Thesis work done on the basis of lab training.

1. Blood bank
Professional donors: Receive monetary returns for blood
donation. They are usually not fit and unhealthy for donation.
Replacement donors: Family members or friends of patients
donate blood in absence or presence of stock.
Voluntary donors: Individuals who are self-motivated to
help unknown patient without any professional benefits. They
are safe donors.
Agglutination reaction
When a particular antigen is mixed with its antibody in the
presence of electrolytes at suitable temperature and PH, the
particles are clumped or agglutinated.
Agglutination is more sensitive than precipitation for
detection of antibodies.
Preparation of 2-5% cell suspension
 It is the basic requirement for all test.
 Arrange the test tubes in serial order.
 Transfer two drops of red cells to labelled test tubes
using Pasteur pipettes with rubber teats
 Add 2 – 5 ml of 0.9% normal saline ( This will be
approximately ¾ th
of a 3 “ test tube”)
 Centrifuge at 200 RPM for 2 minutes.
 Discard the supernant & repeat the process for 3 times.
 To the final button add another 2.5 ml of saline 0.9% &
mix well.
 The colour should be “tomato red”.
 This is the suspension to be used for all serology
procedures.

3. Blood grouping
Objectives:
1. Using the slide agglutination method, determine with 100%
accuracy the ABO group of specimens using reagent
antiseras, blood specimens, and other materials provided. 2. If
applicable, evaluate reagent package inserts / instructional
materials to determine the substance being analyzed, the
principle of the procedure, the expected value, significance of
abnormal results, limitations of the procedure, and
troubleshooting procedures to follow if / when control results
are unacceptable. 3. Utilize lecture notes, textbook and
laboratory information to answer study questions.
Principle: When red cells are mixed with various reagent
antiseras (soluble antibody), agglutination will occur on the
slides containing cells positive for (possessing the antigen) the
corresponding antigen. No agglutination will occur when the
red cells do not contain the corresponding antigen. One
primary application of this principle is blood typing.
Procedure:
1. Set the table with all the materials required. Remember to place
the Monoclonal Antibody (Mab) kit in an Ice tray.
2. Open an Alcohol swab, and rub it at the area from where the
blood will be sampled (finger tip). (Discard the swab)
3. Open the Lancet cover, put pressure at the tip of the finger from
where blood will be sampled (maintain it). Prick the finger tip
with the opened Lancet.(Discard the Lancet)

4. 4. As blood starts oozing out, make 1 drop fall on the three
depressions of the glass slide. (in clinical setup, there will be a
fourth well used as a control).
5. Place a cotton ball at the site where it was pricked. Using the
thumb, put pressure on the area to stop blood flow.
6. Take the Anti-A (blue) bottle, resuspend the content and use
the dropper to place a drop of the Mab in the 1st spot. Place the
bottle back in ice.
7. Take the Anti-B (yellow) bottle, resuspend the content and use
the dropper to place a drop of the Mab in the 2nd spot. Place
the bottle back in ice.
8. Take the Anti-D (colorless) bottle, resuspend the content and
use the dropper to place a drop of the Mab in the 3rd spot. Place
the bottle back in ice.
9. Take a tooth pick and mix the content in each well. Discard the
tooth pick after using in one well (take a new one for the next
well) and wait for the result.
Interpretation: Agglutination (clumping) of the red blood
cells is positive. No agglutination is negative. It is critical to
read the results immediately as false positives can occur when
the mixture begins to dry on the side.
Limitations: 1. Adding red blood cells directly to the drop of
antisera may cause splash back, resulting in contamination of
the red blood cells. 2. Inadequate spreading of the mixture
will make interpretation difficult and may result in
misinterpretation of results. 3. It is critical to read the results
at the end of the designated time.

5. Preparation of blood and components
RBC:
RBC concentrates should be prepared from the whole blood
collected in plastic bag preferably in double or multiple
plastic bag system. Plasma is separated from RBC by
centrifugation or undisturbed sedimentation before expiry
date. The haematocrit should be less than 70%.
Washed red cells:
RBC should be washed with normal saline by automatic cell
washer or centrifugation, for 2-3 times at 4°C±2°C using
laminar bench. Closed system of washing is recommended.
Platelet concentrate:
 It is prepared by centrifugation within 6 hours of
collection at 22°C±2°C using platelet rich plasma or
buffy coat
 Whole blood should contain minimum 4.5X1010
platelets
and are stored at 22°C±2°C with pH 6
 Gentle agitation (60-70 oscillation/minute) should be
maintained throughout the storage using agitator or rotar
with 5-10 cycles/minute. Swirling phenomenon is
checked before use. There should be grossly visible
platelets aggregates.
Leukocytes:
Prepared by buffy coat method; should contain
5X108
leukocytes
Plasma:
single donor plasma:

6. Should be separated from blood upto 5 days after expiry date
which is used only for fractionation
Fresh frozen plasma:
Separated not later than 6-8 hours of collection and frozen at -
30°C
Factor VIII deficient plasma:
It is plasma from which cryoprecipitate has been removed,
stored at 30°C and thawed should be used within 6 hours
Single donor cryoprecipitate:
Stored within 6 hours at -80°C and lower using centrifuge.

7. Work instruction for cross matching
3 methods: Saline method, Albumin method, coombs test
Saline method:
Major: Take 3 drops of patient serum & washed (3-4 times)
and 2 drops of donor’s 2-5% cell suspension in a clean test
tube.
Minor: Take 3 drops of donor serum and washed (3-4 times)
2 drops of patient cell suspension in a clean test tube.
Mix the tube and keep it at 37°C for ½ an hour , Shake &
examine microscopically and macroscopically for
agglutination. If there is no agglutination, blood is compatible
in saline method.
Albumin method:
Major:
3 drops of patient serum (washed 3-4 times) and 2 drops of
donor’s 2-5% cell suspension is taken in a test tube
Minor:
Take 3 drops of donor serum and washed (3 – 4 times), 2
drops patient’s cell suspension in a clean test tube.
Mix it well and incubate at 37°C at water bath for 45 min , Take
it without shaking and remove supernant , add 22% Bovine
albumin, 1-2 drops and incubate at 37 °C for ½ an hour ,
Remove the tubes from water bath and mix well. Examine
under microscope. If there is no agglutination, albumin cross
matching is compatible.

8. AntiHuman Globulin method (Coombs test)
For Major: Take 3 drops of patient’s serum and washed (3-4
times) Donors cells suspension in a clean test tube.
For Minor: Take 3 drops of donor serum and washed (3-4
times) patient cell suspension in a clean test tube. Mix it well
and incubate at 37°C for 45 minutes. Pour out supernatant fluid
and add one drop of Bovine Albumin 22% incubate 30 minutes.
Take out and wash it 3 times in normal saline, remove the
supernatant, add 3 drops of antiHuman globulin serum and mix
well. Keep in room temperature for 10 minutes and centrifuge
at 3000 RPM for 20 seconds and examine under microscope.
If agglutination is absent, this method is compatible.

9. Haemoglobin estimation
Haemo cue method: It is a simple method in which readings
are provided by photometer. A micro cuvette containing the
reagents draws blood automatically and display result in 15 -
45 seconds.
Cyanmethaemoglobin method:
Principle: Blood is mixed with potassium cyanide solution
and potassium ferri cyanide. All except sulphaemoglobin are
converted to Cyanmethaemoglobin. Absorbance is measured
by calorimeter at 540 nm.
Procedure:
1. Take 5 ml of Drabkins solution in a tube.
2. Add 20 micro litre of blood and mix well.
3. Let stand at room temperature for 10 minutes.
4. Read spectrophotometer at 540 nm against a blank
Curve: Absorbance is directly proportional to haemoglobin
concentration
Precaution: Drabkins solution is not pipetted by mouth as
KCN is hazardous.

10. Malarial parasite
Procedure:
Bring kit and specimen to room temperature
Remove test card and assay buffer from foil pouch
Mix anticoagulated blood sample evenly by gentle
swirling to make it homogenous. Dip sample loop and
take sample. Blot the sample in sample well ‘A’
or
Micropipette 5µL of anticoagulated or finger prick
specimen to sample pad ‘A’
Add 5 drops of assay buffer in well ‘B’
Read results at 20 minutes
Discard kit immediately as it is infectious
Interpretations:
Appearance of 2 pink coloured lines shows it is a
reactive malariae
Appearance of 1 pink line shows it is a nonreactive
malariae
Test is invalid in the absence of lines

11. pH meter
Principle:
ELICO pH meter, LI 120/LI 610 operate on the principle of
pH meter, specification and other general information in
logarithmic variation in linear relation to voltage generator at
the glass membrane according to NERNST equation.
Potential which is a function of the free hydrogen ion activity
is measured by glass pH electrode in conjunction with
reference electrode. The developed voltage in relation with
pH,is calibrated according to isopotential of pH sensitive
electrode.
Procedure:
 Fill up a clean dry container with sample.
 Clip the electrode holding clamp at the appropriate
height such that the electrodes are immersed in the
sample.
 Measure the temperature of the sample,set the
temperature compensate control to the measured value of
the temperature of the sample.
Note: ensure
 The electrodes are properly inserted in the sample.
 Allow sufficient time for the electrode to attain
temperature of the sample.
 Use larger volume of sample.
Set the pH/mV switch to pH position and STBY/READ
switch to read and wait for 30 seconds.

12. After the test, set the STBY/READ switch to STBY
position,raise the electrodes,remove the container with the
sample. Wash the electrodes with distilled water and blot
clean with tissue or filter paper.
STBY/READ switch should be in STBY position before
switching the instrument OFF.
Measurement of EMF
Same procedure is done for EMF also.
Set the pH/mV switch to mV position and
STBY/READ switch to READ and wait for 30 seconds.
The value of EMF in mV of sample will be
displayed on the READ OUT.
Salientfeatures:
 Conforms to Bureau of Indian standards (BIS)
 Accurate and highly stable
 Manual temperature compensation
 Elegantly styled
 Small,laboratory version
 Usable for:Potentiometric titrations, oxidation reduction.