This document describes methods for counting red blood cells (RBCs) and white blood cells (WBCs). It discusses collecting blood samples using anticoagulants and various techniques for RBC and WBC counts including using a hemocytometer, electronic counters, and determining hemoglobin concentration. Methods covered include counting cells directly under a microscope, acid hematin reactions, and spectrophotometry. Normal reference ranges for total RBC and WBC counts in different animal species are also provided.

1. Counting of RBC & WBC
Submitted by
Assad Al Imran
19-VMK-57

2. Total Erythrocyte Count
• Total erythrocyte count is a method for determining the functional state
of the erythron.
(Erythron is a term for the mass of circulating erythrocytes plus the
erythropoietic tissue of bone marrow)
• However, this count reflects only the total number of red blood cells
(RBC) in the circulating blood and does not indicate the oxygen-carrying
capacity or amount of hemoglobin (Hb).

3. TECHNIQUES OF ERYTHROCYTE EXAMINATION
• Blood Collection
• Methods:
1. Haemocytometer Method
2. Electronic Methods
3. Using Haemoglobin Determinations
• Acid Haematin Method
• Direct Matching Method
• Oxyhaemoglobin Method
• Cyanmethaemoglobin Method

4. Collection of blood
• The usual procedure in large animals is to collect blood directly from the
jugular vein into a test tube or vial containing an adequate amount of
anticoagulant for the amount of blood desired.
• 5 ml of blood is sufficient for most hematologic examinations.
• An alternate technique in the cow is to utilize the caudal vein or artery as a
source of peripheral blood, and in this instance a clean, dry syringe or
Vacutainer is utilized.
• Blood sampling from the dog is best accomplished by use of a Vacutainer or,
alternatively, a dry syringe into which has been placed one drop of
anticoagulant.

5. • Care must be taken in obtaining blood, as improper collection procedures
may cause cellular distortion.
• Obtaining venous blood from a cat using a Vacutainer or dry syringe may
prove difficult because of the size of the vein and the necessity for restraint.
• Adequate samples can be collected from the jugular vein.
• There are certain advantages in using the jugular vein:
• It is relatively easy to locate,
• There is no excessive resistance to restraint,
• Vein is large and easy to penetrate, and adequate quantities of blood are readily
obtained, even from small animals.

6. • Blood may be obtained from the ear vein of dogs and cats by first cleaning the
surface of the ear followed by placing a thin layer of petroleum over the vein to
be punctured.
• The vein should be punctured by a sharp, pointed scalpel blade or sterile
disposable needle through the petroleum and into the vein.
• The blood forms a drop on the petroleum and is not contaminated with hair or
dander from the ear.
• An adequate flow of blood usually results, and the sample can be either collected
in diluting pipettes or utilized for other techniques.

7. • An alternate technique is to clip a toenail into the vascular area.
• The quantity of blood collected from the ear or toenail is usually inadequate for
use in automated cell counters but may be adequate for haemocytometer counts.
• Blood samples from swine are removed from either an ear vein or the anterior
vena cava.
• In collecting blood, care should be taken to ensure that the needle is of sufficient
diameter.

8. • A needle of too small gauge may cause disruption of erythrocytes and
damage leukocytes.
• Blood should flow smoothly with a minimum of vacuum, and if a
syringe is used, one should avoid pumping the syringe barrel.
• If it is necessary to transfer the blood from a syringe into a test tube,
the needle should be removed, as forcing blood through the needle
may damage cells

9. Anticoagulants:

10. • GREEN: Sodium heparin or lithium heparin used for plasma determinations
in clinical chemistry (e.g. urea and electrolyte determination). Sodium heparin
collection tubes are the classically preferred tube for peripheral blood or bone
marrow for cytogenetic studies. Lithium heparin is considered suboptimal for
cytogenetics.
• Light green or green/gray "tiger": for plasma determinations.
• Purple or lavender: K2 EDTA. This is a strong anticoagulant and these tubes are
usually used for complete blood counts (CBC). Lavender top tubes are
generally used when whole blood is needed for analysis. Can also be used for
some blood bank procedures such as blood type and screen. EDTA tubes are
preferred by most molecular genetics laboratories for molecular genetic studies
(DNA or RNA).

11. • Grey: Sodium fluoride and oxalate. Fluoride prevents enzymes in the blood
from working, by preventing glycolysis so a substrate such as glucose will not
be gradually used up during storage. Oxalate is an anticoagulant.
• Light blue: Sodium citrate. Citrate is a reversible anticoagulant, and these
tubes are used for coagulation assays.
• Dark blue: EDTA. These tubes are used for trace metal analysis.
• Black: used for Erythrocyte Sedimentation Rate (ESR).

12. 1. Haemocytometer method
Equipment necessary for performing a total erythrocyte count
includes:
1. Neubauer Counting chamber.
2. Special coverglass for the counting chamber.
3. RBC pipette.
4. Diluting fluid (Haeyem’s solution or NSS 0.85%).
5. Microscope and lamp.
6. A hand tally for enumeration of cells.

13. For total leukocyte count, the WBC in the area (W) are counted. The total
erythrocyte count is completed by counting all cells in the squares labeled R
Fig: A
Neubauer
Counting
Chamber
focussed on a
microscope
(10X)

14. Fig: A Neubauer Counting Chamber
Fig: An RBC pipette
Fig: Counting Procedure (zig-zag) method (40X)

15. Procedure:
• Blood should be carefully drawn to the 0.5 mark of the RBC pipette
• The diluting fluid should be drawn to the 101 mark to dilute the blood
• The blood and diluting fluid are mixed by shaking the pipette vigorously in a
horizontal position for 2 – 3 minute to ensure complete haemolysis of WBC
• 2 – 4 drops of mixed fluid are discarded at the end of pipette.

16. • The tip of the pipette is touched to the side of the haemocytometer chamber
and drop of a fluid wall run under the coverglass
• Wait for about 2 – 3 minutes as erythrocytes require settling time to assume
a single level
• Total number of cells in 5 squares in the centre of counting chamber is
determined under the high power of the microscope (40X)

17. • Counting is Zigzag manner in all 5
inner squares
• Blue cells are counted
• Gray cells are not counted

18. Counting:
• TEC/μL or mm3 = No. of cells in 5 squares (80 small
squares) x dilution no. x depth x area
• Dilution no. = 0.5 : 100 = 200
• TEC = no. of cells counted x 1/10 depth x 1/5 mm2
area counted x 200 dilution
= No. of cells counted x 5 x 10 x 200/μL or mm3
= No. of cells counted x 10000/μL or mm3

19. 2. Electronic method
• Erythrocytes may be counted electronically.
• The high cost of some units may preclude their use in most veterinary
hospital laboratories.
• However, the accuracy of erythrocyte counts completed by this technique is
much greater than can be attained by the hemocytometer method.
• In some medical laboratories electronic counters are calibrated for human
blood and can be used for animal bloods only if adjusted to account for the
different sizes of animal erythrocytes.

20. Acid haematin method
• These method depend upon the conversion of hemoglobin to acid
haematin, which is usually accomplished by the use of dilute
hydrochloric acid.
• The resulting brownish-yellow mixture is matched with a standard in a
colorimeter or comparator.

21. Procedure:
In these techniques 0.1 N HCl is added to whole blood, and the
mixture allowed to stand until acid haematin has developed.
In this method, the color of the blood and acid mixture is
compared with a standard, and the reading is made either in
percentage of normal or in grams per deciliter of blood.
Since each technique may have a different hemoglobin
concentration as 100 per cent, hemoglobin should always be
reported in grams per deciliter.

22. Sources of error in the acid haematin methods are numerous and
include the following:
(1)Non-haemoglobin substances such as proteins and lipids normally
present in plasma and cell stroma may influence the color of the
diluted blood.
(2) It is difficult to match the sample accurately with a brown glass
standard, although this is probably the simplest glass standard
available.
(3) The variation in ability of individual operators in matching colors
is a common source of error.

23. (4) Each match must be made after the same interval for which the
instrument was standardized; failure to observe this rule introduces
another error.
(5) Some haemoglobin present in blood is in an inactive form as
methemoglobin, sulfhaemoglobin, or carboxyhemoglobin; in acid
solutions these are not converted into haematin and, consequently,
are not included in values obtained by this technique.
For these reasons the acid haematin methods for hemoglobin
estimation are rarely used.

24. Direct Matching Method
Two principal types of direct matching methods are used,
1. Tallqvist hemoglobin scale
2. Dare hemoglobinometer

25. Tallqvist method
• A drop of blood is placed on a piece of white absorbent paper, and
the paper with its drop of blood is inserted in the central perforation
of a serial red color chart.
• The intensity of red is matched with the most suitable color on the
paper scale, and hemoglobin is read as the value that this scale
represents.
This is a rapid, simple, and inexpensive technique and should
only be used as a screening procedure under field conditions, when a
more accurate method is not practical.

26. Dare haemoglobinometer
A drop of blood is placed in a capillary chamber between small glass
plates and matched with a permanent red glass standard.
This has some advantage over the Tallqvist technique, as the
thickness of the drop of blood can be more carefully controlled, but
comparison of red colors remains a difficult problem.
The error for both the Tallqvist hemoglobin scale and Dare
hemoglobinometer is estimated to be between ±10 and 40 per cent.
Both techniques must be considered screening tests only.

27. Oxyhaemoglobin method:
One of the simplest methods of determining oxyhemoglobin employs
the Spencer haemoglobinometer.
This instrument uses a green filter to measure oxyhemoglobin by
light absorption.
Fig: A Spencer haemoglobinometer

28. Procedure:
• A drop of blood is placed on a glass plate, and cells are laked by a
hemolytic agent.
The usual hemolytic agent is saponin, which is dried on the end
of an applicator stick.
• The thickness of the drop of blood is controlled by placing a second
glass plate over the one containing laked blood and pressing the two
together.
• This glass chamber is inserted into the haemoglobinometer, and the
green color is matched with that of a standard.

29. 1. Green colors are somewhat easier to match than other shades,
consequently this technique is relatively more accurate than those
involving other colors.
2. Green also has the advantage that the maximum absorption of
hemoglobin under visual light occurs in the green band of the
spectrum.

30. Cyanmethaemoglobin method:
This is probably the most accurate and widely used technique for
determining hemoglobin concentration
Fig: A spectrophometer
Requirements:
1. Spectrophotometer
2. Sahli’s pipette 20 μL
3. Pipette 5 mL

31. Procedure:
• Take 5 ml of Drabkins solution and to it add 20 μL of blood
• Stopper the tube, mix by inverting several times
• Allow to stand for 5 minutes
• Transfer the sample to cuvette
• Read the absorbance in the spectrophotometer at 540 nm
• Also take the absorbance of the standard solution

32. • A graph can be plotted when a large number of samples are processed
• Hb concentration on horizontal axis and absorbance on vertical axis
Result:

33. Normal values of TEC in different animals:

34. Total Leukocyte Count:
To enumerate the total number of leukocyte (WBC) of a given blood
sample.
Equipment necessary for performing a total leukocyte count includes:
1. Neubauer Counting chamber.
2. Special coverglass for the counting chamber.
3. WBC pipette.
4. Diluting fluid (1% HCl or 1% glacial acetic acid).
5. Microscope and lamp.
6. A hand tally for enumeration of cells.

35. Fig: A Neubauer Counting Chamber
Fig: A WBC pipette
Fig: Counting Procedure (zig-zag) method
(40X)

36. Procedure:
• Blood should be carefully drawn to the 0.5 mark of the WBC pipette
• The diluting fluid should be drawn to the 11 mark to dilute the blood
• The blood and diluting fluid are mixed by shaking the pipette vigorously in a
horizontal position for 2 – 3 minute to ensure complete haemolysis of RBC
• 2 – 4 drops of mixed fluid are discarded at the end of pipette.

37. • The tip of the pipette is touched to the side of the haemocytometer chamber
and drop of a fluid wall run under the coverglass
• Wait for about 2 – 3 minutes as leukocytes require settling time
• Total number of cells in 4 squares on the outside of counting chamber is
determined under the low power of the microscope (10X)

38. • Counting is Zigzag manner in all
4 outer squares
• Blue cells are counted
• Gray cells are not counted

39. Counting:
• TLC/μL or mm3 = No. of cells in 4 squares (64 small
squares) x dilution no. x depth x area
• Dilution no. = 0.5 : 10 = 20
• TLC = No. of cells counted x 10 x ¼ x 20/μL or mm3
= No. of cells counted x 50/μL or mm3

40. Differential Leukocyte Count:
• It represents percentage of various leucocytes in stained smear or bone
marrow
Requirement:
1. Clean slides
2. Leishman’s stain/Wright’s stain/Giemsa’s stain
3. Blood cell couter

41. Procedure:
• A thin blood smear is made from whole blood
• A good smear should be tongue shaped without tails
• After proper drying the smear it is stained and air dried.
• The edge of the smear is examined under oil immersion.

42. • Moving the slide vertically and horizontally, a total of 100
leucocyte are counted using blood cell counter.
• Four fields at edges, centre and tail may be counted.
• Different types of leucocyte are expressed in percentage
• The absolute values are calculated for each of the leucocyte
after enumerating TLC of the sample.

43. Counting:
• For e.g., out of 100 leucocytes counted 56 were neutrophils.
• Then, 56% is the DLC value of neutrophil
• Absolute neutrophil count = 56/100 x 10,000/cu mm
=5,600/cu mm

45. References:
• Veterinary Clinical Pathology 4th Edition - Coles, Embert H
• https://www.slideshare.net/ParasuramanParasuraman/rbc-count-and-
wbc-count?qid=b2bd725d-f9e9-41a6-ac90-
ee6b8e653bb8&v=&b=&from_search=1
• VLD Manual (CVSc)
• Google.com
• https://en.wikipedia.org/wiki/Vacutainer

46. THANK YOU