دكتور عبد الامير introduction to hematology

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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.

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Page 1: دكتور عبد الامير Introduction to hematology

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.

Page 2: دكتور عبد الامير Introduction to hematology

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

Page 3: دكتور عبد الامير Introduction to hematology

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

Page 4: دكتور عبد الامير Introduction to hematology

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.

Page 5: دكتور عبد الامير Introduction to hematology

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

Page 6: دكتور عبد الامير Introduction to hematology

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.

Page 7: دكتور عبد الامير Introduction to hematology

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)

Page 8: دكتور عبد الامير Introduction to hematology

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.

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Page 9: دكتور عبد الامير Introduction to hematology

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Page 10: دكتور عبد الامير Introduction to hematology

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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:

Page 11: دكتور عبد الامير Introduction to hematology

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

Page 12: دكتور عبد الامير Introduction to hematology

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.

Page 13: دكتور عبد الامير Introduction to hematology

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).

Page 14: دكتور عبد الامير Introduction to hematology

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.

Page 15: دكتور عبد الامير Introduction to hematology

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.

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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.

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