VET 204:  Veterinary Clinical Laboratory Techniques
VJ  Macer, LVT
Fall 2003
3 credits

Reading assignment:   Rebar-  Chapter 4  (esp. pp. 30-34)
                                         Tilley-  pp. 248-9, 312-3, 342-3, 415 
        (follow the in-text links for additional information)

Study Guide

Introduction to Erythrocytes

1. Introduction:

   The primary function of the erythrocyte is to transport oxygen (O₂) to every cell in the body.  A secondary function is the transport of carbon dioxide (CO₂) waste away from the cell. Actual oxygen transport is accomplished by hemoglobin, an iron-protein molecule found within the erythrocytes. 

    

2. Kinetics  (Production, Maturation and Destruction of Erythrocytes):

   In a healthy animal, erythrocytes are produced in the bone marrow of the flat bones, including the ribs and pelvis.  Erythropoiesis (erythrocyte production) is dependent upon many factors, including the release of erythropoietin from the kidney in response to cellular hypoxia.  Red blood cells are constantly being produced and destroyed, with individual cells having a life span of 25 (mice) - 155 (cows) days or even longer (depending upon the species of animal).  

   In mammals, erythrocytes are nonnucleated cells and contain limited amounts of ATP for energy.  ATP energy is required for RBC survival and to maintain a pliable cell membrane.  This in turn allows the cells to move through capillaries that are narrower than the diameter of the erythrocyte.  As ATP is used up, the cell membrane becomes more rigid and the hemoglobin molecules become unstable.  Damaged and inflexible cells are destroyed by cells of the monocyte-macrophage system, especially in the spleen and liver. 

   Erythrocytes mature in an orderly progression:

Pluripotent stem cell

Unipotent stem cell Erythropoietin from kidney acts on unipotent stem cell to cause proliferation and differentiation

Rubriblast First recognizable RBC

Prorubricyte

Rubricyte Hemoglobin synthesis peaks Last stage capable of cell division

Metarubricyte Hemoglobin synthesis tapers off and nucleus becomes pyknotic

Reticulocyte Nucleus extruded; hemoglobin decreases & ends; mitochondria & RNA decrease

Mature erythrocyte No hemoglobin synthesis; limited metabolism; cell membrane pliable

Erythrocyte production requires a variety of raw materials, including the B vitamins, vitamins A and E, the minerals iron, copper and cobalt, and protein.  If insufficient quantities are available for RBC production, defective cells may be formed.

3. Morphology of Normal Erythrocytes in Circulation:

   The most commonly seen erythrocyte in circulation is the mature erythrocyte.  Most domestic animals, except for the horse and cow, may also have reticulocytes (polychromatophils) in circulation in low numbers.  Occasionally, metarubricytes (nucleated erythrocytes or NuRBCs) may be seen as well.

   The mature erythrocyte lacks a nucleus in mammals, which allows for greater cell deformity.  Avians, reptiles and amphibians, fish and other vertebrates have nucleated mature erythrocytes of an oval shape; this oval shape replaces the need for cell deformity. Properly stained RBCs will be reddish-orange to light bluish-purple.  

In dogs, sheep and humans, the cell is a biconcave disk, with the center of the cell thinner than the margins.  This results in a central pallor (lighter center to the cell).

In horses and cats, the disk is much shallower, and central pallor generally cannot be discerned.  

Reticulocytes also lack a nucleus in mammals (the cell becomes a reticulocyte the moment the nucleus is extruded from the cell). Reticulocytes are slightly larger than mature erythrocytes  (younger cells are larger than mature cells) and the cytoplasm is bluer  (bluer is newer).  The appearance of the cell varies with the stain used.  If a modified Romanowsky stain  (3-step stain) is used, the cell has an overall blue coloration.  If New Methylene Blue stain is used, an aggregate (clump) of hemoglobin-producing reticulum will be seen.  The presence of reticulocytes in anemic animals is a positive finding, because it means that the animal is producing more erythrocytes in response to demand and pushing them quickly into circulation (e.g. regeneration).

Cats produce two types of reticulocytes.  Cats, like dogs, produce aggregate reticulocytes, and when these cells are seen, they are up to 12 hours old.  Small amounts of reticulum may be retained for several days, however, and these cells are termed punctate reticulocytes.  These punctate cells should not be included in a count of reticulocytes, because you generally want to know if erythropoiesis is occurring now, not if it occurred a week ago.

Cattle rarely release reticulocytes into circulation, but may do so if they are extremely anemic.  Horses never release reticulocytes, regardless of how anemic they may be.

The metarubricyte is the nucleated erythrocyte most commonly observed in circulation.  Metarubricytes should never be present unless reticulocytes are (there should not be a gap in cell types between the mature erythrocyte and the nucleated RBC).  These cells are slightly larger than reticulocytes and the cytoplasm is usually a light pinkish gray, purple or  brown.  The nucleus initially is centrally located in the cell, but becomes progressively pyknotic (condensed) and eccentric, until the nucleus is ready to be extruded from the cell.  

4. Evaluation of the Erythron (quantity & quality of RBCs in circulation):

1. Packed Cell Volume  (PCV, HCT or Hematocrit)-

The packed cell volume is the percentage of whole blood that consists erythrocytes.  There are two primary methods of determining the hematocrit:  using a centrifuge or using an automatic blood cell counter that calculates the value from the total RBC count.   The microhematocrit is the PCV determined by centrifuging whole blood in a microcapillary tube.  The centrifuge separates the blood into layers based on the weight of each component.  Erythrocytes are heaviest, and they pack the bottom of the tube.  The central section is called the buffy coat, and it consists of platelets and white blood cells.  The top portion of the spun tube is plasma, the lightest component.  Most centrifuges require a three-minute cycle, although some machines spin for as few as one minute and as long as five minutes  (check the directions that come with your machine!!).

The primary use of the packed cell volume is the evaluation of RBC status, by determining what percent of the blood is erythrocytes.  The spun tube can also be used to detect microfilariae and to assess the plasma (to determine total solids and interpret the color of the plasma).   Although determination of the PCV is a very accurate procedure, errors may occur that can decrease the apparent hematocrit.  EDTA in excess will shrink erythrocytes up to 10% of their size, which may result in a concurrent decrease in the PCV.  Because erythrocytes are heavy, they may settle quickly to the bottom of the tube, especially in horses and in sick animals...if the blood is not mixed properly, an artificially low PCV will result.  Hemolysis from rough handling of the animal or the blood will also decrease the hematocrit.

Every facility will have their own set of reference ranges that they use to interpret test results.  At Medaille College, the reference ranges are:

 

A variety of factors (other than errors) may affect an animal's PCV.  Factors that may increase the hematocrit include: Excitement-  an excited or frightened animal releases epinephrine as part of their fight-or-flight response.  Epinephrine causes vasoconstriction, which forced RBCs out of the spleen and capillaries and into general circulation. Dehydration decreases the amount of plasma without affecting the number of erythrocytes.  This results in an increase in RBCs relative to the amount of fluid present.  (See diagram at right). Some diseases may cause an increase in the erythron

An animal's PCV may also be low.  A decreased number of RBCs is called anemia.  Anemia is a lab finding, not a sign or a diagnosis.  Many factors may cause anemia  (more on this later!).

2. Hemoglobin

   Hemoglobin is an organic molecule composed of protein, lipids, minerals and vitamins.  It has two primary functions-  gas transport (oxygen and carbon dioxide) and as a buffer to maintain the body's acid-base balance.

   Most of the hemoglobin molecules within an erythrocyte are physiologic hemoglobin--it is capable of transporting the gases required by the body.  Oxyhemogobin is formed by the reversible oxygenation of iron, which results in a collapsing of the molecule around the oxygen, holding it in place until the RBC reaches its target cell.  When the oxygen is released, deoxyhemoglobin is formed.

   Nonphysiologic hemoglobins also exist.  These forms of hemoglobin are incapable of transporting oxygen and, when present in excess, may cause serious and even fatal disease.

   A small amount of nonphysiologic hemoglobin is always present, but easily managed by the body without causing any signs of disease.  When the level of abnormal hemoglobin is too high, however, disease and death may ensue.  

   Cats are different from other animals...and not just in personality and sanity.  The cat's liver has a smaller amount of some biotransforming chemicals, which leads to greater oxidative (damaging) injuries to hemoglobin than occurs in other species of animal, and makes them more susceptible to toxicosis by some drugs.  Acetaminophen, for example, is the most common drug toxicity in cats;  half a tablet may be fatal. 

   Some common forms of nonphysiologic hemoglobin include:

   • Methemoglobin occurs when ferric iron is formed by oxidation of iron (normal oxygen-carrying hemoglobin has undergone oxygenation).  The blood becomes a muddy brown color and the mucous membranes are cyanotic due to inadequate tissue oxygenation.  Oxidizing drugs, such as acetaminophen and ketamine, are common causes for oxidative damage.  The body can usually reverse the effects of oxidation over the ensuing 24 hours, but if damage is on-going, severe methemoglobinemia and disease may result.
   • Sulfhemoglobin is another form of oxidative injury that often occurs with methemoglobinemia.  In addition to acetaminophen, onion ingestion by dogs and the propylene glycol present in soft-moist pet foods may be causes.  Sulfhemoglobin forms small clumps called Heinz bodies that stain bluish-gray with New Methylene Blue stain.  Increased numbers of Heinz bodies are common in cats with diabetes mellitus and hyperthyroidism.
   • Carboxyhemoglobin forms when carbon monoxide replaces oxygen on the hemoglobin molecule.  Hemoglobin has a 200 times greater affinity for CO than oxygen, and reversal takes place extremely slowly.  An animal with carbon monoxide poisoning will have bright cherry-red blood and mucous membranes, and may die of tissue hypoxia even if oxygen is administered.

   Several methods can be used to determine the level of hemoglobin in blood.  The most accurate method is to chemically release the hemoglobin and measure the concentration using a spectrophotometer; this method is used by many automatic blood cell counters and blood chemistry machines.  The hemoglobinometer also uses a chemical reaction to lyse the blood, and then the color is compared visually to similar colors within the hemoglobinometer.  In dogs and humans, the hemoglobin level can be estimated using the PCV index (PCV/3).  Remember that this is an estimate and not an accurate assessment of hemoglobin levels.

   The measurement of hemoglobin is the amount of the molecule present from lyzed erythrocytes in 100 ml (1 deciliter) of blood.  It is sometimes recorded as g%, where % represents 100 ml of blood.  The reference ranges for hemoglobin at Medaille College are:

   Hemoglobin

   Dog 12 - 18 g/dl

   Cat 8 - 14 g/dl

   A decrease in hemoglobin is associated with anemia, esp. iron deficiency anemia.

    

3. Total Erythrocyte Count

   The total erythrocyte count is the number of red blood cells per microliter of blood (a microliter = 0.001 ml).  The count is recorded as millions of cells/ul or cells x 10⁶ /ul.  Canadians record blood counts in cells per liter.

   The best method of counting erythrocytes is to use an automated blood cell counter.  Although erythrocytes can be counted by hand using a hemacytometer, the margin of error is too high (up to +/- 20%) to make the results useful.  A rough estimate of the total RBC count in the dog (and humans) can be obtained using the PCV index (PCV/6).

   A decrease in total erythrocytes is associated with anemia.

   The reference ranges for total RBCs are:

4. Reticulocyte Count:

Reticulocytes are immature erythrocytes.  If they are present, regeneration of RBCs is occurring.  Reticulocytosis is most commonly associated with hemorrhage and hemolysis.

A reticulocyte count should be performed when a dog's PCV is less than 30% and a cat's PCV is less than 20% (i.e. the animal is anemic).  Approximately 0.5-1.0% of the RBCs of a healthy dog or cat are reticulocytes.  

Some referral labs will provide a corrected reticulocyte percentage in animals that are extremely anemic, because this may artificially elevate the reticulocyte count.

5. Erythrocyte Sedimentation Rate (ESR):

The erythrocyte sedimentation rate is the speed at which RBCs settle in anticoagulated blood over a standard time and temperature.  It reflects the amount of inflammatory proteins present on the surface of the erythrocytes.  These proteins make the cells sticky, causing them to clump and settle to the bottom of the blood tube, separating from the plasma.  Dog blood typically will settle 5 - 25 mm per hour;  a dog with inflammation, such as caused by trauma, infection and neoplasia, may have a greater rate of settling.

The ESR is more useful in cattle and people than in small animals.

5. Erythrocyte (Osmotic) Fragility Test:

   This test measures the RBC's resistance to lysis in decreasing amounts of saline.  This test is occasionally ordered for animals with suspected immune-mediated hemolytic anemia and is associated with spherocytes.