Introduction to Hematology

 

Required reading 

Hendrix, “Laboratory Procedures for Veterinary Technicians”, pages 6-26 

Voigt. “Hematology Techniques and Concepts for Veterinary Technicians”, Chapter 1, 2, 3 and 4, pages 3 – 19.

 

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 canine and feline blood

Sites on the Web

http://www.vetmed.wsu.edu/ClientED/lab.asp

An introduction to blood tests.  This is an excellent client education site from the College of Veterinary Medicine at Washington State University.

http://www.bloodline.net

A reference site from the American Society of Hematology.  It contains links to important hematology sites and resources (eg. image database, glossary, articles).  In order to access the site you need to sign up for a free membership.

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. 

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.   

                  plasma&serum.JPG (23077 bytes) 

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 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/µl.  Feline RBC values normally averages 7.5 x 106 cells/µl.  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 µl and the average value for a healthy cat is 12.5 x 103 µl.  (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/ µl.   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
 

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.

hematocrit tube.JPG (9930 bytes)

 

Procedures for PCV 

1.

Fill two hematocrit tubed ľ 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   

 

Hem.& clay.jpg (415126 bytes)                 Hem__centr.jpg (117663 bytes)             PCV_reader.jpg (17983 bytes)

         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.

                    Refract 2.jpg (409594 bytes)                               Load_refract.jpg (21183 bytes)

                        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/µl.   

                For example if the PCV is 42, the RBC count would be 7 x 106 cells/µl

        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/µl) 5.5 - 8.5   (6.8) 5 - 10   (7.5)
WBC (x 103 cells/µl) 6 - 17   (11.5 ) 5.5 - 19.5   (12.5)
Total protein (g/dl) 6 - 7.5   6 - 7.5 
Platelets  (x 105/ µl) 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.