A relative polycythemia may be detected in animals that have decreased plasma, due to dehydration, hypovolemia or shock.  This results in a relative increase in the number of red blood cells-  there has been no production of additional erythrocytes.  The increase occurs because the amount of water that the cells are suspended in has decreased.  A concurrent increase in total solids (dissolved proteins and other chemicals) is usually noted.

Transient polycythemia occurs due to splenic contraction in response to the release of epinephrine by a frightened, angry or excited animal.  Epinephrine causes vasoconstriction, which forces stored erythrocytes out of storage in the capillaries of the spleen and into circulation.  This effect generally lasts from 20 minutes -120 minutes.

Neither relative nor transient polycythemia involves erythropoiesis:  the absolute (actual) number of red blood cells in the body has not increased, only the number counted in relation to the amount of plasma present is higher.  

Absolute polycythemia may occur, however.  An animal that experiences cellular hypoxia will release erythropoietin from the kidney, which will increase the proliferation of RBCs.  Animals that live at a high altitude, such as the dog that moves with its owner from Denver, Colorado, to Buffalo, NY, will have a higher PCV than a native Buffalonian.  There is less oxygen in the air in Denver, so more erythrocytes will have been produced to try to carry oxygen to the cells more effectively.

Animals with respiratory or cardiovascular diseases that compromise oxygen transport may also present with absolute polycythemia, due to the appropriate production of erythropoietin.

Polycythemia vera is an idiopathic stem cell disorder in which inappropriate cell production occurs without increased levels of erythropoietin.  Animals with polycythemia vera often have red mucous membranes, polyuria and polydipsia (PU/PD) and epistaxis.  Inappropriate erythropoietin may occur in animals with acute renal disease or neoplasia. 

2. Anemia (or oligocythemia) is the most common abnormal erythrocyte disorder.  It occurs when the PCV, hemoglobin and total erythrocyte count are below the reference range.  Anemia is not a disease or a sign:  it is a laboratory finding (result).
   
   Some signs frequently seen in animals with anemia include:

   • Dyspnea

   • Anorexia

   • Syncope

   • Gastrointestinal dysfunction

   • Tachycardia

   There are several methods used to classify anemia.  The most common classification systems are based on etiology, bone marrow response, and morphology.

   There are four general causes or types of anemia.  Knowing the reason for the anemia is an important first step in developing a plan of action for treating an anemic animal.  Almost all anemia can be classified as one of the following:

   • Hemorrhagic  (where cells leak out of blood vessels)

   • Hemolytic  (where cells are destroyed within the body)

   • Bone marrow depression  (where the bone marrow is unable to produce adequate erythrocytes)

   • Nutritional deficiency (where the body lacks sufficient raw materials to manufacture the needed cells).  The most common nutritional deficiency leading to anemia in animals is lack of iron.

   Not only is it important to determine why an animal is anemic, it is also essential to know if the animal is responding to the anemia by production of additional erythrocytes.  You need to know the bone marrow response.  An anemic animal should have regeneration of erythrocytes occurring to replace the cells that have been lost or are being lost.  This is the normal bone marrow response.  It generally takes about three days before the bone marrow produces sufficient erythrocytes to be able to detect the increase in the laboratory.  Regenerative responses are most commonly associated with hemolytic or hemorrhagic anemias.

   Animals which have a nonregenerative bone marrow response are not responding appropriately to the decrease in erythrocytes.  This type of response is more common in animals with anemias due to bone marrow suppression (because the bone marrow is incapable of producing sufficient red blood cells) or due to a nutritional deficiency (because there is insufficient raw materials to manufacture the needed cells.

    The morphologic system of anemia classification was developed to provide a way to objectively describe the characteristics of erythrocytes in anemic animals.  These characteristics are often called the erythrocyte indices (singular index), and includes the mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration.

   The mean corpuscular volume  (MCV) is the volume of the average red blood cell in a blood sample.  The MCV is measured in femtoliters (fl).  If the MCV falls within the reference range, the erythrocytes are called normocytic-  the volume is "normal."  This occurs before the bone marrow has had a chance to respond to an anemia and if the bone marrow is suppressed and incapable of responding to the anemia.

   Red blood cells with an increased MCV are called macrocytic.  The volume of these cells is greater than the reference range.  This generally is a good indicator that the anemia is regenerative, with increased numbers of larger reticulocytes present in the sample.  An increased MCV may also occur in cats with anemia associated with feline leukemia virus infection (and is often the source of the client's chief complaint) and in some families of poodles (where it is an insignificant finding). 

   Microcytic erythrocytes have a decreased MCV with volume of the average cell less than the reference range.  Microcytosis is most frequently associated with iron deficiencies in dogs.  It also occurs in some Akitas, where it is not significant.

   The mean corpuscular hemoglobin  (MCH) is the weight of hemoglobin in the average erythrocyte measured in picograms (pg).  It is considered the least useful of the erythrocyte indices, because of difficulties often associated with accurate measurement of hemoglobin.  In general, it adds little knowledge beyond that provided by the MCV.

   The mean corpuscular hemoglobin concentration (MCHC) is the ratio of the amount of hemoglobin (by weight) to the volume of erythrocytes (i.e. the PCV) recorded in grams per deciliter (100 ml).

   Cells with an MCHC within the reference range are normochromic.  Cells below the reference range are hypochromic (they have inadequate hemoglobin).  Hyperchromia does not occur.

   The MCHC is combined with the MCV to describe anemias:

   • Normochromic macrocytic RBCs are characteristic of FeLV

   • Normochromic normocytic cells occurs in a nonregenerative anemia or before the bone marrow responds to the anemia

   • Hypochromic macrocytic RBCs are most commonly seen in regenerative anemias, where many large reticulocytes that have not quite produced all of their hemoglobin are present

   • Hypochromic microcytic cells occur in a variety of conditions, such as iron deficiency anemia, lead poisoning and chronic infections.

The  red cell distribution width (RDW) is a method of measuring variation in the size of erythrocytes (anisocytosis).  There will always be some cell size variation, because RBCs are constantly being created and destroyed.  An increase in the RDW, however, indicates an increase in the number of cells of different sizes:  a regenerative anemia is often occurring.

Most anemias encountered in a veterinary practice will be (thankfully) regenerative anemias-  the animal is able to manufacture more cells to replace those that have been lost.

Unfortunately, there are several anemias that are nonregenerative.  These include the anemia of chronic disease, anemia of chronic renal disease, anemia of endocrine failure and the anemia associated with FeLV infection.

The pathogenesis of the anemia of chronic disease is poorly understood.  We know, however, that animals with chronic infectious, noninfectious and neoplastic diseases often develop a nonregenerative, normochromic normocytic anemia.  It is suspected that iron becomes unavailable for erythrocyte production.  The PCV is frequently mildly depressed, and this anemia is rarely treated;  the anemia generally resolved when the primary (underlying chronic disease) is treated.

The anemia of chronic renal failure also produces a nonregenerative normochromic normocytic anemia.  Fibrosis in the kidney damages the erythropoietin-producing cells, causing hypoplasia of the bone marrow.  This condition can be treated with injections of erythropoietin (such as Procrit(R)) or blood transfusions.

Anemia can occur in animals with hypothyroidism or hypoadrenocorticism.  The hormones produced in these glands are synergistic with erythropoietin.

Feline leukemia virus (FeLV)  infection frequently produces an erythroid hypoplasia in the bone marrow of affected cats.  Up to 70% of anemic cats seen in practice are FeLV positive.

                                  
Alterations in the Morphology of Erythrocytes:

It is normal to observe variations of the morphology of RBCs on a blood smear.  In fact, up to 5% abnormalities can be seen in healthy animals, with up to 10% or more be insignificant findings in healthy cats.

Many morphologic changes are seen in animals with anemia or other blood dyscrasias (abnormalities).  All observed abnormalities should be evaluated and recorded.

Many erythrocytes with morphologic abnormalities are destroyed by the monocyte-macrophage cells within the spleen and liver, and can result in a hemolytic anemia.

It is helpful to develop and use a standard rating system for evaluating the amount of variation in RBC morphology.  The system used at Medaille College is:

+	1+	slight	0-1 / oil immersion field
++	2+	moderate	2-5 / oil immersion field
+++	3+	many	6-15 / oil immersion field
++++	4+	marked / TNTC	>15 / oil immersion field

1. Variation in erythrocyte size:

   A general term for variation in the size of erythrocytes is anisocytosis.  A cells that varies in size from other erythrocytes is an anisocyte.  This term is generally used, however, only when there are cells both larger and smaller than the typical erythrocyte.  

   If some of the red blood cells are larger than most of the other cells, they are called macrocytes (macrocytosis).  A macrocyte is at least one micrometer larger than the normal RBC.  Most macrocytes are immature erythrocytes-- they're reticulocytes.  Macrocytes can also be observed in some poodles, which it is an insignificant finding and the functioning of the erythrocytes is not affected.  Macrocytosis is also seen in cats with anemia associated with feline leukemia.

   Cells that are at least one micrometer smaller than the surrounding cells are termed microcytes (microcytosis).  Most microcytes are also hypochromic, as the cells are unable to complete hemoglobin production.  Animals with iron deficiency or lead poisoning frequently have microcytosis.
   
   

2. Variation in erythrocyte shape:

   A general term for variation in the shape of erythrocytes is poikilocytosis.  A cell that varies in shape from other erythrocytes is a poikilocyte.  This term is generally used, however, only when the cells have a variety of shapes that cannot otherwise be described.  Abnormal shapes may be an artifact, because the easily deformable red blood cell membranes may be distorted by the smear preparation technique.

   One of the most commonly seen shape abnormalities is crenation.  These cells have uniform shallow and slightly refractile (shining when focus is slightly changed) projections around the entire rim of the cell.  Most commonly, crenation is an artifact-  it results from slow drying of the smear, using old blood or a dirty slide.  Crenation may also be pathologic, often as a result of uremia, the accumulation of toxic waste products in the blood that occurs during renal disease.  

   Spherocytes are produced by partial phagocytosis of the cell membrane of erythrocytes.  These cells are microcytic, round (instead of being biconcave disks), and dark.  They are most commonly identified in dogs, because the characteristic central pallor is lost.  Spherocytes also occur as a part of the normal aging process of cells.  The body may lose the ability to recognize the cells and lay down patches of antibody on the cell membrane.  These antibody-coated spherocytes are then selectively removed by the spleen.  Spherocytes commonly occur in animals with immune-mediated hemolytic anemia, an autoimmune disease that occurs when the body begins to destroy healthy erythrocytes. 

   Target cells (codocytes) occur when there is excess cell membrane and the erythrocyte acquires a bell shape.  When viewed on a blood smear, the cell appears to have a colored center, surrounded by a colorless center and outer rim of color, much like the bulls-eye of a target.  These cells may be artifacts, but also occur in animals with liver disease or iron deficiencies.

   Schistocytes (also called schizocytes and keratocytes) are fragments of red blood cells that are damaged in circulation, sliced into pieces by fibrin threads stretched across capillaries.  Schistocytes are always pathologic, and occur in  a variety of diseases including disseminated intravascular coagulopathy (DIC), congestive heart failure and glomerulonephritis.
   
   

3. Variations in erythrocyte distribution:

   The region of a smear that should be evaluated for cell number and morphology is the area where the erythrocytes are one layer thick and just touching, with few large open areas.

   Abnormalities of distribution occur where the cells become attached and form chains or clumps.

   Rouleaux (roo-low) formation is the stacking of erythrocytes in stacks, like coins.  It occurs as an artifact in thick areas of a blood smear, but is rare in the counting area of  healthy dogs.  It is not unusual in cats and is normal in horses. In dogs, Rouleaux formation is increased during inflammation, because fibrinogen, an acute-phase protein, causes the cells to stick together.

   Red blood cells in irregular clumps are autoagglutinated.  Refrigerated blood may develop agglutination, but fresh blood should never agglutinate.  The most common disease that produces autoagglutination is immune-mediated hemolytic anemia, where the antibodies coating the RBCs cause them to form clumps.

   It may be difficult to differentiate between Rouleaux formation and autoagglutination when viewed on a stained smear.  If  a drop of saline is added to a sample in which Rouleaux formation is present, the cells will disperse.  This type of separation will not occur  in autoagglutinated cells.
   
   

4. Erythrocyte inclusions:

   The cytoplasm of mature erythrocytes should be homogenous, with no variation in color.  There a variety of changes that can occur within the cell that will be visible on a stained smear, and these inclusions should be evaluated and recorded.

   A Howell-Jolly body is a small blue-black mass located within an erythrocyte.  Typical Howell-Jolly bodies are 1 - 2 micrometers in diameter.  These are bits of nucleus that are retained in the cell after extrusion of the nucleus.  They are most commonly observed in animals with regenerative anemia, where the cells are rushed from the bone marrow into circulation.

   Heinz bodies (erythrocyte refractile bodies) are masses of defective hemoglobin (sulfhemoglobin) located at the edge of the erythrocyte, projecting beyond the margin of the cell.  When stained with a modified Romanowsky stain, the mass is the same color or lighter than the erythrocyte.  When stained with new methylene blue, however, stain is deposited within the Heinz body, and it becomes a medium blue color.

   Heinz bodies are relatively common in cats, but in other animals, and when more than 10% of the erythrocytes in a cat contain Heinz bodies, it is consider pathologic.  Heinz bodies are formed in response to oxidative injury to hemoglobin resulting from ingestion of acetaminophen, onions, red maple leaves (in horses) or other oxidizers.  Heinz body anemia can be very severe and even fatal if treatment is not provided.

   Basophilic stippling is occasionally seen in animals with regenerative anemias, but is classically associated with lead poisoning.  Small blue granules are scattered throughout the cytoplasm of the RBCs stained with a modified Romanowsky stain.  

   Polychromasia (polychromatophils or polychromatophilic cells) do not appear to contain inclusions when stained with a modified Romanowsky stain-- they are larger than the typical erythrocyte and are bluer than the surrounding erythrocytes.  When stained with new methylene blue, however, these cells are revealed as reticulocytes-  they contain a dark-blue staining reticulum throughout the cytoplasm.  Polychromasia is commonly associated with regenerative anemias.
   
   

5. Parasites in and around erythrocytes:

   There a many different parasites and pathogens that may affect blood.  Dirofilaria immitis, the canine heartworm, lives within the right ventricle and coronary arteries, and may produce microfilaria that circulate in the plasma of dogs.  A Veterinary Technician must be familiar with the biology, testing and treatment of this parasite (but these details are covered in VET 126 Animal Parasitology).

   The  protozoal trypanosomes, including the human and canine parasite Trypanosoma cruzi, are free-living in plasma.

   Haemoproteus and Plasmodium (the organism that causes malaria) are intraerythrocytic parasites.  The organism lives within the red blood cells and infection frequently causes hemolytic anemia as the infected cells are selectively removed from circulation.

   Haemobartonella felis and Haemobartonella canis have recently been reclassified as mycoplasmas (not rickettsias).  These are epicellular parasites-  the organisms is loosely attached to surface of the erythrocyte.  

   H felis is the causative agent of feline infection anemia (FIA) or haemobartonellosis.  The organism is cyclic and spends part of the time attached to the RBC and part of its life cycle free in plasma.  This is important to understand when attempting diagnostic testing of a cat with suspected FIA.  The organism quickly detaches from the erythrocytes when exposed to EDTA anticoagulant or upon refrigeration.  Serial testing over at least four days may be required, and capillary blood should be used, making a smear immediately after collection.  Cats with FIA frequently present with signs consistent with hemolytic anemia, such as lethargy, anorexia, depression (LAD), splenomegaly and icterus.

   There are a number of different rickettsias that infect erythrocytes, producing a hemolytic anemia.  Examples include:

   • Anaplasma marginale  (cattle)

   • Babesia  (dogs, cattle, horses...)

   • Eperythrozoan  (swine)

   • Ehrlichia  (profound bone marrow hypoplasia in horses, dogs, people...)

 Erythrocyte Antigens

1. The biology of erythrocyte antigens

   Antigens located on the surface of the erythrocyte cell membrane produce blood groups, similar to the ABO blood types seen in people.  Knowing an animal's blood type is important for proper administration of blood transfusions, and for mating and parentage testing.

   The dog has eight major blood groups, but most dogs are either CAE 1.1 or 1.2 positive.  Dogs have few naturally occurring antibodies.  Exposure to blood of a different type is required before a dog develops antibodies against that blood type.  The best donor is CAE 1.1 negative.

   Cats have three blood types:  A, B and AB.  These are not, however, the same blood types as people. B positive cats have naturally occurring anti-B antibodies, and transfusion of A positive blood into these cats may result in a transfusion reaction.  The feline blood types tend to be breed dependent.  95% of all domestic short hair and domestic long hair cats are A positive.  B positive blood is most common in the Cornish and Devon Rex and in British and Exotic short hairs.

   Horses have more than sixteen blood types and cattle have at least 11 types.
   
   

2. Blood typing is used to determine what antigens are present on the surface of erythrocytes.  Blood is mixed with reagents to determine if agglutination or hemolysis (or both) occurs.  By identifying which reagents cause a reaction, the blood type can be identified.  This method is used to determine the blood type of people.
   
   

3. Cross matching is another method used to evaluate the presence of antigens on erythrocytes, but the goal is to determine immunologic compatibility of blood for transfusion, not blood type.  One method of cross matching blood is listed:

   • Draw blood from both donor & recipient into separate clot tubes

   • Let the blood clot, then harvest the serum

   • Prepare a washed RBC suspension of both donor & recipient blood-

     • Dip an applicator stick into the cot

     • Add to 1 ml 0.9% saline solution

     • Centrifuge, decant and discard the supernatent

     • Repeat two more times

     • Add 1 ml 0.9% saline solution

   • Perform a major cross match-

     • This tests for antibodies in the recipient serum

     • A mismatch of blood types causes a major transfusion reaction

     • Mix 2 drops recipient serum with 2 drops washed donor RBCs

   • Perform a minor cross match-

     • This tests for antibodies in the transfused blood

     • Mismatch is usually not life-threatening

     • Mix 2 drops donor serum with 2 drops donor RBCs

   • Run controls to ensure proper test technique-

     • Donor-  2 drops donor serum + 2 drops donor RBCs

     • Recipient-  2 drops recipient serum + 2 drops recipient RBCs

   • Incubate all tubes at 37 degrees C for 30 minutes

   • Centrifuge for 2 minutes

   • Evaluation-

     • Examine the supernatent for hemolysis

     • Tap the tubes to see gross agglutination

     • Examine under 100x for microscopic agglutination
       
       

4. Transfusions in practice (oversimplified!):

   In the dog, the first transfusion generally causes no reaction, regardless of the blood type.  However, sensitization to a different blood type will occur over a period of four to ten days, and a subsequent transfusion should be cross matched or typed to prevent a transfusion reaction.  The lifespan of a transfused RBC, even of the same blood type, is usually no longer than 21 dyas.

   In cats, because natural antibodies occur, an initial transfusion may cause a severe transfusion reaction.  Great care should be exercised when transfusing blood into a Devon Rex, Cornish Rex, Exotic Shorthair or British Shorthair, as these breeds are more likely to have the less common B positive blood.
   
   

5. Immunohemolytic disease of the newborn  (neonatal isoerythrolysis) may occur in some animals, including horses, mules and cats.  This is similar to Rh factor in people  The disease occurs when the mother and father have different blood types and the offspring inherits the father's blood type.  In horses, sensitization of the mare to the foal's blood type apparently occurs during parturition.  The mare then produces antibodies against the foal's blood type and transfers these antibodies through her milk as the foal (or kittens) nurses.  The young animal may then develop a hemolytic anemia as the dam's antibodies begin to destroy her offspring's erythrocytes.  Identification of the parents' blood type allows you to substitute milk from a different mare (or queen) for the now-damaging dam's milk.  The first offspring often have no difficulty, but as the dam carries more offspring with a different blood type, she produces more antibodies, and subsequent offspring may become very ill and even succumb to severe hemolysis.