Medical Lab. Techniques

LABORATORY
INVESTIGATIONS
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COLLECTION AND PRESERVATION OF BLOOD
AIM
To collect the blood and preserve it with various anti-coagulants for various
clinical purposes.
APPARATUS
sterile syringe, needle, anticoagulant, cotton, spirit etc. blood can be obtained
by following methods
1.capillary method by finger puncture
2. venous method by venous puncture
PROCEDURE
Collection of blood by capillary method

The usual site used for collection of blood is tip of index finger. Palmar
aspect of thumb, middle or little finger are not used of, because the tendon
sheath of flexor muscles, which is close to the tip of finger can have more
chance to infection . Margin of ear lob, great toe and heel in infants are the
other areas usually used for collection of blood.
Collection method
Area of collection is to be cleaned using cotton soaked in spirit, dry the area,
massage and increase the blood circulation . Sterile the needle, gently prick
the area and discard 3-4 drops of blood, as it contains some tissue fluid.
Collect the subsequent drops by capillary tube or pipette. Some of the routine
examination of blood are Hb estimation, RBC, WBC, total count or
differential count, blood grouping, bleeding time, clotting time, smear for
malarial parasite.
Collection of blood by venous puncture
The site of collection are median cubital vein, external jugular vein, great
saphenous vein, superior sagital sinus, and dorsal venous arch. the last three
sites are for infants.
Collection method
Blood is collected when the patient is in the lying position of sitting along
the side of a table. The puncture area is cleaned by soaking the cotton with
70% alcohol. A tourniquet is applied above the cubital fossa. The needle of
syringe which is sterile is introduced into the veins, such a way that the
syringe is parallel to the vein and 45o to the skin surface. Care should be
taken to prevent counter puncture. When the blood is appeared in the barrel,
withdraw the piston and fill the syringe with required amount of blood.
Needle is taken way with the application of firm pressure at the site of
puncture. Needle is taken out from the syringe and blood is pushed into a
labeled bottle containing appropriate anti-coagulant and mixed gently.
Precaution
• Needle should be sterile to prevent the chance of infections like
hepatitis B,HIV, Syphilis, Malaria etc..
• Avoid the air entering into the vein to prevent air embolism
• Avoid counter puncture to prevent hematoma formation.
Uses and advantages
Blood collected by this method is used for determination of ESR, PCV etc.
Blood testing using whole blood without anti-coagulant are DC, clotting
time etc.
Disadvantages
It is time consuming
Anti-coagulants
Anti-coagulants are substances added to blood in order to prevent coagulation.
coagulation of blood can be prevented by
• Removing calcium by addition of oxalate, citrate and EDTA.
• Inactivating Thrombin and Thromboplastin by the addition of heparin
• Removing fibrin by Defibrination
Main anti-coagulants used are Double oxalate mixture, EDTA, Tri sodium
citrate and Heparin.
i) Double oxalate mixture
This remove calcium by forming insoluble precipitate of calcium oxalate.
Ingredients including Ammonium oxalate (1.2gm), Potassium oxalate (0.8gm)
neutral Formaldehyde (1ml) and Distilled water (100ml).
Ammonium oxalate causes swelling of RBC and Potassium oxalate causes
shrinking of RBC. They have cation balance each other, so the size of the
RBC is unaffected. The amount required is 2 ml of blood. Measure 0.8ml of
solution in a stopper bottle. This can be air dried at room temperature or in
an oven, not more than 60--0c. place 0.5ml of blood into anti-coagulant bottle
and gently mix it well. The oxalate mixture is not recommended for platelet
count or platelet function test. It cause platelets to clump.
Uses
Tests performed by using double oxalate mixture are RBC, WBC, Hb and
ESR determination
Disadvantages
• WBC’s morphology is preserved well, hence not generally used for
blood smear
• Oxalate mixture is not recommended for platelet count, as it cause
the platelets to clump.
ii) Ethylene diamine tetra acetic acid (EDTA)

This is calcium chelating compound. Calcium in the blood is found in an
unionized form and form a soluble compound with EDTA.
4 mg/dl of the solution is prepared. The amount required is 2 mg/dl of blood.
2ml of EDTA solution is taken in a dried bottle which contains 3 mg of dry
chemical and prevent coagulation of 3-4ml of blood. It is used as Di sodium
or Di potassium salt
Uses
Tests performed by EDTA are ESR by Wintrobe’s method, Hb %, RBC,
WBC, PCV etc.
Advantages
• It equates double oxalate mixture in haematocrit studies and is
superior in that, it does not produce any artifacts.
• The blood smear can be produced even after 2-4 hrs.
• Anti-coagulant is ideal for platelet count, since platelet clumping is
inhibited.
Disadvantages
• Excess of EDTA affects both RBC, WBC causing shrinkage and
degenerative changes
• Excess of EDTA causes platelets to swell and disintegrate.
• EDTA is no suitable for using coagulation studies.
iii) Trisodium citrate

This anti-coagulant is used in liquid form. It converts ionized calcium into
unionized soluble complex. 3.8% of Iso citrate is used as an anti-coagulant.
In the ESR determination by Westergren’s method, 1.6 ml of blood is mixed
with 0.4ml of anti-coagulant.
Uses
Used in determination of ESR by Westergren’s method and Prothrombin
time determination at partial Thromboplastin time.
Disadvantage
As it is used as liquid, the blood is diluted and hence it is unsuitable for other
hematological experiments such as Hb% and cell counting
iv) Heparin
Act as an inhibitor for thrombin formation. 0.1-0.2mg/dl of heparin is
necessary to prevent coagulation
Advantages
• It is less toxic and is used for heart surgery and exchange blood
transfusion
• It is used when plasma is urgently required for certain emergency
determination such as blood sugar, urea and electrolyte
Disadvantages
• It is expensive
• It forms a bluish, black ground in smear. It not used for blood and
born marrow smear.
v) Defribrination
It is the process of removal of fibrin, from blood sample. After this process
the blood remains in the fluid stage.
A sample of blood is stained well with glass bead. The clot of fibrin formed
is collected on the bead surface. In this technique fibrinogen is converted
into fibrin and it can be quickly removed. Defibrinated blood contain serum
and blood cells. This test is used for various pathological studies.
ESTIMATION OF HAEMOGLOBIN
Sahli’s acid haematin method
Aim
To determine the amount of Haemoglobin concentration in the given sample
of blood.
Principle
Haemoglobin is converted into acid haematin by the action of HCl. The acid
haematin solution is further diluted with distilled water until its color matches
with exactly that of permanent standard of comparator block. The Hb
concentration is read directly from the calibration tube
Reagent and solution: HCl solution, Sahli’s Haemoglobinometer, pipette,
distilled water
Procedure
By using pipette add.1n HCl in the haemoglobinometer up to the lowest
marking. Drop blood up to 20ul in the sahli’s pipette. Adjust the blood column
carefully without bubbles. Wipe excess of blood on the sides of the pipette
by using a dry piece of cotton Blow the blood into the acid solution in the
graduated tube, rinse the pipette well. Mix the reaction mixtures and allow
the mixture to stand at room temperature of 10 minutes. Dilute the solution
with distilled water by adding few drop of water carefully and by mixing the
reaction mixture until the color matches the color in the comparator . The
lower meniscus of the fluid is noted and reading is noted in g/100ml.
Normal values
Male : 14 –18g%, female: 11.5-16.5g%, infants : 16-18g%
Cyanmethaemoglobin method
Principle
When the blood is mixed with Drabkin’s reagent, containing potassium
cyanide and potassium ferricyanide haemoglobin reacts with ferricyanide
to form methglobin which is converted into slable cyanmeth haemoglobin
concentration and it is compared with known cyanmeth hb standard at 540
nm.
Drabkin’s solution
Potassium ferricyanide 400mg
Potassium di hydrogen phosphate 280mg
Potassium cyanide 100mg
Distilled water 100ml
This is stable at 2-80C
Cyanmeth haemoglobin standard
It is commercially available cyanmeth Hb in solution corresponding to 15g/
100ml Hb.
Equipments : Haemoglobin pipette, test tube.
Procedure
Mark three test tube t, s & b. To the t test tube add 5 ml Drabkin’s reagent
and 0. 2ml blood. To the s test tube take 5ml standard solution and to the b
test tube take 5 ml drabkin’s reagent . Mix the contents of the t test tube
thoroughly and wait for 5 minutes. Read the absorbents t against b at 540
nm.
Result
The Hb count of the given sample of blood done by sahli’s haematin method
is ………g%
Pathological conditions
Hb% increased in polycythemia vera. Hb concentration increased due to
loss of body fluid in severe diarrhea and vomiting . Decreased in pneumonia,
leukemia, chronic hemorrhage and hook worm infections.
ENUMERATION OF TOTAL COUNT OF W.B.C
Aim
to enumerate the number of white blood cells/ cubic mm of blood.
Apparatus
Nautauer’s double counting chamber, WBC pipette, WBC diluting fluid,
spirit, needle and microscope. Nautauer’s double counting chamber, the WBC
containing area on the chamber consist of four big squares in the corner.
Each big square divided into 16 small squares. So totally there are 64 small
square WBC pipette. It is a glass pipette having a narrow stem and bulb. The
bulb is smaller then that of RBC pipette and contains a white bead. There are
three markings, 0.5 below and 11 above the bulb. WBC diluting fluid
composition;- glacial acetic acid 2 cc. gentian violet 1cc, distilled water 100cc.
Distilled water causes hemolysis of RBC, glacial acetic acid destroys
hemolysed RBC and gentian violet stains the nuclear of WBC.
Procedure
Clean the finger tip with spirit and make a deep prick. Such blood up to 0.5
marks. Then such diluting fluid up to the mark 11. Mix the blood thoroughly
by rolling pipette horizontally in the palms. Discard 2-3 drops, clean the
counting chamber and fill the chamber with prepared solution. Air bubbler
should avoided, focus the field under low power, objective and count the
WBC in all the corner squares. So that the WBC in 64 squares are counted.
Knowing this value the total number of WBC in 1 cubic mm of blood can be
calculated.
Result:- the number of WBC in /mm3 of undiluted blood = 12500,
WBC count = 12500/mm3 of blood.
Normal value:- the normal range of WBC count- 4500- 11000/mm3 of blood.
Increase in number of WBC is called leucocytosis.
Decrease in number of WBC is called leucopenia.
Leucocytosis:-
Physiological causes of leucocytosis,
1. After exercise
2. In allergic conditions
3. Excitation
4. Injection of adrenalin
5. In infants, during pregnancy and during menstruation
6. Due to cold and exposure to ultra violet radiations
Pathological cause of leucocytosis
1. Leukemia
2. Inflammatory conditions like acute, infection and tuberculosis.
3. Leucopenia
4. Pathological cause of leucopenia
5. Typhoid
6. Viral diseases
ENUMERATION OF DIFFERENTIAL COUNT OF W.B.C
Aim
To study the blood smear for differential count, cell morphology and
abnormalities.
Principles
The polychrome solution is leishman’s stain containing methylene blue and
eosin. These basic and acidic dyes induce multiple colors, when applied to
cells. Methanol act as a fixative agent and as a solvent. The WBC stained by
acidic dyes described as eosinophills and acidoplilic. The acid component is
nucleus that gets blue to purple shade by the basic dies and they called
basophills. The neutral components are stained by both dyes.
Equipments
Needle, glass slide, Spreader slide, Microscope, Leishman’s stain.
Procedures
Place a drop of blood on the slide and make a tongue shaped smear with the
spreader slide. It is stained by leishman’s stain and dried. First examine the
stained blood film under low power microscope. For screening, select an
area between tail and body of the film, where the R.B.C just touches each
other without overlapping. Place a drop of oil in the selected area. Switch to
the oil immersion elective. Identify various white cell count at least 100
cells and give the % of the cells seen. 100 square are made on the paper and
write the letter N for neutrophil, L for lymphocytes E for eosinophil and B
for basophils.
Results
Neutrophils : 60%
Eosinophils : 2%
Basophils : 0%
Lymphocytes : 38%
Monocyte : 0%
Observations
i) Granulocytes : there are cells with granulated cytoplasm. There include,
neutrophil, eosinophils, and basophils.
1.neutrophils – size 10-12µm. Nucleus:- purple colored, eccentric with heavy
thick chromatin mases. It is divided into 2.5 lobes. Cytoplasm:- abundant
and stains a light pink color. cytoplasm contains small pinkish staining dull
granules.
2. Eosinophils --------------------- size 12- 15µm . Nucleus:- globed contain
dense chromatin masses usually nucleus in is arranged in spectacle shape .
Cytoplasm:- the granules are densely filled in cytoplasm . The granules are
uniform and spherical in size. They are deep orange pink colored .
3. Basophils- size 10-12µm . Nucleus:- indented in several planes . It stains
pale purple color because , it contain less chromatin . It is difficult to see the
nucleus because the cytoplasm granules mask it . Cytoplasm:- it contains
basophilic granules that overlaps nucleus . They are round dark purple to
black color and vary in size.
ii) Non – granulocytes( Agranulocyte)

1) Monocyte: size 10-22 µm. Nucleus:- kidney shaped or horse shoe shaped.
Sometimes it may be round or oval. It stains pale violet and has five chromatin
granular network .
2) Lymphocyte: two forms are observed.
• Large lymphocyte: size 12-16µm in diameters. Nucleus:- dense, oval
or slightly indented. Cytoplasm:- abundant and pale blue colored
• Small lymphocyte : size 10-12µm in diameters. Nucleus:- dark, round
and sometimes indented. Cytoplasm:- it has very little cytoplasm and
often more just around the nucleus.
Normal values
Neutrophils : 40-75%
Eosnophils : 1-4%
Basophils: 0-1%
Lymphocyte : 20-25%
Monocyte : 2-8%
ESTIMATION OF E.S.R.
Aim
To determine the E.S.R in the given sample of blood .
Principle
A normal blood column if kept vertically for sometimes the RBC will settle
down at the bottom . E.S.R is the state of measure of the suspension stability
of RBC . If a vertical column of citrated blood is kept the red cell begins to
settle until they form a packed volume at the bottom of pipette there are
three stages of sedimentation.
1. Stage of aggravation in the first 10 minutes.
2. Stage of rapid sedimentation taking up to 4 minutes.
3. Stage of steady packing of cells
The length of the plasma column at the top of blood column at the end of
first hour in measured in mm. E.S.R also measure the amount of globulin
and fibrinogen if present abnormally.
Methods of estimation of E.S.R
1. Westergren’s method:- westerngren’s pipette is a 30 cm long glass tube
having a diametes of 2.5mm. It is graduated from above downwards from
0.200. The anti coagulant used is 3.8 % sodium citrate. EDTA is also used.
2. Wintrobe’s method : - in this method heamatocrit tube is used . It is a
cylindrical tube opened at one end . The total length is 11cm . The tube is
uniform and has a diameter of 3mm. It is graduated from 0-10. The anti
coagulant is used is sodium citrate, EDTA or double oxalate
Procedure
Westergrein’s method :- blood is collected and mixed with anti coagulant in
the ratio 1:4. The temperature should be 25-28oC. Blood is drown up to the
mark 0. The tube is kept exactly vertical for 1 hour . The height of plasma
column after one hour is noticed.
Result : the E.S.R of the given sample of blood by westergrein’s method is
36 mm/hr.
Normal values
1. Westerngrin’s method :- below 50 years -20mm/hr. 50-65 years - 30
mm/hr, above 65 years - 45 mm/hr
2. Wintrobe’s method :- male 4 mm/hr , female-10 mm/hr, children- 5-
10 mm/hr.
Factures influencing E.S.R

1. Number of cells : high cell count- decrease in ESR, low cell count-
increase in ESR.
2. Shape of cells- tenancy towards spherical form- ESR decrease.
3. Size of cells- increased size- increased ESR.
4. Roulex formation – increases ESR.
5. Plasma protein - fibrinogen increases ESR.
6. Viscosity in plasma- viscosity increases in ESR.
7. Specific gravity of plasma, compared to cell , if this specific gravity
of plasma is less ESR in more.
8. A rise of temperature above 20oC, increase ESR.
9. Position of tube , in standing position ESR is more.
10. ESR increase if the tube is shorter or narrow.
Variations
1. Physiological :-
a. New born- ESR is low.
b. In old age : it is increased
c. ESR is increased during menstruation, pregnancy.
2. Pathological :- ESR is decreased in
a. Polycythemia vera
b. Sickle cell anemia
c. Congestive heart failure
d. Whooping cough
e. Dehydration.
ESR is increased in
3. Anemia except sickle cell anemia
4. Acute myocardial infection
5. Carcinoma
6. Tuberculosis
7. Acute gout.
ESR is rapidly raised in :
1. Temporal arthritis
2. Rheumatoid arthritis
3. Kala azar
4. Multiple myeloma
5. Leukemia
6. Chronic renal disease.
ESR in diagnosis:
1. To distinguish functional from organic diseases.
2. In acute rheumatoid arthritis , acute gout and infective arthritis marked
by raised, while in osteoarthritis remain particularly normal.
3. In myocardial infection , it is raced , while in angina it is not
4. In differentiating stomach cancer from peptic ulcer.
5. It is raised in pelvic inflammation , and not in un ruptured ectopic
gestation.
ESR in prognosis and treatment.
1. In fever, a raised ESR suggest progress of disease
2. In rheumatic fever, it is specially index of persistent rheumatic
infection.
3. In coronary thrombosis, repeated determination serve as a guide of
treating and in management of patient’s activities.
4. In acute nephrites , the great remain high in patients passing into
chronic stage .
ESTIMATION OF PACKED CELL VOLUME
Aim
To determine the packed cell volume of a given sample of blood .
Principle
Packed cell volume (PCV) is the ratio of the volume of formed elements to
the volume of whole blood expressed in %.
Apparatus
Wintrobe’s haematocrit tube filter, oxalated venous blood. Wintrob’s
haematocrit tube is a glass tube having 110 mm length and 3mm diameter,
closed at one end . It is graduated 0-100cm at one side and 100-0 on another
side.
Procedure
In Wintrob’s haematocrit tube , oxalated venous blood is taken without air
bubbler. It is closed with cotton and kept in centrifuge, and rotated at 3000
rev/min for half an hour. Take out the test tube and not the blood cells
packed at the bottom and buffy coat above it . Above the buffy coat , clean
plasma is seen . The packed cell volume can be directly read out from the
haematocrit tube normally buffy coat is of 0.5- 1cm thickness. Wintrob’s
haematocrit tube is also used for determination of ESR.
Normal value
Male : 46+_7%
Female : 42+_7%
Variations
1. PCV increasing polycythemia and luekemia
2. PCV decreased in anemia.
PREPARATION OF PERIPHERAL SMEAR
Blood smear may be made either as slide or as cover glass. The latter method
given more over distribution of cell . But the former is preferred because
slide are easier to handle , less likely broken and do not require mounting
and can be labeled.
Procedure:
i) Preparation of thin smear
Transfer a drop of blood specimen with a pair of application stick to a clean
grease free slide. Place the drop approximately 1 cm from the end of about
5mm diameter. Place the spreader slide at an angle of 30-35. Then pull back
the speeder slide, until it touches the drop of blood and then push forward,
after that dry the smear, the thickness of smear depends upon the rapidity of
pushing the spreader. In the head position of the smear , over lap of RBC are
obtained. the monocyte and polymorphs are predominant at the margin, and
at the tail section. Smaller lymphocytes are more evenly distributed. The
leucocytes others are predominant in the body of smear.
Criteria of good smear
1. Smear should be tongue shaped . The tail end must be smooth and
free from ridges, waves and hole.
2. Smear must have a smooth even surface .
3. The smear should be slightly thicker at the commencement and there
at the tail end.
4. Smear should not exceed to the ridges on to the end of the slide but
should be at least 1-2 inches long.
5. Leukocyte must not be bunched at the edge or at the end .
ii) Preparation of thick blood smear
A large drop of blood is taken at the centre of slide and with the aid of needle
of slide corner. Spread the drop over the slide, about half inch thickness.
After drying keep the slide for 8 hour.
iii) Preparation of cover glass smear
Take two clean dry cover glass (22*22mm). One is held in the left hand
with thumb and fore finger at adjacent corner. A small drop of blood is
touched with a cover glass held in the right hand . This cover glass is quickly
placed on the top of the cover glass in the left hand, after the blood has
spread by capillary method. The cover glass drawn a part with a study, motion
care being taken to keep them parallel dry the film in air, support the cover
glass as the cork is smaller than the cover glass and stain. Then they are
mounted as clean glass slide, the disadvantages of cover slip in that , it is
easily ruptured.
ESTIMATION OF CLOTTING TIME
Aim
To find out clotting time.
Apparatus
Capillary tube (10cm length) stock watch needle etc.
Principle
Clotting time is the time interval between appearance of blood and appearance
of clot.
Procedure
A deep prick is made on the finger tip. Flow the blood into the capillary
tube. Start the stop watch simultaneously. When capillary tube is filled,
place it for 1-2 minutes. After this start breaking the tube at ½ minutes interval
until the fiber in thread is seen between the broken pieces. When the fibrin
thread is appeared, stop watch is stopped. The time taken given clotting
time.
Result:- the clotting time of given sample of blood is 3 minutes.
Normal value:- 5-10 minutes.
Factors preventing in coagulation.
1. By adding substances of biological origin such as prothrombin,
heparin etc.
2. By avoiding contact with water – wettable surface.
3. Removal of Ca2+ ions.
4. Precipitation of fibrinogen.
Factors that fattens coagulation
1. Contact with water wet table surface.
2. Addition of foreign bodies.
3. Addition of thrombin and thrompoplastin , Vit k, CaCl2 etc.
Variations:-
Clotting time is prolonged in hemophilia due to the absence of anti hemophilic
factor. But bleeding time is normal .
ESTIMATION OF BLEEDING TIME
Aim
To find out bleeding time .
Apparatus
Needle, blotting paper, stop watch etc.
Principle
Bleeding time is the time taken between the appearance of blood to the
cessation of blood.
Procedure
Clean the finger tip with spirit and allow it to dry. make a deep prick. Blood
will start flowing. Note the time. With the blotting paper touch the blood
coming after each ½ minutes. A fresh part of blotting paper should be used
each time. Note that the blood spot on the filter paper gets thinner, till it
disappears the bleeding stop. Note the time . This time in interval given the
bleeding time.
Result:- bleeding time of the given sample of blood is 2 ½ minutes.
Normal value:- 2-4 minutes.
Pathological conditions:-
Bleeding time is increases in
1. Thrombocytopenia purpura.
2. Hemophilia.
3. Vitamin K deficiency.
Bleeding time is decreased, when platelet extract is administrated
intravenously.
ENUMERATION OF TOTAL R.B.C COUNT
Aim
To determinate the total number of erythrocyte in the given sample of blood.
Principle
A known volume of blood is diluted and is changed into a chamber of known
dimension and the number of R.B.C s in the distributed blood is calculated.
Apparatus
Hemocytometer, Haeyem’s fluid, spirit, pin etc. Hemocytometer contain
Naubauer’s counting chamber, RBC pipette, and W.B.C pipette. R.B.C
diluting fluid or Haeyem’s fluid isotonic to red cells is it prevent hemolysis.
It contain
• Trisodium citrate – 3.13g.
• Commericial hcho -1ml
• Distilled water -100ml
Procedure
Blood is taken up to 0.5 mark, in the R.B.C pipette and diluting fluid up to
101 mark. It is mixed well and kept for 3 minutes. 2.3 drops of fluid is
discarded, from the pipette, which does not contain any cells. the counting
chamber is charged after 3 minutes. (to allow the cells to settle). The RBC in
the squire are marked ‘R’ in figure under high power. R.B.C count is then
calculated.
Source of errors
1. Diluting fluid should not contaminated with blood cells
2. Keep the counting chamber and cover glass free from dust and dried
blood.
a. Cleaning procedure:- use mild detergents(1% NaHCO3 )
followed by washing with tap water and rinsing with de
ionized water. Dry the haemocytometer, by polishing with
lens paper, tissue paper, or soft cloth. Do not put
haemocytometer in dichromatic solution .
3. some major source of technical errors include improper volume
measurement (specimen & diluted)improper change, of the counting
, use of different pipette.
Result.
The R.B.C count of the sample of blood =4. 25 million/mm3 of blood.
Normal value.
Male:- 4.5- 6*106 cells/µ l
Female:- 4-4.5*106 cells/µl.
Decrease in the number of calculating erythrocyte indicates anemia and an
increased number of erythrocyte indicates the possibility of polycythemia.
BLOOD BIOCHEMISTRY
Serum magnesium
Normal value- 0.8-1.3 milli mole/ litter. Increased in renal in sufficiency.
Decreased in a acute fluid loss from GIT, chronic alcoholism, Chronic
hepatitis, Hyper vitaminosis.
Serum phosphorous
Normal value 3-4 mg/ 100ml. Increased in renal insufficiency,

hypothyroidism, rickets, ostemalacia.
Serum triglycerides
Normal value- below 105 mg/ 100ml. Increased in primary hyper lipo
protenimia, Diabetes mellitus, Hyper Thyroidism, Nephrotic syndrome, Use
of contraceptive pills, Biliary obstructions. Decreased in hyper lipro
protenemia, Mal absorption and Mal nutrition.
Serum bilirubin
Normal value-0.3- 1 mg/100ml. By direct method : 0.1- 0.4 mg/ 100ml , by

indirect method: 0.2- 0.7 mg/100ml. Rise of total serum bilirubin occur in
acute and chronic hepatitis, biliary tract obstruction , ca stones or due to ca
of head of pancreas. Rise of indirect bilirubin occur in hemolytic disease or
reactions. Gilbert’s disease.
Serum calcium
Normal value 9.6- 10.9mg/100ml . Increased in hyper para thyroidism.

(20mg%) hyper vitaminosin (70mg%), multiple myloma, cushing syndrome.
Decreased in hypo para thyroidism, osteo malacia, mal al syndrome , acute
pancreatitis.
Serum cholesterol
Normal value: 150- 200mg/ 100ml. Increased in pregnancy, alcohol and

fatty diet, constipation, Myxoedema, Diabetes mellites, Obesity, Nephrotic
syndrome, Jaundice, Liproprotenemea.
Serum chloride
Normal value 275-350mg/100ml, increase by excess salt intake, over

treatment with saline solution. Decrease in urinary tract obstruction, acute
chronic nephrites with low intake of protein, heart disease, abnormal loss
are in severe diarrhea, vomiting, excessive sweating, treatment diuretic with
renal failure.
Serum sodium
Normal value135- 145mg/dl. Increases in excessive replacement of sodium,

oral or intravenous failure of water retention, diabetes insipidus .decreases
in severe diarrhea , vomiting, Addison’s disease, excessive water intake or
in appropriate ADH secretion.
Urea creatinin ratio
Normal value 21-43mg/100ml. Increases in high protein diet increased

catabolism, fever, burns, steroid therapy, wasting in severe illness, urinary
stress like urinary constipation. Decreases in protein restriction, excessive
vomiting, liver disease with a urea production.
Serum uric acid
Normal value 3-7.5 mg/100ml. Increases in urine synthesis, decreased uric
acid secretion .eg gout over production of uric acid, chronic hemolytic
anemia reduction of renal excretion of uric acid, diuretic, chronic renal failure
starvation, ketosis. Decreases in aspirin therapy.
Serum creatinine
Normal value 0.2- 0.6mg/100 ml . Increases in Hypo para thyroidism,

rheumatoid arthritis, cardiac failure.
Serum alkaline phosphate
Normal value 5-13n/ dl. Increases in severe osteomalacia in osteogenic

carcinoma, metastasis, ca of bones hepatic duct obstruction, obstruction due
to stones, neoplasms, liver disease resulting from drugs like paracetamol,
chloropromazine, hyper parathyroidism, paget’s disease. physiologically
raised in pregnancy, alimentary hyper glycemia, expossure to U.V rays.
Serum acid phosphate
Normal value: 1-5 KA unit. Increases in CA of prostate, with secondary

stage metastasis.
ESTIMATION OF RANDOM BLOOD SUGAR
Trinde’s method
Aim:-to determine the random blood sugar in the given sample of blood.
Principle
Glucose +O2 +H2O glucose Oxidase Gluconic acid+H2O2
H2O2+HBA+AAP peroxidase Qunoine dye + 2H2O
AAP - 4- Amino Antipyrin
HBA - 4- Hydroxy Benzoic Acid
Reagent compositions
Reagent I – glucose.
Glucose oxidase (aspergillin) - 20000 Iu/L
Peroxidase (horse radish) - 3350 Iu/L
4 amino antipyrine - 0.52 m mol/l
4 hydroxy benzoic acid - 10 m mol/l
Phosphate buffer - 110 m mol/l
Also contains non reactive filter and stabilizers.
PH - 7 ± 0.2 at 25oc.
Reagent II - glucose standard.
Glucose standard – 100 mg/dl or 5.55 m mol/l.
Reagent reconstitution
Allow the vial to obtained room temperature. Dissolve the contents of
each vial using glucose diluted with special lipid cleaning agent. Make up
the volume to 200 ml or 500 ml transfer into a clear and dry amber colored
bottle .
Procedure
Take approximately 1 ml of blood mixed with anticoagulant and centrifuged
it for 3 minutes. Thus we can separate plasma from blood . Take 3 test tube
marked T,B,S (Test Blank and Standard) add 100ml working reagent to whole
of them. Add 100 ml of distilled water to test tube marked as B and 100ml
sample to test tube marked as T. Mix well and incubate for 15 minutes at
370C. Read the absorbance of standard and each sample tube against reagent
blank at 510 nm.(500-540) or 510/ 630 nm as biochromatic analyser.
Normal value
Fasting: 70-120 mg/dl
Post prandial: 90-130mg/dl.
Result:- R.B.S of the given sample of blood =152 mg/dl
Fasting blood sugar =120 mg/dl.
Clinical significances
Increased due to diabetes mellitus, in patients receiving glucose containing
fluid intravenously daring increase stress and cerebrovascular accidents.
Decrease on insulin administration as a result of insulinoma, inborn errors
of carbohydrate metabolism or in fasting.
ESTIMATION OF SERUM GLUTAMATE OXALATE
TRANSAMINASE (SGOT or AST)

Aim
to estimate the amount of SGOT present in given sample .
Principle
The oxalate formed by the action of enzyme on appropriate substrate undergo.
Spontaneous de carboxylation , to form pyruvate. This pyruvate react with
2-4 dinitro phenyl hydrogen to give a yellow colored in alkaline medium.
The color intensity measured is calorimetrically at 540 nm.
Reagent
1. Phosphate buffer pH – 7.4
2. SGOT substrate
3. Dentro leavo aspartate - 2.6g
4. á keto glutaic acid - 300g
5. 0.1n NaDH - 20.5ml.
Make up to 100ml with phosphate buffer, adjust pH 7.4. Add a drop of
chloroform and keep it in freezer.
1) DNPH
2) Stock pyruvate standard
3) Working standard
4) 0.4n NaDH
Make 4 test tube (t), standared (s), blank (b) and control (c).take reagent as
follows.
Reagent t s b c
1.substrate 0.5ml0.4ml05ml 0.5ml (warm in water bath at 37oc for 3
mts)
2.serum 0.1ml- - - (mix well and incubate t at 37oc for 16
mts).
At the end of incubation add 0.1 ml serum to (c)
3. Pyruvic acid - 0.1ml- -
4. Phosphate buffer- 0.1ml- 0.1ml
5. DNPH 0.5ml 0.5ml 0.5ml 0.5ml0 (mix well and keep for
20 mts)
6. 0.4 n nadh 5ml 5ml 5ml 5ml (mix well and keep for 10mts and
color read at 540nm)
Result:- 94 Iu/l
Normal value:- 5-20 Iu/l
Clinical significance:- increased in myocardial infection , liver diseases.
ESTIMATION OF SERUM GLUTAMATE PYRUVATE
TRANSAMINASE (SGPT or ALT)

Rietman and frankel’s method
Aim
To estimate the SGPT activity of the given sample of serum.
Principle
The pyruvate formed by the action of the enzyme on the substrate react with
2-4 dinitro phenyl hydrogen (DNPH) at an optimum pH of 7.4 , which gives
brown colored complex in presence of alkali which is stable and can be
estimated calorimetrically at 540 nm.
Reagents
1. Phosphate buffer(ph -7.4)
Anhydrous disodium hydrogen phosphate and 1.89g anhydrous potassium
dihydrogen orthophosphate dissolved in DNPH. And make up to 500ml adjust
pH 7.4. Store in refrigeration with a drop of chloroform.
2. SGPT substrate
• Alanine - 1.78g
• á Keto glutaric acid -30mg
• Phosphate buffer - 50ml.
Make up to 100ml with phosphate buffer.
3. Stock pyruvate standard- 154 mol/ml
141 mg of lithium pyruvate is a dissolved in 100ml of phosphate buffer.
Store in amber colored bottle. Keep in fridge.
4. Working standard- 1.5 n mol/ml
1ml of stocks std dilute to 10ml phosphate buffer.
5. DNPH:- 20g DNPH dissolved in 100 ml hot 1N HCl. Store in amber
colored bottle at room temperature.
6. 0.4n NaOH – 1.0g pf NaOH dissolved in 100ml of distilled water.
Procedure
Make 4 test tubes t, s, b, c and add reagents.
Reagents: t c s b
1. SGPT substrate 0.5ml 0.5ml 0.4ml 0.5ml
2. Serum 0.1ml - - -
Mix and incubate t at 370C for 30 minutes. At the end of incubation time and
0.1ml serum to control.
3. Pyruvate std - - - -
4. Phosphate buffer - - - -
5. DNPH 0.5lml 0.5ml 0.5ml 0.5ml
Mix well and keep it for 10 minutes. Read at 540nm
Normal value: 3.15 Iu/l
Clinical significance
Increased condition infective and toxic hepatitis viral hepatitis, cirrhosis,
pancrratitis
ESTIMATION OF SERUM BILIRUBIN
MALLOY AND EVELYN METHOD

Aim
to determine the amount of total conjugate bilirubin in the given sample of
blood.
Principle
Based on vanderberg reaction bilirubin combined with diazotised sulphanilic
acid to form a purple colored agotilirutin complex. The intensity of a purple
color is a directly proportional consist of two parts - direct and indirect.
Direct bilirubin (conjugated) reacts directly with diazo reagent in aqueous
solution to give diazo compounds with in addition of methanol accelerates
the reaction of in direct bilirubin (unconjugate) in the serum and hence
value for total bilirubin is obtained. Then allow to stand or 30 minutes at
dark room and take reading against blank at 540nm
Reagents
1. Diazo reagents:- Prepare just before use by mixing 10 ml of solution
A and 0.3ml of solution
2. Solution:- Dissolve 0.5g of sulphanilic acid, 7.ml of con HCl, shake
up to 500 ml with distilled water.
3. Solution B:- Dissolve 0.5g NaNO2 in 100m distilled water
4. Diazo blank:- Dilute 7.5ml of con HCl into 100ml distilled water
5. Methyl red stock std:- Dissolve 290mg methyl red in 100ml glacial
acetic acid
6. Working methyl red std:- Take a liter volumetric flask and glacial
acetic acid
7. Working methyl red std:- Take a liter volumetric flask and add 1 ml
of methyl red stock and add to it 500ml distilled water and 5ml glacial
acetic acid. Then add to it 14.4g of hydrated sodium acetate. Dissolve
them and dilute the solution to 1000ml with distilled water. This is
similar to 8 mg% bilirubin
Procedure
For total bilirubin mix 0.2ml serum and 0.5ml diazo reagent. Then 1.8ml
distilled water, 2.5ml methanl are added to it, then keep it is a dark room for
30mts.
For coagulating bilirubin mix 0.2ml serum and 0.5ml diazo reagent. The add
4.5ml of distilled water. It is kept in dark room for 30mts.
Blank:- mix 0.2ml serum and 0.5ml diazo blank. The add 4.3ml distilled
water.
Standard 5ml methyl red working reagent std.
Normal value:
Total bilirubin - 0.2- 0.8 mg%
Conjugated bilirubin - 0-0.2 mg%
Unconjugated bilirubin - 0.2-0.7mg%
Interpretation
Unconjugated bilirubin increases in hemolytic jaundice, conjugated bilirubin
increase in obstructive jaundice and total bilirubin in hepatocellular jaundice.
RHEUMATOID ARTHRITIS TEST (RA FACTOR)
Aim
To detect the presence of RA factor in the given sample
Principle
To demonstrate RF (rheumatic factor) the serum is reacted with IgG coated
(purified human gammaglobulin) later particular, agglutination indicates
presence of RF
Equipments
Graduated serological pipette, calibrated pipette (0.05ml) or capillary pipette
(50ml), glass plates, plenty of the test tube, water path, grease, pencil and
times
Procedure
1. Qualitative
1. Transfer the specimen is pre-labeled test tubes and place them in
560C water bath for 3 minutes. Thus will in activate the compliment
2. Allow all reagents to come to room temperature
3. Arrange the heat inactivated specimen on the front row of a test tube
rack and number them. Enter the number in to record book.
4. Place an empty test tube behind each specimen tube and dispense
0.95ml of buffer in each test tube. To make the transfer of buffer use
11mm serological pipette and draw the buffer to 0.05 mark. the pipette
now holds 1.95ml fluid.
5. Add 0.05ml of test serum to the corresponding buffer solution in the
near test tube. Use separate 1ml pipette (0.01ml of graduation).for
each specimen, mix each tube severely and thoroughly. This given
1:20 dilution of serum.
6. Place one drop of this labeled serum in a space on the divided slide.
Use separate disposable plastic dropper for each serum specimen to
used in the test tube.
7. Include the + ve and – ve control along with the run. This is done
only once a day.
8. Give each square a number corresponding to the specimen. Number
on the tube and mark additional 2 square as +ve and –ve
corresponding to the +ve and _ve controls when they are included.
9. Shake the gamma globulin reagent bottle gently then add one drop
of later suspension to each drop of diluted serum specimen placed in
the sqare.
10. Mix the serum and the latex reagent well with applicator stick or the
tooth pick provided in the kit. Spread the suspension over the entire
surface area.
11. Tilt the slide gently to and fro for 1 minute then note the reaction
reactivity.
12. Reporting
Negative(n) - ----------------smooth homogenous suspension.
Weak positive (wp)- finer agglutination and usually occur within 1
minutes.
Strong positive (sp)- course agglutination and usually within 1
minute.
B. Quantitative
1. prepare the serial dilution of serum in the someway as done other
qualitative serological tests.
a) Take 5 list tube in a rack and add 1.95ml of buffer to test tube
1 and 1ml to others.
b) Add 0.05ml of undiluted serum in tube 1 mix and transfer to
tube 2 and continue the process to tubes. keep 1ml of mixture
in a separate empty tube for further dilution if needed
c) The above will lead to the following serial dilution
i. 1.1:40 ,2.1:80, 3.1:60, 4.1:320, 5.1:640.
2. Perform the slide test with different dilution of the serum use the
same pipette in transferring of one drop of the diluted specimen
(0.05ml) starting from the highest dilution tube 5.1:640, until the
lowest dilution (1;40) is reached in tube1
The RA test is done, in order to detect the presence of rheumatoid factor in
the serum of the patients with rheumatoid arthritis. The factor is non specific
and is formed in other diseases or well both related and non related rheumatoid
arthritis.
ANTI STREPTOLYSIN – O (ASO) TEST
Aim
To detect at the presence of ASO factor in the given sample of blood.
Principle
The ASO test is based on the principle of a neutralization where the anti
body neutralizes the toxin and prevent it from the hemolytic activity on
erythrocytes. In this test, patients serum is first introduced with the
commercial streptolysin O (SLO) Following this suspension of indicates
cells (erythrocytes) are added then incubated and centrifuged. Hemolysis of
the indicator cells is noted by reddish color of the supernatent solution (-ve).
The lack of hemolysis is recognized the clear supernatant solution and
indicates the presence of ASO in patients serum. This is due to the fact that
the hemolysis (so toxin) is neutralized by the antibody, present in the patients
serum, and prevents the lyses of the red cells.
Procedure
Two method: (1) Slide method, (2) Tube method
ASO slide method
The slide latex agglutination test is relatively more simpler than the tube
method test and requires minimal reagents.
Equipments
A.S.O. latex agglutination kit, then contains SLO reagent, ASO coated latex
suspension and control sera ASO +ve and –ve slides.
Procedure
1. Add 0.1ml of SLO reagent into 3 small test tube, previously labeled
are test +ve control (pc) and –ve control (nc)
2. Add 0.1ml of patients serum to the first tube (t) 0.1 ml of ASO +ve
control to second tube. And 0.1 ml of ASO –ve control serum to the
third tube.
3. Mix by shaking and allow to stand for 15 minutes.
4. Following incubation add 1 drop (0.05ml) of each mixture on 3
separated fields marked ,pc, nc on the blank test slide.
5. Share the ASO latex reagent (coated with SLO) to obtain a uniform
suspension and add 1 drop to each field containing the serum to be
tested.
6. Mix the serum with latex suspension thoroughly with the help of
wooden application sticks, glass rods on tooth pick and spread the
mixture over the field.
7. Felt slide back and fort and let the suspension go from end to end of
the periphery.
8. Examine the agglutination using a direct high density spot light at a
distance of approximately 15 cm from the surface of the slide.
9. Agglutination in the test serum indicates the patient has a significant
ASO titer (higher than 100 fold units.) Agglutination should also be
seen in pc serum, but not in the NC serum.
ASO tube method
There is a qualitative procedure.
Reagent
ASO kit, the kit contain buffer, streptolysin (SLO) and counter sera in dry
form. In additional suspension of defribinated rabbit blood is needed. The
cells are worked for 3 times or until supernatant is a clear in normal saline.
With the aid of centrifuge. (2000 rpm for 5mts) and finally 0.5% suspension
is made in saline, which is used on the same way or in the buffer which can
be stored at 40C for a limited period . If supernatant is tinged with the
hemoglobin even after washing 5 times, discard the blood. Citrated human
blood (group O) from the blood can also be used but rabbit blood is preferred.
Equipments:-pipettes, test tube, rack, glass, marking pencil, timer.
Procedure
1. Prepare the initial serum dilution of (1:100 and 1:500)
• Take two test tube and mark them A and B.
• Place them on one side of a test tube rack.
• Add 9.9ml of buffer in tube A, 8 ml in tube B.
• Using 1 ml graduated serological pipette transfer 0.1ml of
the serum specimen into Tube A and mix thoroughly by
blowing into the pipette.
• With the help of 2ml serological pipette transfer 2ml of dilute
serum from tube A to tube B, and mix thoroughly.
2. Arrange 7 test tube in a row and number them from 1-7,use tube
no;1-5 for placing diluted serum and streptolycin control (No. 7)
3. Supernatant tube for red cells, control (no:6) and streptolycin control
(no:7).
4. Place the amount of diluted serum from tube A and B, and the
corresponding amount of buffer in order to give a dilution gradient
of the serum. Note that the tube No:6 (RBC control) gets 1.5 ml,
buffer and tube No:8 (serum control) receives 1.5ml undiluted serum.
The total volume of all tube at the final stage after adding red cells
will be 2 ml, shake gently to mix.
5. Incubate at 370C for 15 minutes, add 0.5ml 6% red cell suspension,
and incubate at 370C for 15mts.then shake gently and continue to
incubate for 45 minutes.
Interpretation:- a titer below 200 shall be considered normal. a single titer
is difficult to interpret and should be followed by filtration of second
specimen obtained two week later. A single test with titer values of 400
Todd units or more is strong evidence for recent streptococcal patient.5
year to age or younger ones are considered sufficient. An increase or decrease
of 2 dilution or more in titer however low in paired sample is significant
for all age. A.S.O titer may not increase significantly in the case of
glomerulo nephritis, an altranative test may be sought. (ALT or ADN–B
False +ve result may be obtained in the case of oxidation of SLO reagent or
use of an in appropriate serum, which inhibit the action of the reagent
(SLO) at a low tube.
Also is recommended for the diagnosis of rheumatic fever.
PHYSICAL EXAMINATION OF URINE
Urine analysis
Volume
the arrange output of urine is 2.25 L/day. Volume may be increased by
excessive water in take, increased salt in take and in diabetes mellitus. Volume
may be decreased due to dehydration, low BP and shock.
Appearance
Normal urine is clear and transparent. It may become turbid when exposed
for a long time due to conversion of urea to (NH4)2CO2 (Ammonium
carbonate). In decreased condition, urine may be cloudy due to presence of
WBC.
Color
Normally urine is straw colored due to the presence of Hemochromes. Bright
red color indicate large amount of fresh blood. Brownish yellow or green
indicate the presence of bile pigments.
Odor
Normally urine is aromatic due the presence of small amount of uric acid.
When urine is allowed to stand still, it given an ammoiacal odor because of
decomposition of urea with the liberation of NH3.presence of ketone bodies
produce fruity smell.
pH
Normal pH is 6-8 .to measure the pH of urine, pH paper are available.
Specific gravity
It is define as the ratio of weight of fixed volume of solution to that at same
volume of water at specified temperature. The urino-meter method of
measuring specific gravity of urine is based on the principle of buoyancy.
Procedure:-mix well the urine and is allowed to attain the room temperature.
Urine is produced in the cylindrical tube to that it is nearly full. If there are
any air bubble, or froth, they are removed using filter paper. Float the urino-
meter in urine and due that the instrument does not touch the sides of they
cylinder. Note the reading.
Normal value:- 1-010-1.025
Pathological conditions:- Specific gravity of urine is decreased in excessive
in diabetes and in diabetes insipidus and excessive perspiration.
FDFGG
CHEMICAL EXAMINATION OF URINE
Protien-Heat Or Acetic Acid Test

This test is based on the principle that protein coagulate on heating, (addition
of few drops of 3% CH3OOH which eliminate the phosphate and carbon).
Procedure
Tell a clean dry test tube with 2/3rd of urine, which is faintly acidic. Heat the
upper part of the test tube till urine boils. If protein or phosphate or carbonate
are present, a white loud like color will appear on the heated portion. Add 2-
3 drops of 3% CH3COOH the cloudiness disappear. Due to the presence of
urine proteins, cloudiness presents.
Observation:- cloudiness presents after the addition of CH3COOH
Result:-Protein is present in the given sample of urine.
Protein urine:- Nephrosis, tuberculosis, cancer, nephritis, polycystic kidney,
fever, toxemia, trauma, severe anemia, aspirin.
SUGAR - BENEDICT’S TEST
Aim
To determine the presence of sugar in the given sample of urine
Principle
Sugar present in urine contain aldehyde group which will reduce the blue
colored CuSO4 to CuO and the color of the reagent.
Procedure
Take 5 ml of benedicts qualitative reagent in a test tube and add & drops of
urine to it. Then toil it for 2-3 minutes allow to cool and note the color.
Observation
The blue color of the solution, gradually changes to green and later on green
colored solution with yellow precipitate was obtained.
Color and range:-
Change of color in solution and precipitate formed.
Clean blue or green with no precipitate : 0%
Green with yellow precipitate : 0.5%
Green to yellow with yellow precipitate : 1%
orange solution with yellow precipitate : 1.5%
Orange to red precipitate : 2% above
Result
The give sample of urine contain 0.5% of sugar.
Pathological conditions: increased glucose in urine is found in diabetes
mellitus, brain injury & myocardial infraction.
Interfering factors
1. Pregnancy and lactation may cause false +ve.
2. Ascorbic acid, creatinine, streptomycin etc may cause false +ve
3. Stress, excitement, testing after heavy meal etc may cause false +ve
KETONE BODIES - ROTHERA’S TEST
Aim
To determine the presence of ketone bodies in the given sample of urine.
Principle
Action of auto acidic acid with sodium nitropruside in the presence of an
alkali produce a purple color.
Procedure
Take 5ml of urine in a test tube. Saturate it with ammonium salphate and
then add crystals of sodium nitropruside and mix well then gently add 1ml
of liquor NH3 through the sides of the test tube so that it former a layer on
the top of urine. If the ketone present a purple (permanganate) color will
form at the function of 2 layer.
Observation
A purple colored ring is formed at the junction of the two liquid.
Result
Ketone bodies are present in the given sample of urine.
Pathological condition
1. Ketone and ketone urea may occur whenever increased amount of
fats are metabolized carbohydrate in take is restricted or diet is rich
in fat.
2. Ketone bodies in urine appear first then in blood.
3. Ketonuria is associated with fever, gastrointestinal disturbances
prolonged vomiting, anorexia, fasting, starvation etc.
4. Drugs that may cause false + ve are levodopa, insulin, pyridine,
ether, paraldehyde etc.
BLOOD - BENZIDINE TEST
Aim
To determine the presence of blood in the given sample of urine.
Principle
Peroxides present in the hemoglobin decomposer H2O2 and liberate nascent
O2 .it oxides benzidine to form a blue colored complex.
Reagents
• Saturated solution of benzydine in glacial acetic acid (48%)
• 3% H2O2.
Procedure
Mix equal volume of benzidine solution and H2O2 in a test tube. Take two
ml of urine in another test tube. Bal it and cool. To it then add equal volume
of benzidine. A blue color indicator the presence of blood.
Observation:- a blue colored complex is formed.
Result:- blood is present in the given sample of urine.
Pathological condition:-
1. Hemoglobin urea is found in extensive burns and crushing injuries;
transfusion reaction compatible blood, fibril intoxification, malaria,
hemolytic anemia etc
2. Drugs which may cause + ve result- bautracin, caumarin, aspirin etc.
3. Interfering factors:-
4. 1.high doses of vitamin C may give false negative results.
3. Drugs causing positive results are bromide, copper, iodine and
oxidizing agents.
BILE SALTS – HAY’S TEST
Aim
To determine the presence of tile salts in the given sample of urine.
Principle
Bile salts when present in the urine lower the surface tension of urine. When
sulphur powder is sprinkled on the surface of urine, it sinks to the bottom of
the test tube. So in case of normal urine, sample, the sulphur powder floats
on the surface of urine.
Procedure
Like about 5ml of urine in a test tube, sprinkle a little dry sulphur powder on
the surface of urine. Observe the sulphur powder, whether it sinks or not. If
it sinks down the bile salts is present. It not bile salts is absent.
Observation:- sulphur sinks to the bottom
Result:- bile salts are present in the given sample of urine.
BILE PIGMENTS- FOUCHET’S TEST
Aim
To determine the presence of bile pigments in the given sample of urine.
Principle
When soluble BaCl2 is added to the urine, Ba2+ combine with SO42- in the
urine to form BaSO4. Any bile pigments if present may adhere to the
precipitate and detected by oxidation of bilirubin (yellow) to biliverdin (green)
and treatment which FeCl2 in the presence of trichloro acetic acid (TCA)
Blue color is given by tile cyanin.
Procedure
Take 5 ml of urine in test tube and add a few drops of MgSO4 and BaCl2.
Mix well and filter paper, on the top of another filter paper, to dry. In the
precipitate on the filter paper, add 2.3 drops of Fouchet’s reagent.
Development of a blue or green color indicates the presence of bilirubin.
Observation:- a green color is developed.
Result:- bile pigment is present in the given sample of urine.
Pathological conditions:-hepatitis, Cirrhosis, Cholecystitis, lymphoma.
MICROSCOPIC EXAMINATION OF URINE
Aim
To identify the microscopic elements present in the given sample of urine.
Procedure
Place 10ml of urine in a conical –u shaped centrifugal tube and centrifuge
for 1500 rpm for 5 minutes. Pour out supernatant liquid. Mix the sediments
remaining in the tube. Transfer one drops into a slide. Apply cover slip and
examine under a low power microscopic with reduced light.
The urine sediments are divided into 2 broad classes organized and
unorganized sediments.
Organized sediments: include
1. R.B.C:-when large number of R.B.C are present in urine it is
pathological. Cells may be large one appearance crenated R.B.C
should be qualified by reporting the number of present/ hpf.
2. Epithelial cells:- Normally a few of there are present in a urine of
marked increase indicates desquamate of surface tissue of urinary
tract.
3. Pus cells:- When they are present in large number (more than 5/hpt).
They include inflammation or infection of urinary trail.
4. Cast
a. Hyaline casts:- They are colorless semitransparent broad
straight and varying in length. They consists of coagulated
protein material. They are present in large number in various
kidney diseases.
b. Granular casts:- They are cast containing granules. They are
coagulated protein in which numerous proteins are embedded.
The granular are due to disintegration of WBC or epithelial
cells which presence of there cells indicate renal diseases.
c. Blood cell casts:-There are clots with visible red cells in
coagulated protein and commonly seen in acute nephritis.
d. Pus cell casts:- There are seen in poly nephrites, where there
is inflammation and formation of pus in kidney. Pus cells are
embedded in protein matrix.
e. Epithelial cell casts:- There consists of coagulated protein in
which epithetical cells from renal tubules are embedded. It
indicates renal diseases.
f. Fatty casts:- There casts contain numerous fat globules, which
indicate renal diseases.
g. Waxy casts:- There resemble hyaline cast, but are much more
rustle, and opaque neither than transparent with a duel waxy
appearance. They are found in later stages of nephritis
Unconjugated sediments:-
A. Crystals found in acid urine:
1. Calcium oxalate:- colorless envelope shaped crystals with
infective corners. More rarely appears a oval shape or biconcave
discs.
2. Lewine crystals:- They are slightly long or oblong resembling
spleen. flat globules with delicate striations.
3. Crystine crystals:- Colorless, highly refractile, thick hexagonal
plate
4. Thyroxine crystals:- Colorless fire needle cluster crossing at
various angles. Clusters may appear as blade in centre.
5. Uric acid crystals:- Usually yellowish brown rheumatic crystals
may be colorless.
6. Sulphur crystals:- There various considerably in shape, most of
them are like, sheath of needle. It may be clear or brown, in color
usually appears with concentric finding.
7. Cholesterol crystals:-- Large flat plate with one or more corners
cut off. They may occur in nephritis or lipid nephrosis
B. Crystals found in alkaline urine:-
1. Ammonium magnesium phosphate:-
2. They are colorless and appear like feathers or leaf
3. Calcium carbonate:- They are small dumb ball shaped and
dissolved in 10% acetic acid
4. Ammonium carbonate:- They are yellow spherical bodies usually
with radical and concentric striations
5. Calcium phosphate:- Often form large irregular usually granular
colorless plates. Small cells may be mistaken for squamous
epithelial cells.
FG
Digitally signed by Dr. Abraham
DN: CN = Dr. Abraham, C = US, O = www.
ayurvedicmedicinalplants.com, OU = Ayurveda
Reason: I am approving this document
Location: Kerala

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LABORATORY

Collection of blood by capillary method

ii) Ethylene diamine tetra acetic acid (EDTA)

iii) Trisodium citrate

ii) Non – granulocytes( Agranulocyte)

Factures influencing E.S.R

Normal value 3-4 mg/ 100ml. Increased in renal insufficiency,

Normal value-0.3- 1 mg/100ml. By direct method : 0.1- 0.4 mg/ 100ml , by

Normal value 9.6- 10.9mg/100ml . Increased in hyper para thyroidism.

Normal value: 150- 200mg/ 100ml. Increased in pregnancy, alcohol and

Normal value 275-350mg/100ml, increase by excess salt intake, over

Normal value135- 145mg/dl. Increases in excessive replacement of sodium,

Urea creatinin ratio

Normal value 21-43mg/100ml. Increases in high protein diet increased

Serum uric acid

Normal value 0.2- 0.6mg/100 ml . Increases in Hypo para thyroidism,

Serum alkaline phosphate

Normal value 5-13n/ dl. Increases in severe osteomalacia in osteogenic

Serum acid phosphate

Normal value: 1-5 KA unit. Increases in CA of prostate, with secondary

TRANSAMINASE (SGOT or AST)

TRANSAMINASE (SGPT or ALT)

MALLOY AND EVELYN METHOD

CHEMICAL EXAMINATION OF URINE

Protien-Heat Or Acetic Acid Test

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