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Hematology
Hematology
Hematology
There are several factors to consider when determining the appropriate blood collection volume
and technique. These include:
Hematoma or thrombus
o Enter vessel at an angle of 30 degrees or less
o Use a gauge of needle smaller than the vein
o Apply pressure until bleeding has stopped (1+ minutes)
Pain at blood collection site
o Use a needle of smaller gauge than the vein
o Practice on vein models prior to live animal
Infection at blood collection site
o Use sterile single-use devices only
o Clean work surfaces with disinfectant
o Wear gloves, wash hands
o Contact a qualified veterinarian for treatment recommendations if any of the
following are noted.
Heat, pain, swelling first noted at the insertion site of the blood draw,
purulent material draining from the insertion site.
Induration (hardening) of the vessel c. Pyrexia, local or systemic
infections, septic shock.
Clean the counting chamber and cover slip with soft tissue paper and cover the rule part of
neubauers chamber with cover slip.
Transfer the diluting fluid on watch glass or Petridis.
Mix well the blood on blood vial.
Suck the blood up to 0.5 mark of RBC diluting
pipette. Clean the tip of the pipette with cotton.
Suck the diluting fluid up to 101 mark of the
diluting pipette.
Mix well the blood and diluting fluid inside the
diluting pipette by keeping it on the palm.
Discard 2-3 drops of solution.
Keep 1 drop of the mixed solution on the side of
the cover slip (charging)
The fluid get well throughout the counting area
of neubauers chamber.
If air bubbles are formed remove the counting
chamber and cover slip and repeat the same Fig: neubauer’s counting chamber (source: www.wikipedia.org)
process again.
Leave the solution for 2 minutes.
At first observe the diluted blood on 10x on
microscope and after that observe on 40x.
Count the RBC present on 5 medium squares (4 of 4 corners and one on the middle)
While counting the RBC touching the right side and down side should not be counted.
Total RBC counted should be multiplied by 10000 and is expressed on cubic ml.
CALCULATION
Calculation
W*50
F. Hemoglobin estimation
There are various methods that can be used to estimate the
hemoglobin of an animal in a laboratory. Some of the widely
used methods are:
Principal: Converting Hb into acid hematin has a dark brown colour. The solution developed
is diluted with water and the colour developed after dilution is mathched with colour of the
standard tubes.
Material required:
Sahli’s haemoglobinometer
Pasteur pipettes ( one for Hcl and one for distilled water)
Stirrer
0.1 N- Hydrochloric acid
Distilled water
Comparision tube
Pipette ( haemoglobin pipette with rubber tubing and mouth piece)
Procedures:
N/10 hydrochloric acid is put in the hemoglobinometer up to the mark 20 below.
Draw fresh or oxalated blood up to 20 c mm. mark of the hemoglobinometer pipette.
Immediately mix it with N/10 hydrochloric acid of the hemoglobinometer.
Mix it properly and allow it to stand in the comparator for 10 minutes for
the conversion of hemoglobin to acid haematin.
Now add distilled water drop by drop with the help of dropper and mix with
stirring rod till the color matches well the fixed colour in the comparator.
The mark tallying with the upper level of diluted acid haematin denotes the level
of hemoglobin. This is expressed in terms g per 100 ml of blood.
Cyanmethemoglobin method:
Principle: Blood is diluted in a solution containing potassium cynide and potassium ferricynide,
which converts Hb to cyanomethemoglobin later by potassium cyanide. The absorbance of the
solution is then measured in a spectrometer at a wavelength of 540 nm or in a colorimeter using a
yellow green filter.
Material required:
Hb pipette
Spectrometer
Reagents : Drabkin’s solution with pH 7.0 -7.4
Procedure:
Take 5 ml of Drabkin’s solution in a test tube.
Mix the blood sample by gentle inversion and draw 0.02 ml of blood into the
Hb pipette.
Wipe the outer surface of the pipette to remove excess blood.
Place the pipette into the tube containing Drabkin’s solution and slowly expel the
blood into the solution.
Mix well and let it stand undisturbed for 5 min.
Measure the absorbance of this solution at 540 nm in a spectrometer after adjusting
the OD at 0 by using Drabkin’s solution as blank.
Calculate the hemoglobin concentrate using a standard curve.
References
https://www.hematology.org/education/patients/blood-basics
SEER Training Modules, composition of the blood. U. S. National Institutes of Health, National
Cancer Institute. 21, apr, 2021<https://training.seer.cancer.gov/>.
https://www.phlebotomy.com/blood-collection-sites-precautions-wall-atlas.html
https://www.euro.who.int/WHO-guidelines-on-drawing-blood-best-practices-in-phlebotomy-
Eng.pdf
https://animal.research.uiowa.edu/iacuc-guidelines-blood-collection
https://ouv.vt.edu/content/dam/ouv_vt_edu/sops/large-animal/sop-bovine-blood-collection.pdf
McCurnin, D., and Bassert, J. Clinical Textbook for Veterinary Technicians (5th ed.).
(Philadelphia, PA:Saunders Elsevier 2002)