VET 204:  Veterinary Clinical Laboratory Techniques
 

Study Guide

Reading assignment:   Rebar on methods of blood collection and testing  (Chapter 3)

Introduction to Hematology

1. Hematology is a division of medicine that uses laboratory procedures to evaluate blood and blood-forming organs.
   

1. Blood Composition:

Blood consists of cells, water and dissolved substances, such as glucose, sodium and albumin.  The water solution is plasma.  If clotting factors are removed from plasma, it is called serum.

3. Hematopoiesis:

   Hematopoiesis is the formation and development of blood cells.  There are many locations in the body that contribute to cell blood cell formation; these sites are either medullary (within the bone marrow) or extra-medullary (of non-bone marrow origin). 

   Within the embryo, the yolk is initially responsible for all the blood cells, followed by the liver and spleen in the fetus.  Near parturition, the bone marrow throughout the body begins forming cells.  As the animal matures, the long bones, like the femur and humerus, cease cell production except at the distal ends, and hematopoiesis is concentrated in the flat bones of the skull, ribs and pelvis.  Lymphocytes are produced in lymphoid tissues in the thymus and lymph nodes.  

   The kidney produces erythropoietin, a chemical that stimulates erythrocyte production and maturation.  The liver stores iron and other cell components, and plays a pivotal role in erythrocyte destruction and hemoglobin processing.  The spleen along with other parts of the monocyte-macrophage system, phagocytizes old and defective blood cells. 
   
   

4. Blood Collection and Handling

   The goal of the technician in collecting blood is to ensure that the sample is of highest quality.  If the sample is of poor quality, the results will not be reliable.  

   There are several rules of thumb to keep in mind when collecting blood-

   • Produce minimal trauma (to both you and the animal!!)  Damaging tissues surrounding the blood vessels can stimulate the clotting mechanism and cause contaminants to leak into the blood sample.  An fearful or angry animal will release epinephrine, which can increase the values for several different parameters.
   • Use proper procedures. You need to know and adhere to optimal practices in order to ensure a high quality sample  (see below).
   • Obtain the sample before treating the animal or allowing it to eat.  Drugs and food can also some test results.
   • Collect more than you think that you need!  If your equipment fails, or the specimen tube tips over, spilling part of the sample, you may have to call the client to bring their animal back for another blood draw...and most animals do not appreciate a return trip to your practice!!

   Blood can be collected using a needle and syringe or by using a blood collection system that consists of a special needle and a vacuum tube.  Samples collected by needle and syringe should be transferred into a vacuum tube for processing and storage.  It is essential that the needle be removed from the syringe before transferring the blood to the tube.  Passage of cells through the metal needle damages the cells.  This is unavoidable when removing the blood from the patient--but should be minimized by removing the needle to put the blood in the tube.

   Blood collection tubes come in a variety of sizes, from microtubes that hold as little as 10 microliters of blood up to 10 milliliters.  The larger the tube, the greater the vacuum.  Tubes should be filled to capacity--if a blood collection system is used, the tubes will fill with the proper amount.  If a needle and syringe is used, the tube used should be filled by the sample collected.

   There are many other errors that can occur before, during and after blood collection.  If you are familiar with what can cause a poor quality sample, you will be less like to collect one.

   Hemolysis is destruction of erythrocytes and may be due to collection error or a pathologic process.  Simply handling an animal roughly may result in hemolysis.  Calmness of animal, restrainer and collector are important!!  Rough handling of the syringe (using too much pressure when aspirating the blood into a syringe) or using a large blood tube with a corresponding large vacuum can lyse red blood cells.  Excessive mixing of the blood in the tube, storing the blood for a prolonged time and very high or very low temperature can also lyse erythrocytes.  Lipemia (the presence of increased lipids in the blood) makes red blood cells more fragile and more likely to lyse.

   Lipemia may be pathologic or may be due to diet.  Mammals should be fasted 6 - 12 hours prior to collection.  Cats eat a high fat diet and mobilize fats readily.  They are almost always lipemic.

   Excitement from fear, anger, happiness or exercise causes the release of epinephrine from the adrenal glands.  Epinephrine moves cells from the margins of the blood vessels and from the spleen into circulation.  This movement may damage erythrocytes, resulting in a hemolytic sample.

   Slow collection of blood, trauma at the collection site and inadequate mixing of the sample may result in clotting of the blood.  Even prolonged venous stasis, due to holding the vein off prior to blood collection, can initiate coagulation.  Clots in the blood interfere with counting of the blood cells, producing invalid results.

   Make sure that you label sample tubes immediately and provide complete information.  The tube should include at least:

   • Owner name
   • Animal name
   • Date
   • Time
   • Physiologic state  (i.e. excited or unexcited)

In order to perform examinations with blood, it must be kept in a fluid state.  Inserting a needle into the blood vessel and removing the blood initiates the coagulation process.  The best sample for making smears to evaluate blood cell morphology is whole blood with no additives--the smear should be made with blood dropped onto a microscope slide from the syringe (with the needle removed).  Frequently, however, the blood cannot be examined immediately and must be preserved for several hours prior to testing.  This blood will need the addition of an anticoagulant. 

The most frequently used anticoagulant for hematology is ethylene diamine tetra-acetic acid, better known as EDTA.  Blood collection tubes containing EDTA have a lavender colored stopper and label.  EDTA chelates (combines with) calcium, an essential clotting factor.  It is the best preservative for mammalian cells and does not interfere with staining of the cells.  It is important to match the size of the blood collection tube to the sample:  in excess, EDTA can shrink erythrocytes up to 10%, falsely decreasing the packed cell volume (hematocrit).  Prolonged exposure to EDTA can cause morphologic changes in WBCs, making them appear to be toxic.

EDTA effectively preserves different blood components differing lengths of time.  Keeping times for some common tests are:

Packed cell volume Hemoglobin Total RBCs Reticulocytes RBC indices Total WBCs Differential leukocyte count Total platelets Smear, unstained Smear, stained

24 hours 24 hours 24 hours 1 hour 4 hours 24 hours 1 hour 1 hour 2 hours Forever

Heparin is an anticoagulant that binds to prothrombin, another clotting factor.  This is sold as a tube with a green stopper.  It is more frequently used with nonmammalian blood.  The primary disadvantages are that it interferes with staining of blood cells and can cause clumping of white blood cells and platelets.

Sodium citrate is an anticoagulant that is used for coagulation studies and the blood tubes have a light blue stopper.

 

2. Any test is more accurate if an animal is "normal."  The more "abnormal" an animal is, the more inaccurate the test will be.
   
   

3. The Complete Blood Count  (CBC)

   The CBC is a panel (profile) of tests performed on blood or plasma that describes the quantity and quality (i.e. morphology) of blood components.

   There is some variation of what constitutes a CBC, but essential components include:

   • Number of erythrocytes  (either total RBC count or hematocrit)

   • Total leukocyte count

   • Differential leukocyte count

   • Total solids (plasma protein)

   Some other tests that may be included are:

   • Hemoglobin

   • RBC morphology

   • RBC indices

   • Platelet evaluation

   Blood cells can be counted by hand, using a commercial Unopette system, or using an automated blood cell counter.  In our area, many of the veterinary hospitals own an automated counter.  The remaining practices count cells by hand or send blood to a referral lab to have a CBC performed.  

   Hand counts have a margin of error of 10 - 20% or even more.  The leukocyte counts are most reliable (with an error of +/- 10%) and RBC counts are least reliable (+/- 20⁺  %).   Automated cell counters are more rapid, more accurate (error of 1 - 2%) and more precise.  These systems are more expensive ($5,000 - $50,000) and require greater training and expertise to ensure that the machines are operating properly.

   Blood counts are recorded as cells per microliter (also called cells/mm³ ) or cells/L (Canadian). It's impossible to actually count 6 million erythrocytes in a microliter of blood however--we test a smaller sample and calculate the results, similar to counting the pulse in 15 seconds and multiplying times 4 for the BPM.  The steps involved in counting cells are:

   • Dilution of specimen (so the cells are at a countable concentration)

   • Taking a sample of the diluted specimen

   • Counting the cells in the diluted specimen 

   • Conversion of the sample results to cells/microliter