Blood sample collection methods

There are several factors to consider when determining the appropriate blood collection volume
and technique. These include:

 The species to be sampled

 The size of the animal to be sampled
 The age and health of the animal to be sampled
 The minimum volume required for analysis
 The frequency of sampling necessary
 The training and experience of the personnel performing the collection
 The suitability of sedation and/or anesthesia

The sample volume selected should always be the

minimum volume of blood which satisfies
experimental needs. Appropriate restraint
(physical or chemical) should be employed to
minimize risk of injury to the animal and
personnel.

 Restrain animal in squeeze chute,

halter and secure lead to the stanchion
with a quick-release knot (Figure 1)
with head elevated and jugular vein
exposed.
 Clip (optional) and swipe with antiseptic gauze to remove superficial dirt and debris.
This may also assist in visualizing raised vein.
 Occlude jugular vein by applying pressure at the base of the jugular groove and visualize
raised vein (Figure 2).
 With bevel up, insert needle firmly into skin and into vein at 20° angle (Figure 2).
 If using vacutainer, once needle inserted, stabilize needle and push the vacutainer tube
into hub. If you have hit the vein, blood will flow freely into tube. Multiple tubes can be
filled by removing filled tube and replacing with fresh tube.
o NOTE: Do not pull needle out of vein with vacutainer tube still attached as this
will release vacuum in vacutainer.
 If you have missed the vein, carefully reposition needle, with vacutainer attached, until
vessel penetrated. Vessel is fairly deep and may roll away from needle. Typically no
 more than two to three attempts should be
made at a time to minimize distress to the
animal and potential damage to the vein.
 Alternately, you can use needle and syringe.
Break the seal on the syringe by gently
pulling back before using. Clear air, and with
needle attached to syringe, insert firmly
needle at 20° angle, and aspirate syringe to
confirm insertion and collect blood (Figure
3).
 Once collection complete, remove vacutainer
tube, then, applying pressure over injection
site, remove needle.
 Dispose of needle in approved Sharps
container.
 In order to ensure adequate hemostasis, apply pressure with gauze for 30 to 60 seconds.
 Serial samples can be taken by alternating sides, and by moving insertion sites cranially,
as long as there is no hematoma formation.

Blood can be collected via the coccygeal vein (Figure 4)

 Restrain animal.
 Raise the tail vertically until it is horizontal to the ground.
 Locate the groove lying in the ventral midline of the tail.
 Swab the site with antiseptic.
 Midway along the body of a coccygeal vertebra, insert the needle perpendicularly to the
surface of the skin to a depth of a few millimeters.
 Withdraw blood sample and remove needle.
 Dispose of needle in approved sharps container.
 In order to ensure adequate hemostasis, apply pressure with gauze for 30 to 60 seconds

Different types of vacutainer for blood collection

 for preparation of serum vacutainer without anticoagulant (having red cap)

 for hematological tests vacutainer with anticoagulant EDTA (having violet cap)
 for biochemical examination vacutainer tube with heparin (having green cap)

Animal species Blood collection site Needle size(gauge)

Horse Jugular vein 18-19 gauge
Cattle and Jugular vein 18-19 gauge
buffalo
 Sheep and goat Jugular vein 20 gauge
Swine Ear vein or anterior venacava 20 gauge
Poultry Wing vein or jugular vein 21-22 gauge
Dog Cephalic or cephanous vein 20-22 gauge
Cat Cephalic or cephanous vein 20-22 gauge
Rabbit Ear vein 22-23 gauge
Mice Orbital sinus Microhematocrit tube

A. Precautions while taking and handling of blood samples

Precaution while taking blood sample:

 Be sure to be well informed about the patient.

 Know the test required before commencing with the venipuncture.
 The site to be puncture should be free from hematoma and edema.
 Proper sterilization procedures should be done before puncturing the vein.
 Do not puncture in the same vein that an IV fluid is inserted as the components of
the iv fluid would contaminate your specimen.
 Check that syringe is not clogged by pulling and pushing the plunger. Check the
needle of any factory defect. Do not; however, open the pack, not until ready
puncture as this may lead to contaminations.
 Allow the animal to sit down or lie down in a comfortable position.
 Scrub with the antiseptics like spirit etc.
 Allow the alcohol to dry before puncturing. Inserting the needle while the wet
would be painful.
 The bevel should be facing upwards to allow the smooth flow of blood into the
barrel of the syringe.
 When blood starts to flow into the barrel, the plunger should not be pulled fast to
avoid clotting before the blood could be transferred to an appropriate container.
 Transfer the blood immediately after extraction to an anti-coagulated tube for
plasma and to a plain tube for serum. Vacutainer tubes can be used to facilitate
extraction and preservation of blood.
Potential Adverse Effects, Mitigation, or Treatmen

 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.

B. Total count of RBC

Materials required

 Neubauer’s slide counting chamber along

with cover slip.
 RBC diluting pipette (with red grain)
 RBC diluting fluid (Haems fluid) or (Decies
fluid)
 Watch glass or small Petri dish
 Microscope

Fig: neubauer’s slide (www.wikipedia.org)

Procedure for counting RBC

 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

R= total RBC counted on 5 secondary square chamber

Total RBC present on 1 cu mm or 1 microlitre of blood= R*10,000

C. Total count of WBC

Materials required:

 WBC diluting pipette (containing white grain inside)

 Counting chamber/ neubauers chamber
 Cover slip
 Microscope
 WBC diluting fluid
 Procedure for counting WBC

 Cover the rule part of counting chamber with cover slip.

 Mix the blood with anticoagulant properly and suck the blood up to 0.5 mark of WBC
diluting pipette.
 Discard the extra blood above 0.5 mark
 Suck WBC diluting fluid up to 11 marks.
 Mix well the blood and WBC diluting fluid by keeping the WBC diluting pipette within
2 palms.
 Discard 2-3 drops of fluid and place one small drop of fluid at the edge of cover slip and
let the fluid to spread throughout the rule area and leave it for 1 minute.
 With the help of microscope observe it under 10 x power. The condenser of microscope
should be kept down so that the cells can be seen clearly.
 Count the WBC on 4 large squares present on 4 corners while counting WBC never
count the WBC on the down and right side.

Calculation

W= total number of WBC counted on 4 large squares.

WBC present on 1 cu mm or 1 microlitre blood =

W*50

D. Differential count of WBC

Principal of differential leucocyte count (DLC):
The polychromic staining solution (Leishman stain) contains methylene blue and eosine. These
basic and acidic dyes induces multiple colours when applied to cells. Methanol acts a fixative
and also as a solvent.

Specimen for DLC:

 EDTA anti-coagulated venous blood or free flowing capillary blood.
Requirements for DLC:
 Microscope slide and a glass spreader slide
 Cedar wood oil (immersion)
 Leishman stain/ Giemsa stain
 Buffer solution (PH : 7.0)
 Staining rack
 Cotton and tissue paper
 Pipette
 Timer
 Cell counter
Before performing the differential leukocyte count, a thin smear of blood should be made on
glass slide. The smear should be thin, leveled and should not contain any bubbles.
 Let the smear dry on air.
 Number the smear for identification
 Fix the smear by dipping on methanol solution for 5 minutes.
 Dip the smear on the giemsa stain solution of 1:10 (1 part of giemsa stain mixed
with 10 parts of water) for 30 minutes.
 Now wash the slide with tap water let it dry.
 Observe under oil immersion lens (100x)
 Count at least 100 blood cells.
 The different type of cells visible by this process is eosinophils, basophils,
neutrophils, monocytes, lymphocytes.
 The number of cells is denoted in percentage.

E. Collection of blood serum

 For the collection of serum the blood should be collected on the vacutainer with red cover.
The tube should be Kept on the room temperature for 1-2 hours on inclined position. The
blood should clot. After the blood clots the tube should be kept on the refrigerator
overnight for clot retraction which makes easier for the collection of serum.
 On the next day the tube should be taken out from the refrigerator. With the help
Pasteur pipette the serum should be transferred to the serum vial.
 The tube should be centrifuged for the further collection of serum. The colour of serum
is slightly yellow.
 The vials containing serum should be kept on refrigerator. For longer storage period
the serum may be stored on dip fridge.

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:

a. Method based on the development of colour:

 Sahli’s or acid hematin method (commonly used)
 Cyanmethemoglobin method (commonly used)
 Oxyhemoglobin method
 Alkaline hematin method
b. Measurement of oxygen combining capacity
 c. Measurement of iron content

Sahli’s or acid hematin method:

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.

Normal level of hemoglobin in different animals

Animal Hemoglobin level (g/100ml)
Cattle 11.3
buffalo 12.9
horse 11.5
goat 10.9
sheep 14.4
pig 11.0
Dog 13.0
cat 12.0
Diminished level is observed in case of anaemia. Increased level is observed in case of
polycythaemia and haemoconcentration.

G. Basic interpretation of data of blood test/analysis

1. Lymphocytosis: increased number of lymphocytes in blood is known as lymphocytosis.

Lymphocytosis is seen during viral infection, tuberculosis, brucellosis, hypothyroidism,
following vaccination, leukemia, adrenocortica insufficieny etc.
2. Lymphopenia: decreased number of lymphocytes in blood is known as lymphopenia.
Lymphopenia is seen during canine distemper, infectious canine hepatitis, corticosteroid
therapy, hypothyroidism, coxiella burnetti infection, F.M.D, mucosal disease etc.
3. Neutrophilia: increased number of neutrophils in blood is known as neutrophilia.
Neutrophilia is seen during septicaemic disease, uremia, gout, coronary thrombosis,
pyogenic infection, acute inflammation, cancer, arthritis, pyometra, post-surgical
 operation, pregnancy, calf diphtheria, rheumatic fever etc. the condition in which the
immature neutrophils are present in blood is known as shift to left.
4. Neutropenia: the condition in which the number of neutrophils decreases is known as
neutropenia. Neutropenia is seen during the infection of diseases such as pasteurellosis,
bovine viral diarrhea, infectious canine hepatitis etc.
5. Eosinophila: increased number of eosinophil is known as eosinophilia. Eosinophilia is
seen during parasitic infection, skin diseases, anaphylactic reaction etc.
6. Basophilia: the condition in which the number of basophils increases in blood is known
as basophila. Basophilia is seen during pox, sinusitis, cirrhosis etc.
7. Monocytosis: increased number of monocytosis in blood is known as monocytosis.
Monocytosis is seen during brucellosis, tuberculosis, carbon tetra chloride poisoning etc.

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)