دكتور عبد الامير نضري

1. Introduction to Hematology
Objectives
After completing this unit you should be able to:
1. Define hematology
2. List the components and functions of blood
3. List the units of measurement for RBC counts, WBC counts, platelet
counts, plasma proteins
4. Describe the functions of organs associated with the circulatory system
5. List the components of a complete blood count (CBC)
6. Given a sample of blood, perform the tests to determine the PCV and
plasma protein values
7. Estimate erythrocyte and hemoglobin values given the PCV
8. List the PCV and plasma protein values for normal farm animals ,
canine and feline blood
Introduction
Hematology is the study of blood and an important part of clinical pathology and the
diagnostic process. It includes not only the examination of the cellular and fluid
potions of blood, but also includes a study of the tissues that form, store and circulate
blood cells. A veterinarian uses the results of hematology tests to help determine the
health of an animal. These results are used in conjunction with the history, physical
exam and other laboratory findings. In this unit you will be introduced to the
components of blood and the procedures involved in a complete blood count.
Components of blood
Blood is a tissue consisting of cells within a fluid matrix. Blood creates an internal
environment which directly or indirectly baths all cells of the body and protects it
from the external environment.
Blood contains red blood cells (erythrocytes), white blood cells (leukocytes) and
platelets (thrombocytes). Figure 2.3 on page 10 of Voigt illustrates the cellular
components of blood. Red blood cells contain hemoglobin and are responsible for
carrying oxygen from the lungs to the cells throughout the body, as well as carbon
dioxide from the tissues to the lungs for excretion. White blood cells are either
“granulocytes” (contain granules in the cytoplasm) or “agranulocytes” (do not contain
granules in the cytoplasm.) Granular leukoytes include neutrophils, eosinophils and
basophils. Agranulocytes include lymphocytes and monocytes. WBCs are a critical
component of the immune system. Platelets are cell fragments from large
multnucleated cells (megakaryocytes). Platelets are important in blood clotting or
hemostasis.

2. The clear to pale-yellow fluid portion of blood is called plasma. Five to ten percent
of the plasma consists of proteins. The majority of the proteins are albumin, globulins
and fibrinogen. Albumins transport numerous substances in the blood and are the
main determinant of the osmotic pressure. Globulins (alpha, beta and gamma) are
important in transport and immunity. Fibrinogen is important in blood clotting and
the inflammatory cascade. If blood is allowed to clot, the clotting factors are removed
from the plasma and the remaining fluid portion of the blood is called serum.
Functions of blood
The three main functions of blood are transportation, regulation and defense. Many
of these functions will be covered in detail in other units.
Transports Oxygen and nutrients to cells in the body
Carbon dioxide and waste materials from cells in body
Hormones from glands to target organs
Regulates Body temperature
Water balance
pH
Electrolytes
Defense Phagocytosis of foreign invaders
Involved with immunity
Blood clotting
Blood values
The amount of blood present in an animal averages 7 % of body weight (20 – 50
ml/lb). A 100 pound animal would have about 3150 ml of blood. As a general rule
0.2 ml of blood/ lb of body weight can be safely removed from a healthy animal

3. without detrimental effects. The blood volume, then, of a 100 lb dog would be: 45 kg
X 0.7 = 31.5 kg of blood, and since 1 kg = 1 liter, this is equivalent to 3,150 mL of
blood.
Red blood cells are the most abundant cells in the blood. The average red blood cell
count in dogs is 6,800,000 red blood cells per micro liter of blood. This value is
routinely written as 6.8 x 106 cells/᪽l. Feline RBC values normally averages 7.5 x
106 cells/᪽l. The life span of a canine red blood cell is 120 days, thus in a healthy
animal red blood cells are constantly being produced and destroyed. It is estimated
this replacement occurs at the rate of 35 million cells per second. RBCs are produced
in the bone marrow in response to the hormone erythropoietin. They are destroyed in
the spleen when they are too old, or damaged.
White blood cell values in an animal are constantly changing depending on the
degree of disease and stress present. The average white blood cell count in a healthy
dog is 11.5 x 103 ᪽l and the average value for a healthy cat is 12.5 x 103 ᪽l. (The
difference between 103 and 106 is a magnitude of 1000. Therefore, there is normally
1 white blood cell for every 1000 red blood cells in healthy animals.) The life span of
white blood cells vary from a few hours to years, depending on the type of the cell,
the physiology of the animal, and other factors. WBCs are produced in the bone
marrow, mature in lymphatic tissues, bone marrow and spleen, and circulate through
the blood and tissues where they are destroyed.
Platelet values in normal animals can vary a great deal with referenced “normal
ranges” from 350,000 to 500,000 platelets /ul. The values can also be written as 3.5 -
5 x 105/ ᪽l. Platelets are produced in the bone marrow, and either used up in the
clotting cascade, or destroyed and filtered out of the blood in the spleen.
Plasma protein values range between 6 to 8 gm/dl for adult domestic animals and 4
to 6 gm/dl for younger animals. Plasma protein is an important indicator of the
patient's hydration status, and over all health. It is one of the most important
diagnostic values you can obtain.
Tables 2-4, 2-5 and 2-6 on pages 71 and 72 of the Hendrix text contains “normal”
reference hematologic values for domestic animals.
Organs associated with circulatory system
Many organs are associated with the circulatory system. They include:
Heart: Pumps blood throughout the body
Lungs: Gas exchange: Oxygen and carbon dioxide
Liver: Produces clotting factors and albumin
Removes waste material from blood

4. Spleen: Blood storage
Removal of dead and damaged RBC’s
WBC production in the lymphoid tissues of the
spleen
Bone marrow: RBC and WBC production
Complete blood count
A complete blood count (CBC) provides information to the veterinarian that can be
used to determine the health of an animal. A CBC should contain, at a minimum, the
following information
PVC – Packed Cell Volume
Plasma protein, also referred to as Total Protein, or
Total Solids
Total WBC count
Blood smear examination
Differential
RBC morphology
Reticulocyte count when patient is anemic
Platelet estimation
Hemoglobin concentration
Estimated RBC count
RBC indices
Complete blood counts are done manually and with automated equipment. Some
determinations, such as the differential, are better made manually. Other
determinations such as hemoglobin concentration are better made using
instrumentation. This course will emphasize manual procedures for performing a
CBC. Automated procedures will be briefly covered. In this unit you will learn to
perform a PVC and a plasma protein determination.
Packed cell volume (Hematocrit)
The PCV, also known as the hematocrit, is one of the most common blood test
performed in a veterinary clinic. PCV is the percentage of RBC in the blood. It is
easy, inexpensive, reliable, and provides valuable information. The term “packed cell
volume” describes the principle behind the test. Blood is drawn into a capillary tube,
being careful to not overfill the tube. The tube is centrifuged and the cellular
components are”packed” into the bottom of the capillary tube. The red blood cells
make up the red layer at the bottom of the tube, the buffy coat contains leukocytes and
platelets and the clear to yellowish top layer is plasma.

5. Procedures for PCV
1. Fill two hematocrit tubed ᪽ full with blood containing
anticoagulant. If blood is obtained directly from the patient, use a
hematocrit tube containing an anticoagulant.
2. Wipe the tube with a kimwipe
3. Place finger over the “non-blood” end of the tube and push the
opposite end into a clay sealant 3-4 times
4. Place the hematocrit tube in a centrifuge with the clay end toward
the periphery
5. Centrifuge for 2 – 5 minutes. (3 minutes at 15,000; 5 minutes at
10,000)
6. Place centrifuged hematocrit tube on a reader with the top of the
clay sealant at the 0% mark and the top of the plasma layer at 100%
7. Read the % of RBC which is read at the top of the RBC layer, do
not include the buffy coat
8. Note and record the color of the plasma (ex. Clear and transparent,
white and cloudy, etc)
9. Note an increase or decrease in the size of the buffy coat. The buffy
coat is usually less than 1 mm wide. Also, note the color of the
buffy coat which is normally white

6. Filling capillary tube Centrifuge with
Hematocrit reader
clay sealant in background hematocrit tubes
After the hematocrit is read, the plasma in the capillary tube is used to measure the
concentration of plasma proteins in the blood. A refractometer or TS meter is used
for the measurement. The refractometer (TS meter) measures the total solids present
in a solution. The principle behind the test is that light rays bend when they travel
through a solution. The amount of bending is proportional to the concentration of
solids in the solution.
Procedure for plasma protein determination
1. Break the hematocrit tube above the buffy coat
2. Place a drop of the plasma on the glass plate of the refractometer and
close the plastic cover. The drop should be obtained from the
non-broken end of the tube so that fragments of glass do not get
on the refractometer.
3. While holding the plastic cover in place, point the refractometer
toward a strong light source.
4. The horizontal line between the bright and dark area is used to read
the scale for TS. Be sure this line is sharp and in focus. If it is fuzzy
or blurred, you may need to add more sample to the glass surface, or
be sure there are no air bubbles between the cover and the glass
surface.
5. Record the value for plasma proteins (TS) -- don't forget the units of
measure!
6. Clean the refractometer using distilled water and lens paper or chem
wipes -- DO NOT use running tap water to clean your
refractometer. Be sure it is thoroughly dry before using again.

7. Opening plastic cover Loading
refractometer
of refractometer with plasma
Estimation of values
PCV values can be used to estimate the RBC count and hemoglobin value of the
blood.
To estimate RBC count divide the PCV by 6 and record as value x 106 cells/᪽l.
For example if the PCV is 42, the RBC count would be 7 x 106 cells/᪽l
To estimate hemoglobin value divide the PCV by 3 and record as g/dl
For example if the PCV is 42, the hemoglobin value would be 14 g/dl
SUMMARY OF CANINE AND EQUINE VALUES (MEANS)
Canine (Mean) Feline (Mean)
PCV (%) 37 - 55 (45) 24 - 45 (37)
RBC (x 106 cells/᪽l) 5.5 - 8.5 (6.8) 5 - 10 (7.5)

8. WBC (x 103 cells/᪽l) 6 - 17 (11.5 ) 5.5 - 19.5 (12.5)
Total protein (g/dl) 6 - 7.5 6 - 7.5
Platelets (x 105/ ᪽l) 2 - 9 3 - 7 (4.5)
Hemoglobin (g/dl) 12 - 18 (15) 8 - 15 (12)
Assignment:
1. Practice doing PCV's under the guidance of your mentor. Estimate the
RBC count and hemoglobin values.
2. Practice doing plasma protein determinations under the guidance of your
mentor.
3. Once your are confident with your technique in doing a PCV and plasma
protein determination, perform the tests on two samples and report your
findings on Lab Report #1. You will find the lab report form for lab # 1 in
the What's New Section of the website. Don't forget to answer the
questions included in the lab report.
4. Email Dr. Durham a copy of your lab report #1 -- Be sure to put "Lab
Report # 1" in the subject line of your email so it is easily
recognizable.
Skip to main content
Cornell University Veterinary Medicine
SEARCH:
AHDC Cornell
Sign in | Register
Test and Fee Search
 My Account

9. o Register
 Tests & Submissions
o Test & Fee Information
o Sample Guidelines
o Forms & Submission Information
o Shipping
o Supplies
o Vet Support Services
 Results
o Standard Results
o NYSCHAP Results
 Lab Sections
o Anatomic Pathology
o Avian Disease
o Bacteriology/Mycology
o Clinical Pathology
o Comparative Coagulation
o Endocrinology
o Molecular Diagnostics
o NYSCHAP
o Parasitology
o Referrals
o Quality Milk (QMPS)
o Serology & Immunology
o Toxicology
o Virology
 Technical Resources
 Research
 People
 About
 Giving
Clinical
Pathology

10.  Clinical Pathology Testing
 Test Info & Protocols
 Research/Development
 Educational Resources
 ClinPath Residency
 About the Clinical Pathology Section
 Personnel
 Contact Clinical Pathology
 Dept of Population Medicine & Diagnostic Sciences
 College of Veterinary Medicine
 News & Announcements
 Contact AHDC
CVM Home > AHDC > Sects > ClinPath > Sample > Test
Samples for Hematology
 General information
 Making a blood smear
 Clotted samples
 Non-mammalian hemograms
General information
In general, hematology testing is performed on EDTA- (purple top tube)
anticoagulated blood. This is the only type of anticoagulant that can be assayed with
our hematology analyzer, therefore all hematology tests performed with this analyzer
(routine hemograms, red and white cell counts, etc) will only be done from EDTA
tubes. Heparin (green top tube) is not recommended as an anticoagulant for cell
counts, because the cells clump in heparin, invalidating counts. Citrate (blue top tube)
is not recommended due to the dilution of the blood by the liquid citrate. These
guidelines should be followed for collecting blood for hematology tests:

11.  A full EDTA tube should be submitted. Partially filled EDTA tubes affect the
cells because EDTA is hypertonic (e.g. echinocytes will form in underfilled
EDTA tubes and red cells shrink, decreasing the mean cell volume and
increasing the mean cell corpuscular hemoglobin concentration). EDTA tubes
should ideally be more than half full.
 Ensure that the blood is mixed promptly with the EDTA to avoid sample
clotting. This is especially pertinent with microtainers. This should be done by
rolling the tube between your palms or gentle inversion several times. Do not
shake the tube!!
 Microtainers should be avoided. If only a small amount of blood can be
collected, e.g. from a young dog or cat, or very sick animals in which multiple,
sequential samples are going to be collected, the blood should be collected
into a microtainer. The microtainer should be full. Full microtainers are
required, because if we have too little blood, we may not be able to perform
other tests that may be required, e.g. diluting the sample, checking counts etc.
 The tubes should be labeled with the patient identification and owner name at
the minimum. A request form with pertinent history details should be
submitted concurrently with the sample, e.g. dog administered oxyglobin.
 If there is going to be a delay between sample collection and submission,
always make 2-3 blood smears from the sample and submit with the EDTA
tube (see making a blood smear below).
o Smears should be submitted unfixed, unstained, with the EDTA blood
for any hemogram or test involving counts or blood smear
examination. We do not charge any extra for these blood smears, and
we always (provided smear quality is sufficient) do our blood smear
examination from the submitted smears.
o We request these smears is because changes occur in cells when they
are stored for more than a few hours. Platelets begin to clump, white
cells become pyknotic and undergo nuclear swelling so that many
neutrophils look like bands when they actually are not. The red cells
may lyse. Red cells also consistently swell in vitro, such that old
samples (usually > 24 hours) have macrocytic hypochromic red blood
cells.
 Some hematology samples, e.g. packed cell volume and total protein by
refractometer, can be performed on heparin or citrate anticoagulants. We can
also perform cell counts on these anticoagulants, however this will only be
done on specific research samples or on individual patients, after consultation
with the Clinical pathologist on duty. In these cases, our automated
hematology analyzer will not be used for counts; instead we will use bench
methods, including an impedance-based cell counter for white and red cell
counts and a hemocytometer for manual leukocyte or platelet counts. Note that
for a fecal occult blood, we need feces, not blood!
 EDTA blood should be kept refrigerated until submission or mailing and
should be mailed on a cold pack, but should be kept out of direct contact with
the pack (insert paper towels between the blood and the icepack). Direct
contact may cause freezing of red cells, with subsequent hemolysis.
Furthermore, blood smears should not be refrigerated (causes cell lysis) or
exposed to formalin (alters staining characteristics).
Making a blood smear

12. When there is going to be a delay between sample collection and submission, e.g.
samples shipped to the laboratory or collected after hours, always make 2-3 peripheral
blood smears. We have provided tips and an illustration for making a good blood
smear below.
Tips for making a good blood smear
 Clean slides: It is imperative to use clean high-quality glass slides with clean
edges. Touching the edges of the spreader slide will affect the quality of the
smear.
 The size of the drop: If the drop is too large, the smear will be too long and
thick. A small drop may not be fully representative of the blood.
 Speed of spreading action: The speed at which the spreader slide is moved is
very important. If you move it too fast, the smear is too short and all the cells
are at the feathered edge. If you go too slow, the smear is too long (lacks a
feathered edge).
 Angle of the spreader slide: The angle determines the length of the smear.
An angle of approximately 30-40° is optimal. If you use a larger angle (45°),
the smear is very short. If you use a lower angle, the smear will be too long.
Maintain this angle through the duration of the spreading action.
 Even contact: Even contact between the two slides is essential throughout the
smear preparation process – do not add much downward pressure onto the
spreader (top) slide.

13. Illustration on how to make a peripheral blood smear (wedge smear).
A: Use clean slides with a frosted end. Place a drop of blood on this slide as follows
(we recommend the use of a microhematocrit or capillary tube rather than the pipette
shown the image). Fill a capillary tube at least 3/4 full with well-mixed blood; then
hold your finger over one end to prevent it flowing out. Holding the tube horizontally
over the slide, release the pressure of your finger from the end, and tilt the tube
slightly toward the vertical to allow a controlled amount of blood to flow out of the
tube and onto the slide. Place a drop of blood approximately 4 mm in diameter on the
slide, approximately 0.5 cm from the frosted area.
B: Pick up a second clean slide and hold it by placing your first two or three fingers
on one edge of the slide and your thumb on the opposite edge; the slide in your hand
is the spreader slide. Do not touch the spreading edge (short non-frosted end) with
your hands. Place the spreading end of the spreader slide at a 30–40 degree angle on
the slide in front of the blood droplet. The entire short edge of the spreader slide
should be in complete even contact with the lower slide. Using your other hand, pin
the lower slide to the countertop to prevent it moving. In one smooth motion, draw the
spreader slide back through the entire drop of blood (C).

14. C and D: Once the blood spreads along the edge of the spreader slide (this occurs
quickly), push the blood forward along the length of the lower slide. It is very
important to relax your wrist and maintain a constant smooth motion and the same
angle for the spreader slide when spreading the drop of blood as well as consistently
even contact (with very slight downward pressure) between the two slides.
E: If the drop size and speed/angle of the spreader slide are correct, you will run out
of blood before reaching the end of the slide, thus producing a “feathered edge” and a
smear that is no more extends no more than ¾ along the length of the slide. If your
smears do not look like the example shown above, look at the table below to identify
the fault(s) and the cure(s).
Common blood smear faults and their cures
FAULT CURE
Smear too short or small Use a larger drop of blood and/or
Decrease the angle of the spreader slide and/or
Decrease the speed of the spreader slide.
Smear too long, extends to end
of slide with no feathered edge
Use a smaller drop of blood and/or
Increase the angle of the spreader slide and/or
Increase the speed of the spreader slide.
Smear has waves and ridges Relax the wrist holding the spreader slide (too much
downward force causes the spreader slide to skip)
and/or
Increase the speed of the spreader slide.
Maintain even contact between the two slides and a
smooth motion while pushing the blood forward
Only part of the drop was picked
up by the spreader slide
Draw spreader slide completely back through the
drop before pushing forward. If one side of the drop
was left behind, the edge of the spreader slide was
not in contact with the stationary slide - relax the
wrist holding the spreader slide.
Smear too thick Use a smaller drop of blood and/or
Decrease the angle of the spreader slide and/or
Increase the speed of the spreader slide.

15. Smear too thin Use a larger drop of blood and/or
Increase the angle of the spreader slide and/or
Decrease the speed of the spreader slide.
Clotted samples
If blood has clotted in the EDTA tube, the sample will not be analyzed. Clotting
affects our automated hematology analyzer adversely and also invalidates cell counts
in an unpredictable fashion. For CUHA, we make every effort to notify the
clinician/technician/student that a sample has clotted so that a new sample can be
drawn from that patient. Furthermore, as soon as we know the sample is clotted, the
test is cancelled in the computer. For samples submitted through the Animal Health
Diagnostic Center, we cancel hemograms or tests involving counts if the sample is
clotted. However, if a blood smear is provided with the sample, we will add on a
blood smear examination, which can provide valuable information.
Non-mammalian samples
Only small amounts of blood can be collected from these species, necessitating the
use of microtainer tubes. Similar to mammals, EDTA is the preferred anticoagulant
for non-mammalian hematology. However, there are certain species of birds, e.g.
cranes, and reptiles, e.g. turtles, whose blood hemolyzes on contact with EDTA. This
hemolysis invalidates the PCV and affects assessment of red blood cell morphology
during blood smear examination. For these species, blood can be collected directly
from the needle into citrate anticioagulant. However, the correct citrate to blood ratio
must be maintained, i.e. 1 part citrate to 9 parts blood. Ideally, the citrate should be
placed into the syringe and the appropriate volume of blood withdrawn directly into
anticoagulant. For example, to collect 1 ml blood, 0.1 ml citrate is placed into a
syringe and 0.9 ml of blood is taken from the patient (collect blood up to the 1 ml
mark). If less blood is collected, you will have to resample, hence make sure you can
obtain the correct amount of blood. We require at least 500 μL of blood for
performing a hemogram, hence you can collect only this amount of blood, which is
achievable in most non-mammalian patients. The correct amount of citrate to blood
must be maintained because citrate dilutes the blood; this dilution must be corrected
for when evaluating the hemogram (i.e. each value should be multiplied by 1.1 for a
1:9 citrate:blood ratio). We do not make this correction in our reports. Heparin is not
recommended as an anticoagulant because leukocytes and thrombocytes clump,
invalidating WBC counts and differential cell counts.
Samples for Coagulation Tests
As of September 1, 2001, the Clinical Pathology Lab will no longer be performing
coagulation tests; these tests will instead be offered exclusively by the Comparative
Coagulation Laboratory in the Diagnostic Lab at Cornell University. The Clinical
Pathology lab will continue to offer the Fibrinogen by Heat Precipitation test.

16. Test Components Specimen Comments
Fibrinogen by Heat Precipitaton FIB EDTA tube (>1/2 full) Large animals only
This is performed on EDTA blood only (lavender top tube). Fibrinogen is quite stable,
although hemolysis and lipemia will interfere with the test.
Corne
ll
University
©2010 Cornell University College of Veterinary Medicine
Ithaca, New York 14853-6401
Last Update 8/21/2012