VTNE: Basics of Laboratory Procedures 1

Laboratory procedures are a significant part of the veterinary practice. This is such a significant topic and so central to any veterinary practice that we were forced to divide it into two chapters. This guide discusses hematology, coagulation and bone marrow testing, cytology/histology, clinical chemistry, and immunology/serology. Another guide on Fatskills covers the second half of lab work, parasitology, urinalysis, and microbiology.
The ability to perform and interpret laboratory information is necessary to diagnosis and treatment of animals because they are unable to tell the veterinarian what their symptoms are and evaluate how they 'feel.' The ability to bring together and evaluate information obtained from laboratory tests is important to communication between the doctor, the technician, and the client.
Knowledge of subjects such as blood science, testing, clinical chemistry, and immunology are central to a modern veterinary practice. Hematology is the study of blood and blood-related diseases, and testing the blood is one of the most common ways of discovering the health of an animal. These tests are generally fairly simple to perform and inexpensive, which is why hematologic testing is one of the keystones of any veterinary practice.

Key laboratory procedures terms covered here:
acidosis
agglutination
alkalosis
anticoagulant
azotemia
chemotaxis
ejaculate
hematochezia
hematologic indices
hematopoiesis
hemoglobin
hemolysis
icterus index
leukogram
lipemia
malabsorption
malassimilation
maldigestion
marginating pool
mean corpuscular hemoglobin (MCH)
mean corpuscular hemoglobin concentration (MCHC)
mean corpuscular volume (MCV)
melena
negative feedback
pellet
phlebotomy
plasma
polychromatophil
polydipsia
polyuria
reticulocyte
serology
serum
supernatant
turbidity
uremia
Vacutainer®
venipuncture
viscosity

This guide will include questions that focus on the first part of laboratory procedures, covering six primary concepts:
- hematology
- coagulation testing
- bone marrow testing
- cytology and histopathy
- clinical chemistry
- immunology and serology

Hematology
Hematology can provide the veterinary practice with valuable information about the animal's ability to carry oxygen, organ function, and immunologic status. Calculations based on hematologic values allow us to classify anemias and the animal's ability to respond to invaders. The ability to perform and interpret the CBC (complete blood count), hematologic indices, and leukogram are integral to the operation of a veterinary practice.

Hematologic Testing
Proper interpretation of blood work requires good phlebotomy (blood drawing), knowledge of appropriate blood tubes and appropriate storage techniques, and the ability to make a diagnostic blood smear. Failure in any of these areas can lead to artifacts that can interfere with proper interpretation of results and can invalidate any results obtained.

Phlebotomy: Drawing a Good Blood Sample
Phlebotomy is the art (and science) of getting blood from a vein, usually for the purposes of diagnostic blood testing. Drawing an adequate blood sample begins with knowledge of the appropriate quantity of blood to be obtained. Blood can be drawn from an animal up to 1/4 of the total blood volume. Blood tubes are labeled with the quantity of blood needed to fill the tube. They are maintained in a vacuum so that the appropriate amount of blood will be added to the tube when a Vacutainer® system is used. This is especially important in tubes containing an anticoagulant. Inappropriate quantities of blood added to anticoagulant tubes can result in artifacts on the blood smear and in dilution for packed cell volume (PCV) and total protein (TP) measurements. Drawing blood with a needle and syringe is common in practice, but may cause problems with hemolysis (red blood cell rupture and release of hemoglobin), which will interfere with many chemistry results. In general, it is best to take too much blood than too little.

Blood Tubes
Blood tubes have tops that designate the additive (or lack of an additive) and the purpose of the tube. Most tubes are used to separate the liquid from the cellular components of the blood. The resultant liquid is either plasma or serum, depending on whether it is allowed to clot prior to centrifugation. If it is allowed to clot, it is called serum and does not contain the clotting proteins. The total protein (TP) of serum will be slightly lower because of this. Plasma still contains fibrinogen.




Performing phlebotomy properly without causing artifact is one of the most important skills for a veterinary technician in practice (or research).

The primary sites for venipuncture are:
Cat and dog: jugular vein, cephalic vein, lateral saphenous, common saphenous/femoral vein (cats only)
Horse: jugular vein, facial vein, tail vein
Cow: jugular vein, tail vein

The most important part of venipuncture is atraumatic technique. This requires the ability to visualize the vein and a basic knowledge of anatomy. Visualization can be improved by clipping the area and wetting it with isopropyl alcohol, which will cause blood vessel dilation. The needle should follow the path and direction of the vein. The vein should be held off between the venipuncture site and the heart to increase the back pressure of blood in the vein and allow it to stick out. You should not try to fish for the vein because this will cause tissue damage with the release of tissue thromboplastin, which will cause platelet activation and clotting in the syringe and the blood tube. As stated, Vacutainer® systems should be used if possible. If coagulation studies must be performed, a Vacutainer is essential. If Vacutainers are not available, care must be taken not to collapse the vein by using excessive back pressure when pulling back on the syringe. Collapsing the vein will also cause tissue damage and increase platelet activation.
Use of appropriate needle and syringe size is also important when performing phlebotomy. All materials needed for venipuncture should be collected prior to beginning the process. The needle should be the maximum size that can comfortably fit into the vein without causing damage to the vessel wall. Use of a very small needle will cause hemolysis by increasing turbulence inside the needle and disrupting the red blood cell membranes. Hemoglobin in the plasma is also possible from certain diseases (blood parasites and autoimmune hemolytic anemia, for example), so iatrogenic hemolysis (hemolysis caused by bad technique) may cause a misdiagnosis of another problem.

Blood Storage
In general, blood should be processed as quickly as possible after being drawn and centrifuged. Many chemistry values (such as ammonia and glucose, for example) will be invalidated by prolonged time between blood draw and processing and blood cell morphology will also be affected. Platelets will aggregate and lyse after 6 hours of storage. Ideally, whole blood should be kept refrigerated and processed within 6 hours. Plasma should be frozen if not processed within 6 hours. Serum should be kept refrigerated and processed within 8 hours.

Artifacts
As stated, hemolysis and platelet activation are the most common artifacts seen from improper blood handling. Lipemia (increased fat and cholesterol in the plasma) may also interfere with machine blood counts and several chemistry tests. Lipemia may be caused by metabolic diseases (hypothyroidism, hyperadrenocorticism, diabetes), be an inherited problem (congenital hypertriglyceridemia), or be caused by eating prior to blood draw. For this reason, animals should be fasted for 10 to 12 hours prior to drawing blood for analysis. Postprandial lipemia usually occurs within 2 to 3 hours of a meal.

Making a Good Blood Smear
With the advent of better machine blood cell counters, many veterinarians and veterinary technicians have lost the art of making and reading a good blood smear. This technique is extremely important. Without hand evaluation of blood smears, many pathologies will be missed including parasitism, hematopoetic cancers (leukemias), certain toxicities (lead, zinc, maple leaf, acetaminophen) and other cellular inclusion diseases (Ehrlichia, anaplasma, hemobartonella, distemper). In general, a blood smear should be performed if any abnormality on the machine CBC is seen or if the animal is ill.
A good blood smear has three parts: The body, the monolayer, and the feathered edge. No matter which technique is used, the smear should look like a flame, with the base being made of the body and the feathered edge as the flickering outer portion of the flame. The sides of the smear should not make contact with either edge, and the monolayer should be of adequate size to analyze for the white blood cell differential.
The key to making a good smear is the size of the drop. It should be small enough that the contact with the spreading slide does not bring the blood to the slide edges, but big enough that the smear is adequate size. The slide for the sample should be clean and free of scratches and the spreading slide should have a smooth edge for making contact with the blood drop.
The drop of blood is placed on the sample slide approximately one-fifth of the length of the slide away from the edge. The edge of the spreader slide is placed on the sample slide at a 45-degree angle and is backed into the blood drop and smoothly pushed across the length of the slide. The blood will be drawn along with the spreader slide to make the characteristic flame shape. The body is the portion of the smear closest to the original drop. This section is the thickest area and cannot be evaluated because the cells are piled on top of one another, making individual morphology (cell shape and staining characteristics) difficult. The monolayer is the diagnostic area. This region is thinner and contains cells in a single layer (red, white, and platelets) that can be evaluated for cellular morphology. The feathered edge is where the heaviest items are seen, including clumps of platelets, parasites (trypanosomes and microfilaria), and destroyed red blood cells. This area should not be used to evaluate white blood cell morphology, but should be used for platelet estimation in cases of artifactual platelet clumping.
Once the smear is made it should be stained with a Romanowsky type stain (an alcohol-based stain) such as Diff-Quick. Diff-Quick has three parts: The fixative (methyl alcohol), the eosin stain (which stains the proteins of the cell red), and the basophilic stain (methylene blue stain) which stains the nucleic acids of the cell blue. Artifacts can result from staining. These include the following:
water artifact—a refractile artifact (highly reflective bubbles in the cytoplasm of cells) resulting from water contamination of the alcohol fixative; may be mistaken for parasitic infection 

stain precipitate—a purple aggregate of small crystals that may be mistaken for platelet clumping; usually found in a different focal plane on the microscope than the cells
Both of these artifacts can be prevented by appropriate maintenance of the Diff-Quick stain by changing out the alcohol and stains and cleaning the staining jars regularly. Staining solutions and alcohol fix should not be topped off with fresh solution because this will increase artifacts.

Red Blood Cell Indices
Evaluation of anemias and polycythemias is within the scope of practice of veterinary technicians. Such an evaluation will result in a laboratory diagnosis. The method for making laboratory diagnosis of anemias is to perform hematologic index calculations. Laboratory diagnosis is the responsibility of the technician while medical diagnosis is the responsibility of the veterinarian.

Hematocrit
The hematocrit (HCT) is arguably the most important piece of laboratory information obtained from the patient. A total of ten items of interest may be obtained from the HCT: The icterus index (II), packed cell volume (PCV), buffy coat (BC), total protein (TP), and occasionally microfilaria can be seen by direct examination. Estimated red blood cell count (RBC), estimated hemoglobin (HB), mean cell hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and mean cell volume (MCV) can be obtained by calculations based on the HCT.
The HCT is performed by collecting blood into a microhematocrit tube approximately two-thirds full, sealing one end with clay, and placing the tube into a balanced high-speed microhematocrit centrifuge. The tube is spun for 3 to 5 minutes on high speed. This separates the various blood components (serum, white blood cells and platelets, and red blood cells).

Icterus Index
The icterus index (II) is the color of the serum. Normal serum is clear to straw colored (in herbivores, it may appear yellow to orange because of the amount of beta carotene or vitamin A in the diet). Other colors may indicate disease or iatrogenic problems with blood handling. The serum color is evaluated by placing the tube in front of a white piece of paper and assessing the color.



Packed Cell Volume
The packed cell volume (PCV) tells us the percentage of red blood cells per milliliter of whole blood. It is an estimate of anemia but should not be evaluated without also checking the total protein (TP). The PCV is read by placing the hematocrit tube against a Read-o-crit scale. The top of the clay and the top of the serum column are lined up with the bottom and top lines of the scale and the interface between the buffy coat and the red blood cell column is located. That line corresponds to the PCV.
The reason the PCV should not be evaluated by itself is that it depends on the hydration of the animal. If an animal is dehydrated, there will be less water and thus less serum in the whole blood. The total protein will be elevated because the protein will be more concentrated. The PCV will, therefore, appear increased. If the PCV is elevated in the face of a high total protein, that is not a real value. If it is elevated and the TP is low or normal, that is a real value and pathology should be suspected.

Buffy Coat
The buffy coat (BC) is a rough estimate of the white blood cell count. It is made up of white blood cells and platelets and should be the smallest component of the HCT. It should be 1 to 2% of the column and should be measured when the PCV is measured. If microfilaria are present due to heartworm disease or Dipetalonema, they can be visualized at the BC serum interface by examining the tube at low power under the microscope. Buffy coat smears may be made, which will allow visualization of white blood cell parasites such as Ehrlichia. The technique is the same as for a normal blood smear except that the sample will consist of the buffy coat material only.

Total Protein
The total protein (TP) concentration must be evaluated along with the PCV, as described. Most of the proteins in the blood are made in the liver.
The highest concentration of protein in the blood is albumin. Albumin is the protein that controls osmotic pressure (maintenance of blood volume inside the blood vessels). In general, a drop in TP means a drop in albumin concentration. The most common cause of decreased albumin concentration is liver disease.
The second highest concentration of proteins in the blood is fibrinogen. Fibrinogen may cause an increase in TP in animals with inflammation (especially cattle).
The rest of the TP is made up of the globulins, including the immunoglobulins.

Estimates
Two estimates can be obtained from the PCV: The red blood cell estimate and the hemoglobin estimate. The red blood cell estimate may be vastly different from the actual count and should not replace it. The hemoglobin estimate may have to be used in animals with severe lipemia or hemolysis because both of these artifacts will interfere with machine calculations of hemoglobin concentrations.
RBC est = PCV/6 × 106/µl
HB est = PCV/3 g/dl (grams per deciliter)

Mean Cell Hemoglobin Concentration and Mean Corpuscular Hemoglobin
Mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH) are the two calculations that will estimate the amount of hemoglobin present in the blood. Although the MCHC is more accurate, the MCH is still used in practice. These calculations are important because, along with the MCV, they will help us to give a laboratory diagnosis of an anemia. Young red blood cells contain less hemoglobin than adult red blood cells. This means that they will not be able to carry as much oxygen as adult cells. On cellular morphology, they will appear blue-tinged by the stain because they still contain ribosomes for hemoglobin manufacture. These cells are called polychromatophils because they vary in color from the adult RBCs. A regenerative anemia will have a decreased MCH and MCHC because there will be more polychromatophils present resulting in a decreased hemoglobin concentration. This is called a hypochromic anemia (low-color anemia). The hyperchromic (too much hemoglobin) state is not possible.

The calculations for MCH and MCHC are:
MCH = HB × 10/RBC pg (picograms are one-trillionth of a gram)
MCHC = HB × 100/PCV g/dl (grams per deciliter)

Mean Corpuscular Volume
The mean corpuscular volume (MCV) estimates the average size of the red blood cell. This is important for similar reasons to the MCH and MCHC. Young red blood cells are larger than adult red blood cells so, if a regenerative anemia is present, the MCV will be increased. This is called macrocytosis. If we have very old red blood cells or the cells are being damaged (fragmentation hemolysis), we may have a low MCV. This is called microcytosis.
To review, if we have a low PCV with a low MCHC and a high MCV, we would call that a hypochromic, macrocytic anemia, which would generally indicate a regenerative anemia. If, on the other hand, we have a low PCV with a normal MCHC and a high MCV, we would call that a normochromic, macrocytic anemia, which would probably indicate something like an iron deficiency anemia (inadequate production of hemoglobin because there are low iron stores).
Although machines can calculate these values, the machine will not be able to read the hemoglobin concentrations in cases of hemolysis and lipemia. The ability to calculate HB estimates, the MCHC, and the MCV may help the veterinarian diagnose a case. The MCHC and MCV results should always be checked against a reticulocyte count (see Reticulocyte Counts).

Hematologic Inclusions and Abnormalities
Blood cell morphology (shape and staining characteristics) can be very important to the diagnosis of various diseases including viral, bacterial, and parasitic disease. Toxicities can affect cellular morphology as well.

Red Blood Cell Inclusions
The function of the red blood cell is oxygen distribution. Oxygen is carried on the hemoglobin molecule, which consists of proteins (heme and globin) bound to ionic iron. The reason for blood's red color is oxidation of the iron ion. It literally rusts the iron, making it red.
Normal blood cells in most mammals are anuclear (having no nucleus), round, and biconcave (indented on both sides). Camellid (alpaca, camels, vicuna, llama) blood is elliptical in shape. Erythrocytes stain red/orange with Diff-Quick stain in the adult form. As stated, young red blood cells are called polychromatophils and stain various shades of blue with Diff-Quick. Polychromatophils are generally larger than adult red blood cells and are generally not biconcave. Very young circulating red blood, metarubricytes, cells may still have a nucleus. They usually have a dark blue cytoplasm and a dark, condensed nucleus. The nucleus is expelled from the cell when it becomes a polychromatophil. Incomplete expulsion results in Howell-Jolly bodies, which are nuclear remnants in adult red blood cells.
Reptile, avian, and fish red blood cells are elliptical and nucleated. The nuclei are dense and oval and the cytoplasm is pink. The polychromatophils have more open nuclei and bluish cytoplasms. Metarubricytes and other young red blood cells look similar to those of mammals.

Viral Inclusions
Distemper virus results in intracytoplasmic inclusions in all blood cells and epithelial cells. The inclusions generally stain pink and are round and varied in size.

Bacterial Inclusions
Hemobartonella/Mycoplasma organisms are atypical bacteria that lack cell walls.
Anaplasma marginale is the most prevalent tick-borne disease in livestock. It is a rickettsial bacterium and an obligate intracellular parasitic bacterium. The bacterium produces toxins that eventually destroy the blood cells (extravascular hemolysis).



Parasitic Inclusions
Most of the parasitic inclusions, or hemosporidians, infect red blood cells exclusively. Leukocytozoon also infects white blood cells. They have varying pathogenicity depending on the species and most are fairly host specific. Appearance on examination of the blood smear is laid out in the following table.


Toxic Inclusions
Various toxins can cause inclusions in red blood cells. These may be confused with some of the bacterial and parasitic inclusions and must be differentiated.



White Blood Cell Inclusions
The function of the white blood cells is in immunity. Some white blood cells directly phagocytize invaders (monocytes and macrophages), others have granules that help to digest foreign invaders (neutrophils, eosinophils, basophils), and still others manufacture proteins and mediators that destroy invaders (lymphocytes). White blood cells are divided into two groups: granulocytes and agranulocytes, depending on the presence or absence of granules in the cytoplasm. The granulocytes include neutrophils, eosinophils, and basophils. The agranulocytes include lymphocytes and monocytes.
The granulocytes have condensed, pinched-off nuclei in the adult form (segmented). In the younger forms, the nucleus is smooth and shaped like a tube or band. In very young forms, the nucleus is round and indented (metamyelocyte). All three forms contain granules.
Neutrophils are the cells involved in acute inflammatory processes. The granules of neutrophils are neutral to stain (do not stain with Diff-Quick), so the cytoplasm is clear. Neutrophils are the most numerous white blood cell in all species except cattle. Numbers increase in any inflammatory or infectious process (neutrophilias) because neutrophils respond to cell mediators from tissue damage and bacterial cell wall products. Decreases in neutrophils are called neutropenias. Neutrophils are very short lived in the blood stream (8 hours).
Birds and reptiles do not have neutrophils; their equivalent is the heterophil. Heterophils have round nuclei and a mixture of red/eosinophilic and clear granules. The function is the same as that of neutrophils.
Eosinophils are involved with allergic and parasitic diseases. The granules of eosinophils stain with eosin (they are red in Diff-Quick-stained samples). The granules may be large and round (horses), rod shaped (cats), or small and round (dogs and cattle). Eosinophil granules contain substances that directly destroy parasites and others that counteract the effects of basophils, such as histaminase. Eosinophils are usually equal in numbers to monocytes on differential cell counts. They tend to last for 8 to 14 hours in the blood stream. Elevations are called eosinophilias; decreases are called eosinopenias.
The granules in eosinophils from birds and reptiles can vary in color from red to lavender or blue (common in snakes, cockatoos, and owls). The function of these cells is the same as for mammalian eosinophils.
Basophils are also involved in allergic and parasitic diseases. Both eosinophils and basophils respond to IgE, the immunoglobulin associated with allergic disease. The granules of basophils stain with the basophilic stain (they are purple on Diff-Quick-stained samples). The granules are generally small and round and may fill the entire cytoplasm, making it difficult to see the nucleus. In cats, the granules are very small and appear as lightly dusted purple or just a diffuse purple/lavender color to the cytoplasm. Basophils contain several vasoactive amines such as histamine and heparin, which are involved in anaphylactic reactions. They also contain eosinophil chemotactic factor, which calls eosinophils to the site of degranulation (eosinophils counteract some of the damaging effects of basophil degranulation). Basophils are the least numerous of all the white blood cells. Basophil elevation (basophilia) is commonly seen in heartworm disease.
The agranulocytes are both the largest and the smallest white blood cells. Agranulocytes look the same in birds and reptiles as in mammals. They do not contain granules. They are very long lived in the circulation. Lymphocytes may remain in the tissues and circulation for years. Monocytes can go into tissues and back into the circulation (granulocytes are trapped in the tissues and cannot go back into the circulation). Most lymphocytes are found in the lymph nodes. Most monocytes convert to macrophages, which are embedded in the tissues (Kuppfer cells in the liver, Langerhans cells in the skin, etc.). Granulocytes are only seen in the tissues in inflammatory responses.
Monocytes are very large cells (about five red blood cells can fit inside a monocyte). The blast (young) forms look very similar to the mature cells. Monocytes have a bluish grey cytoplasm and a pink-purple nucleus with open chromatin (light can come through the nucleus) that varies in shape. The cytoplasm often has vacuoles and looks foamy. The nuclear-to-cytoplasmic ratio (how much cytoplasm vs. how much nucleus) is usually 1:1 (the nucleus has the same area as the cytoplasm). Monocytes are involved in chronic inflammatory processes and in processing antigens for presentation to lymphocytes. They will be increased in viral infections and chronic inflammatory disease (such as fungal infections or granulomas). Monocytes' main purpose is phagocytosis. Monocytes are often seen with red blood cells, bacteria, or other organisms in their vacuoles. An increase is called monocytosis; a decrease is called monocytopenia.
Lymphocytes are usually very small cells that are almost the same size as a red blood cell in their inactive form. The cytoplasm is light (active form) or dark blue (inactive form) and the nucleus may be either condensed (inactive form) or contain open chromatin (active form). There may be a perinuclear halo (lighter area in the cytoplasm around the nucleus) that corresponds to the Golgi apparatus that is actively making proteins (either immunoglobulins or complement) in active cells during an immune response. Lymphocytes are the second-highest number of white blood cells in the circulation in all but cattle, where they are the most numerous.
Lymphocytes are grouped into B cells and T cells depending on where the cells are labeled. B cells are labeled in the bone marrow in mammals and in the bursa of Fabricius in birds. T cells are labeled in the thymus gland. B cells are responsible for humoral immunity (making immunoglobulins) and T cells are responsible for cell-mediated immunity (making complement and causing direct death of organisms and cancer cells). Most of the circulating lymphocytes are T cells. In FIV (feline immunodeficiency virus), SIV (simian immunodeficiency virus), and HIV (human immunodeficiency virus), the T cells are depressed in number. This leaves patients open to viral infection and cancers.
The trigger for lymphocyte and monocyte increases is any antigenic stimulation. An antigen is any protein or glycoprotein that is perceived as foreign to the body. The monocyte phagocytizes the invader and presents a modified portion of the invader on its surface. This is then handed off to a B lymphocyte, which makes a specific antibody to that particular protein and clones itself so that more can be made. A T lymphocyte would make a receptor on its surface that binds to the protein so the T lymphocyte can release substances to destroy it (complement). Each B and T lymphocyte is specific to a particular protein.



Platelet Inclusions
Mammalian platelets are cell fragments that are made from cell budding of the megakaryocyte in the bone marrow. They are basically packages of coagulation mediators, calcium, and inflammatory mediators. Platelets appear as small granular purple bodies between cells in the monolayer of stained slides.
Birds, reptiles, and fish have thrombocytes, which are cells that have the same functions as platelets. Thrombocytes have dark, condensed nuclei and refractile-appearing cytoplasm on Diff-Quick stain.
Platelets (and thrombocytes) function in primary hemostasis, which is the formation of the unstable clot. The unstable clot forms and degrades within 2 to 3 minutes of blood vessel injury. Platelets are drawn to the site of a blood vessel injury by mediators produced by the blood vessel lining (vascular endothelium). Once there, they bind to the subendothelial collagen by a bridge of von Willebrand factor (vWF) and factor VIII. Binding stimulates the platelets to aggregate (come together) and adhere (become sticky). They also produce platelet chemotactic factor, which calls more platelets to the area. They initiate the secondary clotting cascade which eventually forms the stable fibrin clot.
Defects of primary clot formation can occur with too few platelets (thrombocytopenia) or decreased platelet function (von Willebrand disease, collagen deficits, uremia, etc.). Platelet counts generally must be less than 80,000/µl in order to cause clotting deficits. More information on secondary coagulation and coagulation testing can be found in the section on Coagulation Testing.
Anaplasma platys (formerly Ehrlichia platys) is a rickettsial organism that specifically invades platelets, causing infectious canine cyclic thrombocytopenia (ICCT). The morula can be seen in the platelet cytoplasm as a uniform purple inclusion. A. platys causes profound thrombocytopenia leading to bleeding disorders and fevers in dogs (epistaxis, echhymosis, petechiae, etc.). It is carried by the brown dog tick (Rhipicephalus sanguineus). It may be difficult to diagnose on blood smear because of the similarity in color to the platelet. ELISA, IFA, and polymerase chain reaction (PCR) testing are available and accurate for diagnosis.

Noncellular Hematologic Parasites
Some of the parasites seen on blood smears are extracellular. These are usually fairly large organisms and are usually seen on the feathered edge of the blood smear.

Hemoflagellates
Hemoflagellates are trypanosomes. Trypanosomes are protozoal organisms with a flagellum and an undulating membrane. They are generally transmitted by reduviid bugs or flies. They live between the blood cells, but the amastigote stage lives in the tissues and causes damage. Trypanosoma cruzi causes Chagas disease, and the amastigote stage infests muscle tissue (including cardiac muscle) and can cause cardiomyopathy. Buffy coat smears should be made if trypanosomes are suspected.

Filarial Nematodes
Filarial nematode testing can be broken down into microfilarial testing (visualization of the microfilarial stage in the blood) or serology/ELISA (testing for the presence of adult worms or the antibodies to adult worms in the blood). Testing for all but the presence of antibodies requires presence of adult worms in the heart and pulmonary vessels and will not show prepatent infections. Since heartworms migrate for a prolonged period of time in the larval form (4 to 6 months), significant damage can occur in animals prone to respiratory difficulty from heartworm migration. In cats, a combination of antibody and antigen testing may be required to obtain a diagnosis because of the low worm burden (usually only 3 to 5 adult heartworms are present in cats) and lack of microfilaria in the systemic circulation (see the Immunology/Serology section for more on ELISA testing).
Direct Microfilaria Testing: Heartworm microfilaria are usually present at high numbers in the systemic circulation in the late morning. They are at lower numbers at the time of mosquito feeding (dusk and dawn) because they are generally in the capillaries of the skin at that time. Microfilarial testing can be performed either as a direct test or as a concentration test. Direct testing is usually performed on a very small sample size and gives a high percentage of false negatives. Examples of direct tests are checking the hematocrit tube at the buffy coat/plasma interface and examining the prepared and stained blood smear at the feathered edge. Another method is examination of a drop of fresh blood that is coverslipped and examined under the microscope using the 10× objective. Because of the small sample size examined and inconsistencies of movement in microfilaria, false negatives and misdiagnosis of heartworm disease in animals infected with Dipetalonema is common.
Concentration Microfilaria Testing: A better method of diagnosis of microfilaria is a concentration method. The most commonly used concentration method in practice is the Difil test. In this test a filter is placed in a special chamber. One cc of blood is mixed thoroughly with 9 cc of lysing solution and the mixture is then forced through the filter and rinsed. The filter is placed on a drop of heartworm stain and examined under a coverslip on 10× objective. The best method for microfilaria testing is the Modified Knott's technique. One cc of blood is mixed with 9 cc of 2% formalin (which relaxes the microfilaria) and centrifuged on low speed for 5 minutes. The supernatant (liquid portion of tube contents) is poured off and the pellet is resuspended and stained with methylene blue. A drop of the resuspended pellet is placed on a glass slide, coverslipped, and examined.



Reticulocyte Counts
Reticulocyte counts should be performed in any case of anemia to confirm regeneration of red blood cells (the ability of the bone marrow to make new red blood cells in response to a crisis). Reticulocytes are simply polychromatophils that have been stained with new methylene blue stain (NMB). NMB stains nucleic acids. Since polychromatophils are still producing hemoglobin, the ribosomes (which contain RNA) are still present. The ribosomes stain blue with NMB. Red blood cells containing these blue granules on NMB stain are called reticulocytes. They can be either punctate (one or two granules in the cell) or aggregate (multiple granules in the cell, usually in a clump). Punctate reticulocytes are closer to the adult stage of the red blood cell. Aggregate reticulocytes are younger and newly released from the bone marrow.
Bird and reptile reticulocytes have the ribosome granules in a ring around the red blood cell nucleus.
The presence of reticulocytes indicates a regenerative anemia and can confirm an elevated MCV and low MCHC. Horses will not release reticulocytes into the systemic circulation, so retics in a horse would be extremely abnormal. Evaluation of anemias in horses should be by serial PCV measurements to assess response. Increasing PCVs indicate regeneration.
Cats release punctate retics into the systemic circulation without anemia. Presence of aggregate reticulocytes is significant and indicates a regenerative anemia; therefore, only aggregate reticulocytes should be counted in cats.
In all animals other than cats and horses (including birds and reptiles), punctate and aggregate reticulocytes should both be counted. Any animal exhibiting anemia should have a reticulocyte count performed. Reticulocyte staining is performed by adding equal amounts of NMB to whole blood in a 1:1 ratio. This should be mixed thoroughly and allowed to incubate for a minimum of 10 minutes. A blood smear should then be made from the mixture and allowed to dry before examining under oil immersion (100× objective). One thousand red blood cells are then examined and the number of reticulocytes are recorded.

The observed reticulocyte count percentage is then performed:
Observed reticulocyte % = (# of reticulocytes/1000 red blood cells) × 0.1 (units in %)
To get the absolute reticulocyte count, the observed reticulocyte percentage is multiplied by the animal's red blood cell count:
Absolute reticulocyte count = observed reticulocyte count × red blood cell count (units in #/µl)
The corrected reticulocyte % corrects for the degree of anemia and gives a more true indicator of the regenerative nature of the anemia:
Corrected reticulocyte % = (observed reticulocyte % × PCV of the patient)/mean PCV of the species (units in %)
A mild regenerative anemia generally has a corrected reticulocyte of 1 to 8%, a moderately regenerative anemia generally has a corrected reticulocyte of 9 to 15%, and a marked response is >15%.

White Blood Cell Counts
The relative number of the various white blood cells on a blood smear can give a rough idea of the type of process occurring in the body. As stated, neutrophilia indicates an inflammatory response, eosinophilia and basophilia indicate an allergic or parasitic disease, monocytosis and lymphocytosis indicate antigenic stimulation. The ability to perform and analyze white blood cell counts and differentials is key to helping the veterinarian establish a diagnosis and is an integral part of veterinary technology.

Technique
Most white blood cell counts are performed on machines that have been specifically calibrated for animals. Manual cell counts must still be performed on avian and reptile blood because the red blood cells are nucleated and impedence counters measure nuclei when performing white blood cell counts. Because the Unopette® system is no longer being made, the technique for manual counts will not be discussed. It is also important to note that, in some species (cats), platelets may be misidentified as red blood cells by the impedence counters, especially in cases of megaplatelets (exceptionally large platelets). Nucleated red blood cells are also counted as white blood cells in mammalian blood. These are all reasons that a blood smear should be performed when possible.

Calculations
The three primary types of white blood cell counts are differential count, absolute differential, and corrected white blood cell count and differential.
 

Differential Count
When performing a white blood cell evaluation, the slide should be examined on 40× (high dry) to evaluate cell numbers and at 100× for specific white blood cell morphology. A total of 100 white blood cells should be counted and the numbers of segmented neutrophils, band neutrophils, eosinophils, basophils, monocytes, and lymphocytes should be counted. Once 100 cells have been counted, you will have a percentage of cells out of 100 cells that are each of the individual cell types.

Absolute Differential
Cell type percentage is not useful when trying to interpret disease processes. Correct evaluation of white blood cell counts requires absolute values, which are the actual numbers of cell types per microliter of blood. This calculation must be performed on each individual cell type. Adding all of the absolute values together should equal the total white blood cell count. The calculation is as follows:
Absolute differential = % of cell type × white blood cell count (units # cells/µl)
Corrected White Blood Cell Count and Differential
If more than five nucleated red blood cells are seen among 100 white blood cells, you must correct for their presence. As stated, nucleated red blood cells (metarubricytes) are recognized by cell counters as white blood cells. Correction for their presence will change the differential and may make the difference between a leukocytosis and a leukopenia. The calculation is as follows:
Corrected white blood cell count = (observed white blood cell count × 100) ÷ (% nucleated red blood cells + 100)
Once the corrected white blood cell count is performed, the absolute differential should be performed using that white blood cell count instead of the original white blood cell count.

Leukograms
Now that the differential is calculated, it should be checked against the normal range for the species. The absolute differential should be recorded in the record along with an assessment (leukocytosis/leukopenia, neutrophilia/neutropenia, eosinophilia/eosinopenia, basophilia, monocytosis/monocytopenia, lymphocytosis/lymphopenia) and any morphologic abnormalities of the red or white blood cells should also be recorded. There are specific signs associated with elevations of the white blood cells that are called leukograms. There are three basic leukograms: inflammatory, stress, and physiologic and they are each caused by different mechanisms. All three basic types show neutrophilias. Specific leukograms can point the veterinarian in a certain direction and aid diagnosis.



Coagulation Testing
As described in the platelet section, coagulation is a complex process. If one or more of the steps cannot be performed, the whole process breaks down. Coagulation consists of three parts: vasoconstriction, primary hemostasis, and secondary hemostasis.
Vasoconstriction occurs immediately on disruption of the blood vessel wall. It mostly occurs in arteries and arterioles because of the amount of smooth muscle present in the vessel wall. This is stimulated by epinephrine release from the nervous tissue surrounding the vessel and also by release of two chemicals from the platelets: thromboxane and serotonin. Serotonin is a neurotransmitter similar to dopamine and stimulates vasoconstriction as well as increasing pain at the site of injury (to prevent further damage to the area). Thromboxane is a prostaglandin that stimulates vasoconstriction and activates platelets. Note: aspirin inactivates thromboxane and causes increased bleeding.
Secondary hemostasis is a series of enzymatic reactions that causes the formation of the stable fibrin clot. This involves a series of 12 factors, calcium, vitamin K, and fibrinogen. All of the clotting factors (except for a small part of factor VIII) are made in the liver. Vitamin K is needed for the production of factors II, VII, IX, and X in the liver. Vitamin K is a fat-soluble vitamin that is stored in the liver. Severe liver disease will cause decreases in the clotting factors and prolonged bleeding in animals. The same symptoms may be caused by anticoagulant rodenticide poisoning. Warfarin, Coumadin®, coumarin, and brodifacoum all act by blocking vitamin K activity. Symptoms of vitamin K toxicity do not occur for days to weeks after ingestion of the rodenticide because the clotting factors last for a long time in the body.

The 12 clotting factors make one final product called thrombin. Thrombin causes the conversion of fibrinogen to fibrin. The fibrin is then acted on by factor XIII which causes cross-linkage of the fibrin strands to make a mesh which is the stable fibrin clot. This closes the defect and prevents blood loss. Heparin acts as an anticoagulant by causing increased thrombin destruction.
Once the clot is formed, it must be broken down so that blood flow can be restored. This occurs by the activation of plasmin, which breaks the fibrin clot into pieces that can then be recycled in the liver. The pieces of fibrin are called fibrin split products. Fibrin split products can decrease clotting by blocking thrombin activity.
Coagulation testing is a detective story. Some factors such as vWF (von Willebrand disease), factor XII (hemophilia A), factor IX (hemophilia B), and factor VIII (may be included in von Willebrand disease) may be checked for by genetic or protein testing directly. In an emergency situation in clinical practice, waiting 3 to 5 days for a result from the laboratory may mean death for the patient. Luckily, many laboratory tests are available to help us diagnose disease.

Platelet Counts and Estimates
Platelet counts can either be performed by a machine or manually. Hemacytometer platelet counts are generally unavailable at present and will not be discussed here. If platelet counts are suspect from the machine, a manual estimate may be performed on the monolayer. If 8 to 10 platelets are seen per oil immersion (100×) field, that is considered adequate. If decreased platelets are seen, the feathered edge of the slide should be examined for platelet clumps.

Buccal Mucosal Bleeding Times
Buccal mucosal bleeding time (BMBT) is an indicator of primary hemostasis (platelet function and presence of vWF). The procedure involves an awake animal. The lip is pulled back and a simplate (a standardized double lancet) is used to make two small lacerations in the mucosal membrane. A piece of filter paper is used to blot the blood from the lacerations every 10 seconds. Do not touch the wound with the filter paper as this will disrupt the clot. The normal time for clot formation is 1 to 3 minutes for dogs and cats. Increased BMBT indicates thrombocytopenia or platelet dysfunction. This can also be used as a screening test for von Willebrand disease. Aspirin therapy will cause increased BMBT.

Activated Clotting Time
The activated clotting time (ACT) is an indicator of secondary hemostasis (specifically the intrinsic and common pathways). It evaluates the ability of the blood to clot in the presence of a clot activator (diatomaceous earth). The procedure involves getting a venipuncture (see Hematologic Testing) and drawing the blood into a grey-top tube containing diatomaceous earth. The tube should be warmed to body temperature prior to drawing blood and maintained at body temperature throughout the test. Once the blood and the diatomaceous earth are thoroughly mixed, timing begins. The tube should be placed in a warm water bath or hot block and checked after 30 seconds by tilting the tube. After the initial check, the tube should be checked every 5 seconds until the blood becomes a gel. Timing stops at this point. In normal cats, the ACT should be <65 seconds. In dogs, between 60 and 90 seconds. Anything affecting factor XII or thrombin formation (heparin, vitamin K deficiency, liver failure, hemophilia A) will prolong the test.

Activated Partial Thromboplastin Time
The activated partial thromboplastin time (APTT or aPTT) also tests the intrinsic and common pathways of secendary hemostasis but is more accurate than the ACT. Blood should be drawn atraumatically into a blue-top (citrated) tube and centrifuged at low speed to separate the plasma. The plasma is drawn off and placed in a plain red-top tube and labeled citrated plasma. The plasma should be frozen and run within 4 to 6 hours. The APTT is usually sent out to a reference laboratory. The plasma is incubated with a platelet substitute that causes activation of Factor XII. Calcium is then added and fibrin formation is measured. Normal is usually between 14 to 20 seconds, depending on the laboratory. Prolonged times are abnormal. The pathologies are the same as for the ACT.

Prothrombin Time
The prothrombin time (PT) tests the extrinsic and common pathways of secondary hemostasis. Citrated plasma is again used and is added to a thromboplastin-calcium mixture and the time to formation of fibrin is again measured. The reason that this measures the extrinsic portion of the cascade is that no part of the platelet is needed to begin the cascade. The citrated plasma should be assessed within 2 hours of collection and frozen. The normal PT is 7 to 10 seconds. Prolonged PT may be a result of liver disease, vitamin K deficiency, or factor VII deficiency.

Thromboplastin Time
The thromboplastin time (TT) tests for decreased fibrinogen and is a reference laboratory test. It tests the conversion of fibrinogen to fibrin by the conversion of thromboplastin to thrombin.

Fibrin Split Products
In disseminated intravascular coagulation (DIC), there is excessive clot formation throughout the body in the small blood vessels. This occurs because of tissue damage from some other mechanism initiating the extrinsic clotting cascade (heat stroke, trauma, shock, etc.). All of the clotting factors and cofactors are used up because of the microclots, and fibrinolysis begins so that blood flow can be reestablished. Fibrinolysis begins with the activation of plasmin. Plasmin breaks down the fibrin and causes release of fibrin split products (FSPs) that normally would be broken down and recycled in the liver. In DIC, the levels of FSPs overwhelm the system and result in bleeding because FSPs inhibit the action of thrombin.
Fibrin split products can be tested at the reference laboratory on a latex agglutination test. The blood is collected in a special latex agglutination tube. The serum is added to latex particles coated with special antibodies that bind to the FSPs. The resulting antigen antibody complex is then examined to evaluate the severity of the disease.

Bone Marrow Testing
Bone marrow testing is an underutilized technique in veterinary practice. Bone marrow evaluation can provide answers for many unexplained changes in CBC values and staging of cancers. Indications for performing bone marrow aspirates are thrombocytopenias, neutropenia without sepsis, pancytopenia (both red and white blood cell decreases), leukemias (cancer of young blood cells), polycythemia (red blood cell increases without elevated total protein). and suspicion of metastatic cancers (cancers from non–blood cell origin) such as lymphoma, melanoma, or mast cell tumors.
A CBC and reticulocyte count should be performed at the same time as bone marrow studies for comparison. This allows a full picture of the blood composition to be formed and provides better diagnostic value.
Evaluation of bone marrow consists of assessing the myeloid:erythroid (M:E) ratio (the number of white blood cell precursors divided by the number of red blood cell precursors), the number of bone spicules, the fat-to-cellular ratio (space taken up by fat cells divided by the space taken up by cells), the iron stores, the number of megakaryocytes (platelet precursors), and the differential (number of various stages of myeloid and erythroid precursors).
The M:E ratio should be roughly one (there should be one white blood cell precursor to each red blood cell precursor). The myeloid series is considered to be only the granulocyte series (young neutrophils, eosinophils, and basophils) and does not include lymphocytes and monocytes. The myeloid series has blue-gray, light cytoplasm and open chromatin in the nucleus until they become segmented. The erythrocytic series has dark blue cytoplasm and the more mature (rubricyte and metarubricytes) cells have very dark, condensed chromatin.
Bone spicules appear as dark blue areas in the bone marrow that have a large number of cells around them. Fat appears as empty spaces in the bone marrow that do not have cells or stain. There should be a one-to-one ratio of cells to fat. Iron stores appear as very dark, blue-black or brown areas. Iron is stored as hemosiderin in the macrophages of the bone marrow. Lack of iron will prevent erythropoiesis.
Megakaryocytes appear as large purple to blue cells containing multiple nuclei. They are the largest cell in the bone marrow. Young megakaryocytes have fewer nuclei and are more basophilic (more blue in color) than mature megakaryocytes. There should be 3 to 5 megakaryocytes per bone spicule.
Greater than 90% of the erythrocytic precursors should be approaching maturity (rubricytes and metarubricytes). Greater than 75% of the myeloid precursors should be approaching maturity (myelocytes, metamyelocytes, and bands).
Sites for bone marrow aspiration and biopsy include the proximal humerus (best for most small animals), the wing or crest of the ilium, and the proximal femur in small animals. In cattle, the dorsal ends of T10-12 are used. In horses, the sternum is the best site (usually performed at euthanasia). Because the bone is highly innervated by pain fibers, sedation is recommended (required in cats) and adequate local anesthesia is mandatory.

Bone Marrow Aspiration
There are several types of bone marrow aspiration needles. The Rosenthal needle is the most commonly used in practice. It is a beveled, 16–18 ga needle with a solid stylet. Care must be taken to make sure the stylet is seated correctly to prevent cortical bone getting stuck in the opening.

The Illinois sternal-iliac needle may also be used. This needle has a depth gauge to prevent the needle from going too deep into the bone.
Bone marrow aspiration must be performed aseptically. The area must be clipped and prepped as for a surgical procedure and sterile gloves should be worn. Local anesthesia should be injected along the entire route of the aspiration needle down to the bone, 5 minutes before the procedure begins. The skin over the site of insertion is incised with a #11 or #15 scalpel blade and the needle is used to penetrate the subcutaneous tissue and muscle to the level of the bone. The needle is then rotated in a clockwise-counterclockwise rotation until the cortex is penetrated. The stylet is removed, a 12–20 cc syringe is placed on the hub of the needle, and negative pressure is applied to the plunger. A flash of material should be seen in the hub. More than a flash indicates blood contamination and may invalidate the sample. Negative pressure is released and the needle is removed from the animal. The syringe is removed from the needle, filled with air, and reattached to the needle. The material is expressed onto a slide or into a lavender-top tube and a squash prep is made immediately. Bone marrow coagulates extremely quickly on exposure to air. Squash preps are made by placing a drop one-quarter the length of the slide from the end, covering with another slide at a 90-degree angle, and drawing the slide to the opposite end of the drop slide. This provides a cytology sample.

Bone Marrow Biopsy
Bone marrow aspiration may not give an adequate sample because it is only sampling a small number of cells. Bone marrow biopsy allows a core sample to be obtained that is suitable for both cytology and histology.
The sites used for obtaining a core biopsy are the same as for an aspirate. The needle used is called a Jamshidi needle. The bevel of the needle is curved inward slightly to prevent the core from being pulled out of the needle when it is withdrawn. It has a stylet similar to the Rosenthal needle. The technique is the same as for an aspirate until the needle penetrates the cortical bone. On penetration of the bone marrow cavity, the stylet is removed and the needle is advanced 1 to 2 cm further into the bone marrow and rotated clockwise and counterclockwise. This is called stirring the sample. The needle is then withdrawn by twisting in one direction. The needle is then inverted and the stylet is inserted into the needle end, pushing the core onto a slide. Half of the sample should be placed into fixative for histology and half made into a squash prep.

Cell Maturation Series
All blood cells derive from the pluripotential stem cell (PPSC). This cell is a mesenchymal cell that has not differentiated or matured. It can form any of the blood cell precursors.
The cell maturation series of the erythrocytic cell line is as follows:
PPSC > rubriblast > prorubricyte > rubricyte > metarubricyte > polychromatophil > red blood cell
The erythrocytic series are all varying shades of blue. They start out large and shrink with each change in cell type. The rubriblast and prorubricyte have very dark blue cytoplasms and open chromatin and prominent nucleoli (areas within the nucleus where the genes are located). These cells can still divide. The rubricyte has no nucleoli and the nucleus is becoming condensed. There is a slight pinkinsh tinge to the cytoplasm. The metarubricyte has a very condensed, pyknotic nucleus and a definite pinkish color. The polychromatophil has no nucleus.


Blood Cell Maturation

The myeloid series is the same for neutrophils, eosinophils, and basophils. The only difference is the granules that each cell contains. Granules are seen in even very young cells (promyelocyte) making identification of the cell line possible even in young cells. The cell maturation series of the myeloid cells is as follows:
PPSC > myeloblast > promyelocyte > myelocyte > metamyelocyte > band > segmented
The myeloid series are all lighter in both the cytoplasm and the chromatin than the erythrocytic series. The myeloblast and promyelocyte are both actively dividing larger cells with prominent nucleoli in the nucleus. The myeloblast, promyelocyte, and myelocyte all have round nuclei. The metamyelocyte has an indented nucleus (like a kidney bean). The band has a tube-shaped nucleus and the segmented granulocyte has pinches in the nucleus (generally 4 to 5).
Lymphoblasts may also be seen in the bone marrow. These lymphocyte precursors are larger than mature lymphocytes and have very open chromatin (they are a lighter pink than mature lymphocyte nuclei) and prominent nucleoli.

Cytology and Histopathology
Cytology can provide quick answers and is relatively noninvasive. Most techniques may be performed in an awake patient, while histopathology requires anesthesia and a longer turnaround time for results because of the requirements of adequate tissue preparation. Cytologic samples may be read in the veterinary clinic while the client waits in the exam room. The disadvantages of cytology are the small sample size and the difficulty of analysis if the technician is not familiar with the normal appearance of a specific tissue.
Histological analysis gives a much larger sample size that can be processed for various testing procedures including fluorescent antibody testing and special staining. The normal architecture of the various layers of tissue are preserved. Pathologists trained in tissue analysis read histologic samples and analyze the samples for the practice so there are fewer mistakes.

Cytological Techniques
The ability to perform cytology in practice can improve patient care, increase the bottom line of the clinic, and help to initiate treatment of patients sooner. Proper staining of samples is very important to cytological techniques. Improper staining or stain artifacts can invalidate results. There are three basic types of cytologic stains: Romanowsky stains, nucleic acid stains, and Papanicolaou stains.
Cells seen on cytology may be divided into three categories: epithelial, mesenchymal, and round cells. Being able to differentiate cells into these categories can aid in establishing a laboratory diagnosis of neoplasia. It is also important to divide a sample into inflammatory or noninflammatory categories.






Tumors of the epithelial cells are called -omas (e.g., epithelioma) if they are benign, carcinomas if they are malignant. A glandular benign tumor is called an adenoma; a cancer is called an adenocarcinoma. One of the hallmark signs of adenocarcinoma is acinar formation, which is a group of epithelial cells with a duct in the middle.
Benign tumors of mesenchymal cells are also called -omas (e.g., fibroma, hemangioma). Malignant tumors are called -sarcomas (e.g., fibrosarcoma, hemangiosarcoma).
Inflammatory samples have neutrophils and possibly macrophages present in the sample. Septic samples are inflammatory specimens with bacteria present. Eosinophilic infiltrates can mean allergic inflammation, autoimmune disease, or parasitic infestation. In the cat, lymphocytic infiltrates can indicate antigenic stimulation or allergic or autoimmune stimulation.
Noninflammatory samples would be tumors/neoplasms or dysplasias (abnormal architecture or placement of cell types in the sample). Tumors usually have rapidly dividing cells. The nucleus is usually bigger than the cytoplasm (increased nuclear-to-cytoplasmic ratio), there may be nucleoli, and there may be mitotic figures present (spindle formation in the nucleus) in cells that should not be dividing. In dysplasias, the cells usually appear normal for the tissue type, but are arranged in a different pattern.

Fine Needle Aspiration
Fine needle aspirations are very quick and easy ways to obtain cells from solid or fluid-filled lesions. The area to be aspirated should be prepped with alcohol and a 22–20 ga needle on a 6–12 cc syringe should be inserted into the mass. Negative pressure should be applied. The plunger should then be released, the needle should be backed out slightly (not out of the lesion completely), and redirected. The procedure should be repeated two to three times. Negative pressure should be released and the needle should be removed from the lesion. The syringe is removed from the needle, filled with air, and reattached to the needle. Air is forced through the needle and the sample is expressed onto the slide. A squash prep is made (see Bone Marrow Aspiration) and air-dried. Air-drying will partially fix the sample onto the slide. The sample is then stained, air-dried, and examined under the microscope.

Impression Smear
Impression smears can be made either from wet ulcers or from cut surfaces of solid masses. The surface of the lesion or mass is blotted with a piece of gauze and the slide is touched to the surface. The slide is then air-dried, stained, and examined under the microscope. Wet ulcers may have secondary contamination by bacteria, so results may be suspect.

Scrapings
Scrapings can be performed on dry ulcers or areas of scaly skin. A #10 blade is drawn across the lesion at a 90 degree angle. Saline may be used ito prevent cutting the tissue if the slide will be stained. Oil should be used if the objective is to check for ectoparasites.

Swabs
Swabs may also be used on wet ulcers, on mucous membranes, or in fistulous tracts. Secondary contamination of lesions is common, so bacterial presence on these samples should be suspect. A sterile cotton swab is moistened with sterile saline and drawn across the lesion or inserted into the fistulous tract. The swab is then rolled over the surface of a slide, air-dried, and stained.

Fluid Testing
The accumulation of transcellular fluids in body cavities is abnormal. Samples of the fluid can aid in diagnosis of disease process and may be therapeutic in cases of abdominal and pleural effusions to increase patient comfort and decrease respiratory distress.

Techniques
Proper preparation of the patient and technique is an important part of the job of the veterinary technician. Although abdominal and thoracic aspirates and tracheal washes are within the veterinary technician's scope of practice, chest tube placement is not. It is important to check your practice act because rules in various states are very different.

Abdominal Aspiration: Abdominal aspiration, or abdominocentesis, should be performed in cases of unexplained ascites (fluid accumulation in the abdomen), abdominal trauma, and puncture wounds. It is important to take fluid samples from all four quadrants (right cranial, left cranial, right caudal, left caudal) to ensure proper sampling. The area should be clipped and prepped and sterility should be maintained throughout the procedure. An over-the-needle catheter should be used if possible to help prevent bowel penetration. The sample is drawn with a 6 cc syringe and placed into a lavender-top and a red-top tube for further analysis. The sample should be tested for total protein, creatinine, BUN, and bilirubin as well as cytology. If septic inflammation is seen, the red-top tube should be submitted for bacterial or fungal culture. Cases of ureteral or bladder rupture will have elevated creatinine and urea (BUN) in the fluid. Gall bladder rupture will cause increased bilirubin. Cells that are only found in the peritoneal and pleural spaces are called mesothelial cells. Mesothelial cells are large, epithelial type cells that may be multinucleated. Mesothelial cells have brushed borders that look fuzzy on cytology. The cytoplasm is moderately basophilic. Mesothelial cells are often confused with cancer cells because they may be multinucleated or mitotic (dividing).

Thoracic Fluid Aspiration (Thoracentesis or Thoracocentesis): There is always a small amount of pleural fluid in the chest. This allows proper expansion of the lung lobes and maintenance of negative pressure in the chest. Overproduction or abnormal fluids in the pleural space cause restrictive lung disease by interfering with negative pressure. The lungs are not able to expand because the pressure in the pleural space is the same as the pressure in the alveoli. Aspiration of pleural fluid in these cases is therapeutic (makes the animal feel better) as well as diagnostic. Hemothorax (blood), pyothorax (pus), chylothorax (chyle), and hydrothorax (serum) can all be causes for thoracocentesis. Pleural fluid aspiration should be bilateral in animals with a complete mediastinum and bilateral disease. Some animals may have incomplete mediastinums and may only require thoracocentesis on one side to achieve results on both sides of the chest. The technique is similar to abdominocentesis. The patient is aseptically prepped and sterile gloves are worn. A bleb of local anesthetic is placed under the skin and a sterile over-the-needle catheter is prepared. A stopcock is attached to the syringe and an extension set is attached. The over-the-needle catheter is advanced perpendicular to the thoracic wall, withdrawn slightly, and advanced further at a 45-degree angle until a pop is felt. The pop indicates penetration of the parietal pleura. The needle is withdrawn from the catheter and the extension set, three-way stopcock, and syringe are attached. Fluid is then drawn off. Care should be taken to avoid introducing air into the pleural space as this will also cause restrictive lung disease. Cytology, triglyceride, glucose, and lactose levels and culture and sensitivities should be performed on pleural fluid. Elevations in lymphocytes and triglycerides indicate thoracic duct leakage.

Tracheal Wash or Lavage. Tracheal washes are performed in animals with coughs, increased lung sounds, suspicious thoracic radiographs, and anything else that causes disease of the lower respiratory tract (trachea, bronchi, bronchioles, alveoli). They will not be diagnostic for restrictive lung diseases or upper respiratory tract pathology. There are two types of tracheal washes: The percutaneous or transtracheal approach, and the endotracheal approach. The transtracheal approach yields more accurate evaluations because the animal will still have a cough reflex, so material from the lower airways will have a higher possibility of being collected. In the endotracheal approach, the animals are anesthetized, so the cough reflex is suppressed. This is the approach usually used with cats. In the percutaneous technique, the area over the larynx is clipped and scrubbed. The animal is awake for the procedure. Local anesthetic is infiltrated subcutaneously at the site of the needle insertion. The animal is restrained either sitting or standing with the neck extended. A jugular catheter is then inserted into the cricothyroid ligament (the junction between the cricoid and thyroid cartilages is palpable). The catheter is advanced through the needle to the level just above the tracheal bifurcation (this distance should be premeasured). One to two ml of sterile saline per 10 pounds of animal is injected into the trachea. When the animal coughs, the material is aspirated. The sample should be placed in a lavender-top tube for cytology and a red-top tube for culture and sensitivity. The cytology sample should be centrifuged and the pellet suspended and prepared as a squash prep. The endotracheal technique requires anesthesia and endotracheal tube placement. The endotracheal tube must be sterile and small enough to allow easy passage into the trachea. Care should be taken to avoid contamination of the tube by touching the oral and pharyngeal surfaces. The cuff should be inflated and the animal placed in lateral recumbency. A jugular catheter is advanced down the endotracheal tube until it is outside of the tube, the stylet is removed, and 1 to 2 ml sterile saline per 10 pounds of body weight is placed into the trachea. Negative pressure is applied to the syringe to aspirate some of the material. If the animal is at a very low plane of anesthesia, coughing may be caused by rolling the patient from side to side. Normal cells seen on tracheal washes include ciliated columnar cells (long, rectangular cells with the nucleus more toward one end of the rectangle and hairs at the other end) and goblet cells (columnar cells filled with a purple substance called mucin) from the trachea and cuboidal cells (square cells) from the bronchi. Occasional mast cells, monocytes, and neutrophils may also be seen, but they should be fewer than 1 cell/HPF.

Fluid Types
Fluids may be obtained from arthrocentesis, cerebrospinal taps, or body cavities. Fluids give a lot of information, especially in reference to the information obtained from a chemistry panel and CBC on the blood. The technique for obtaining fluids from body cavities has been described in the previous section.
Joint fluid evaluation is important in differentiating various joint diseases. Joints should be evaluated in cases of fever of unknown origin. Any joint aspiration should be performed with a sterile prep and gloves to prevent introduction of pathogens into the joint fluid. The carpus, tarsus, and stifle are the usual joints aspirated. Joint fluid should be relatively thick and slick (this is called viscosity) and have a high protein content. Viscosity can be assessed by how long a string of joint fluid can be from the tip of the needle to a slide. Two to three cm is normal. Joint fluid should be colorless and clear. Inflammatory processes will increase cloudiness (turbidity) and protein concentration and decrease viscosity. On cytology, joint fluid should be mostly mononuclear cells. Normal cell counts are less than 500 cells/µl. Noninflammatory or degenerative processes such as osteoarthritis cause cellularity to be 500 to 5,000 cell/µl. Increases in neutrophils over 10,000 cells/µl indicate inflammatory or septic (bacterial) processes in the joint. Increases in eosinophils usually indicate autoimmune disease.
Cerebrospinal fluid collection is usually performed by the veterinarian, but is within the technician's scope of practice in most states. The animal must be anesthetized when this technique is performed and all instruments as well as the technician and the veterinarian must be maintained in strict asepsis. Trauma to the spinal cord and brain herniation may occur when performing this procedure.
Evaluation of cerebrospinal fluid should include color, clarity, total protein, culture and sensitivity, and cytology. The fluid should be clear and colorless with a very low cellularity (less than 9 cells/µl) with mostly mononuclear cells (lymphocytes). Increased protein levels indicate pathology. Increases in neutrophils usually indicate bacterial or viral infections. Increases in lymphocytes indicate viral or degenerative diseases. Increases in eosinophils indicate parasitic or fungal diseases.
Fluids obtained from body cavities (the peritoneal and thoracic cavities) are called effusions. Effusions come in three basic types: The transudate, the modified transudate, and the exudate. Differentiation of effusions is by the protein concentration and cellularity. The fluids do not always fit well into the assigned categories, so there may be some overlap in evaluation.



Histopathology
Histopathology samples are generally larger than cytologic specimens and the normal tissue architecture is maintained. Tissue samples are obtained by surgical excision of part or all of a mass. This is called biopsy. Biopsies are performed on suspicious growths (neoplasias), abnormal areas (dysplasias), and abnormal tissue locations or sizes (anaplasias and hyperplasias). Asepsis must be maintained when obtaining biopsies, and normal tissue should be included in the sample so comparisons may be made. Pathology reports should include the location of the lesion on a body map, and the size, shape, color, and texture as well as a history of the animal.
Histopathology is usually performed by veterinary pathologists and should be sent to reputable laboratories. Generally, biopsies are placed in buffered formalin for preparation. If electron microscopy is needed for viruses or other reasons, glutaraldehyde should be used as a fixative. Some studies require frozen samples or other preparation, so careful reading of laboratory requirements is important.

Clinical Chemistry
Clinical chemistries are usually run on animals as part of preanesthetic testing, as part of a diagnostic profile for disease, and to determine the effectiveness of therapy on a system. Samples may be either whole blood, serum, or plasma depending on the test. If plasma is required, heparin is the anticoagulant of choice because it does not interfere with mineral concentrations (especially calcium). EDTA, calcium oxalate, and sodium citrate form insoluble complexes with calcium that will affect levels. They will also bind to potassium in some cases. Always use the correct tube for analysis. Sodium fluoride (gray-top tube) is the anticoagulant of choice for blood glucose determination because it acts as a glucose preservative, but it should not be used for other tests.

Most chemistries can use serum for analysis and this is the preferred method if the test can use either serum or plasma. Serum must be spun as soon as possible after clot formation (30 to 120 minutes) to make sure the blood cells have minimal exposure to the serum. This will prevent further changes of values because of blood cell metabolism and degradation of proteins.
Sample volume is also important. Enough blood to run three assays will account for the possibility of technician, instrument, or reagent error. The possibility of dilution studies or add-on testing also exists, so having extra volume available is a good idea. Always fill the tube to a minimum of 75% full to ensure proper dilution with the anticoagulants and proper tube performance in clot activator tubes. Remember: The PCV is the percentage of red blood cells per milliliter of fluid, so if you draw up 10 cc of blood in a patient with a PCV of 45%, 4.5 milliliters will be cells and 5.5 milliliters will be serum or plasma.
Proper phlebotomy is as important to serum chemistries as it is to coagulation studies. Hemolysis (red blood cell lysis) and lipemia (triglycerides in the plasma/serum) will interfere with many tests by causing interference with spectrophotometric (requires the transmission of light through a sample) and colorimetric (determines the color of a sample or chemical reaction) analyses.
Proper history taking is also very important to evaluation. Young animals will have increased serum calcium, phosphorus, and alkaline phosphatase levels because of increased bone growth. Corticosteroid therapy (prednisone) will increase alkaline phosphatase and liver enzymes. Phenobarbital (seizure medication) will also increase liver enzymes. Dehydration will increase the total protein, potassium, sodium, and chloride. Low dietary protein can cause decreased total protein, low albumin, and low blood urea nitrogen (breakdown product of protein metabolism). Fluid therapy may either dilute electrolytes or increase concentrations if they are oversupplemented. Antibiotics can cause changes in some chemistries. In general, blood should be drawn prior to the initiation of therapy and then be used to monitor the animal's response.


Clinical chemistries can give a glimpse into the workings of the organ systems that may be affected by disease. Some chemistries test the organ's ability to manufacture products. These are called function tests. Others test cellular integrity, or leakiness, of the cells. These are enzyme tests. Enzyme tests generally test for tissue damage, while function tests test the effective biomass of an organ (how much of the organ is able to do the job).
Most tests may be grouped together into organ systems and this is how clinical chemistries should be approached in practice. When evaluating chemistry panels, organ-related tests should be evaluated together. This avoids confusion and enables the technician to make sense of the values and decide whether or not a particular test value may have been affected by problems in another organ system. Since many of the enzymes can be produced by more than one system, this is important to the evaluation of a chemistry profile.
In this section, the tests will be discussed as they relate to systems. Ancillary tests that relate to the function or health of the specific system will also be discussed.

Liver
The liver is one of the most important organs in the body. All of the blood from the intestines comes through the liver prior to returning to the heart. The liver processes all the nutrients, detoxifies the blood, and filters bacteria. Processing of nutrients includes lipid packaging and distribution to the rest of the body, manufacturing proteins from amino acids (coagulation proteins, albumin, globulins), formation of glycogen from glucose, and conversion of maltose and fructose to glucose. It is also the primary storage site for iron, copper, and fat soluble vitamins. Detoxification includes excretion and elimination of ammonia in the form of urea (ammonia is a breakdown product of protein metabolism) and detoxification of drugs and toxins produced by intestinal bacteria. The liver also synthesizes bilirubin from the metabolism of hemoglobin from red blood cell destruction and bile acids, which aid fat metabolism.
Different pathologies will affect different enzymes and function tests depending on the type of disease.

Liver Enzymes
Some liver enzymes will be elevated when any changes occur in the liver leading to cellular damage and cell death. Cellular damage makes hepatocytes leaky. Enzyme assays check whether leakage is occurring. This is usually not quantitative (is not an assessment of how many cells are damaged). Liver enzymes will be increased with infectious, inflammatory, neoplastic, toxic, degenerative, and traumatic changes. The main enzymes associated with hepatocyte damage are ALT (alanine aminotransferase), and AST (aspartate aminotransferase).

ALT is used as a screening test for hepatocellular disease. Most of the enzyme comes from the liver hepatocytes, but some is also manufactured in the muscles, kidneys, and pancreas. In dogs, cats, and primates it is a very good indicator of liver disease. In ruminants, ALT is not a good indicator; SDH (sorbitol dehydrogenase) is better. This test should be performed on serum. Lipemia and hemolysis will cause false elevations in this enzyme. The serum should not be frozen.
AST is not liver specific and should only be evaluated if comparisons to muscle enzyme values are made—CPK (creatinine phosphokinase) levels. If muscle enzymes are elevated along with elevations of AST, then the increase in AST is probably from muscle damage, not liver damage. Hemolysis and lipemia will cause elevations in this enzyme as well.
SDH is hepatocyte specific and is the most useful liver enzyme test in large animals. It is very unstable, so serum tests must be performed within 12 hours.
GDH (glutamate dehydrogenase) is another hepatocyte specific liver enzyme test in ruminants. It is also used in birds.
Other liver enzymes will be elevated due to cholestasis (bile flow problems). Biliary obstruction can be either extrahepatic (outside of the liver) or intrahepatic (within the liver). Causes of elevations may be inflammation, infection, neoplasia, cholelithiasis (gallstones), or drugs. The cholestatic enzymes are GGT (gamma glutamyltranspeptidase), and ALP (alkaline phosphatase).
GGT is considered a liver-specific enzyme and is the most sensitive indicator of cholestasis in most species. It is only manufactured in the liver. Care must be taken to separate the red blood cells as quickly as possible because they may decrease the enzyme levels in the serum.
ALP is another sensitive indicator of cholestasis. Production of ALP is increased when there is increased pressure within the biliary system (bile backup), inflammation of the bile ducts (pancreatitis and diabetes), or the presence of drugs such as corticosteroids and phenobarbital. It is not as sensitive an indicator in cats as it is in dogs because it remains in the system for a very short time in cats (i.e., has a short half-life). The major source of ALP is the liver, although bone, the intestinal mucosa, and the kidneys also produce the enzyme. This enzyme assay is not affected by hemolysis.

Liver Function Tests
Liver function tests assess the functional integrity of the liver (ability to synthesize products). They also test the hepatic portal system (blood flow from the intestines to the liver). Tests that are considered primary hepatic (liver) function tests include total protein and albumin (both are measures of the ability of the liver to manufacture proteins), clotting factors, bilirubin (measures the ability of the liver to break down hemoglobin), ammonia (elevations of ammonia will indicate a decreased ability of the liver to convert ammonia to urea), and bile acids tests.
The total protein has been discussed in the Hemocrit section, and the clotting factors in the Coagulation Testing section. Albumin and other protein level evaluation will be discussed further in the GI Function Tests section. In general, it is important to remember that the liver is the manufacturing center and processing center for proteins. If the liver is nonfunctional, all proteins except the immunoglobulins will be decreased (immunoglobulins are made in the lymphocytes).
Albumin is exclusively manufactured by the liver. Its function is to prevent plasma loss from the capillaries by exerting oncotic pressure. Loss of albumin will result in edema. Decreased albumin will also cause decreased calcium levels because most of the calcium in the serum is bound to albumin. Most of the total protein is albumin. Albumin testing should be performed on serum.
Severe liver disease will cause coagulation defects because of the lack of clotting factors and vitamin K storage. The PT and APTT can be used to assess secondary coagulation (clotting factors). These tests should be run on citrated plasma.
Bilirubin is the breakdown product of the heme portion of hemoglobin. The liver normally clears hemoglobin from the blood by conversion to unconjugated/indirect bilirubin. Unconjugated bilirubin is then converted to conjugated/direct bilirubin which is found in the serum. Unconjugated bilirubin levels increase with excessive red blood cell destruction (prehepatic disease). Conjugated bilirubin levels increase with both hepatocellular damage and cholestasis. The serum chemistry usually gives the total bilirubin and the conjugated bilirubin levels. The unconjugated levels can be determined by subtracting the direct bilirubin from the total bilirubin. Bilirubin will be affected by hemolysis, lipemia, and light exposure. Light will decrease the unconjugated bilirubin level by 50% per hour. Hemolysis will decrease bilirubin concentrations with some assays. Bilirubin levels will not be affected by problems with the hepatic portal circulation.
Ammonia is the breakdown product of protein metabolism. Ammonia is very toxic to most of the tissues in the body, especially the central nervous system. The liver normally metabolizes the ammonia into urea. Abnormal processing of ammonia can be due to loss of portal circulation (portosystemic shunt disease) or loss of liver tissue (cirrhosis). Increased levels of ammonia will cause seizures and coma, usually in relation to feeding times. The animal eats protein, breaks it down, and the levels of ammonia rise and the animal will become comatose or have a seizure. Once the ammonia is cleared, the animal's mental status may improve. This test must be run on heparinized plasma (green-top tube) within 1 to 3 hours of phlebotomy because it is very unstable.
Serum bile acids (SBA) are synthesized in the liver from cholesterol. Most bile acids that are excreted by the liver are reabsorbed into the portal circulation and recycled, so there is a constant level maintained in the blood stream. SBA are normally stored in the gallbladder and eating a meal causes the release of bile acids into the intestines. Bile acids function in fat absorption and metabolism. Generally SBA are measured in serum samples after a 12-hour fast (preprandial). The animal is then fed and the test is repeated in 2 hours (postprandial). If pre- and postprandial levels are elevated, there is a problem with hepatic function.
Other tests that can help with evaluation of liver function are blood glucose, cholesterol levels, and triglycerides. Liver failure is often associated with mild to moderate hypoglycemia (low blood sugar) because of the inability of the liver to manufacture or break down glycogen. Cholesterol and triglycerides also rely on the liver and the bile acids for transport. Cholesterol and triglycerides may be elevated in cholestasis.

Kidneys
The kidneys function in water and electrolyte conservation and elimination, acid base balance, elimination of urea and creatinine, conservation of glucose and protein, maintenance of blood pressure, activation of vitamin D, and red blood cell production (erythropoetin production). Renal failure indicates that the kidneys are no longer able to maintain these functions. In order to call kidney disease renal failure, over 75% of the kidney must be nonfunctional. Uremia (azotemia) occurs in renal failure and is the clinical syndrome associated with the loss of function.
Since the kidney's ability to concentrate urine is controlled by the pituitary and adrenal glands, these function tests will be covered in the appropriate section on hormone testing. Azotemia, or elevation of waste products in the blood, and function tests should always be evaluated along with the urinalysis because the urinalysis is often more sensitive to renal disease than the chemistries. In fact, azotemia will not increase to diagnostic levels until the kidney is >75% damaged.

Kidney Enzymes
BUN (blood urea nitrogen) and Cr (creatinine) are both products of protein metabolism. They are both excreted as waste products by the kidney. High concentrations will occur in animals with renal disease.
Creatinine is derived from muscle breakdown. It is filtered and excreted by the kidney and is maintained at fairly constant blood levels. The advantage of Cr over BUN is that it is not affected by dietary protein. Creatinine is also more specific for kidney function than BUN. Neither Cr or BUN is affected by hemolysis.
The BUN, as stated in the previous liver section, is the endpoint of protein breakdown. Increases of the GFR (glomerular filtration rate, the amount of urine produced by the glomerulus) will reduce the BUN in the circulation. Decreased GFR will increase the BUN in the circulation. Increase in waste products is called azotemia. There are three types of azotemia: prerenal, renal, and postrenal.



Kidney Function Tests
Kidney function tests assess the ability of the kidneys to concentrate urine. This is under the influence of two hormones: aldosterone and antidiuretic hormone (ADH).
Antidiuretic hormone is produced by the hypothalamus and stored in the posterior pituitary gland. It targets an area in the renal tubules called the distal convoluted tubules and collecting ducts, and increases the resorption of water by those tubules. Failure of antidiuretic hormone can cause polyuria and polydipsia. This can be tested by water deprivation and vasopressin tests.
Aldosterone is a hormone produced by the adrenal cortex. It controls the reabsorption of sodium at the distal convoluted tubule. Water follows sodium, so water is conserved as well. Decreased aldosterone will result in polyuria and polydipsia because sodium is allowed to leave the kidneys. This occurs in hypoadrenocortism.
The best way to measure the ability of the body to concentrate its urine (and retain water) is to measure the specific gravity (s.g.) of the urine. The specific gravity indicates the density (particles per volume) of urine. Large particles such as proteins and glucose have a greater effect on the s.g. than electrolytes. A specific gravity of 1.000 is the s.g. of distilled water. Urine with an s.g. of 1.001–1.012 is considered isosthenuric, which means that the animal is unable to concentrate its urine. An s.g. >1.016 in a dog and >1.030 in a cat means that the animal has the ability to concentrate its urine. Normal concentrating ability means an s.g. >1.030 in the dog and >1.040 in the cat. Urine s.g. should always be considered in the face of TP (hydration status) and urine protein.
Other tests that should be evaluated with kidney problems are phosphorus, calcium, sodium, potassium, chloride, and total CO2. Since electrolytes are cleared or reabsorbed in the kidneys and the kidneys are responsible for acid–base balance, there may be changes in these levels, especially in severe renal disease.

Electrolytes
Electrolytes are the charged ions that circulate in solution in the plasma. Positively charged ions are called cations (sodium, potassium, calcium, and magnesium) and negatively charged ions are called anions (chloride, bicarbonate, phosphate). Electrolytes may be further broken into groups by whether they are found within the cells (intracellular ions) or in solution in the plasma (extracellular ions). The major intracellular ions are potassium, magnesium, and phosphate. The major extracellular ions are chloride, sodium, and calcium. This is important because, in cases of hemolysis, the intracellular ions may be elevated in the serum tests.
Electrolytes are usually expressed in mmol/L or mEq/L, which are concentrations based on molecular weight (how much individual atoms weigh). Electrolytes function in nerve and muscle activity, as cofactors for enzymes, as acid–base regulators, and in the maintenance of water balance.
Sodium (Na+). Elevated sodium levels (hypernatremia) are caused by diabetes insipidus (loss of water without sodium loss because of ADH deficiency), excessive sodium supplementation, hyperadrenocorticism (water loss without sodium loss), and dehydration. Hyponatremia (sodium decreases) can be caused by excessive diuretic use (loop diuretics such as furosemide will cause loss of sodium), and chronic vomiting.
Potassium (K+). 98% of the body's potassium is found within the cell, so the serum levels do not reflect the severity of potassium decreases. Hyperkalemia (elevated potassium) is seen in renal failure, urethral obstruction, hypoadrenocorticism (aldosterone deficiency causing sodium loss and potassium conservation), or due to oversupplementation. Hypokalemia can result from fluid loss, use of loop diuretics such as furosemide, chronic vomiting, and hyperadrenocorticism.
Magnesium (Mg+2). Less than 1% of the total body magnesium is available for testing because 60% is found in the bones and 40% is contained within cells. Of the available 1%, one-third is bound to albumin. Serum must be used for testing. Hypermagnesemia can occur secondary to increased supplementation, decreased renal output, hypoadrenocorticism (aldosterone deficiency), chronic renal failure, and diabetic ketoacidosis. Hypomagnesemia can be caused by anything that causes decreased albumin levels, chronic diarrhea, or poor gastrointestinal absorption. Magnesium absorption is affected by calcium and phosphorus absorption.
Calcium (Ca+2). Calcium is a major component of bone (99% of the body's calcium is bound in the bone). Of the other 1%, over half is bound to albumin. The rest is free in the serum (ionized calcium) and is the active form. Calcium measurement must take into account both the bound and the unbound calcium.

The formula for corrected calcium level is as follows:
Corrected total calcium = total serum calcium – (serum albumin + 4)
This formula is only valid for dogs and should be used if the albumin level is high or low. Calcium measurements should only be performed on serum. Hemolysis will not affect results.
Calcium is regulated by three different mechanisms. Vitamin D is a calcium carrier and facilitates calcium uptake across the intestinal mucosa. Parathyroid hormone (PTH) is required for maintenance of calcium balance. PTH is made in the parathyroid glands and stimulates calcium mobilization from bone, kidney resorption of calcium in the distal convoluted tubule, and gastrointestinal absorption of calcium. All these mechanisms increase serum calcium concentrations. Calcitonin works against PTH by decreasing bone resorption of calcium. Calcitonin does not affect kidney or gastrointestinal uptake of calcium. Calcitonin will decrease serum calcium levels. Hypercalcemia (increased levels of calcium in the serum) can be caused by bone tumors and septic osteomyelitis because it causes osteolysis (bone destruction), which releases calcium. Hyperparathyroidism stimulates the production of PTH, which also causes hypercalcemia. Hypocalcemia (low blood calcium) is usually caused by hypoalbuminemia (low albumin levels).



Phosphate (PO4–2). Phosphate is the primary anion of intracellular fluid. It is the ionic form of phosphorus. 80% of the phosphorus in the body is found in the bones along with calcium. 14% is found in the muscles and only 1% is found in the serum. Serum should be tested for phosphorus levels. Hyperphosphatemia can occur with renal failure, hypoparathyroidism (decreased PTH) and hemolysis (intracellular anion). Hypophosphatemia can be caused by metabolic alkalosis, neoplasia, and hyperparathyroidism (increased PTH).
Soft-tissue mineralization is a problem that can occur with imbalances of calcium and phosphorus. A calculation that can be used to determine the severity of disease and the likelihood of soft-tissue mineralization is as follows:
Total serum calcium × serum phosphorus
If the product is greater than 60, there is an increased risk of soft-tissue mineralization.
Chloride (Cl–). Hyperchloremia (excessive chloride) is caused by dehydration, renal failure, and metabolic acidosis (blood pH <7.35). Hypochloremia is caused by increased sweating (especially in horses), vomiting and diarrhea, and metabolic alkalosis (pH >7.45).
Bicarbonate (HCO3–). Metabolic acidosis occurs when bicarbonate levels fall, causing the pH of the blood to drop. This may be caused by renal failure, ketoacidosis, or hyperparathyroidism. Compensation for metabolic acidosis is hyperventilation (the animal is trying to cause respiratory alkalosis). Metabolic alkalosis occurs when the bicarbonate levels increase, causing the blood pH to rise. This may be caused by Addison's disease (hypoadrenocorticism) or anesthesia.

Proteins
Protein levels have been discussed in previous sections. Protein evaluation is performed by use of a refractometer in practice (see Red Blood Cell Indices). If serum is measured, the protein will reflect only the albumin and circulating globulin fractions, not the clotting proteins (fibrinogen). Plasma contains all the proteins. Other methods of protein evaluation include serum electrophoresis. Electrophoresis shows relative concentrations by the molecular weights, sizes, and charges of the proteins and allows evaluation of individual protein types.
Protein levels can be an indicator of hydration in animals and should be evaluated along with PCV, urinalysis, and the chemistry. Hemolysis and lipemia can affect refractometer results.





Pancreatic Testing
The pancreas acts as both an exocrine and an endocrine gland, which means that it affects both digestion and metabolism. Diseases of the pancreas may affect the liver and vice versa because of the close proximity of the organs. Ascending infections from the gastrointestinal tract may also cause diseases of the pancreas. The cells of the exocrine portion of the pancreas surround ducts. These are called the acinar glands. Between these rows of cells in the pancreas lie the islets of Langerhans, which contain the cells that have endocrine function. Although the cells are not truly mixed together, inflammation of the exocrine cells (pancreatitis) can lead to damage of the endocrine cells (diabetes mellitus). For this reason, the endocrine pancreas is included here rather than in the Hormone Testing section.

Endocrine Testing
The endocrine pancreas regulates glucose metabolism. Two hormones are produced by the islets of Langerhans: glucagon and insulin.
Glucagon is made by the alpha cells in the islets of Langerhans. Glucagon acts on the liver (hepatocytes) to convert stored glycogen into glucose and stimulates gluconeogenesis (the formation of glucose from amino acids). This has the net effect of raising blood glucose. The secretion of glucagon is stimulated by decreased glucose in the blood.
Insulin is made by the beta cells in the islets of Langerhans. Insulin acts on the cell membranes of all the tissues in the body to increase cell uptake of glucose. This lowers glucose in the blood. The secretion of insulin is stimulated by rising glucose levels. When insulin levels fall, blood glucose rises; when insulin levels rise, blood glucose falls.

Serial Blood Glucose Testing
Blood glucose tests can be performed on either whole blood, serum, or plasma. If plasma is used, it should be collected in a sodium fluoride (gray-top) tube. If serum is used, the blood glucose will be low because blood cells use glucose for metabolism and the cells are in contact with the serum for >30 minutes prior to spinning and separating. If whole blood is used with a glucometer, care must be taken to ensure that the glucometer is calibrated properly for animal blood. Some of the quick glucometers will give falsely low values for canine and feline blood samples. Call the manufacturer to make sure the glucometer you are using is appropriate for your species. Normal blood glucose is usually 70 to 120 mg/dl across species.
Hypoglycemia (low blood glucose) can occur due to technician error, insulinoma (cancer of the beta cells of the pancreas that secrete insulin), insulin therapy, hypoglycemia of toy breeds, liver disease, starvation, and septicemia (bacterial infection in the blood).
Hyperglycemia can occur from diabetes mellitus (most common cause), glucocorticoid usage (prednisone), hyperadrenocorticism (overproduction of glucocorticoids by the adrenal gland), pancreatitis, or administration of glucose-containing fluids.
Serial blood glucose monitoring every 2 hours can establish a glucose curve, which is used for monitoring insulin therapy. A glucose curve is essential for beginning insulin therapy because it explains the way insulin works in a particular animal. The blood glucoses are charted on a graph with the glucose concentration on the vertical axis and the time after insulin on the horizontal axis. A line connects the dots and a curve is established that tells the rise and fall of glucose for that particular animal on that particular form of insulin. If the glucose levels fall and rise within a 6-hour period, the animal will probably need twice-a-day insulin therapy. If the levels fall and rise in a 12- to 14-hour period, once-a-day insulin will be sufficient.
Note: Stressed cats will get a stress-induced hyperglycemia that may increase their blood glucose levels from normal to 400 g/dl, so establishment of glucose curves may be difficult in some cats.

Fructosamine Levels
In animals that have been on insulin therapy and are well controlled, fructosamine assays may be used to monitor glucose levels. Fructosamine is a glycosylated protein (a protein bound to glucose). It is actually albumin bound to glucose in an irreversible bond. This test will establish whether the animal has had adequate glycemic control (control of glucose levels) over a two- to three-week period. Because the protein binding is irreversible, the levels will not be affected by stress-induced hyperglycemia, so it is a much better monitoring technique than serial blood glucoses in the cat. This test is good as long as blood protein values (TP) is normal. If the animal is hypoproteinemic, the test will show false low values. Serum should be used.

Glycosylated Hemoglobin Levels
Glycosylated hemoglobin is hemoglobin irreversibly bound to glucose. This test has the advantage of being an even longer-term monitor of glucose control (2 to 3 months), so it is good for animals that have been stable on insulin for several months. This test should be performed on EDTA whole blood (purple-top tube).

Exocrine Testing
The exocrine function of the pancreas is digestion. The pancreas secretes many different products into the proximal small intestine including lipase (digests fat), amylase (digests sugars and starches), trypsin (digests proteins), chymotrypsin (also digests proteins), and nucleases (digest RNA and DNA). It also secretes bicarbonate, which makes the pH of the intestines more alkaline (high pH). The action of all these enzymes together is called the digestive activity. High levels of the enzymes in the blood mean that the pancreas has become leaky, which could lead to pancreatitis (inflammation of the pancreas). Low enzyme production by the pancreas can lead to maldigestion, or inefficient breakdown of food in the intestines. Pancreatic exocrine testing should be performed on serum when available.

Diagnosis of pancreatitis is based on clinical history (consumption of high-fat diet and obesity in dogs, stress in cats), physical exam (vomiting with abdominal discomfort), clinical chemistries (amylase, lipase, PLI), and ultrasound. Damage from pancreatitis may lead to exocrine pancreatic insufficiency (EPI), which causes maldigestion syndrome, or to diabetes mellitus. In dogs, clinical signs are much more severe than in cats, but the damage caused is the same. It is estimated that 40% of cats with diabetes have some evidence of either chronic pancreatitis or damage from acute pancreatitis at necropsy.
EPI is characterized by a normal CBC, normal serum chemistries, and normal urinalysis in an animal with chronic weight loss, diarrhea, and large-volume feces. German Shepherds are predisposed to EPI genetically, but it is possible in any breed. Diagnosis is by clinical signs, TLI, and PLI.

Amylase
Amylase breaks down starches into glucose. The pancreas is the major source for amylase, but the kidneys, liver, and GI tract also contribute to the total. Because it has so many sources, amylase is not a very sensitive indicator of pancreatic function. Amylase testing is usually done by adding a dye to the sample that binds to starches to form a color complex. As amylase reacts with the starches, the color decreases. The color is measured by a spectrophotometer. Because it is a colorimetric test, hemolysis will lead to false elevations while lipemia will lead to falsely low values. Amylase values should be examined along with lipase, kidney, and liver values. If the amylase is elevated and elevations of BUN and Cr are seen, the test is probably a false positive. If lipase is elevated at the same time, it is probably pancreatic in origin.

Lipase
Lipase breaks down fats into fatty acids. Most lipase is derived from the pancreas, so it is a more sensitive indicator of pancreatic activity than amylase. Serum samples are recommended for evaluation. A greater than threefold increase above normal usually indicates pancreatic disease. This test will also be affected by hemolysis and lipemia.

Pancreatic Lipase Immunoreactivity
The pancreatic lipase immunoreactivity (PLI) test is an extremely sensitive test of pancreatic dysfunction. It has an 82% specificity for pancreatitis. The test is actually a test of the lipase specifically derived from the pancreas. Because there is a slight difference in structure between cat and dog lipase, two different tests have been developed. The cPLI (canine PLI) is actually a tabletop ELISA test. The fPLI (feline PLI) currently still must be sent to the laboratory.

Trypsinlike Immunoreactivity
Trypsin breaks down proteins. Both trypsin and trypsinogen (its precursor) are specifically derived from the pancreas. Like lipase, trypsin is species specific. Trypsinlike immunoreativity (TLI) is a good indicator of functional pancreatic mass, so can be helpful in diagnosing both EPI (low levels) and pancreatitis (high levels). Because trypsin is cleared through the kidneys, levels may be increased with renal disease.

Gastrointestinal Function Tests
The gastrointestinal (GI) system is responsible for final breakdown of food, absorption of nutrients, and excretion of wastes. Intestinal function tests demonstrate the ability of the organs to perform their jobs.
Different things can cause gastrointestinal disease. Maldigestion (as stated in the section on Pancreatic Testing) can occur from a lack of gastric secretions or pancreatic or GI enzymes. Lack of hydrochloric acid release from the stomach prevents the denaturing (unfolding) of the long protein molecules and inhibits the onset of protein digestion. EPI due to pancreatic dysfunction prevents initiation of carbohydrate and lipid and the continuation of protein digestion. Intestinal mucosal disease prevents the secretion of the intestinal enzymes responsible for completion of digestion. Malabsorption is the failure of materials to cross the mucosa into the blood stream. This is usually caused by small intestinal bacterial overgrowth or other diseases that cause thickening of the bowel mucosa. Malassimilation is a combination of maldigestion and malabsorption.
Symptoms associated with gastrointestinal disease are a result of one or more defects in digestion. Steatorrhea is excess fat in the feces caused by a lack of fat digestion or absorption (lipase deficiency). Creatorrhea is undigested muscle fiber in the feces. This may be caused by a failure of protein digestive enzymes. Amylorrhea is undigested starch in the feces caused by lack of carbohydrate digestion. Melena (black, tarry stool) indicates upper GI bleeding (digested blood) from the small intestines or stomach. Hematochezia (frank, or visible, blood in the stool) indicates lower GI bleeding from the cecum, rectum, or colon.
All animals with GI disease should have a gross, direct, and flotation fecal exam performed. Other fecal evaluations should be performed if the answers are not diagnostic. Ancillary tests include microscopic exam, fecal protease/gelatin digestion, and fecal occult blood. Blood tests should include cobalamin and folate testing as well as assessing exocrine pancreatic function.

Microscopic Examination of the Feces
A number of in-house tests may be used to differentiate diseases and aid in establishing therapy immediately prior to receiving confirmation from the laboratory. They are not highly accurate and should not be used as the sole diagnostic test.
Gross examination of the feces includes recording its color, texture, volume, odor, and consistency. Greasy-looking, foamy stools may indicate excess fat in the stool, for example.
Several microscopic stains can help differentiate the various materials found in the stool. Fats can be stained with Sudan III or IV. If more than 2 to 3 globules of orange-red are present per 40× field, a lipase deficiency is suggested (maldigestion). The stained slide is then heated. If there are still globules of orange-red present, free fatty acids are present in the stool indicating malabsorption of fats. High-fat diets may cause false positives.
Carbohydrates can be assessed using Lugol's iodine. Iodine binds with starch, forming blue/black granules. If a lot of granules are present, this suggests amylase deficiency (maldigestion). This test may cause a false positive if the animal is on a low-quality dog food.
No stain is required for muscle fiber visualization, but Diff-Quick may be used to make them more visible (do not stain feces in the same Diff-Quick stain used for cytology). If 2 to 3 fibers are seen per 40× field, it is probably a protease deficiency (may be pepsin, trypsin, or intestinal proteases).

Gelatin Digestion Test
The gelatin digestion test is also called the fecal protease test. This test is diagnostic only for pancreatic proteases (chymotrypsin and trypsin). The feces are mixed with gelatin. If trypsin is present, the gelatin will dissolve. The test can also be performed by mixing the feces with bicarbonate and placing a piece of undeveloped x-ray film (which has gelatin on a plastic base) in it. If the gelatin dissolves (the film clears), trypsin is present and active. This is not a very accurate test.

Cobalamin and Folate
Intestinal bacteria synthesize and secrete vitamins that are absorbed into the systemic circulation. Bacterial overgrowth will increase the levels of these vitamins in the blood.
Cobalamin is vitamin B12. It is normally protein bound and needs a pancreatic enzyme in order to be absorbed in the ileum. Decreased serum cobalamin is seen in distal small intestinal mucosal disease (ileum) and increases are seen with bacterial overgrowth and EPI.
Folate is vitamin B9. Absorption normally occurs in the proximal small intestine. Decreased levels are seen in small intestinal mucosal disease (proximal small intestine) and increases are seen with bacterial overgrowth and EPI.
Both vitamin levels are checked at the same time. If only cobalamin is decreased, the disease is in the ileum. If folate is decreased, the disease is duodenal/jejunal. If both are increased, bacterial overgrowth or EPI is present.

Other Tests
The fat absorption test may also be a gauge of GI function. The animal is fasted for 12 hours, then a baseline sample is drawn. Triglycerides are measured. Peanut or corn oil is given orally, then hourly blood samples are drawn to check for lipemia. Lipemia should be present by 4 hours after the oil dose. This means that lipid has crossed the intestinal mucosa.
The same type of test may be performed with D-xylose or other carbohydrates. If the blood concentrations increase after 4 hours, there is carbohydrate absorption.

Hormone Testing
The endocrine glands of the body control homeostasis (the ability of the body to maintain proper functions). The endocrine glands all work in concert with each other in a series of feedback loops. Certain hormones raise substances in the body and other hormones will decrease their levels. It is a series of checks and balances.

Thyroid
The thyroid gland is responsible for metabolism and calcium regulation (calcitonin; see Electrolytes). Thyroid hormones cause activation of the mitochondria, which causes increased energy to be produced in the form of ATP. The hormones of the thyroid gland include T3 (triiodothyronine), T4 (tetraiodothyronine), and calcitonin.
Thyroid hormone release is under the control of the anterior pituitary gland (thyroid-stimulating hormone, or TSH) and the hypothalamus (thyrotrophin-releasing factor, or TRF). Excessive levels of T4 will cause feedback inhibition (decreased production) on both the hypothalamus and the anterior pituitary gland.

T4 and fT4
T4 accounts for 80% of the total hormonal output of the thyroid gland. Most (99%) is bound to albumin. The other 1% is called free T4 (fT4) and is the form that can enter cells. T3 is actually the bioactive form that causes changes in metabolism. A small amount is manufactured by the thyroid gland, but most is converted to T3 within the cells. This conversion is called deiodination.
Thyroid hormone increases metabolic activity. It is responsible for maintenance of body temperature as well as protein, carbohydrate, and lipid metabolism. It encourages protein synthesis, decreases carbohydrate conversion to fat, and stimulates lipid breakdown. Hypothyroid animals are usually obese while hyperthyroid animals are very thin.
Serum levels of T4 and fT4 are measured by radioimmunoassay. The requirements are dependent on the particular laboratory. T4 can also be checked using an ELISA test. This is not adequate for evaluation of hypothyroidism (decreased T4, T3, and/or elevated TSH), but is diagnostic for hyperthyroidism.
T3 and fT3 levels are usually produced in such low amounts that they are difficult to measure, so are typically not evaluated.

Thyroid-Stimulating Hormone
TSH is produced by the pituitary gland. High TSH levels usually indicate hypothyroidism. Low TSH numbers can indicate hyperthyroidism because T4 will cause negative feedback on the pituitary. This test must be run at a reference laboratory.

Adrenal Gland
The adrenal gland is responsible for production of several hormones. The adrenal cortex secretes sex hormones, mineralocorticoids (aldosterone), and glucocorticoids. It is responsible for metabolic hormonal control of glucose utilization (along with insulin and glucagon), and maintenance of mineral balance in the kidneys. It also acts as a secondary source for sex hormones. The adrenal medulla secretes epinephrine which is responsible for flight-or-fight mechanisms and sympathetic responses. The medullary and sex hormones will not be discussed in this section.


Aldosterone is produced by the zona glomerulosa of the adrenal cortex. It is a mineralocorticoid (a mineral-controlling hormone produced by the adrenal cortex) specific for the control of the retention of sodium in the distal convoluted tubule of the kidney nephron. Sodium is retained in exchange for potassium and hydrogen ion, which helps to maintain the acid–base balance of the kidney. Because water always follows sodium, water is also retained in the body. Retention of water increases blood pressure.
Glucocorticoids are produced by the zona fasciculata of the adrenal cortex. Glucocorticoids are responsible for regulating glucose metabolism. They inhibit protein synthesis and can increase protein breakdown (catabolism). They have an anti-insulin effect and can decrease the animal's response to insulin (insulin-resistant diabetes). Increases in glucocorticoids results in a potbellied appearance due to muscle loss, increased glycogen storage in the liver (which can cause elevations in alkaline phosphatase), and decreased immune competence (because of old, hypersegmented neutrophils in the circulation).
Control of hormone secretion by the adrenal cortex is under hypothalamic (corticotrophin- releasing factor, or CRF) and anterior pituitary gland (adrenocorticotrophic hormone, or ACTH) control. ACTH causes activation of all the hormones of the adrenal cortex (aldosterone, glucocorticoids, and sex hormones). Negative feedback inhibition is caused by excessive levels of glucocorticoids in the circulation. This is important in therapy of animals with corticosteroids such as prednisone. If glucocorticoids are administered, the production of ACTH will be suppressed. Suddenly discontinuing glucocorticoids can result in exceedingly low levels of glucocorticoid and mineralocorticoids being released from the adrenal glands, which may result in life-threatening elevations of potassium. For this reason, the production of glucocorticoids and ACTH are tested as indicators of adrenal cortical function, not aldosterone production.

Baseline Cortisol
Cortisol (glucocorticoid) is the substance tested for all adrenal tests. A baseline cortisol level provides a starting point for adrenal testing, but does not give diagnostic information. Low cortisol levels may indicate Addison's disease (hypoadrenocorticism). High cortisol levels may indicate hyperadrenocorticism, but may also indicate stress.

ACTH Stimulation Test
The ACTH stimulation test is a screening test. It works by stimulating the adrenal gland to produce glucocorticoids (cortisol) by giving an injection of an ACTH analog. Exaggerated elevation of the cortisol level indicates hyperadrenocorticism. Failure to increase the cortisol level indicates hypoadrenocorticism. The procedure for testing is as follows. A baseline cortisol level is taken. ACTH is injected into the animal. The cortisol level is then taken 2 hours after the injection is given.
This test is also used to monitor Lysodren® therapy (0,p' = ddd or mitotane), or trilostane therapy for hyperadrenocorticism.
The ACTH stimulation test should be performed along with a high-dose dexamethasone suppression test to help initially diagnose whether an animal has pituitary-dependent or adrenal-dependent hyperadrenocorticism.

Low-Dose Dexamethasone Suppression Test
The low-dose dexamethasone suppression test (LDDS) is also a screening test for hyperadrenocorticism. In theory, dexamethasone should suppress production of glucocorticoids because of negative feedback inhibition of ACTH. The procedure is similar to the ACTH stimulation test.
Baseline cortisol is obtained and dexamethasone (0.01 mg/kg in dogs, 0.1 mg/kg in cats) is injected into the animal. Postinjection samples are taken at 4 to 6 hours and again at 8 hours post injection. There should be a decrease in the cortisol level after dexamethasone injection.

High-Dose Dexamethasone Suppression Test
The high-dose dexamethasone suppression test (HDDS) is used to distinguish between hyperadrenocorticism of pituitary origin and adrenal origin. The dose of dexamethasone is ten times higher (0.1mg/kg in dogs, 1.0 mg/kg in cats) than in the low-dose test, but the technique is the same. If the post-test cortisol is less than 50% of the pretest value, the animal probably has a pituitary tumor. If it is greater than 50% of the baseline, it is probably an adrenal tumor. Most dogs have pituitary-dependent hyperadrenocorticism. Most ferrets have adrenal-dependent hyperadrenocorticism. Since adrenal tumors can usually be removed, performing the HDDS along with the ACTH stimulation test is important diagnostically.

Reproductive Testing
Reproductive testing is extremely important in production animals and also in breeding stock in small animals. Failure to produce semen or offspring can mean the difference between maintaining that animal in a herd or culling. By evaluating reproductive status, the farmer or breeder can improve the pregnancy rate, milk production, and litter size of the animal and prevent infertile animals from being a drain on valuable resources such as feed. Reproductive testing also allows artificial insemination to be successful, thereby decreasing the numbers of sexually transmitted diseases that occur in companion and production animals.

Female
Control of reproductive status in animals is under the influence of the hypothalamus (GnRH) and the anterior pituitary (FSH and LH), causing ovarian release of either estrogen or progesterone. The release of the various hormones is associated with certain cellular changes on vaginal swabs.
The hormones that are usually tested are estradiol (estrogen) and progestins (progesterone). FSH may also be tested. FSH is produced by the anterior pituitary gland, which causes formation of a follicle on the ovary. This follicle produces estrogen, which rises until the follicle formation is complete (mature follicle). When estrogen is at its peak, LH is released from the anterior pituitary gland. LH causes the follicle to rupture, releasing the egg. The ruptured follicle transforms into a corpus luteum (yellow body) under the influence of LH and begins to produce progesterone. During this time, the estrogen levels are decreasing rapidly. If the animal becomes pregnant, progesterone continues to be produced by the corpus luteum until the end of pregnancy. If the animal does not become pregnant, the corpus luteum dissolves under the influence of prostaglandin F2 alpha (PGF2a), progesterone levels fall rapidly, and the ovary becomes quiet. Estradiol levels are usually at their peak just prior to ovulation (proestrus). Progestin level elevations are an indicator of pregnancy.
The estrous cycle generally has four phases: proestrus, estrus, diestrus, and anestrus. Specific changes seen on vaginal smears made during each phase are associated with the hormonal, physical, and behavioral changes in the animal. Vaginal smears can be used for timing breeding as well as diagnosis of some pathologies, and are much less expensive than hormonal testing. The animal should be restrained in a standing position and the vulva should be cleaned prior to swabbing. A sterile and lubricated vaginal speculum is then inserted into the vulva and advanced into the vagina. A sterile, moistened cotton swab is then gently rolled against the vaginal wall, removed, and rolled on a clean slide. The slide is then air-dried, Diff-Quick stained, and examined under the microscope at 10× and 40×. The cellular changes will be evaluated and the stage of estrus recorded.


Abnormal cytologies can include nonepithelial cell invasion (cancers) and inflammatory cells (neutrophils) in large numbers during anestrus (pyometra).

Male
Semen testing of males is important to limit sexually transmitted diseases, improve genetics of the herd, and increase chances of conception. Some dog and cattle breeds have congenitally low sperm counts or inadequate ejaculate, but the genetics of the animal may be so desirable that they must be used as breeding stock. Artificial insemination can improve conception rates.
Semen is extremely fragile and must be handled carefully when performing laboratory tests. All materials used in semen handling must be dried thoroughly and warmed to body temperature (98.6° Fahrenheit) prior to use to maximize sperm motility and prevent damage. Water and disinfectants will kill sperm quickly. All materials should be assembled prior to sample collection so that the sperm is not exposed to environmental conditions. Materials needed are artificial vagina, collection tubes, slides, coverslips, stains, and diluents.
Sperm evaluation should include all of the categories because each test assesses different attributes of the semen that may be important to fertility of the animal.
The ejaculate is the entire semen volume. This may be measured at the time of collection. It consists of three fractions: The sperm-free, sperm-rich, and sperm-poor fractions. The sperm-free fraction is the accessory sex gland secretions. The sperm-rich fraction is mostly sperm cells. The sperm-poor fraction contains sperm and prostatic secretions. The sperm-poor fraction should not be collected in dogs, horses, or pigs because it will dilute and decrease the quality of the sperm in these species. All other species should have all three fractions collected. The gross appearance of sperm should be recorded as color and opacity (thickness). The color is usually white to gray. Blood contamination will change the color. The opacity is usually recorded for those animals with highly concentrated semen (ruminants). Other species have more translucent semen and the opacity may be difficult to evaluate.
Sperm motility is the movement of the sperm in the sample. Motility indicates how much activity is present in the sperm. Strong swimmers make it more likely that the sperm will get to the oviduct of the female and penetrate the egg.
Wave motion is one method of motility assessment. This is performed by placing a drop of semen on a warmed slide and examining on 10× to evaluate the amount of movement present. This activity depends on high sperm concentrations, so it works best on ruminant semen.
A true motility test should be performed in animals with more dilute semen. This tests the motility of individual spermatozoa. A drop of semen is diluted with a drop of warmed physiologic saline (0.9% saline) on a warmed slide and covered with a coverslip. The drop is examined at 100× and individual spermatozoa are examined to see whether they travel. They are classified by the amount of movement, rapidity of movement, and whether they travel in a straight line (linear movement). The percentage of good swimmers is then estimated. This test is subject to poor sample handling, so it should be repeated if bad results are obtained, to rule out error.
Sperm concentration is also an important measure of fertility. This is a quantitative test (not subjective). 0.5 ml of semen is diluted with solution to give a 1:200 dilution and mixed thoroughly. The solution is used to charge the hemocytometer chambers. Wait 10 minutes, then count the spermatozoa in the erythrocyte counting area (large central square) at 400×. Multiply the number by two million. The solution may be either 9g sodium chloride in 1 liter of distilled water mixed with 1 milliliter of formalin, 3% chlorazene or 12.5 g sodium sulfate with 33.3 milliliters of glacial acetic acid in 200 milliliters of water (Gower's solution). The first solution can be made in the clinic.
The live-to-dead ratio allows you to assess the percentage of living versus dead sperm in a sample and can be an indicator of fertility. The sperm is stained with a vital dye (dye that stains only living cells) such as eosin/nigrosin. This stain is prepared by adding 1 g of eosin B to 5 g of nigrosin in 3% sodium citrate dihydrate. The stain and the slide are both warmed and one drop of the dye is added to one drop of semen and allowed to incubate for 3 seconds. A push smear (similar to the blood smear technique) is made and allowed to dry. The slide is examined at 40× and oil immersion. Live sperm will appear white against the dark background, dead sperm will appear pink. 200 spermatozoa will be counted. The number of live versus dead will be counted (use a cell counter) and a percentage is obtained.
Morphology is assessed on the same slide as used for the live-to-dead ratio. 500 spermatozoa are assessed for normal morphology of the head, midpiece, and tail. Morphological abnormalities are broken into primary and secondary abnormalities.


All abnormalities should be noted. It is important also to note that other types of cells should not be seen in the semen. If they are present, their quantity should be recorded. If neutrophils are seen, the semen should be submitted for culture and sensitivity.

Pituitary Testing
The pituitary gland is also known as the master gland. It has two components: The anterior pituitary (also called the adenohypophysis) and the posterior pituitary gland (also called the neurohypophysis). The anterior pituitary gland produces hormones that stimulate other glands to produce their hormones (FSH, LH, TSH, ACTH). Problems with the anterior pituitary gland will cause problems with their target glands. Decreases in production of the stimulating hormones may be from primary pituitary dysfunction or secondary to overproduction of hormones from the target glands. Testing for these hormones has been discussed in the previous sections.
The posterior pituitary gland is actually a storage depot for hormones produced by the hypothalamus. These hormones direct milk letdown and parturition (oxytocin) and urine retention/water elimination (ADH). ADH is released in response to a reduction in blood pressure and causes the distal convoluted tubule of the nephron (functional unit of the kidneys) to conserve water. This results in an increase in blood volume, which increases blood pressure. Failure of ADH will cause diuresis (increased urine production) or polyuria (excessive urine production). This can happen due to diabetes insipidus (a pituitary gland dysfunction), nephrogenic diabetes insipidus (a failure of the kidneys to respond to ADH), or a hypothalamus problem where there is no production of ADH. Urine specific gravity, the water deprivation test, and the vasopressin test can be used to determine whether this function is intact.
The specific gravity has been discussed under Kidneys, and will be discussed further under Urinalysis.

Water Deprivation Test
The water deprivation test is an excellent test of renal tubular function. It should be used only for animals with polyuria/polydipsia (PU/PD) or in animals with a specific gravity <1.012. If the animal is azotemic (high BUN and/or Cr), the test should not be performed because it will damage the kidneys even more. Baseline data should be obtained prior to the test including body weight, PCV, TP, SG, and BUN. Food and water should then be withheld. The body weight, PCV, TP, SG, and BUN are monitored every 4 to 6 hours until there is a 5% loss in body weight. Body weight loss indicates fluid loss. The USG should increase with water deprivation because the urine should concentrate in response to the water loss. A failure to concentrate the urine is due either to a lack of ADH or to an unresponsive kidney.
In some cases a syndrome called medullary washout can occur. This occurs when the animal is getting overhydrated by either drinking too much (psychogenic polydipsia) or getting too many fluids. In this case the electrolytes are not at a high enough concentration in the blood or kidneys to cause urine concentration. The urine specific gravity will remain low despite adequate or increased ADH levels. In this case, a gradual water deprivation test should be performed. Water is given in very limited amounts over a 3 to 5 day period. This allows the electrolytes to come back to normal levels. Once the solute levels are normal, the normal water deprivation and vasopressin tests can be performed if they are still indicated. This test should only be performed with close patient monitoring.

Vasopressin Test
The vasopressin test differentiates between unresponsive kidneys and hypothalamic/pituitary disease. The animal is given an injection of vasopressin (synthetic ADH) and the urine concentration (SG) is monitored every hour for 2 to 4 hours. If the urine concentrates (the SG increases), there is a lack of ADH production and the disease is localized to the pituitary or the hypothalamus.

Immunology/Serology
Because immunoglobulins are specific to specific substances (antigens), they can be used by the veterinary practice to diagnose diseases and rapidly identify problems that must be treated immediately. Many tests may be performed in-house (agglutination test, Coombs test, ELISA), but others may need to be sent to a reference laboratory.

Enzyme-Linked Immunosorbent Assay
This testing method allows the practice to rapidly diagnose many diseases in-house with the manufacture of tests like the SNAP® test by IDEXX and other quick-test systems. Some ELISA tests must still be performed in the laboratory because they have not been made into quick-test assays.

Direct Testing
Direct ELISA tests test for the presence of antigen. Direct tests have the specific antibody for the antigen of interest present in the well (filter paper or plastic well). The specimen is then added to the well. Any antigen present in the sample will bind to the antibody and stick to the well. The well is washed to remove any other unbound substances and a second antigen-specific antibody is added to the well. This will bind to the antigen again. An antiantibody (an antibody that binds to that species' antibodies) is added to the well. This antibody has a label that causes a color change. The well is washed again to remove any unbound antibody. The amount of color formed will correlate to the amount of antigen present in the sample.
This test is very specific. False positives are very rare. Examples of direct tests are canine heartworm tests, giardia, feline leukemia, and parvovirus. There are many others.

Indirect Testing
Indirect tests test for the presence of antibody to a specific antigen. In this case, the specific antigen is present in the well. The sample is added and the antigen-specific antibody binds to the well. The sample is washed and an antiantibody with a label is added to the well. This is then washed a second time. Color change correlates to the amount of antibody to the antigen present in the sample.
This test is very sensitive. This means that false negatives are rare. However, animals that have recovered from the disease may still show up positive, so care must be taken when interpreting these tests. Examples of indirect tests are the feline immunodeficiency virus, feline infectious peritonitis, and feline heartworm antibody test.

Agglutination Tests
The theory behind agglutination tests is the formation of insoluble complexes (complexes that cannot be separated by washing with saline) of antibodies and antigen. Because antibodies are specific, they will only react with the substance that is being studied. These complexes can be seen either with the naked eye or by microscopy.

Coombs Test
The Coombs test is used in animals that are suspected of having immune-mediated hemolytic anemia (IMHA), or auto immune hemolytic anemia (AIHA). This disease occurs when the body produces antibodies to the red blood cells, causing clumping and destruction (hemolysis). In many cases, these clumps of red blood cells (autoagglutination) are seen when examining the CBC. In other cases, Coombs testing is necessary.

The patient's cells are washed with saline after separating them from the plasma to remove any free-floating antibodies. Coombs serum containing anticanine antibody (antibody specific for dog antibodies) is then added to the sample. If agglutination (clumping) occurs, that indicates that the red blood cells have antierythrocyte antibody attached to them. This is called a Coombs positive test, and the animal should be treated for IMHA.

Blood Typing
Blood typing is also a serologic test based on the formation of antibody:antigen complexes. In dogs, DEA (dog erythrocyte antigen) is the most important test. Animals that are DEA– will have life-threatening transfusion reactions to DEA+ blood. In cats, type A blood will cause life-threatening reactions when given to type B cats.
Blood testing for both breeds involves mixing blood with the antibodies located on the test card surface. Positive agglutination will occur in dogs with DEA+ blood. Cats have one well with type A antibody and one well with type B antibody. All animals should be tested prior to receiving a blood transfusion.

Brucellosis Testing
Brucellosis testing is the reaction of Brucellosis organisms with the antibodies in the serum to form complexes that clump on the card. It is commonly performed in canine fertility testing (both males and females) and on milk from dairy cattle.

Polymerase Chain Reaction
Polymerase chain reaction (PCR) is not really an immunologic test, but it can increase the amount of protein available for immunologic testing. It is the formation of multiple copies of a piece of DNA in order to amplify the ability to detect it in a sample. This will allow the formation of protein by reverse transcription that may be analyzed by immunology or serology. It has become very common to use PCR for viral testing and hereditary diseases.

Agar Gel Immunodiffusion
Agar gel immunodiffusion (AGID) uses the same principles as agglutination testing. The antibody:antigen complex is seen as a line in the agar between the sample (patient antibody) and the test antigen. The test antigen is placed in a well in the center of the plate. The patient's serum and positive and negative controls are placed in wells surrounding the center well. Since agar is porous, the serum and antigen will diffuse through it. Where they meet, a white line of precipitate is formed.
This test is used for equine infectious anemia (EIA) in horses, pseudorabies virus, bovine leukemia virus (BLV), and Johne's disease in ruminants.

Fluorescent Antibody Testing
Fluorescent antibody testing (FAB) can be used both as a diagnostic test for disease and as a protein labeling test in the pathology laboratory. An antibody specific to the protein or organism to be identified is labeled with a fluorescent marker that will glow yellow or green under black light. This is added to the specimen and examined under the fluorescent microscope. It can be used to identify certain viruses and bacteria. This is called immunohistochemistry because it uses immune complexes to find things in the tissues.

Titers
Serologic titers measure the concentration of a specific antibody in the serum. Serial dilution is performed by continuing to dilute the serum until there are no complexes formed with the antigen to be tested.
Serum titers can be very important in vaccination. A higher titer means that there is more antibody available to fight off a specific infection. Vaccines that cause higher titers are generally more effective at protecting the animal from disease.
Titers measure only humoral immunity (immunity from antibodies), not cellular immunity, so a low titer does not necessarily mean low immunity. Some vaccines stimulate humoral immunity better, some stimulate cellular immunity. It is difficult to measure cellular immunity in the laboratory.