ESTIMATION OF HEMOGLOBIN
Equipments required:
Sahli's haemometer
20 cu mm micropipette
Graduated dilution tube (present in haemometer)
Spirit or 70% alcohol
5N/10 HCI (1 ml conc. HCI + 9 ml distilled water) Cotton
.Glass rod
&EDTA
Distilled water
Procedure: The Sahli's haemometer contains an empty dilution tube in centre which is filled with N/10
HCI till the 20% or 2 marks. The pipette is then rinsed with EDTA. The finger tip is cleaned with spirit.
70% of alcohol or absolute alcohol and gentle prick is given with needle. As soon as blood oozes out, the
first drop is removed and then blood is sucked with the help of pipette upto the 0.05 ml ring mark. This
blood is then added to the central dilution tube where already N10 HCI is present. The two are mixed
with glass rod and left for 10 minutes. Then add distilled water to this mixture drop by drop till the color
in dilution tube matches with the colored glass in the adjacent tubes. Take out the dilution ube and note
the content of hemoglobin on gm% scale.
Principle: When the blood is mixed with N/10 HCI, the red blood cells get ruptured and the hemoglobin
content in them comes out into N/10 HCI. The haemoglobin gets converted into acid haematin whose
colour is compared with non- fading coloured tubes present in Sahli's haemometer. The most
appropriate matching colour indicates the haemoglobin content of blood sample. This is known as
colour index method." Normal values(g/dl): Men(13-18); women(12-16.5).
BLOOD CELL COUNTS
1. RED BLOOD CELL (RBC) COUNT
Equipments required:
1. Haemocytometer with neubauer's counting chamber
2. Neubauer's slide
3. RBC pipette with red bead
4. Square cover slip
5. Cotton
6. Absolute alcohol
7. Disposable needle
8. Hayem's fluid: It is made by mixing 0.5 gms of Sodium Chloride (NaCl) with 2.5 gms of Na,SO, (Sodium
Sulphate)+ 0.25 gms of Mercuric chloride in 100 ml of distilled water
Procedure: Rinse the RBC pipette with EDTA. Sterilize the tip of finger with a cotton swab dipped in
absolute alcohol
and give a prick with disposable needle. Discard the 1st drop and suck the blood into the pipette upto
the 0.5 mm mark Dilute the blood by sucking Hayem's fluid into the same pipette upto 101 mark, thus
the blood gets diluted 200 times. This blood is mixed in the bulb of the pipette by stirring it; this diluted
blood is then released under the cover slip placed over the counting chamber. The blood moves inside
the cover slip due to capillary action.
The RBC's are counted from the blood. They occupy the central chamber and any of the 5 squares
randomly out of the total 25 squares. Each small square is further divided into 16 small squares and thus
5 small squares will be counted. Supposing the total number of RBC's counted in 5 squares is 400 then
the total number of RBC's is counted as follows:
Total no. of RBC's in 1/10 mm' blood
Total number of RBC's in 1 mm³ of blood5×400×10/1 = 40,000 = 4million
Principle: The Hayem's fluid contains NaCl which is isotonic to cells thus does not allow hemolysis of
cells. The mercuric chloride in the fluid fixes the RBC's so that damage to them is prevented. The dilution
of cells causes them to come to a level to which they can be easily counted. Inference: The normal cell
count is 4.2-6 million. The count less than it cause anemia and more than cause's polycythaemia.
2. WHITE BLOOD CELL (WBC) COUNT
Equipment required:
1. Haemocytometer
2 WBC pipette with white bead
3. Disposable needle
4. Rectified spirit or absolute alcohol
5. Cotton
6. EDTA
7. Concentrated HCI for cleaning the haemocytometer
B. Turk's fluid
Procedure: Rinse the WBC pipette with EDTA. Clean the finger tip with spirit and pick it with disposable
needle. As soon as the blood oozes out, discard the 1st drop and suck the blood into the bulb upto 0.5
mm3 mark. Then suck Turk's fluid upto mark and mix it with blood sucked into the pipette by making it
flow into the bulb of the pipette. Thus, the blood gets diluted 20 times. Put a cover slip on the counting
chamber of the haemocytometer and touch the tip of pipette with its edge-the fluid flows automatically
into the space between the cells are studied under suitable magnification and the number of WBC's in
all four corner chambers are counted each of which is subdivided into 16 small squares.
130/ 4 =32.50
Calculations: Supposing total number of WBC's counted in all 4 squares is ,so mean value is 130/ 4
=32.50
Total number of WBC's ie., 32.50 x dilution factor ie., 32.50 x 20
This is the value obtained in 1/10 cu mm of blood
So to calculate WBC's in 1 mm' of blood ie. 32.50x20x6500 WBC's
Principle: The dilution of blood causes the decrease in number of WBC's to such a level that they can be
counted easily. Turk's fluid contains a stain called gentian violet which stains the white blood cells and
the acetic acid present in Turk's fluid haemolyses the RBC's.
3. PLATELET OR THROMBOCYTE COUNT
A platelet count is a test to measure how many platelets we have in our blood. Platelets help the blood
clot & hence determination of platelets is requested in the investigation of bleeding disorders.
Thrombocytopenia is often associated with prolonged bleeding and poor clot retraction. It occurs in
aplastic anemia, megaloblastic anemia and in immune thrombocytopenia. Thrombocytosis is found in
polycythaemia vera, following splenectomy and in chronic myelogenous leukemia.
Specimen: EDTA- Anticoagulated blood (fasting not necessary). A plastic syringe is preferred for blood
collection.
Capillary blood also can be used, it gives lower results compared to venous blood.
Requirement:
1. Microscope
2. Improved Neubauer's counting chamber
3. RBC pipette
4. Platelet diluting fluid
Procedure:
1. Mix the blood specimen carefully.
2. By using RBC pipette draw blood upto 0.5 mark.
3. Wipe excess blood on the outside of the pipette.
4. The diluting fluid is drawn up to mark 101.
5. Mix the contents in bulb thoroughly.
6. After 5 minutes, discard the first drop, then transfer a small drop on one side of counting chamber.
7. Place the filled mounted counting chamber under a Petri dish with a moist filter paper. Let it stay
undisturbed for 15 minutes.
8. Place the counting chamber carefully on microscope stage. Under low power magnification focus red
cell counting area. Move to view the corner square of red cell area and change to high power objective.
9. Keep the condenser down and reduce the light by adjusting the diaphragm. The platelets will appear
like highly refractile particles.
10. Count platelets in all 25 small squares. The area covered by 25 squares in equivalent to 1 [Link].
Calculation: Platelets per cumm (ul)
Number of platelets countedx Dilution/ Volume of fluid
Normal range: 250,000-50000/cu mm (ul)
Equipments required:
1.3: BLEEDING TIME
1. Blotting paper
2. Stop watch
3. Disposable needle
4. Cotton swab soaked in ethyl alcohol
Procedure: Finger is cleaned with swab dipped in absolute alcohol and prick is made on the finger with a
needle. The drop of blood that comes out is soaked on a blotting paper till the blood stops coming out.
This is known as bleeding time and is usually 1 to 3 minutes.
Equipments required:
CLOTTING TIME
1. Wright's capillary tube
2. Cotton swab soaked in ethyl alcohol
l 3. Disposable needle (4) Stop watch.
Procedure: Clean the finger with sterile swab and make a prick with sterile disposable needle. The blood
enters into the capillary due to capillary action. The capillary is left horizontally on the table and then
start the stop watch. Break a 8-10 mm long piece of tube after every few minutes, carefully pulling away
the broken pieces till a thread of blood starts forming between the broken ends. As soon as thread
appears, stop the watch and record the time taken by blood for clotting. It should be 2-6 minutes.
PROTHROMBIN TIME
This is the time taken by platelet poor citrated plasma to clot after adding calcium and tissue (brain)
thromboplastin.
This is an important screening test for defects of the extrinsic and common coagulation pathway since
the intrinsic pathway is bypassed by adding tissue factors and calcium.
Method: 1. 0.1 ml of brain extract is mixed with 0.1 ml plasma in a glass test tube placed in a water bath
at 37°C. 2. After 1 min 0.1 ml of warm 0.1 M CaCl, is added to the above mixture and stop watch is
simultaneously started.
3. The end point is obtained by tilting the tube in a regular consistent manner and stopping the stop
watch, the moment a clot appears. 4. The test must be performed in duplicates with the test and control
plasma and the average values obtained.
1.4: BLOOD GROUPING (DETECTION OF BLOOD GROUPS)
Discovery of ABO blood grouping in 1900 by Karl Landsteiner was a milestone in the history of
immunohaematology.
He reported the presence of two antigens ie., A and B on the surface of human red blood cells. Based on
this discovery,
he divided RBC's into three groups A, B and O. Very soon in 1902 a fourth group AB was discovered by
Decastello and Sturli. According to the ABO group system the blood of each individual carries an
antibody directed against the antigen which is absent from person's RBC's. Principle: The test is based
on haemagglutination reaction human RBC's possessing A or B antigen will agglutinate with the
corresponding antibody. Agglutination of RBC's with anti-A antibody is a positive result and indicates
presence of A blood group. While agglutination with Anti-B antibody shows B blood group while
agglutination with both A and B shows AB blood group absence of agglutination with A and B antibodies
shows absence of both A and B antigen and is known as O blood group (Fig.1.3).
Procedure: Take a clean glass slide and mark three circles on it labeling them as A, B and AB. Place one
drop of appropriate blood grouping reagent at the above marked area. Place one drop of oxalated
whole blood to each of the marked area. Mix well with separate applicator sticks and spread it over an
area of approximately 2 cm diameter, tilt the slide back and forth for 2 minutes. Then, observe
macroscopically for evidence of agglutination.
RHESUS SYSTEM: The Rh factor is also a type of agglutinogen which was first seen in blood of Rhesus
monkey. In human beings some possess this factor while others lack it. Those possessing this
agglutinogen are Rh +ve while those without it are Rh-ve. Unlike blood groups the corresponding
antigen is completely absent in the blood. In other words, Rhesus negative blood does not automatically
contain the corresponding antibody. Procedure: One drop of blood is put on a clean slide and then one
drop of Anti Rh antisera is added. The two are mixed with applicator and the slide tilted to and fro for 2
min. Presence of agglutination indicate Rh+ve and absence indicates Rh-ve group.
CROSS-MATCHING (OR CROSSMATCHING) BLOOD
Cross-matching in transfusion medicine, refers to the testing performed prior to a blood transfusion in
order to determine if the donor's blood is compatible with the blood of a recipient, or to identify
matches for organ transplants. There are two basic principles behind cross matching: donation must be
harmless to the donor & the blood donated must be harmless to the recipient.
Donor Screening. For screening, the first important thing is history. The donor should not be too young
(less than 18 years) or too old (more than 60 years). Questions regarding present state of health, recent
illness, operations if any. H/ O malaria, syphilis, jaundice, tuberculosis, heart disease, diabetes, any
medicines use should be taken.
Physical examination includes temperature, blood pressure, pulse, heart or lung disease, pregnancy etc.
should be done. Investigations required include Hb, Malaria, parasite detection,
VDRL, microfilaria test, HBs Ag test, grouping and typing of donor. Compatibility testing. The indication
of whether a given donor blood is safe to transfuse depends not on grouping or typing but whether
there is agglutination or lysis when they are mixed together. The procedure used to determine
compatibility of donor and recipient's blood is called cross match. It should be done in two parts, major
cross match and minor cross match. The method commonly used is open slide method.
Procedure:
1. Label one tube with patient's name or number.
2. Label another tube with donor's name or number.
3. Half fill each tube with saline.
4. Label one slide for cross match, drawing a line down the middle to separate the two sides. Put the
patient's number on left side and identify it by putting P. This side is major side. Put the donor number
on right side and identify it by pulling D on that side. Draw two circles one on each slide with wax pencil.
5. Make 5% cell suspensions in each of the patient's and donor blood labeled tubes.
6. To the major side (P) add one drop of patient's serum and one drop of donor's cell suspension. To the
mino rside (D) add one drop of donor's serum and one drop of patient's cell suspension.
7. Mix by gently rotating the slide and incubate at room temperature for 10-15 minutes and examine
macroscopically and microscopically for agglutination.
BLOOD CHEMISTRY
A. BLOOD SUGAR ANALYSIS: Glucose is a major carbohydrate present in blood. Its oxidation in cells is the
source of energy for the body. Increased levels of glucose are found in diabetes mellitus,
hyperparathyroidism, pancreatitis, renal failure. Decreased levels are found in insulinoma,
hypothyroidion, hypopitutarism and extensive liver disease. Various methods commonly used are:
GLUCOSE OXIDASE METHOD
Principle: Glucose is axidised to gluconic acid and hydrogen peroxide in presence of glucose oxidase.
Hydrogen peroxide further reacts with phenol and 4-amino antipyrine by catalytic action of peroxidase
to form a red colored quinoneimine dye complex. Intensely of color formed is directly proportional to
the amount of glucose present in the sample.
Reagent used:
Glucose reagent Glucose standard
Procedure: Pipette into clean dry test tubes labeled as Blank (B), Standard (S) Test (T)
Mix well and incubate at 37°C for 10 minutes or at room temperature (25°C) for 30 minutes. Measure
the absorbance of standard (Abs.s) and test sample (Abs.T) against the blank within 60 minutes.
GLUCOSE TOLERANCE TEST
Procedure: Instruct the patient to fast overnight and collect a urine specimen and fasting blood
specimen for sugar determination. Dissolve 50 gms of glucose in about 200 ml of water and obtain blood
and urine specimens at 30 minutes,2 hr, 1 and 2 hours after giving glucose. Determine the blood sugar
level on each specimen and do a urine sugar on each specimen. The results can be depicted on a curve.
The shape of the curve is determined by,
• The capacity of the body to secrete adequate amounts of insulin.
The release of counter regulatory factors. The results can be shown on a graph. The fasting blood sugar
should not be above 100 mg/ml. The maximum blood sugar should not be more than 160-180 mg/ml
and the maximum is generally reached within 1st hour. The blood sugar should have returned to normal
fasting limits after 1 hour or at least by 2 hours. There is usually no sugar in any of the urine specimens.
In diabetes, mellitus the fasting levels are already elevated but it rises to a peak much higher reaching
upto 400 mg/dl and it takes 3-4 or more hours to return to fasting level.
B. UREA ESTIMATION: Urea is the end product of protein metabolism. It is synthesized in the liver from
the ammonia produced by catabolism of amino acids. It is transported by blood to the kidneys from
where it is excreted. Increased levels are found in renal disease, urinary obstructions shock, congestive
heart failure and burns. Decreased levels are found in liver failure and pregnancy
Principle: Urease Hydrolyses urea to ammonia and CO,.The ammonia formed further reacts with a
phenolic chromogen and hypochlorite to form a green colored complex. Intensity of color formed is
directly proportional to amount of urea present in the sample.
Procedure: The contents required are Buffer Reagent (L,), enzyme reagent (L), chromogen reagent (L),
urea standard (S).The wavelength/filter is 570 nm (Hg 578 nm)/yellow before testing urine is diluted 50
times by adding (1 +49) distilled water. Pipette into clean dry test tubes labelled as Blank (B), Standard
(S) and Test (T)
Mix well and incubate for 5 minutes at 37°C or 10 minutes at room temperature (25°C) chromogen
reagent (L). Mix well and further incubate for 5 min. at 37°C or 10 [Link] room temperature (25°C).
Measure the absorbance of the standard (Abs.S) and test sample (Abs.T) against Blank within 60
minutes. Normal urea levels lie between 14-40 mg/dl
C. CREATININE ESTIMATION: Creatinine is the catabolic product of creatinine phosphate which is used by
skeletal muscle. The daily production depends on muscular mass and is excreted out of the body entirely
by the kidneys. Elevated levels are found in renal dysfunction, reduced renal blood flow (shock,
dehydration, congestive heart failure). Decreased levels are found in muscular dystrophy.
Principle: Picric acid in alkaline medium reacts with creatinine to form an orange colored complex with
the alkaline picrate. Intensity of color formed is directly proportional to the amount of creatinine
present in the sample.
The contents used are picric acid reagent (L,), Buffer reagent (L), Creatinine standard (2mg/dl). The
wavelength used is
520 nm (Hg 546 nm) green filter. The steps are as follows: 1. Deproteinisation of specimen: For this
pipette into clean dry test tube picric acid reagent (á,) 2 ml anda sample 0.2 [Link] well and centrifuge
at 2500-3000 rpm for 10 min to obtain clear supernatant. Then pipette into clean dry test tubes labelled
as Blank (B) Standard (S) and Test (T).
D. URIC ACID ESTIMATION: Uric acid is the end product of purine metabolism. It is excreted by the
kidneys and intestinal tract by microbial degradation. Increased levels are found in gout, arthritis,
impaired renal function, and starvation. Decreased levels are found in Wilson's disease, Fanconi's
syndrome and yellow atrophy of liver.
Principle: Uricase converts uric acid to allantoin and hydrogen peroxide. The hydrogen peroxide formed
further reacts with phenolic compound and 4 aminoantipyrine by the catalytic action of peroxidase to
form a red colored quinonemine dye complex intensity of color formed is directly proportional to the
amount of uric acid.
Normal reference values of uric acid are 3.4-7 mg/dl in males and 2.5-6 mg/dl in females. The following
reagents are required;
Buffer reagent (L,),Enzyme reagent (L) and uric acid standard (8 mg/dl). The wavelength used is 520 nm
(Hg 546mm) yellow green. Pipette into clean dry test tubes labelled as blank (B), standard (S) and Test
(T).Mix well and incubate at 37°C for 5 minutes or at room temp. for 15 minutes. Measure the
absorbance of the standard (Abs. S) and test sample (Abs T) against blank within 30 minutes.
E. BILIRUBIN ESTIMATION: Bilirubin is formed from the heme portion of aged or damaged RBC's. It then
combines with albumin to form a complex which is not water soluble. It is known as unconjugated
bilirubin. In the liver, this bilirubin complex is combined with glucuronic acid to form a water soluble
conjugate known as conjugated or direct bilirubin.
Elevated levels of bilirubin are found in liver disease excessive hemolysis, obstruction of biliary tract and
in drug induced reactions. Principle: Bilirubin reacts with diazotized sulphanilic acid to form a colored
azobilirubin compound. The unconjugated bilirubin combines with sulphanilic acid in the presence of
caffeine-benzoate accelerator. The intensity of color formed is directly proportional to the amount of
bilirubin present in the sample.
The normal reference value of total bilirubin is mg/dl and direct is 0.2 mg/[Link] reagents required are
direct bilirubin reagent (L), Direct nitrite reagent (L), Total bilirubin reagent (L). Total nitrite reagent (L),
Artificial standard (S). The procedure uses wavelength filter 546 nm/yellow green.
Direct bilirubin assay: Pipette into clean dry test tubes labelled as Blank 8 (ml) Mix well and incubate at
room temperature for exactly 5 min. Measure the absorbance of test samples (Abs.T) immediately
against their respective blanks. Total bilirubin assay: Pipette into clean dry test labelled as Blank (B) and
Test (1)
Mix well and incubate at room temperature for 10 [Link] the absorbance of test samples (Abs.T)
immediately against their respective Blanks.
[Link] ESTIMATION: Cholesterol is a component of cell membranes and a precursor of steroid
hormones and bile acids synthesized by body cells and absorbed with food. There are four classes of
Lipoproteins: HDL, LDL, VLDL, Chylomicrons.
Principle: The test determines cholesterol levels after enzymatic hydrolysis and oxidation. The
colorimetric indicator is dye which is generated from H-amino antipyrine and phenol by hydrogen
peroxide under catalytic action of peroxidase.
The wavelength used is 505 nm. For it pipette into clean dry test tubes the following:
Pipette into test tubes Distilled water
1.6: BLOOD CULTURE
It is required in certain diseases where bacteremia forms an important part. Bacteremia occurs
transiently in pneumococcal pneumonia, bacterial meningitis, urinary tract infections, enteric fever and
generalized salmonella infections. The chance of isolating bacteria from culture depends on the stage of
disease at which culture is done. e.g., in typhoid fever, bacteria can be isolated during early stage only.
The blood is collected using perfect aseptic conditions and inoculated into enriched aerobic and
anaerobic culture media. The site for blood collection is well-swabbed with cotton moistened with spirit
and about 15 ml blood is withdrawn. The protective covering from culture bottle is removed. The mouth
of bottle flamed and the needle is inserted into the culture bottle. Three bottles of broth should be
incubated, one for aerobic cultivation, one for anaerobic and the third for incubation in 5-10% CO,. Once
collected the cultures should be incubated at 37°C immediately and left overnight at appropriate
temperature. Subcultures are made every 2 days either until growth is found or until 2 weeks have
elapsed. If there is no growth after 2 weeks, then a report of no growth is sent.
1.7: SEROLOGICAL AND IMMUNOLOGICAL-TESTS
1. WIDAL TEST
Widal test is an agglutination test employed in the serological diagnosis of enteric fever (typhoid &
paratyphoid) caused by [Link]&[Link] (A & B). The specific antibodies are usually detectable in
patient's blood at the end of first week of infection. The test is named after a French physician &
bacteriologist Georges FernandIsidore Widal developed this test for diagnosis of typhoid. who first
Principle: The serum of a patient is tested for 'O' & 'H' antibodies by using antigenic suspensions
[Link]'O'(somatic Ag) &[Link]'H' (flagellar Ag) respectively. For paratyphoid testing, the antigen
suspensions used are [Link]'AH' & Preparation of antigens: [Link]'O' antigen & [Link]'H' antigen
are prepared from S.typhi901 strain. 'O' antigen for [Link]'BH'. [Link] A&B are not taken as
they cross react with [Link]'O' antigen. 'H' antigen is prepared by treating overnight broth culture with
0.1% formalin.
Methods: Widal test may be conducted in two ways;
1. WIDAL (slide agglutination/micro agglutination) TEST:
Procedure:
1. In each four rings of the slide, labeled as 'O','H','AH' & 'BH' add one drop of serum.
2. Add corresponding antigen drop (one) in each ring of the glass plate.
3. Mix the antigen suspension and the diluted serum drop in each ring by using separate applicator stick.
4. Slowly rock and tilt the glass plate and observe for 3 minutes.
5. Record the degree of agglutination as 4+,3+, 2+, 1+ or no agglutination (negative) (Fig.1.6,a). Note:
The positive specimen confirmed by slide test should be subjected to quantitative tube agglutination
test.
WIDAL (tube agglutination /macro agglutination) TEST: Procedure: In this test two types of tubes are
used:
Round-bottomed Felix tubes.
b) Conical-bottomed Dreyer's tubes.
1. For each serum sample, arrange 4 rows (2 rows of Felix tubes & 2 rows of Dreyer's tubes) of 6 tubes
each in a widal rack.
2. Take 6 tubes in other rack for preparation of master dilution.
a). Keep 7ml normal saline in tube 1 & 3.5 ml in other 5 tubes.
b). Add 0.5 ml of test serum in tube 1 & mix well.
d. Take 3.5 ml from tube 1 & transfer to tube 2. Mix well & continue this transfer till last tube.
3. Transfer 0.5 ml from master dilution tube to each of the corresponding vertical rows in test rack.
Place 0.5 ml normal saline in each of tubes in 7th row to serve as control.
4. Add 0.5 ml of [Link] O antigen to each of 7 tubes in first horizontal row.
5. Add 0.5 ml of [Link] H antigen to each of 7 tubes in second horizontal row.
6. Add 0.5 ml of [Link](H) antigen to each of 7 tubes in third row.
7. Add 0.5 ml of [Link](H) antigen to each of 7 tubes in fourth row to obtain dilution of 1:30, 1:60,
1:120, 1:240, 1:480 & 1:960. 8. Shake the rack well to mix & incubate the rack in thermostatically
controlled water bath maintained at 37°C for overnight incubation.
Reading: The control tubes must be examined first, where they should show no agglutination. The
agglutination of 'O 'antigen appears as a 'matt' or 'carpet or granular disc-like pattern at the bottom of
[Link]'O' antigen containing tube. The agglutination of 'H' antigen appears as loose, wooly or cottony
(Fig.1.6,b).
Interpretation: The highest dilution of serum that produces a positive agglutination is taken as titer.
1. Agglutination appears by the end of first week. The titers increase during second, third & fourth week
after which it gradually declines.
2. Single test is usually of not much value. Demonstration of rising titer of antibody by testing two or
more sera specimen is more meaningful than single test.
3. Patients already treated with antibiotics may not show any rise in titer, instead there may be fall in
titer.
4. Patients who have received vaccine against salmonella may give false positive reaction.
5. Individuals who had suffered from enteric fever in past sometimes develop anti-salmonella antibodies
during an unrelated or closely related infection. This is called anamnestic response with temporary rise
in 'H' titer only.
6. Antigen suspensions with fimbrial antigens may sometimes give false positive reactions.
2. VDRL (VENERAL DISEASE RESEARCH LABORATORY) TEST Clinical significance: It is the most widely used
precipitation (flocculation) test used in diagnosis of venereal disease by a spirochete, [Link]. It is a
simple & rapid test requiring very small quantity of serum. Method: Slide test (Qualitative serum slide
test) philis caused Principle: Since complement present in serum interferes with the flocculation
reaction, it is inactivated by keeping serum at 56°C in water bath. Reagin (antibodies that appear in
syphilis) in serum reacts with the VDRL-antigen of lipid coated with cardiolipin) and forms, floccules.
particles Requirements
Procedure
Pipette about 0.5 ml serum in a small test tube (10x100mm).
Keep it in the warm bath at 56°C for inactivation for 30 minutes. 3 Cool to room temperature (25°C5°C).
minutes.
The temperature of water bath should be maintained exactly at 56°C. If the temperature increases
above 56°C, serum may get coagulated and then it cannot be used for the test.
Pipette in the cavity of the glass slide (or in the marked circular area), 0.05 ml inactivated serum.
5 Shake the bottle of working VDRL antigen gently, add one drop (1/60 ml) to the cavity (containing
serum).
6. Rotate the plate on the rotor immediately for 4 minutes (or rotate by hand 10 times in 5 seconds in 5
cm diameter circle).
7. Examine visually in bright day light and confirm the results by observing under low power objective.
Results: Presence of clumps indicate positive test where as uniformly distributed crystals indicate
negative test. Repeat the steps 1 to 5 by using a negative and one positive control [Link] as
follows:
3. RA TEST
It is used for in vitro detection of rheumatoid factor in serum by quantitative and semi quantitative latex
slide test.
Rheumatoid arthritis is an auto-immune disorder, where antibodies are produced against self antigen
IgG. These autoantibodies are termed as rheumatoid factor which is immunoglobulin of predominantly
IgM class which combines with FC portion of immunoglobulin IgG molecules RF is present in sera of 80%
cases of rheumatoid arthritis. Principle: RA test antigen consists of polystyrene latex particles coated
with specially modified preparation of human gammaglobulin (IgG) in order to avoid non-specific
agglutination. The suspension of coated latex particles agglutinate when mixed with serum containing
rheumatoid factor. Collection of sample: No special precaution of the patient is required. Serum is used
and serum sample stored at 2-8°C after collection.
Procedure: Qualitative method In this method, a special slide marked as test is used. Using a dropper
one drop of test serum is put within the circled area marked as test shake the vial gently and add one
drop of latex gammaglobulin reagent to the above drop and mix well rock the slide gently to and fro for
2 minutes and examine for agglutination. Read the test results within 2 minutes. Clearly visible
agglutination indicates positive result.
Semi-quantitative Method. To determine the titer ie, the highest dilution showing visible agglutination
prepare serial doubling dilutions of test serum using normal saline from 1:2, doubling upto 1:128. 1.
Place one drop (40 in) of each dilution of serum within the respective marked area of the special slide
provided with the kit.
2. Add one drop of thoroughly mixed latex gamma globulin reagent and mix well with disposable
applicator stick. Use separate sticks for each serum dilution.
3. Gently rock the slide to and fro and observe agglutination for 2 min.
1.9: EXAMINATION OF BONE MARROW
Bone marrow biopsy is an important method for investigation of hematopoietic disorders. There are two
main methods:
1. Aspiration biopsy (needle): In this bone marrow is aspirated through a wide bore needle e.g. Salah
needle, Klima needle (Fig.1.8,a,b). 2. Trephine biopsy: in which biopsy is obtained by a specially designed
trephine e.g., Socker-Nordin needle, Turbel's needle.
(c): Collection of bone marrow biopsy
Indications: Diagnostic and prognostic indications are present.
Diagnostic:
Megaloblastic anaeima
Iron deficiency anaemia for knowing Iron stores.
Sideroblastic anaemia
Leukaemia
Kala-azar
Secondary deposits in bone Lysosomal storage diseases,Gaucher's disease etc.
PUO
Prognostic:
Acute leukemia
Agranulocytosis
Aplastic anemia
Sites for aspiration The first piece of body of sternum
Posterior Iliac crest
Medial aspect of upper end of tibia in children less than 2 years of age. Lumbar spinous process.
The usual aseptic precautions are taken, after aspiration immediately transfer the contents onto a slide
and suck of as much blood as possible with a needle and then make a smear with the remaining yellow
irregular shaped fragments.
Dry the smear and fix the slide with acetone free methyl alcohol and then stain it with either May-
grunwalds stain or Giemsa stain for 15 minutes. Wash in buffered water for 5-7 minutes and dry it.
Under low power examine the fragments to access degree of cellularity. The cells are dark blue while fat
is round or vacuolar. A normal smear contains 40% fat cells and 60% marrow. The following things are
noted:
1. The types of erythropoiesis whether normoblastic or megaloblastic and ratio of myeloid to erythroid
cells is noted.
2. Leucopoiesis: Number and maturity of developing granulocytes, abnormal cells, toxic granulation
Plasma cells: They are oval or round in shape nucleus in eccentric and has large dense mass of chromatin
arranged in wheel shaped fashion, cytoplasm is dark blue in colour. Megakaryocytes: Note the number
and morphology and look for platelet formation or budding. Megakaryocytes are large cells 35-1601 in
diameter nucleus is lobed, round or ring shaped and stains purple. The cytoplasm is abundant, light blue
or pink and filled with fine purplish granules.
Myeloid: Erythroid ratio: It is determined by counting 300-500 nucleated cells in the marrow and noting
the ratio. Normal ratio is 3-5: 1. Foreign Cells: Gaucher's, Niemann pick and metastatic tumor cells may
be present.
Parasites and bacteria: Microfilaria, Leishmania Donovani bodies, malarial parasites, tubercle bacilli, etc.
may be seen.
1.10: COLLECTION OF BLOOD FOR CLINICAL TESTING
Blood serves as the most important kind of specimen and the bacteria found in the circulating blood
(bacteremia) are associated with the various diseases (septicemia). It must be noted that the blood as a
specimen is collected by venipuncture during acute phase of the disease and before any antibiotic
therapy.
Procedure of blood collection: 1. Clean the skin over the venipuncture site in a circle 6mm in diameter
with 70% alcohol. 2. Apply 2% iodine solution to the cleaned area and saturate the entire area with it,
allowing it to remain on the skin for at least one minute which should be keenly noted.
3. Insert the needle into vein and withdraw blood.
4. Clean the site again with 70% alcohol, after collecting the blood specimen; introduce it directly into
special blood collection bottle through the hole in the cap without touching the needle with any
exposed surface. Anticoagulants like sodium polyanethol sulphate (SPS) should be used. The amount of
blood collected (approximately 10 ml for adults and 5 ml for pediatric patients) is diluted at least 10
times its volume with liquid medium like Nutrient Broth. Cold medium should not be used and the
bottles should be allowed to stand for at least 30 minutes at room temperature prior to inoculation.
1.11 TYPES OF BLOOD PRODUCTS/COMPONENTS
With advancements in types of blood bags and component preparation techniques, whole blood from a
single blood donor can be divided into three or four blood components preserving the function of
individual component by storing them at desired temperature and issuing specific component as per
patient's need. Following are the types of blood Products.
Whole Blood (WBC)
Packed Red blood cells (PRBC) in CPDA-1
SAGM-PRBC (Packed Red Cells in additive solution
Paediatric PRBC units (Volume 80-100ml)* Platelet Concentrate (PC)
Fresh Frozen Plasma (FFP) Cryopoor Plasma (CPP)
Cryoprecipitates (Cryo) Apheresis Platelets (SDAP)
Paediatric PRBC units are prepared by dividing adult PRBC unit into three parts using sterile connecting
device and transfer bags
