Clinical Pathology

1.Urine analysis.

Urine is one of most easily obtained specimen examined in the lab. It is formed in
the kidney, as a product of ulra filtration of plasma in renal glomeruli followed by
reabsorbtion of water and solutes in tubules and active secretion of some
substances by tubules.

Use of urine analysis:

1. Diagnosis of renal disease and urinary tract.

2. Diagnosis of systemic disease like hormone disorder ,congenital disorders.
Liver disorder (jaundice),Diabetes.
3. Pregnancy diagnosis.
Characteristics of normal urine:

Physical appearance- Clear ,pale yellow color due to urochrome, aromatic odour
due to volatile organic acids.

PH – acidic (6.0) but on standing alkaline due to release of ammonia from urea by
urease enzyme which is initiated by bacterial contamination.

Chemical composition: Water – 95%

Soluble waste product – urea, uric acid, creatinine.

Electrolytes – Na.K,.Cl.,Po4. So4

Proteins – amino acids, enzymes, purine

Hormone – keto steroid, aldosterone, estrogen, catecholamines.Pituitary
gonodotropins.

Biogenic amines and serotonin metaboliles.

Collection of urine:

Methods:

1. Collect midstream voided urine in wide mouth glass or plastic graduated

container after cleaning the external genitals (clean specimen)]
2. In bed ridden patients – collect urine in bed pan or urinal
3. Collect urine by catheterization (for culture_ - but danger of introducing
infection to bladder if aseptic techniques are not followed.
4. Neonates and infants – special urobag with adhesive stripes are available for
urine collection.
5. Supra pubic puncture and get urine sample.

Types of urine specimen

1. Random urine – Easy to get, but variable in solute concentration.

2. Post prandial sample – Used for post prandial measurement of protein and
sugar.
3. Early morning first urine – ideal specimen
Advantage of early morning sample –

 Low Ph - preserve cell and casts well

 Increased osmolality reflects concentration ability of kidney
  fasting specimen helps in diagnosis of diabetes
 For confirmation of pregnancy.
 Overnight growth of bacteria helps in diagnosis of UTI.
4. Evening urine – for estimation of urobilinogen. collect urine after 4pm since
it is excreted maximally in 2-4pm
5. 24hours urine – early morning first urine is discarded and collect all urine
in large ,transparent, wide mouth ,capped plastic clean container of 3-5
liter Capacity for 24hours including early morning urine of next day (8 a.m
to next day 8 a.m). It is used for quantitative estimation of solutes and
concentration of tubercle bacilli.
6. Timed specimen – urine is collected in timed interval like after 2hours,
4hous, 8hours. It is also used for quantitative tests. Urine should be
examined within 1-2hours.
Changes occur in standing urine.

1. Cells (RBC, WBC, epithelial) are destroyed due to hypotonicity of urine.

2. Casts degenerate
3. Alteration of PH
4. Bacterial contamination that split urea to ammonia-- alkaline urine
5. Ketones are evaporated.
6. Pigments are oxidized
7. Glucose is metabolized.
Urine Preservatives

Definition: Substances used to preserve cells in urine if present, prevents

bacterial growth, stabilize solutes.
 Types of preservation:

1. Refrigeration – If urine is kept at 5oc that maintains PH & prevents bacterial

growth.Preserved for 8hours – for microscopic cell count analysis,Can be
kept for 24hours – for chemical analysis.
2.Chemical preservation- Using formalin, chloroform, toluene, Thymol, boric
acid, sodium carbonates, sodium fluorides, con, HCL.

Use –

1. It is used when there is delay in examining urine.

2. For 24hours urine specimen analysis.
3. When samples has to be transported or mailed to distant lab for
examination.
1. Toluene – It is the best of all preservation.
Dose – It is added in sufficient quantity to form thin layer on the surface of urine.

Advantages – It prevents air entry and oxidative damage. Best preservative for
chemical constituents.

Demerits – It interfere with protein examination by SSA method.

2. Boric acid – dose 5g/120ml of urine. 50g/24hours urine.

Merit – Best for testing chemical constituents. Don’t interfere with protein,
sugar, ketone body tests.

3.Con.HCL – dose – 10ml/24hours. Best for hormone analysis calcium, urea, uric
acid, creatinine analysis.

4.Formalin – 40% formalin dose – 1drop/ 30ml urine

 10ml /24hours urine

Advantages – It preserves cell and cast well

Demerit – Since it is a reducing agent. It interfere with copper reduction test. But
does not interfere with clinistix method.

5.Thymol – dose – 5mm size crystal/100ml urine

Merit – It prevents bacterial growth and preserves cell well

Demerit – interfere with sugar, acetone test and protein test by heat and
acidification method.

6.Sodium carbonate – Used for urobilinogen, porphyrins determination.

Dose – 2.5ml is added to brown glass container before urine collection that
maintain alkalinity..

7.Sodium fluoride – Used for glucose estimation since it prevents glycolysis by cell
and bacteria.

Dose – 0.5g/24hours urine.

8.Chloroform – Can be used in place of formalin. Dose 50drops/24hours urine.

Routine examination of urine.

a. Physical – volume, color, odour, transparency, specific gravity, osmolality

b. Chemical – Ph ,proteins ,reducing sugar, ketones, pigments, blood.

c. Microscopic – cells, casts, crystals, micro organism.

d. Culture – For micro organism.

Physical examination:

1.Volume – The normal range of urine volume is 1200 = 2000ml/day with volume
excreted during day is 3-4times than that excreted during night.

Abnormal – Increased volume of urine excreted >2liter/day = polyuria.Seen in

physiological – excessive coffee intake, cold climate ,increased intake of fluid
.Pathological – diabetes insipidus, diabetes mellitus.

Decreased volume of urine excretion – oliguria <500ml/day.It is seen in –

physiological – warm climate, decreased intake.Pathological – renal failure,
obstruction to urinary tract.

Total suppression of urine output – anuria -seen in severe renal disease.

2.Color

Normal – pale yellow colour due to presence of urochrome and urobilin.

Concentrated urine is deep yellow ,urine become dark color on standing due to
oxidation of colorless urobilnogen to colored urobilin.

Abnormal:

Reddish brown – increased urobilinogen and porphyrin in urine

Bright red – increased fresh blood.

Pink – small amount of blood.

Smoky brown – blood pigments.

Brownish yellow or green – bile pigments.

Milky white – chyluria due to filariasis.

3.Odour:

Normal – mild aromatic odour. Standing urine become ammoniacal smell because
of decomposition of urea with liberation of ammonia.

Abnormal – Mousy odour – phenyl ketonuria

Sweet fruity odour – ketone bodies increased in urine.

Maple syrup odour – maple syrup urine disease

Ammoniacal odour – UTI

4.Appearance – Normally it is clear.

Abnormal – It is cloudy or turbid. Causes of turbidity

 Presence of Po4 in alkaline urine – cleared by adding acetic acid.

 Urate Precipitate in acid urine – disappeared by heating.
 Presence of pus cells ( >200/cmm), RBC(>500/cmm), bacteria,fungi – cleared
by centrifuge
 Presence of fat or chyle – cleared by adding ether or chloroform
  Mixing with sperm, prostatic fluid in male genital secretion, menstrual blood
in female.
5.Reaction – PH of normal urine is 4.6 – 8.0

1. It is determined by strips of litmus paper.

In acid urine – blue litmus turns red.

In alkaline urine red litmus turns blue

In neutral urine – both red and blue litmus changes towards purple.

2.By using PH paper (nitrazine paper) which contains acid base indicator methyl
red and bromophenol blue. on dipping, the color change from orange – green –
blue develops as the PH rised from PH 5-9.

6.Specific gravity

It gives us indication of the amount of solutes in solution in that urine.

Normal – 1.0.15 – 1.025 in 24hours urine.

Substances influenced specific gravity – Urea Na, Cl, Po4 (physiological),

pathological -- protein, sugar.

Method a.– Instruments used for determination specific gravity is called

urinometer. It is a weighted cylinder which floats in urine. It has a scale in the
stem reading from 1.000 to 1.060 with division of 0.001 – 0.002. it is calibrated for
certain temperature of 150c – 200c. Urine is poured into a cylinder or conical glass
vessel so that it is nearly full. If there are bubbles or froth they can be removed
with filter or blotting paper. Urinometer is floated in the urine and care is taken to
see that it does not touch the sides & bottom of the container. Noting the lowest
part of meniscus and specific gravity is directly read from it.

If volume of urine is insufficient –

a. Dilute urine with equal volume of distilled water. Then multiply the last two
digits of reading by two to get correct value.
Correction of specific gravity:

1.Temperature correction – any change in temperature influence specific

gravity. increased temperature – urine rises &expands – lower specific gravity

Decreased temperature – urine contracts – increased specific gravity.

Any change in temperature of 3oc – if increased temperature – add 0.001, if

decreased 3oc temperature – subtract 0.001.

Example:

Temperature of urine is 32oc. calibration of urinometer is at 20oc

Uncorrected specific gravity = 1.011 & temperature difference is 12oc

Corrected specific gravity -1.015 (By adding 0.004)

2.Albumin correction – presence of albumin increases specific gravity

300mg/100ml urine rises 0.001 specific gravity.

For 1g% of albumin – 0.004 is deducted.

<0.5g% of albumin – no need of correction.

3.Glucose correction – for 1g% of glucose – deduct 0.003

4.Dilution correction

Biochemical analysis

Protein, sugar, ketone, bile salts, bile pigments, blood in the urine is tested by
various chemical tests.

1.Tests for detection of proteins. Heat coagulation test, sulpho salicilic acid,
nitric acid test (hellers test) dipstick method.

a.Heat coagulation test:

Principle – Proteins are coagulated when subjected to heat at acid PH.

Procedure - Take urine in ¾ of test tube and heat the top portion.Watch for
cloudiness in the upper column .Add 3drops of Glacial acetic acid.

Result - Presence of protein is indicated by presence of turbidity or Precipitate

in the upper column which is compared with that of lower column. Addition of
acetic acid dissolves Precipitate due to phosphates and facilitates protein
precipitation.

 No coagulum - Negative
 Traces of turbidity –Trace
 Definite turbidity without granules – 1+
 Turbidity with small granules – 2+
  Turbidity with large granular clumps -3+
 Opaque coagulant – 4+

b.Sulphosalicylic acid test

Principle – Proteins are coagulated by Sulphosalicylic acid.

Procedure - Take 4-5 ml of urine in the test tube and add 2-3 drops of 20%
aqueous solution of Sulphosalicylic acid .Mix thoroughly.

Result - Presence of protein is indicated by presence of turbidity

c.Heat test for Bence Jones Proteins (BJP)

BJP is a homogenous monoclonal protein produced by a single colony of

Plasma cells.

Principle – BJP has peculiar thermal characteristics that it precipitates when

heated to 50—60C.But resolubilizes at 90-100C and again precipitate on
cooling to 56C.Since other proteins are precipitated on heating,it is better to
filter the urine after boiling to avoid the masking of BJP by other proteins.

Procedure - Take urine in the ¾ of the test tube and add 2-3 drops of 10%
acid to PH 3.5 and gently heat in a beaker containing water with a
thermometer. Observe the turbidity upto the temperature 90C.Then cool the
beaker till the temperature is 60C.

Result - Presence of protein is indicated by presence of turbidity at 40C and

flocculent will form at 60C and disappears at 90C.Precipitate reappears at 60C.
2.Test for detection of glucose.

Non specific cu reduction test (benedicts test)

Enzymatic reagent strip test – clinistix.

Benedicts test:

Principle –When Soluble cupric ions of CuSo4 is heated in an alkaline solution,

it is reduced to yellowish to red insoluble cuprous ions of cuprous oxide by
urinary reducing substances.

Procedure -Take 5ml of benedicts reagents in a tube and heat it to check for
auto reduction. Then add 8drops of urine and heat it for boiling.Presence of
sugar is indicated by change of color from blue to green, yellow, orange, red.

Result –

 Blue color - Negative

 Light green turbidity –Trace
 Green precipitate – 1+
 Yellow precipitate – 2+
 Orange precipitate -3+
 Red precipitate – 4+

3.Test for ketone bodies – (acetone, acetoacetic acid,, beta hydroxy butric
acid) Rotheras test (For acetone & acetoacetic acid) , Gerhardts test For only
acetoacetic acid))
Rotheras test

Principle – Alkaline Nitroprusside turns into purple color by acetone &

acetoacetic acid.

Procedure -Take 3ml of urine in a test tube and saturate it with NH4SO4
crystals. Transfer clear supernatant to other tube, add small single crystals of
sodium nitro prusside. Mix well.layer a drop of liquid ammonia over it.Watch
for the formation of colored ring at junction of liquids.

Result – Purple ring formation at junction indicates positive for ketone bodies.

4.Test for blood.

Chemical test (benzidine test), microscopic examination, reagent strip test.

Benzidine test

Principle –The peroxidase like activity of Hemoglobin splits hydrogen peroxide

into water and nacent oxygen which oxidizes benzidine into a colored
compound.

Procedure -Saturate 3ml of glacial acetic acid with benzidine powder in a test
tube. Take clear supernatant fluid in other test tube, and add 2cc of urine.
Watch for color change. The add 1ml of Hydrogen peroxide (H2O2).

Result -Appearance of green or blue colour indicated presence of Hb & RBC in

urine.

 Green color - 1+
 Greenish blue –2+
  Blue – 3+
 Deep blue – 4+

Yellow precipitate – 2

5.Test for bile salts.

Hays test

Principle –Bile salts lower surface tension

Procedure - Spray or sprinkle sulphur powder on the surface of urine taken in

wide mouth tube or Container. Observe the granules.

Result - If sulphur granules sinks to bottom that indicates presence of bile

salts.

6.Test for bile pigments – Bilirubin, urobilinogen

a.Bilrubin in urine detection-foam test, iodine test, methylene blue test, nitric
acid test, fouchets test, icto test (Diazo tablet test)

Fouchets test

Principle –Barium chloride precipitates phosphates which entrapped &

concentrates bilirubin.Addition of Fouchets reagent (Ferric chloride
,Trichloracetic acid and water) oxidizes bilirubin to colored biliverdin (green)
bilicyanin (blue).

Procedure -Take 10ml of urine in a tube add 5ml of BaCl2 (10%). White
Precipitate is formed .filter it. Spread the filter paper on another dry paper and
add two drops of fouchets reagent.Observe the color change.
Result - Green or blue colour indicates positive for presence of bilirubin.

b.Urobilinogen in urine detection – Ehrlich test, reagent strip test.

Ehrlich test

Procedure - 10ml of fresh urine is taken in a test tube and add 1ml of Ehrlich
reagent(Para dimethyl amino benzaldehyde with con.HCL in water).Wait for 5
minutes

Result –

 Appearance of Cherry red color indicates increased urobilinogen .

 Faint pink colour – normal urobilinogen.

Microscopic examination of urine:

It is done in the urine sediment which is obtained by centrifuging 10ml of the

urine at moderate speed (1500rpm for 5minutes). Place a drop of urine
sediment over glass slide, mount with cover slip and examine under
microscope.

Precautions:

1. Urine should be fresh. In case of 24hours urine, it should be properly

preserved.
2. Urine should be mixed well before doing microscopic examination.
3. There should be no air bubble under the coverslip.
 4. Condenser is lowered and light is cut down considerably.
Normal urine microscopic findings.

0-2 RBC/hpf, <5 WBC/hpf , 1-2 epithelial cell/hpf , Few crystals no casts and
micro organism .

Pathological constituents of urine sediment.

a.Unorganized sediment

Crystals – in acid urine – uric acid, calcium oxalate crystals, leucine, tyrosine,
sulpha crystals

-in alkaline urine – triple Po4, calcium phosphates, calcium carbonate,ammonium

biurate.

b.Organized sediment

Casts (Tubular shape) – hyaline, waxy,granular, epithelial casts, pus cell casts,
fatty casts.

Cells – RBC, pus cell, Epithelial cell,Spemetozoa

Micro organism –Bacteria, fungi,Parasite

Chyle in urine seen a fat globules.

Crystals is acid urine:

 a. Uric acid and urate crystals. Urate – reddish brown granular deposits .it is
dissolved by heating and by addition of NaOH. Uric acid – Rhombic, flat,
prism, ovoid form, colored crystals (reddish brown)
b. Calcium oxalate – Seen as colorless octahedral crystal resemble as
envelope.
c. Cystine crystal – colorless hexogonal plates.
d. Leucin crystal – seen as yellow colored spheres with radial and concentric
striations.
e. Tyrosine crystal - in the form of fine needles arranged in sheaves with a
marked constriction in the middle.
Crystals is alkaline urine:

a. Triple Po4 or ammonium magnesium PO4 – seen as colorless, coffin lid or

leaf like.
b. Calcium PO4 – colorless prism arranged in rosettes or stars.
c. Calcium carbonate – colorless spheres or dumbbell shape.
d. Ammonium biurate – yellow spheres covered with fine or coarse spicules.

Organized sediment:

Casts are coagulated protein which is cylindrical in shape with rounded or broken
ends ,varying in length and formed in the kidney.

Type:

a. Hyaline cysts – colorless and semitransparent.

b. Granular cast – casts containing granules – fine ,coarse.
 c. Epithelial casts – coagulated protein with epithelail cells.
d. Blood cell casts – Casts in which red cells are embedded.
e. Pus cell casts – protein cast with disintegrated WBC
f. Fatty casts – casts with fat globules.
g. Waxy casts – similar to hyaline cast but it is opaque , dull. Waxy
appearance.
h. Pigmented casts – casts containing pigments like bilirubin, melanin.
2.Other – RBC, WBC epithelial cells.

3.Micro organism – bacteria, fungi, parasite.

Addis count – It is quantitative measures of urine sediment .12hours sample with

formalin as preservative is preferable .Measures the volume. Centrifuge 10ml of
the urine for 5minutes at 1800rpm and prepare urine sediment with distilled
water .after discarding supernatant charge the neubauer chamber with sediment
and count cells in all 9 squares.

Calculation = N = S x N x D x V

10 x A

V = volume of urine 12hours.

A = Area

D= depth

N =number of cell type or cast.

S = volume of urine centrifuged divided by 10

N= 1 x N x 10 x V

10 9

Normal range -Casts = 0-5,000/12hours

RBC = 0-5,00,000/12hours.

WBC = 0-10,00,000/12hours urine.

Use of addis count – to assess the progress of patient with nephritis.

 2.CSF analysis
Definition:CSF (Cerebrospinal fluid) is the ultra filtrate of plasma found in the
subarachnoid space surrounding brain and spinal cord.

Normal volume: 150ml. it is kept constant by reabsorbtion of CSF and active

secretion and ultra filtration in choroid plexus.

Normal CSF pressure – it is monitored by glass monometer attached to LP needle.

70-150mm of CSF.

Method of CSF sample collection– it is collected by Physician by lumbar puncture

– a procedure in which needle of about 10cm length and 1-1.5mm bore with
internal stillet is inserted into subarachnoid space at lower end of spinal cord.
Short needle are used for small children.

Collection – It has to be collected in three separate sterile dry test tubes. II nd

tube has more quantity nearly 2.5ml. Don’t draw CSF more then 6-8ml. First tube
is used for microbiologic examination. II nd tube for biochemical analysis. III rd
tube for cytological analysis ( pathological)

Tests to be done in CSF:

1.Pathological – Gross, microscopic – cytologic examination.

2.Biochemical – Protein,Glucose,Chloride,LDH

3)Microbiologic culture and stains. – AFB, GS, India ink for cryptococcus.
4)Serologic examination – Test for syphilis – VDRL, TPI, test.

Gross examination:

1)Specific gravity – Normal 1.003.Not routinely done.

2)Color – Normal is clear and colorless, looking like distilled water. It does not clot
on standing.

It is noted by comparing the color of CSF and with tube containing distilled water
against a sheet of white paper.

Abnormal color – a) Reddish- It may be due to trauma or subarachnoid

hemorrhage ( SAH)

Trauma – a)first tube CSF is bloody and color intensity decreased in the third tube.
b) Pressure Normal, c) coagulation may occur, d) Xanthochromia absent.

SAH – a)all three tubes are equally blood stained, b)pressure increased, c) no
coagulam since defibrination occur in vivo, d) Xanthochromia present.

b)Yellow colour – Othterwise called xanthochromia. It may be due to hemorrhage

,jaundice,Tumor .

Previous Hemorrhage(2-12hours) – lysis of red cell – Hb – bilurubin

Deep jaundice – conjugated bilurubin (infant)

Tumor – blockage of CSF circulation – yellow coloration

3) Turbidity – It is seen when there is increased number of cells 400-500(RBS or
WBC) or bacteria increased or both. If less number of cells are present in CSF –
there is no grossly visible turbidity.

4)Coagulum: Normal CSF does not clot on standing .In pathological state, if you
allow CSF to stand overnight, firm clot will be formed. This is due to increased
protein content of CSF.

It is seen in

a)Brain tumor with increased protein

b)Traumatic tap with fresh blood in CSF

c).TB meningitis – cobweb like clot forms and AFB may get caught in the clot.

CYTOLOGIC EXAMINATION:

Quantitative study & Qualitative morphological study.

Quantitative study - Normal CSF contain no RBC, 0-5 WBC (small lymphocytes).
since number of cell are very less – It is better to examine in undiluted form .

Neubauer chamber with 9 areas or 18 areas (counting on both sides)

Or Fuchs Rosenthal chamber with 16 squares counting .Cell count should be done
immediately and not beyond 30minutes Since there will be lysis and detoriation
of cells that lead to inaccurate results.
Diluting fluid:

a)1% Methylene Blue/Toludine Blue or 1% crystal violet – 9 part CSF + 1 part

stain.it stains WBC and not lyse RBC.

b)Diluted acetic acid – 0.1g crystal violet.

-1ml glacial acetic acid

- Distilled water 50ml

- 7% phenol few drops.

It is used for Hemorrhagic CSF. it lyses RBC.

Techniques of dilution.

9:10 dilution – it is done by draw diluting fluid upto 1 mark in WBC pipette and
CSF 11 mark. Mix well discard stem portion and load the chamber with bulk
content.

Calculation:

a)RBC & WBC count in undiluted CSF

Neubauer chamber = N x 10( depth) x 1 / 9(area)

= 1.11 x N

Fuch Rosenthal chamber = N x 5( depth) x 1 / 16(area)

= 0.3125 x N

Count RBC ( note for crenation) ,Count WBC &do chamber differential count
b)In diluted CSF – depth 1/10mm, dilution- 9:10

Neubauer chamber – N / dilution x depth x areas

N/ 1/10 x 9/10 x 9 = N x 10 x 10 / 9x9 = 1.2346 x N

Fuch Rosenthal chamber =– N/ dilution x depth x areas

N/ 2/10 x 9/ 10 x 16 = N x 5 X 10 / 9x16 = 0.347 x N

Qualitative or DC

a)Chamber differential count – identify Neutrophils & lymphocytes by their

nucleus in the chamber after charging, count 100 WBC and read as percentage.

b)Leishman stained smear – centrifuge the sample. Smear made from sediments.
Air dry and do leishman stain.

If you add 1-2 drops of bovine albumin to CSF cell sediment that preserve cells
and retain morphology well.

Abnormal: Increased Neutrophils in CSF – Seen in acute pyogenic meningitis,

aseptic meningitis, associated with viral meningitis ,brain abscess,

Increased lymphocytes – TB meningitis,neuro syphilis, non infectious CNS disease.

 3.CAVITY FLUID:

It is the fluid aspirated from various body cavities. It includes pleural fluid,
peritoneal or ascitic fluid, pericardial fluid, synovial fluid.

Normal – Cavity contains less amount of fluid which keeps the surfaces moist and
lubricated so that movement of adjacent surfaces occur with minimal friction.

Abnormal – If increased in volume of fluid is there, it is called effusion.

Collection of sample – Samples are obtained by percutaneous punctures and

should be transported to lab immediately.

Storage changes – Cells disintegrate, bacteria grows, change in chemical

composition. So fluid should be examined immediately or to be kept in
refrigerator.

Tests done in cavity fluids:

Pathological – Gross & Microscopic examination

BioChemical – protein ,glucose,Chloride,LDH

Microbiological – Gram stain, culture.

Difference between transudate and exudates.

Characteristics Transudate exudates

1.Appearance Clear cloudy

2.Color straw yellow yellow to red

3.Odour Nil May have if septic

4.Specific gravity <1.018 >1.018

5.Protiens <2g% >2g%

6.Glucose 10-20mg less than blood Very low

glucose

7.Cells less High count

8.Bacteria none +
Gross Physical Examination

 Volume – Measure the volume of fluid received & noted

 Colour – Note whether fluid is colorless,yellowish,reddish,greenish brown
 Appearance – Clear or turbid
 specific gravity &odour if volume is more- Not routinely done

Microscopic Examination

a.Cell count:

Diluting fluid – If cloudy -use RBC diluting fluid for counting RBC

 WBC diluting fluid for counting WBC

If clear fluid – No diluton is needed.

Use Neubauer chamber for cell counting using the formula

N / DilutionxDepthxArea

b.Differential Count – Centrifuge the fluid – smear prepared from sediment. a).
Dry it – Do leishman stain and DC

c.Cytological study -Fix it in alcohol – stain with pap stain or Hematoxlie & Eosin
stain - Malignant cells screening by Pathologists.

Specific test for synovial fluid:

1.Clot test – Take 1-2ml of fluid in dry test tube and examine after 1hour for
presence of clot. If clot is present – it indicates inflammatory damage to synovial
membrane.
2.Test for viscosity – Place a drop of fluid over thumb with a help of finger touch
the drop and lift the finger if fluid is viscous. It will stretch. Note the distance of
stretch.

Since above procedure involves technicians hand, other method is tried .fluid is
slowly expressed from syringe through needle. It stretch and form string of atleast
4cm before it breaks. If fluid has decreased viscosity it will not stretch or form
long strings. Decreased viscosity - < 1cm string length – seen in inflammatory
condition due to decreased hyaluronic acid.

3) Mucin clot test or ropes test.

Add 1ml of synovial fluid into 20ml of 5% acetic acid in a small beaker. It measures
the hyaluronate in the fluid, Result is reported as

 Compact clot with clear fluid – good

 Soft clot with turbid solution – fair
 Friable clot with cloudy solution – poor
 No clot and flakes in cloudy suspension – very poor.
4)For cell count – WBC diluting fluid is unsatisfactory because acetic acid in
diluting fluid Precipitate the hyaluronate So 0.1N HCl may be used instead.

5)Examination for crystals – Wet film study and stained smear study for cloudy
urate crystal within cytoplasm of phagocytes which is seen in gout.
 4.SPUTUM

Definition – It is the secretion of trachea &bronchial tubes. If excesses it is

expectorated by coughing.

Collection: Early morning sputum is ideal. Ask the patient to cough (not to spit
from mouth which contains saliva and collect it in a closed sputum cups or wide
mouth bottle with covers.

Tests done in Sputum:

Pathological – Gross & Microscopic examination

Microbiological – Gram stain, AFB, Culture.

Gross/Macroscopic examination

1)Volume – It varies according to non productive cough, productive cough.

2)Consistency – It may be serous, frothy, mucoid, purulent, seropurulent,

Hemorrhagic.

3)Odour – Normally it is odourless. In chronic infection, lung abscess,

bronchiectasis (where retained sputum in dilated bronchus undergoes
decomposition) foul odour is present.

4) Color:

If sputum is mucoid – colorless and translucent. purulent – yellowish white or

greenish.
Coal miners, smokers – grey to blackish due to pigment ladden macrophages.

Blood in sputum is called haemoptysis. It may be due to fresh blood (red colour)
or rusty sputum which is reddish brown in colour due to alleration in Hb

5) Miscelleneous – Large objects present in the sputum is visible macroscopically.

They are

a)Bronchial casts – It is a branching fibrin structures that are coughed out.

Seen in bronchitis and pneumonia.

b)Broncholiths.- It is calcified bits of necrotic lung tissue seen is TB and

fungal infection of lung.

c)FB – pins, glass beads, nuts, which are accidentally aspirated.

d)Sulphur granules – Yellow granules made up of colonies of fungus. Eg

Actinomycosis of lung

e)Parasite – Round worms, lung fluke (paragonimus westermani)

Microscopic examination: - Using unstained sputum – stained sputum.

Unstained sputum.

Smear is made and coverslip is placed over it and examined

a)Elastic fibers – It appears as wavy refractile bundles.

It is seen also by concentration technique – 10ml of sputum + equal volume of
NaOH. Then centrifuge and sediment is smeared and examined for elastic fibres.
It is seen in any destructive disease of lung.

b)Curshmann’s spiral – Wiry spiral structure with a central thread. Seen in

asthma.

c)Charcot–Leyden crystals – Fine needle shaped or Hexogonal colorless crystal

20-30 mm length eg.asthma.

It originates from disintegration of eosinophil. Stains strongly with eosin. It is not

seen in fresh sputum. Seen only in sputum when sits for sometimes.

d)Pigmented cells: Pigmented macrophages present in sputum that ensures the

specimen is sputum and not saliva.

e)Fungus – Look for branching mycelia.

f)Parasites – Paragonimus westermani , echinococus granulosus. Strongyloides

stercoralis, hook worm. Entamoeba histolytica, ciliated bronchial epithelail cells
(mobile in wet film) should not be mistaken for ciliated protozoa.

Stained sputum smear:

1)Dry the smear and fix it by flame and stained with GS, AFB.

2) Staining with methylene blue or Toludin Blue

3). Pap stain for cytological study of cancer cells after fixation.

4). Leishman or wrights stain for

  PMN – pyogenic infection of lung
 Eosinophil – asthma, parasitic infection of lung
 Lymphocytes – early TB
 Macrophages – presence confirm the sputum sample
 Epithelial cells – columnar cell (ciliated) from tracheobronchial tree ,flat cell
from alveoli, Squamous cell from throat.
 RBC – if excess – bleeding into lungs or bronchi .
Concentration method for TB in sputum:

Specimen – 24hours sputum collected in sterile bottle.

Method – Mix equal volume of sputum with 6% H2SO4,.leave it for 20minutes.

centrifuge for 30minutes at 3000rpm. Discord the supernatant. Wash the
sediment with distilled water three times. Then make a smear with sediment.

Smear made from sediment – dry – flame fixation – Ziehl neelsan stains. Or
culture from sediment

This method is not adopted for fungus since it is killed by acid used.
 5.SEMEN ANALYSIS:

Definition – Fluid composed of secretion of seminal vesicle, prostate, cowpers

glands and spermatozoa which are formed in seminiferous tubules of testis.

The secretion depends on adequate secretion of androgenic hormone from testis.

In vasectomy – semen secretion is present but lack spermatozoa.

In testicular atrophy – No hormone – no semen secretion.

Indications:

1)To determine the fertility of men in case of infertility.

2) To check the effect of vasectomy.

3)In medico legal situation such as dispute about paternity of a child.

Collection: Specimen is collected into clean dry bottle after 48hours- 5days of
sexual abstinence. It should be brought to lab immediately. It is not tested
immediately since it is viscous. It liquefied within 15-30minutes normally by the
action of enzymes in fluid. Then it is examined.
Tests done in Semen:

Pathological – Gross & Microscopic examination

BioChemical – Fructose

Gross/Macroscopic examination:

1)Volume – It is measured is small graduated cylinder. normal 4-5ml. If it is

<1.5ml it is abnormal.

2) Viscosity– Semen normally liquefied within 30minutes. If viscous after

30minutes it interferes with sperm motility in female genital tract to fertilize
ovum.

3)Color:

Normal – grey white; cloudy opaque

After long period of abstinence – yellow

Haemorrhagic – reddish or reddish brown.

4) Reaction PH – seminal vesicle secretion is acidic ,whereas prostatic secretion is

alkaline. So normal PH is 7.8 (7.2 – 8.9) motility is decreased in acidic PH.

Microscopic examination:

1)Motility – Place a drop of liquefied semen on a glass slide put a coverglass over
it .seal the edge with Vaseline to prevent evaporation and drying .examine under
high power. We will see numerous spermatozoa running and swimming in all
directions across the field.
Observe & count the percentage of the fastly moving,slowly moving,immotile and
non progressive movement of sperms

Normal – 60-80% motility (actively moving).Only 20% are sluggish.

If <50% motility – cause infertility.

Slide may be observed after 3hours, 6hours 12 hours 24hours upto 3hours there
is no reduction in motility. Then progressive loss of motility which completes by
12 hours.

More non motile sperm >50%– Astenospermia

Non motile sperm should be differentiated from live or dead sperm by doing
Eosin test. Dead sperm head takes up the stain whereas live sperm head is
colorless.

If more than 50% sperms are dead - Necrozospermia

2)Sperm count:

Diluting fluid

- Na2Co3 - 3g (counteract mucin and allows even dilution of viscous fluid)

- Phenol or formalin – 1ml (preservation, kill sperm and prevent motility)
- Distilled water – 100ml

Dilution: 1 in 20 dilution using WBC pipette or bulk dilution (0.02ml semen +

0.38ml diluting fluid)
Charge the chamber. count sperm in 4 corner square after settling of sperm. Since
number of sperm is expressed per ml of seminal fluid. The usual formula (which is
expressed per microliter) is modified by multiplying with 1000.

Sperm/ 1ml = N x 20 x 10 / 4 x 1000 = N x 50.000

Normal count – 60.000.000 – 150.000.000

<60.000.000 – oligospermia.

After 2-4 weeks of vasectomy - no sperm – azoospermia.

3)Morphology:

Smear made from semen – dry – heat it gently for fixing dried smear onto slide.
Remove mucous which interfere with staining by washing in diluting fluid (contain
Phenol and Na2CO3) Then wash in buffered distilled water.

Staining:

1)Leishman stain as for blood smear.

2)Or 0.25% aqueous basic fuschin for 5minutes.

3)Carbol fuschin dilution with equal volume of 95% ethanol – 3minutes

Water wash – 3minutes with loefflers methylene blue diluted with 2 parts of
water – water wash 1minute – dry.

Morphology normal:
50-70 micron length, oval head,(3.6x2-3 ) stained as light blue, nucleus as dark
blue. Small neck , body, long tail which occupy 90% of total spermatozoa length.
body and tail are stained as light red or pink.

Abnormal morphology:

Head – small size – giant size, double head.Abnormal shape, irregular distribution
of chromatin and vesicular chromatin of nucleus.

Neck – absence, enlargement, bifurcation

Tail – Short, absent, double.

Normal semen – Should have not more than 20% abnormal firm.

Teratozospermia - >50% abnormal morphology

Others: Look for presence of other cells like RBC, WBC, epithelial cell, immature
round germ cell from testis.

If WBC increased – infection – GS – other bacteria.

 AFB – TB
Results

1. Volume
2. Viscosity/Liquefaction time
3. Color
4. PH
5. Morphology
 6. Motility
7. Sperm count
8. Dead sperm
9. Other cells.

6. Stool analysis

Motion is the excretory product of Gastro intestinal system .Normal motion is solid to
semisolid, yellowish brown in color without odour.
Indications
1.To diagnose the causes of infections either parasitic or bacterial dysentery
2.To identify the presence of blood
3.To identify the presence of reducing substances
Sample – Stool minimum of 2g collected in a clean wide mouth container
Tests done in Stool
Pathological – Gross - color,volume,consistency, PH reaction,mucus & blood

Microscopic examination - Pus cells,red blood cells,Fat bodies

BioChemical – Reducing substances, Occult blood, Fat

Microbiological – Wet mount study for Ova,cyst ,Gram stain, culture.

.
Gross/Macroscopic examination
Colour -Yellow colour is normal.
 Green color – Ingestion of Spinach.
 Reddish brown – Large amount of fruit ingestion.
 Red colour – Beetroot ingestion, Fresh blood.
 Clay colour – Obstructive jaundice.
 Tarry black – Hemorrhage in the stomach /upper intestine.
 Bright red to brown – Bleeding in rectum.
 Red streaks of blood on the surface of feces- Fissure & hemorrhoids.
Consistency-Soft & formed – Normal.
 Hard – Habitual constipation.
 Soft liquid – Diarrhoea.
 Small numerous, largely mucus & blood with small amount of fecal material –
Dysentry.
 Rice watery without any fecal matter – Cholera.
Reaction -Neutral PH is normal.
 Alkaline – Excess protein ingestion.
 Acidic – Excess carbohydrate ingestion.
Mucus-Small quantity of mucus is Normal.
 Excess quantity – Intestinal infection.
 Entirely mucus without feces & streaks of blood – Dysentry,
Ileocolitis,Intussusception.
Blood –It is absent normaly.
 Formed stool with blood streaks – Lesions in anus,rectum & sigmoid
colon.
 Liquid stool with bright red color blood ,mucus,pus – Bacillary
dysentery,Ulcerative colitis.
Microscopic examination.
  Sample – Stool minimum of 2g collected in a clean wide mouth container.
 Saline preparation -Glass slide, coverslip,saline,Microscope,Stick or Tooth pick.
 Iodine preparation – A drop of Lugols iodine .
Procedure
 Saline preparation
1. Place a small drop of saline over a glass slide.
2. Take a small portion of stool from representative area using stick or head of tooth
pick & keep it over the slide.
3. Mix well the sample with saline using stick.
4. Put a coverslip over the sample mixture.
5. The smear is thin enough to allow the news paper print to be read through the
saline smear.
6. Now the smear is ready for examination under microscope.
Note-Ova & larvae of helminths are well seen & identified by saline preparation
 Iodine preparation
The same procedure is repeated using a drop of Lugols iodine
Note.
It helps in detection, identification & differentiation of the cysts of Entamoeba group
of organisms.

The following things to be searched under microscope to detect abnormalities -Pus

cells,RBC,Macrophages,Fat globules,Bacteria,Yeasts & moulds,Protozoa – Motile
forms (Trophozoites) & non motile cyst forms of Entamoeba, Trichomonas
vaginalis,Giardia lamblia.

Chemical examination
Reducing substances – Benedicts test
Occult blood – Benzidine test