Veterinary Hematology 101; 2005 Abstract

Veterinary Hematology is more than just blood cells. Blood, highly functional and truly definitive, does much more than provide for the transport of cellular metabolites and waste products. Blood is made up of four major components: Plasma, Red Blood Cells, White Blood Cells, and Platelets.

Each component possesses its own diagnostic significance in the veterinary clinical setting.

Without blood, you have no viable mammal life. Therefore without blood, the clinician has no valid clinical picture of disease-state. To appreciate the value of blood, the clinician should examine each individual component of blood separately to gain an appreciation for the clinical significance and diagnostic value of veterinary hematology in their practice. Anemia will also be discussed.

Plasma

Plasma carries blood and blood proteins. Aside from a high water content, plasma also contains dissolved salts, calcium, sodium, magnesium, and potassium. Plasma contains clotting factors and on exposure to air it will clot. Serum is the clear fluid that separates from clotted whole blood and clotted plasma. Plasma comprises approximately 20% of the animal body's extracellular fluid. Most plasma protein molecules are too large to pass through the capillary walls into the interstitial space. The small amount of protein that can pass through the capillary walls is primarily taken up by the lymph nodes and eventually returned to the circulation.

The Majority of the plasma proteins are produced in the liver. Plasma proteins form three major chemical groups (fractions) and have varying functions:

*albumin - approximately 60%

*fibrinogen - approximately 4%

*globulins - approximatly 36% over three subfractions (IgA, IgB, & IgG)

The relative proportions of plasma proteins can vary in certain diseases and these variations can be clinically useful in determining proper IV therapy. Albumin is the smallest of the plasma proteins and easily passes through capillary walls. In kidney disease, large amounts of albumin are excreted through damaged kidney tubules and can be detected in the urine.

Functions of the plasma proteins include:

Intravascular collid osmotic pressure. Maintains fluid and electrolyte levels.

Transport of insoluble substances allowed by protein binding processes

Contribution towards the plasma viscosity

Inflammatory response via microbe fighting antibodies

Protein storage reserve

Clotting

Protection from infection via plasma gamma globulins

Plasma also contains inorganic ions, which are important in regulating cell function and maintaining homeostasis. As an example, depletion of potassium may occur following severe diarrhea and vomiting. Potassium is an essential element of cell excitability. Sharp decreases in potassium will cause muscle weakness and cardiac abnormalities. Similar problems may cause sodium depletion. Subtherapeutic sodium levels in the plasma will result in the volume of extracellular fluid to decrease which will lead to a drop in blood pressure causing lethargy, dizziness, weakness and fainting.

Plasma carries a wide range of substances including dissolved gasses left over from the respiratory exchange cycle (mostly CO2). Blood carries oxygen because it does not have an affinity for plasma related to its water solubility.

Nutrients, the most abundant being glucose, are carried in the blood plasma as a source of fuel for cellular metabolism. Amino acids, fatty acids, triglycerides, cholesterol and vitamins are also carried by plasma. Urea, uric acid, creatinine from the kidneys, bilirubin from the gall bladder and other waste materials are also transported by plasma. Plasma proteins carryhormones, such as cortisol and thyroxine. The plasma also carries certain drugs and ETOH.

Platelets

Platelets are the result of cellular fragments shed from the megakaryocyte while in the bone marrow. Platelets considered cell fragments rather than actual cells, play a critical role in blood clotting. When an injury to the body occurs, a chemical substance is released at the site of injury.

Platelets are able to quickly adhere to this chemical and begin to form alliances with other platelets and clotting factors. This alliance is the body's defense against bleeding to death.

Platelets are also significant in forming diagnostic clues to the blood smear and can be useful at guiding the clinician in care planning, treatment and further diagnostic steps. Platelet morphology together objective data can be indicative of bleeding disorders and leukemia.

RBCs

Red Blood Cells, seemingly basic, are created and have the sole purpose of keeping the mammal alive by carrying oxygen to the tissues and white blood cells out of the bone marrow and into circulation. Red Blood Cells along with other blood components are present in nearly every portion of the body. When there is not enough blood in the body, anemia occurs and the animal begins to have clinical signs. It becomes imperative that clinicians immediately identify the etiology of anemia in order to help define or refine treatment. In doing so, the clinician will examine the blood smear and available objective data in order to quickly determine whether the anemia present as defined by a low pack cell volume (PCV) is one of production, consumption, sequestration or destruction. We will be discussing the cellular size, shape, color and other diagnostically significant data present in various states of anemia to aid the clinician in accurate slide evaluation.

WBCs

The white Blood Cell (WBC) plays an important role in the animal body by providing our bodies

with a weapon to fight against infection and disease. The primary function of the WBC is served mostly after it leaves the marrow and enters the blood stream after being carried by the RBC from its site of formation in the marrow, to its site of labor in the blood stream. There are five types of white blood cells seen in blood and each has different roles to perform.

The Neutrophil

The neutrophil, in conditions of health and certain disease, is usually the most common granulocyte found in blood. The cytoplasm of the neutrophil contains three differing types of granules. It is these granules that result in it being termed a granulocyte. Neutrophils generally have segmented or hyper-segmented nuclei giving them the appearance of being mutlinucleated.

In fact, they are not multinucleated as a thin strand of chromatin connects each lobe of the prominent dark purple, multilobed nucleus. At times, this chromatin strand can be visualized by most microscopes, when care is taken to look for it. Sometimes however, the strand becomes obscured by parts of the nuclei itself as a result of cell orientation and smear technique.

The three type of granules seem in the cytoplasm of the cell perform specific functions.

Primary granules are non-specific and contain lysosomal enzymes, defensins, and some lysozyme. The granules are similar to lysosomes. They stain violet in color when prepared with Wright's stain or Diff Quik. The enzymes produce hydrogen peroxide, which acts as a powerful antibacterial agent.

Secondary granules, found in the cytoplasm of the neutrophil, stain neutrally a light pink. They contain collagenase, which helps the cell to move through connective tissue, and deliver lactoferrin, which is toxic to bacteria and fungi.

Tertiary granules have only recently been appreciated as a granular component to granulocytes.

They are thought to produce proteins, which help the neutrophil to stick to other cells and hence aid the process of phagocytosis.

Neutrophils, once they arrive at an area of infection, respond to chemicals (called chemotaxins which are released by bacteria and nectrotic tissue cells) and travel towards the area of highest concentration of infection or necrotic tissue. Once they arrive at their destination, they begin the process of phagocytosis in which the offending cells are engulfed and destroyed by powerful enzymes. This process requires much energy, so the glycogen reserves of the neutrophil are soon depleted and the neutrophil promptly dies soon after the phagocytotic process. When neutrophils die, their contents spill out into the blood stream and remnants of their enzymes cause liquefaction of closely adjacent tissue. This results in an accumulation of dead neutrophils, tissue fluid and abnormal materials that is known as pus.

The Eosinophil

Eosinophils appear as the most colorful portion of blood and as a primary function, provide for a defense against the larvae of parasitic worms and unicellular organisms. Eosinophil granules contained in the cytoplasm contain a substance called MBP (major basic protein) which is toxic to many parasitic larvae. Eosinophils have surface receptors for the antibody Immunoglobulin E (IgE). These receptors are not found in other white blood cells and are believed to be of importance in their role at fighting parasitic infection.

The number of Eosinophils in peripheral blood circulation increases in some allergic conditions.

Numbers of Eosinophils increase in the peripheral blood smear when nasal and bronchial mucosal linings are irritated in asthma, brochitis, hay fever and certain adverse drug reactions.

Eospinophils are believed to neutralize the effect of histamine.

Eosinophils also have a marked tendency in number to be highest in the morning and lowest in the afternoon3 in the canine and feline.

The Basophil

The Basophil is rarely seen on periphral blood smear in cats and dogs is defined by its large cytoplasmic granules, that usually obscure the nucleus of the cell. They are similar to mast cells and become mast cells upon leaving the blood and entering the tissues.

Both basophils and the mast cell contain selective receptors for IgE that is produced in response to various allergens.

Response to specific allergens by the basophil is quick and results in expulsion of the cells granular contents, which contain histamines and vasodilating agents. This is another reason that contributes to the basophil not being readily present in the peripheral blood smears of cats and dogs. The result of a basophilic response creates an immediate state of hypersensativity in the animal. This can result in hay fever, asthma, urticaria, and most seriously, anaphylactic shock.

The Monocyte

The Monocyte is the largest cell type seen in peripheral blood smears. The nuclei is not multilobular, but appears deeply indented (kidney bean shaped) or U-shaped (horse-shoe shaped). The chromatin appears reticulated. The cytoplasm of monocytes contains many lysosome granules which give it a standard grayish-blue color in most instances. Monocytes form part of a cellular alliance that has been described as the monocyte-macrophage system3. This system is made up of bone marrow precursor cells, such as monoblasts and promonocytes, as well as circulating monocytes containing free and fixed tissue macrophages.

Monocytes become tissue macrophages and remove dead cell debris when they leave the bloodstream. They also attack organisms and certain fungi. Organisms and fungi affected by the monocytes are those which cannot be destroyed by the neutrophil. Unlike neutrophils, monocytes are able to regenerate the contents of their lysosomes and thus live longer. Cell types that are derivatives of the monocyte include: Kupffer cells of the liver, sinus cells of the spleen and lymph nodes, pulmonary alveolar macrophages, as well as free macrophages in the synovial, pleural and peritoneal fluid.

The Lymphocyte

Types of Lymphocytes include the Small Lymphocytes, K-Lymphocyte, B-Lymphocytes and (Helper) T-Lymphocytes.

Small lymphocytes are produced in the lymph nodes and spleen are similar to all the lymphocyte types but differ in the location in which they sequester for their function.

Helper T-Lymphocytes originate in the thymus and produce long living T-Cells which become Killer T-Cells or K-Cells which mediate antibody dependent cell cytotoxicity (tumor rejection).

B-Lymphocytes are localized in the corticomedullary region of the lymph nodes and are made up of cells of the germinal centers of the cortex in lymph nodes, in the red pulp of the spleen, and in the submucosal regions of the stomach and respiratory tract.

Lymphocytes, distinguished by having a deeply staining purple nucleus that is sometimes eccentrically located, usually contain a relatively small amount of cytoplasm. The small ring of the cytoplasm contains numerous ribosomes and readily stains blue with Wright's Stain or Diff-Quik. Small numbers of granules may also be noted in the cytoplasm randomly.

Lymhpocytes increase in number as a response to viral infection. The small lymphocyte will approximately the same size as the normocytic RBC. The cytoplasm is often not visible because it is obscured by the nucleus of this cell. This cell is definitively round under examination and lacks "divets." There can be variations in the size of the lymphocyte in the k-9/f-9 with the small type usually being the predominant type. In the small lymphocyte the chromatin is usually so coarse that it is masked. The medium and large forms of the lymphocyte often appear smudged.

Lymphocytes will increase in number with restraint: physical or chemical, and you will usually notice a corresponding increase in PMNs.

Anemia

Anemia is defined as a below standard hematocrit (HCT). A species specific hematocrit1 is as follows: Dog: 37-55, Cat: 24-45, Horse: 32-52, Porcine: 24-46, Bovine: 24-46. There are further variations of this data available that are further differentiated on the basis of age and sex. The author uses a combination of sources, which he has found through experience to be clinically reliable and accurate.

Most anemic conditions (except hemorrhage anemia) can be ordered to have an etiology of consumption, production, destruction or sequestration and further differntiation of anemic types are considered by ascertaining variation is size, shape, color.

Anemias of consumption include the hemolytic anemias and those created by disease conditions should as DIC and parasites where platelets and other clotting factos are consumed. Some anemias which sequester platelets and blood to the spleen, have also been placed in this classification, but etiologic differentiation has been found to be of clinical significance.

Anemias of destruction such as Autoimmune Mediate Hemolytic Anemia (AIMHA) exist when the body's own antibodies destroy its own red blood cells.

Whether or not you use your own in-house clinical lab or send your specimens out to a reference lab, this lecture will bring you back to the basics and help you remember that which you may have forgotten in school. Many clinicians find this lecture and format helpful to expand on basic knowledge and clinically apply what they see either under the microscope or on the lab report.

Not having adequately available time to donate towards the lab, more clinicians are relying upon technicians to interpret laboratory results being unsatisfied with the time investment required to await the return of results from distant reference labs. Clinics and hospitals are using this author's methods for training their staff to facilitate the clinician having more time to provide for patient and client care, thus increasing value added service and customer satisfaction.

1. OW Schalm, et al. Veterinary Hematology, 3 Ed., Lea & Febiger 1975

2. Bernard Feldman, DVM various works

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Biography of Lon Bartoli, BSN, AHT, VCLS

Lon Bartoli, the author of Veterinary Hematology 101:A Pocket Reference Guideã and several abstract form titles, is currently pursuing his lifelong dream of working in emergency services and teaching.

As a professional firefighter I & II/EMT-I, Lon Bartoli holds a bachelor's degree in Business Management as well as a bachelor's degree in Nursing Science. He has worked in veterinary medicine, (human) critical care nursing, EMS, business management, the insurance industry and fire science. Since 1991, he has been arduously involved in clinical laboratory science and is now considered by many in the profession as a guru in the clinical laboratory setting.

Lon has been recognized as the Montana Coordinator for the Back To Sleep - SIDS foundation which provided public service information geared towards helping to decrease the number of SIDS.