).
• Immediately afterwards gastric secretions are aspirated at 15-minute
intervals for 1 hr.
 MAO : is determined by titrating each of the four specimens and averaging
the results. (Normal MAO= 12-26 mEq/hr)
 PAO : is sum of two consecutive,15-minute samples showing highest
acidity, multiplied by 2.(after stimulation)
• Normal PAO = 1-20 mEq/hr.

80. Interpretation of results :
1) Volume : Normal = 20-100 ml
• Causes of Increased volume of gastric juice:
• Delayed emptying: Pyloric stenosis
• Increased secretions: Zollinger-Ellison syndrome,duodenal
ulcer.
2) Color : Normal gastric secretion – colorless
• Red -- Fresh bleeding from ulcer, trauma, cancer
• Brown -- Old haemorrhage
• Yellow to green – Bile regurgitation
3) pH :
• Normal gastric pH= 1.5-3.5
• Increased pH- pernicious anemia(>7)due to absence of Hcl.

81. 4) Basic acid output (BAO): N= upto 5 mEq/hr
• Duodenal ulcer = 5-15 mEq/hr
• Zollinger-Ellison syndrome = > 20 mEq/hr
5) Peak acid output (PAO): N = 1-20 mEq/hr
• Duodenal ulcer = 20-60 meq/hr
• Zollinger-Ellison syndrome = > 60 mEq/hr
• Achlorhydria = 0 mEq/hr
6) BAO/PAO : N = < 0.20 (or < 20%)
• Gastric ulcer =0.20-0.40 (20-40%)
• Duodenal ulcer = 0.40-0.60 (40-60%)
• Zollinger-Ellison syndrome = > 0.60 (> 60%)

82. Other test for gastric acid secretions
These tests are named after the stimulant used for MAO.
Histamin: It was the first standard stimulant used for gastric acid
secretion test.Subcutaneous injection of histamin phosphate
(0.04mg/kg) is given to stimulate gastric acid.
Pentagastrin: It is currently most preferred agent administered in
dose of 6μg/kg.
Tubeless analysis : A resin bound dye, Diagnex blue is given
orally.The release of dye by the action of gastric acid and its
appearance in urine indicates the presence of gastric acid.
Fractional test meal : In the past,test meals(eg.Oat meal gruel,
alcohol) were administered orally to stimulate gastric secretion
and determine MAO or PAO.

83. Hollander’s test (insulin hypogastric analysis)
• This test was used for confirmation of comleteness of vagotomy
earlier but no longer recommended due to risk associated with
hypoglycemia.
• Principal : Hypoglycemia is a potent stimulus for gastric acid
secretion and is mediated by vagus nerve.This response is
abolished by vagotomy.
• Procedure : After determining BAO,Insuline (0.2units/kg) is
administered I/V. and acid output is estimated every 15-min for
2hrs(8-post stimulation sample).
• Result : Vagotomy is considered complete if after insulin-induced
hypoglycemia (Blood glucose < 45mg/dl) , no acid
output is observed within 45 minutes.

84. Tests for gastrin
 S.Gastrin levels : Normal fasting value= 20-150 pg/ml by RIA
• S.Gastrin levels Increased in:
1. Atrophic gastritis (low gastric acid secretion)
2. Zollinger-Ellison syndrome (High gastric acid secretion)
 Gastrin Provocation test :
1. Secretin test : I/V injection of secretin(1unit/kg) is given.
if S.gastrin levels rise by > 50% of basal value in 5-15
minutes,it is diagnostic of Zollinger-Ellison
syndrome(Gastrinoma).
2. Calcium infusion test : I/V infusion of calcium (5mg/kg/hr)is
given for 3 hrs.
Rise in S.gastrin level by > 50% of basal value is diagnostic of
Zollinger-Ellison syndrome(Gastrinoma).

85. OTHER
BODY FLUIDS

86. Sweat test
• The sweat test measures the concentration of chloride that is
excreted in sweat. It is used to screen for cystic fibrosis.
• For normal salt reabsorption to occur, individual ions of sodium
and chloride must be taken from the sweat and moved back
into cells of the sweat duct.
• These ions are moved by transporters called ion channels. For
sodium, there is a sodium channel; for chloride, there is a
chloride channel called CFTR.
• For sweat to be produced with the proper concentrations of
sodium and chloride, sodium channels and chloride channels
(CFTRs) must work properly.
• In cystic fibrosis(AR),the CFTR chloride channel is defective,
and does not allow chloride to be reabsorbed into sweat duct
cells. The concentration of chloride in sweat is therefore
elevated in individuals with cystic fibrosis.

87. • The sweat test is usually done on the forearm, but may be done
on the thigh of infants and children.
• First Step - Stimulating Sweat: Place two electrodes
containing a sweat-inducing drug called pilocarpine on the skin.
A small electric current is delivered through the electrodes to
help the pilocarpine stimulate sweat.
• Second Step – Collecting Sweat: After about 10 minutes,
stop the current and remove the electrodes.The test site is
carefully cleaned and dried, then a piece of preweighed filter
paper is placed over the test site and covered with parafilm to
prevent evaporation. Sweat is collected for 30 minutes. The
filter paper is retrieved and weighed to determine the weight of
sweat collected.
• People with cystic fibrosis have high levels of salt but the
amount of chloride is the factor that determines the outcome of
the test.

88. • Chloride Ranges in Infants:
< 30 = normal
30 – 59 = borderline
≥ 60 = positive for CF
• Chloride Ranges in Children and Adults:
< 40 = normal
40 – 59 = borderline
≥ 60 = positive for CF in children, but could be
normal for adults.
• If an adult has a result close to 60 mmol/liter, genetic
testing may be done to confirm the diagnosis.

89. Saliva
• Saliva testing is a diagnostic technique that involves laboratory
analysis of saliva to identify markers of endocrine, immunologic,
inflammatory, infectious, and other types of conditions.
• Saliva is a useful biological fluid for assaying steroid hormones
such as cortisol, genetic material like RNA, proteins such as
enzymes and antibodies.
• Saliva testing is used to screen for or diagnose numerous
conditions and disease states, including Cushing's disease,
anovulation, HIV, cancer, parasites, hypogonadism, and
allergies.
• Most saliva testing is performed using enzyme-linked
immunosorbent assay (ELISA), polymerase chain reaction
(PCR), high-resolution mass spectrometry (HRMS), or newer
technologies such as fiber-optic-based detection.

90. • late-night salivary cortisol testing is a suitable alternative to
serum cortisol testing for diagnosing Cushing’s syndrome,
reporting that both sensitivity and specificity exceeded 90%.
• Early morning salivary cortisol to be “as good as serum” as an
Addison’s disease screening technique.
• Saliva also contains some antibody molecules that apparently
derive from plasma.
• Thus measurements are sometime performed on saliva to
detect antibodies against infectious agents .
• Testing of saliva for antibodies against HIV has been
practiced widely in sexually transmitted disease clinics.
• A 2011 study demonstrated that HBV surface antigen saliva
testing using ELISA had a sensitivity of 93.6% and specificity of
92.6%.
• Genetic testing requires DNA from the patient that can be
obtained from buccal cells in saliva or from swabs of the interior
of the mouth.

91. Tears
• Tear is the watery secretion of the lacrimal glands that
clean and lubricate the eyes.
• Average basal tear volume = 5-9 μL
• Flow rate of tear =0.5-2.0 μL/minute
• Test for assessment of tear secretion :
• Schirmer's test : determines whether the eye
produces enough tears to keep it moist.
• Indication:
• Dry eyes(keratoconjunctivitis sicca)
• Excessive watering of the eyes(epiphora)

92. Test procedure: Both eyes are tested at the same time.
• A topical anesthetic is placed into the eye to prevent tearing
due to the irritation from the paper.
• Place a small strip of filter paper inside the lower eyelid
(conjunctival sac).
• The eyes are closed for 5 minutes.
• The paper is then removed and the
amount of moisture is measured.
• A young person normally moistens 15 mm of each paper strip.
• Result :
1. Normal - ≥15 mm wetting of the paper after 5 minutes.
2. Mild - 14-9 mm wetting of the paper after 5 minutes.
3. Moderate - 8-4 mm wetting of the paper after 5 minutes.
4. Severe - <4 mm wetting of the paper after 5 minutes.
Persons with Sjögren's syndrome moisten < 5 mm in 5
minutes

93. Tear film breakup time(BUT) test
• This test is done to determine dry eyes.
• Take a glass rod dipped in the 2% fluorescein solution & apply
on the inferior temporal bulbar conjunctiva.Patient is asked to
blink 3-4 times in order to distribute the fluorescein evenly over
the cornea.Examine the cornea under cobalt blue light on
slit lamp.
• The dry area is indicated by the
presence of a black spot.
• The time between the last blink and
the first appearance of a dry spot
should be recorded in seconds as
the tear film breakup time.
• Repeat 3 times & take average.
• BUT ≥ 20 seconds is Normal

94. SUMMARY
• Body fluids are diverse, with variation in
physical appearance, properties, cell types,
and cell counts.
• In general, studies of body fluids are most
helpful to assess inflammation,infection,
malignancy, and hemorrhage.

95. • The study of body fluids presents challenges
to the laboratory.
• Analysis involves multiple departments of
the
laboratory and specialized knowledge of
each type of body fluid.
• Hematology is important in examining the
cells and crystals found.

96. • Microbiology can help detect infectious agents in
a nearby body cavity or membrane, and
immunological tests and other miscellaneous
tests can also provide the physician with critical
information.
• Further consultation with pathology required for
the identification of tumor cells and other
abnormal cells.
• Thus it’s a muldisciplinary & multispeciality field
of medical science.

97. References
• Henry’s Clinical Diagnosis and Management by Laboratory
Methods,22nd ed.,2011,Elsevier Saunders, Philadelphia.
• Graff’s Textbook of Routine Urinalysis and Body Fluids,2nd ed.,
2011, Lippincott Williams & Wilkins,Wolters kluwer,Philadelphia.
• Urinalysis and Body Fluids; Susan King Strasinger & Marjorie
Schaub Di Lorenzo; 4th ed., 2001; F. A. Davis Company •
Philadelphia.
• Williams Obstetrics, 23rd Ed.,2010, The McGraw-Hill Companies
• Delmar’s Manual of Laboratory and Diagnostic Tests,
Rick Daniels; 2010, Delmar,Cengage ;USA
• Various internet references