Clinical Chemistry and Interpretive Cytology by jlsmith165

Clinical Chemistry & Interpretive Cytology Lab Notebook

4 - unit 2 - hematology key terms 10 - blood cells & cell morphology 20 - order of draw for commonly used collection tubes 22 - packed cell volume - hematocrit 24 - blood film preparation 29 - unit 3 - hemostasis key terms 32 - coagulation cascade 36 - common coagulation disorders of dogs 37 - canine von Willebrand disease 38 - drugs associated with thrombocytopenia in dogs and cats 39 - conditions that may result in disseminated initravascular coagulation 40 - unit 4 - immunology key terms 44 - summary of the complement system 46 - canine and feline blood types 47 - possible causes of urticaria and angioedema in dogs and cats 48 - reasons for false-negative intradermal skin test reactions 49 - reasons for false-postivie intradermal skin test reactions 50 - molecular diagnostic test for veterinary pathogens 51 - types of hypersensitivity reactions 52 - unit 5 - urinalysis key terms 56 - routine urinalysis procedures 57 - urine color chart 58 - urine values for dogs and cats 59 - urine artifacts 60 - urine cells 61 - urine crystals 62 - urine casts 63 - urine infectious agents 64 - unit 6 - clinical chemistry key terms 70 - plasma sample preparation 71 - serum sample preparation 72 - plasma protein functions 73 - conditions associated with altered serum protein 74 - acid-base balance 75 - major electrolytes in body fluids 76 - unit - cytology key terms 80 - cytologic patterns 84 - cytologic patterns table

T e r m s K e y H e m a t o l o g y 2 U n i t 4

A absolute volume : the number of each type of leukocyte in peripheral blood; this is calculated by multiplying the relative percentages from the differential by the total white blood cell count acanthocyte : an erythrocyte with spiny projections of varying lengths distributed irregularly over its surface agranulocytes : the white blood cell group that has no visible cytoplasmic granules; lymphocytes and monocytes anemia : a decrease on the oxygen-carrying capacity of the blood; usually a result of decreased RBC circulating numbers anisoctytosis : a variation in the size of RBC

anticoagulants : any substance that inhibits or prevents clotting anulocyte : bowl-shaped RBC aplastic : referring to bone marrow when all blood cell types are decreased or absent apoptosis : the death of single cells by a process involving shrinkage, rapid fragmentation, and the engulfment of the fragments by neighboring cells and macrophages aspiration biopsy : aseptically prepare site and insert a hollow needle through bone and into bone marrow, using a syringe a sample of liquid is withdrawn atypical lymphocyte : a general term ;used to describe a lymphocyte with morphologic abnormalities, including azurophilic granules, increased cytoplasmic basophilia, overly abundant cytoplasm, or a larger and more convoluted nucleus than seen in normal lymphocytes autoagglutination : the clumping or agglutination of an individual’s cells by that individual’s own serum, usually because of the presence of autoantibodies

basophil : a granular leukocyte with an irregularly shaped, relatively palestaining nucleus that is partially constricted into two lobes and with cytoplasm that contains coarse bluish-black granules of variable size basophilic stippling : erythrocytes that are characterized by small, blue-staining granules; this represents the presence of residual RNA buffy coat : the layer of material above the packed erythrocytes after centrifugation; it consists primarily of leukocytes and thrombocytes

C E F D G

chronic granulomatous inflammation : characterized by increased macrophages

chronic inflammation : a hyperplastic condition characterized by increased numbers of plasma cells, mature lymphocytes, and mast cells

complete blood count : a group of tests that evaluate the cells that circulate in blood, including red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs); a CBC can evaluate overall health and detect a variety of diseases and conditions, such as infections, anemia and leukemia.

codocyte : an erythrocyte that is characterized by an increased membrane surface area relative to the cell’s volume

erythropoietin : the hormone that stimulates erythropoietic activity in the bone marrow ethylenediaminetetraaccetic acid : an anticoagulant that binds calcium

core biopsy : core sample of marrow

coverslip smear : a method of preparing a blood film with the use of two coverslips

chronic polygranulomatous inflammation : characterized by increased macrophages and neutrophils citrate : any salt of citric acid; citrate salts are used as temporary anticoagulants for studies of blood coagulation

erythropoiesis : the production of erythrocytes

echinocyte : an erythrocyte with multiple small projections that are evenly spaced over the cell circumference

eosin : a type of pink to red acid dye that is a component differential stains; primarily used for the routine staining of blood films

dacrocyte : an abnormal erythrocyte that is shaped like a teardrop

dÖhle body : small, gray-blue areas that represent ribosomes and that are seen in the cytoplasm of some immature and toxic granulocytes drepanocyte : a morphologic abnormality of erythrocytes that is characterized by sickleshaped cells

fibrinous inflammation : typically involves the infiltration of the bone marrow with fibrin exudate without the presence of inflammatory cells

eosinphil : a granulocyte with granules that have an affinity for the acidic components of stains

erythrocyte indices : calculated values that provide the average volume and hemoglobin concentrations of erythrocytes in a peripheral blood sample erythrocyte sedimentation rate : the rate at which erythrocytes fall in their own plasma; may be detected by automated analyzers or manual methods

granulocytes : any cell with distinct cytoplasmic granules

H heinz body : round structures of erythrocytes that represent denatured hemoglobin and that appear as a pale area when stained with Wright’s stain hematopoiesis : the production of blood cells and platelets hemoglobin : the oxygen-carrying pigment of erythrocytes, which is formed by developing erythrocytes in the bone marrow; it is a type of hemoprotein that contains four heme groups and globin heparin : an acid mucopolysaccharide that is present in many tissues, especially the liver and lungs, and that has potent anticoagulant properties heterophil : a leukocyte of avian, reptile, and some fish species that contains prominent eosinophilic granules; functionally equivalent to the mammalian neutrophil

histogram : a graphic display of a frequency distribution that is represented by a series of rectangles that divide the data into classes; the height of a rectangle indicates the number of values that are contained in that class (class frequency), and the width of each base represents the size of the intervals into which the classes have been divided howell-jolly body : basophilic inclusions of young erythrocytes that represent nuclear remnants hypercellular : an increased number of cells compared to normal in an area of the body hyperchromatophilic : a term that refers to a cell that appears darker than normal on a peripheral blood sample hypocellular : a decreased number of cells compared to normal in that area of the body hypochromasia : the presence of erythrocytes with decreased staining intensity as a result of a decrease in hemoglobin concentration hyposegmentation : a term that is used to describe the nucleus of a leukocyte with fewer than the normal number of nuclear lobes

icteric : description of plasma that is deep yellow; seen in animals with liver disease or hemolytic anemia illinois sternal needle : needle type used for bone marrow collection impedance analyzer : a type of analyzer that counts particles based on their displacement of electrolyte solution when the particles pass through an aperture; the magnitude of the displacement creates an electrical signal that allows particles (e.g., cells) to be classified on the basis of their size

J K

jamshidi needle : needle type used for bone marrow collection

karyolysis : the degeneration of dissolution of a cell nucleus karyorrhexis : the fragmentation of a cell nucleus keratocyte : in hematology, an abnormally shaped erythrocyte that appears to have horns

L laser flow cytometry : an analyzer that involves the use of focused laser beams to evaluate size and density of solid components left shift : the presence of increased numbers of immature cells in a peripheral blood sample leptocyte : an erythrocyte that is characterized by an increased membrane surface area relative to the cell volume leukemia : a condition characterized by the presence of neoplastic cells in the blood or bone marrow leukemoid response : the exhibition of blood counts (particularly leukocytes) and sometimes other clinical findings that resemble those of leukemia leukocytosis : the presence of increased numbers of leukocytes in the blood leukopoiesis : the production of leukocytes

lipemic : the presence of fatty material in plasma or serum lymphocyte : a leukocyte that is involved in the inflammatory process and that also has roles in humoral and cellmediated immunity lymphopenia : the presence of decreased numbers of leukocytes in a peripheral blood sample lymphoproliferative disease : a set of disorders characterized by the abnormal proliferation of lymphocytes into a monoclonal lymphocytosis

M macrocytosis : a condition in which a cell is abnormally large mean corpuscular hemoglobin MCH : an expression of the average hemoglobin content of a single cell in picograms that is obtained by multiplying the amount of hemoglobin (in grams) by 10 and then dividing the number by the number of erythrocytes (in millions)

mean corpuscular hemoglobin concentration MCHC : the concentration of hemoglobin in the average erythrocyte (or average weight of hemoglobin to the volume in which it is contained) mean corpuscular volume MCV : an expression of the average volume of individual red cells in cubic microns that is obtained by multiplying the hematocrit percentage by 10 and then dividing that number by the number of erythrocytes (in millions) megathrombocytes : abnormally large platelets that are usually newly formed; seen in greater numbers during an increase in platelet production methanol : methyl alcohol methemoglobin : the form of hemoglobin that contains oxidized iron; inefficient at oxygen transport

microcytosis : a cell that appears much smaller than normal microhematocrit : a term that refers to use of capillary tube and a high-speed centrifuge to determine the packed cell volume monocyte : a precusor cell representing a stage in the development of the tissue macrophage; after a monocyte leaves the bloodstream and enters tissue at a site of inflammation, it becomes an activated macrophage myeloproliferative disease : BABESIA : any of a genus Babesia of sporozoans parasitic in mammalian red blood cells and transmitted by the bite of a tick

methylene blue : a stain component that is buffered to an alkaline pH, and it stains the acidic components of the cell, such as leukocyte nuclei

N PQ O R new methylene blue : a supravital stain; when used to stain reticulocytes the organelles clump into visible granules that are referred to a reticulum

neubauer rulings : a specific pattern of precise markings on a hemotocytometer slide that facilitates the counting of leukocytes, erythrocytes, and platelets in the blood and o f all cells in other fluids

neutrophil : a leukocyte that functions to phagocytize infectious agents and cellular debris; plays a major role in the inflammatory process

nonregenerative anemia : a deficiency of the red blood cells where the bone marrow is not increasing red blood cell production and releasing reticulocytes

nucleated erythrocyte : an immature red blood cell that still contains a nucleus

packed cell volume : the ratio of red blood cells to total plasma volume

pancytopenia : a decrease in the numbers of all blood cell types pelger-huët anomaly : an inherited anomaly that is characterized by the appearance of bilobed neutrophils in a peripheral blood sample plasma : the fluid portion of the blood

neutrophilia : an abnormal increase in the number of neutrophils seen in a peripheral blood sample

oxalate : an anion of oxalate acid oxyhemoglobin : oxygen that is bound to hemoglobin

platelet : irregular, discshaped fragments of megakaryocytes in the blood that assist with blood clotting

pluripotent stem cell : a cell capable of differentiating into one of many cell types poikilocytosis : any abnormal cell shape polycythemia : an increase in the number of circulating red blood cells, also known as erythrocytosis

quantitative buffy coat analysis : uses differential centrifugation and staining to provide an estimation of cellular elements

reactive lymphocyte : have increased basophilia in the cytoplasm; they may have more abundant cytoplasm, and they sometimes contain larger and more convuluted nucleus red cell distribution width RDW : a measurement of the range in the volume and size of erythrocytes

regenerative anemia : a type of anemia where the bone marrow acts as it is supposed to by releasing reticulocytes ( immature red blood cells) in an attempt to make up for the absence of red blood cells reticulocyte production index : also called a corrected reticulocyte count, is a calculated value used in the diagnosis of anemia reticulocytes : immature erythroctyes that contain organelles (ribosomes) that are lost as the cell matures romanowsky stain : a stain made from water-soluble eosin, methylene blue, and absolute methanol and used in parasitology, hematology and cytology rosenthal needle : fine needle a very thin, highly flexible steel needle with a narrow inner core used to biopsy bone marrow rouleaux : an arrangement of erythroctyes that appears as a column or stack; seen in healthy horses, and may also be present on blood smears from healthy cats and pigs; may also be an artifact of blood being held too long before being prepared for blood smear

S schistocyte : RBC fragments, usually formed as a result of the shearing of RBC via intravascular trauma serum : the fluid portion of blood after it has been clotted; it does not contain cells or coagulation proteins smudge cell : a leukocyte that has ruptured sodium fluoride : best known as a glucose preservative; also has anticoagulant properties spherocyte : an intensely stained erytrocyte that has reduced or no central pallor stomatocyte : an erythrocyte with a linear area of central pallor

target cell : a leptocyte with a peripheral ring of cytoplasm surrounded by a clear area a dense, central, rounded area of pigment

thrombocytes : platelets; cytoplasmic fragments of bone marrow megakaryocytes thrombopoiesis : the production of platelets thrombopoietin : primarily produced in liver endothelial cell, but it is also released from cells in the kidney and other sites

vacutainer : a glass tube with a rubber stopper from which air can be removed to create a vacuum; usually used to draw blood

torocyte : “punched-out” cells which are generally considered artifacts that result from improper smear technique toxic granulation : darker-colored granules that can be seen under a microscope in neutrophils; granules are often larger and more abundant than normal granules; their presence is non-specific and usually signals a bacterial infection or inflammation

wedge smear : a common technique of blood smear preparation wright-giemsa stain : a specially prepared mixture of methylene blue and eosin in methanol, used to stain cells; commonly used in hematology for peripheral blood smears and in cytopathology for fine needle aspirations and body fluids wright’s stain : mixture of eosin and methylene blue dyes; used primarily to stain peripheral blood smears, urine samples, and bone marrow aspirates 9

B l o o d

C e l l s

C e l l

M o r p h o l o g y

erythrocyte : transports oxygen from the lungs to the tissues and carbon dioxide from tissues to the lungs for expulsion

lymphocyte : small (mature) cells have round nuclei with smooth, dense chromatin and a small rim of light blue cytoplasm; lymphocytes must be distinguished from nucleated red blood cells

monocyte : often larger than neutrophils and are usually the largest leukocyte, but there are no consistent species differences; nucleus can be round to kidney-shaped to pseudo-lobulated

neutrophil : segmented neutrophil, or heterophil in birds and reptiles; the predominant granulocyte and the predominant leukocyte in health; mature neutrophils are released from marrow

eosinophil : nuclei of mature eosinophils are shorter and less segmented than neutrophil nuclei; the cytoplasm is pale blue; species variation exists in the number, size, and granule shape

basophil : cells may contain many small deep purple granules that obscure the nucleus; canine basophils are difficult to recognize since many do not have readily apparent granules

platelets : thrombocytes in mammals are anucleate cells; contain small pink-red granules; shed in the blood from megakaryocytes in bone marrow, key players in hemostatic process

platelet clumping : clumps can be granular or granular; clumps of degranulated platelets must be distinguished from small flecks of fibrin; will affect platelet counts

acanthocyte : (or spur cells) are spherical cells with blunttipped or club-shaped spicules of different lengths projecting from their surface at irregular intervals

agglutination : occurs in immune-mediated disorders in which antibody coats the erythrocyte,resulting in the bridging and clumping of RBCs; will not disperse when RBCs are mixed with saline

basophilic stippling : small, regular, basophilic granules; most commonly represents aggregates of reticulum; seen with regenerative anemia, acute lead toxicity, bone marrow disorders

dacryocytes : are sickle-shaped erythrocytes that occurs in certain deer breeds (excluding reindeer and Montjac deer), antelope, some breeds of sheep, goats, antelope, mongoose and genet

eccentrocytes : hemoglobin is concentrated at one pole of the cell; the other pole is a small area of unstained cytoplasm bound by a distinct membrane

echinocytes : spiculated RBCs; the term crenation (“burr” cell) is also used to refer to cells of this type, particularly when the spicules are sharp and the echinocytes are an artifactual change

elliptocytes : elongated RBC. There are three types: Type I is a slightly oval-shaped cell, type II is a more rounded to oval shaped cell and type III is an elongate elliptical cell

heinz bodies : pink or clear inclusions; most common in cats due to their unique hemoglobin structure and relative lack of RBC-reducing enzymes; represent precipitates of oxidized hemoglobin

howell-jolly bodies : basophilic nuclear remnants that are seen in young erythrocytes during response to anemia; may be seen after the removal of the spleen or with splenic disorders

hypochromasia : pale red blood cells with larger areas of central pallor due to reduced hemoglobin content; frequently seen with iron-deficiency anemia

poikilocytes : umbrella term to describe erythrocytes with abnormal shape; subclassified by specific shape changes, some of which have fairly unique diagnostic significance

rouleaux : involves the grouping of erythrocytes in stacks; seen with increased fibrinogen or globulin concentrations; may be present in healthy horses, cats and pigs

schistocytes : red blood cell fragments; generally taken to reflect mechanical injury to erythrocytes; a wide variety of forms may be observed.

spherocytes : erythrocytes that form a sphere rather than the discoid shape; appear as cells that are smaller and more dense than red blood cells and have a reduced area of central pallor

target cells : (codocytes or leptocytes) have a “lump” of hemoglobinized cytoplasm within the area of normal central pallor, causing them to resemble a “bullseye” target

torocyte : cells that have increased central pallor but a normal thick rim of hemoglobin; the increased central pallor is an artifact; could be mis-identified as hypochromic red blood cells

O r d e r o f D r a w f o r Co m m o n ly U s e d Co l l e c t i o n T u b e s 20

Tubes need to be collected in a specific order to avoid potential contamination of samples with additives from other tubes. Tubes that contain citrate additives are drawn first.

additive

primary use

sodium citrate

coagulation studies

glass: no addivite plastic: silicon-coated

serum for blood chemistry

gel separator and clot activator

serum for blood chemistry

heparin

plasma for blood chemistry

EDTA

hematology

potassium oxalate or sodium flouride

coagulation testing glucose testing

P a c k e d

C e l l

V o l u m e

H e m a t o c r i t

PACKED CELL VOLUME (PCV): This is a directly measured value obtained from centrifuging blood in a microhematocrit tube in a microhematocrit centrifuge. The PCV is measured as the height of the red cell column in a microhematocrit tube after centrifugation (see image at right). It is the quickest and most readily available measure of the red blood cell component of blood. Unlike the HCT, this measurement is affected by plasma trapping and how the red blood cells pack within the column. Examination of the “crit tube” can also provide subjective information about the color and clarity of the plasma ( icterus, hemolysis, lipemia), and the size of the “buffy coat” (which contains WBC and platelets). Additionally, one can score and break the tube as desired to remove the plasma for refractometric protein estimation, or to extrude the buffy coat for smear-making. The “buffy coat smear” has the advantage of providing a concentrated preparation of nucleated cells, which can be useful if looking for low-incidence infectious agents (e.g., Anaplasma organisms in leukocytes).

M a k i n g

W e d g e

S m e a r

1. place a small drop of blood at the end of a clean slide using a microhematocrit tube of the end of a wooden applicator stick; place this on a flat surface,or suspend it in midair between the thumb and the forefinger 2. hold a second slide (the spreader slide) at a 30-degree angle, and pull it back into contact with the drop of blood, spreading the blood along the edge of the spreader slide; push the spreader slide forward with a rapid, steady, even motion to produce a blood smear that is thick at one end and that tapers to a thin, feathered edge at the other; the blood smear should cover about three quarters of the length of the slide 3. air dry the smear by waving the slide in the air; this fixes the cells to the slide so that they are not dislodged during staining 4. label the slide at the thick end of the smear; if the slide has a frosted edge, the label may be written there 5. after drying, stain the smear with Wright’s stain or Romanowsky-type stain; follow manufacturer’s staining directions 6. after staining, rinse the slide with distilled water; allow the slide to dry upright, with the feathered edge pointed upward, this allows the water to drip off the slide and away from the smear

Fe at he red E d g e • • • •

clumpled platelets large cells microfilaria leukocyte distribution

• • • •

platelet number estimate leukocyte number est. morphologic evaluation data validation

Monolayer or Count Area

B o dy o r Th i c k Ar e a • rouleaux formations • agglutination • cell clumping

Tr o u b l e s h o ot i n g S ta i n i n g P r o b l e m s problem

solution

excessive blue staining (RBCs may stain blue-green color) prolonged stain contact

decrease staining time

inadequate wash

wash longer

specimen too thick

make thinner smears, if possible

stain, diluent, buffer, or wash water too alkaline

check with pH paper and correct pH

exposure to formalin vapors

store/ship preparations separate from formalin

wet fixation in ethanol or formalin

air dry smears before fixation

delayed fixation

fix smears sooner, if possible

surface of the slide was alkaline

use new slides

excessive pink staining insufficient staining time

increase staining time

prolonged washing

decrease duration of wash

stain or diluent too acidic

check with pH paper and correct pH

excessive time in red stain solution

decrease time in red stain solution

inadequate time in blue stain solution

increase time in blue solution

mounting coverslip before preparation is dry

allow preparation to dry completely before coverslip

weak staining insufficient contact with one or more stain solutions

increase staining time

fatigued (old) stains

change stains

another slide covered specimen during staining

keep slides separate

uneven staining variation of pH in different areas of slide surfaces

use new slides, avoid touching surface

water allowed to stand on some area of the slide

tilt slides close to vertical to drain water from surface

inadequate mixing of stain and buffer

mix stain and buffer thoroughly

precipitate on preparation inadequate stain filtration

filter or change the stain(s)

inadequate washing of slide after staining

rinse slides well after staining

dirty slides used

use clean, new slides

stain solution dries during staining

use sufficient stain; do not leave it on slide too long

miscellaneous overstained preparations

destain with 95% methanol and restain

refractile artifact on RBCs with Diff-Quik stain

change the fixative 27

T e r m s K e y H e m o s t a s i s 3 U n i t 28

A D B C

activated clotting time : a test of the intrinsic and common pathways of blood coagulation that involves the use of a diatomaceous earth or kaolin tube to initiate clotting activated partial thromboplastin time : a test of intrinsic and common coagulation pathways; an intrinsic pathway activator is added to plasma, and the time taken for clot formation is measured

d-dimers : a protein fragment that is formed from the breakdown of fibrin

buccal mucosa bleeding time : a test that uses a standardized shallow incision into the buccal mucosa of the upper lip to evaluate primary hemostasis

clot retraction : a crude but simple test that allows for the evaluation of platelet number and function and intrinsic and extrinsic pathways

dissemiated intravascular coagulation : an acquired secondary coagulation disorder that is characterized by the depletion of thrombocytes and coagulation factors; also referred to as consumption of coagulopathy and defibrination syndrome

P F M H

phosphatidylserine : a phospholipid and is a component of the cell membrane; plays a key role in cell cycle signaling, specifically in relation to apoptosis; found in mammalian blood

fibrin degradation products : protein fragments formed from the breakdown of fibrin

fibrometer : an instrument that measures clot formation by mechanical detection of the clot by a moving probe

hemophilia : a genetic abnormality of hemostasis that results from the deficient production of certain coagulation factors hypercoagulable : characterized by abnormally increased coagulability hypocoagulable : characterized by abnormally decreased coagulability

mean platelet volume : a machine-calculated measurement of the average size of platelets found in blood and is typically included in blood tests as part of the CBC microparticles : present in the blood of healthy animals and are increased in various diseases, including cardiovascular disease, diabetes, sepsis and cancer; they are small membrane vesicles derived from activated cells and apoptotic cells

PIvKA : in vitamin K deficiency, des-carboxylated proteins are formed that are functionally defective because they cannot bind calcium and phospholipid; these abnormal coagulation factors are called proteininduced by vitamin K absence

plateletcrit : (reported as a %) is analogous to the hematocrit and reflects the mass of platelets; this result is not usually reported on most hemograms and is infrequently used in laboratories platelet-large cell ratio : (P-LCR) is defined as the percentage of platelets that exceed the normal value of platelet volume of 12 fL in the total platelet count prothrombin time tests : a one-stage test for detecting certain plasma coagulation defect that are caused by a deficiency of factors V, VII, or X

platelet distribution width : a measurement of platelet anisocytosis calculated from the distribution of individual platelet volumes

monovette : an enclosed blood collection system that allows the user to draw blood from the patient using the syringe or vacuum method reducing the rate of hemolysis and vein collapse

T thrombin : an enzyme that is formed from prothrombin, calcium, and thromboplastin in plasma during the clotting process; thrombin causes fibrinogen to change to fibrin, which is essential during the formation of a clot thrombocrit : (or plateletcrit) is the percentage of blood volume occupied by platelets, and is an assessment of circulating platelet mass thrombocytopenia : a condition that involves a decrease in the number of circulating platelets

thrombocytosis : a condition that involves an increase in the number of circulating platelets thromboelastography : a method of testing the efficiency of blood coagulation; it is a test mainly used in surgery and anesthesiology thrombopathia : a condition in which there is a deficiency of clotting in which there is a deficiency of clotting ability for reason other than thrombocytopenia

V von Willebrand disease : an inherited disorder that is characterized by the abnormally slow coagulation of the blood as well as spontaneous epistaxis and gingival bleeding; it is caused by a deficiency of a component of factor VIII; excessive bleeding is common after injury or surgery von Willebrand factor : a blood glycoprotein involved in hemostasis, specifically, platelet adhesion

) W r

v (

s i s

F a c d

Both vWF and platelets are needed to form a primary platelet plug, the end result of primary hemostasis

facilitates platelet adhesion

e l

carrier protein for factor VIII

o e e T

r P

a m

v o

t o

circulates in plasma

synthesized in endothelium

s t F n o t u

produced in the liver circulated in plasma in an inactive form

o f o

coagulation factors:

activated through coagulation cascade

While primary hemostatis provides a temporary platelet clump “band-aid” to the damaged vessel wall, secondary hemostasis involves the formation of a stable fibrin clot.

Common Inherited Coagulation Disorders of Dogs 36

disorder

breeds affected

prothrombin deficiency

cocker spaniel, beagle

factor VII deficiency

beagle, malamute

factor VIII deficiency

many breeds (hemophilia A)

factor IX deficiency

many breeds (hemophilia B)

factor X deficiency

cocker spaniel

factor XI deficiency

great pyrenees, english springer spaniel

factor XII deficiency

poodle, shar pei

D i s e a s e W i l l e b r a n d v o n C a n i n e

type 1 variably reduced levels; all multimer sizes proportionality reduced; most common and recognized in > 70 breeds; hemorrhage tendency variable, often with surgery or trauma

type 2 disproportionately low activity; deficiency of high molecular weight multimers; larger and more effective multimers absent; bleeding can be severe

type 3 complete deficiency (< 1% plasma von Willebrand factor); most severe in that all multimers absent

C a t s a n d D o g s i n

T h r o m b o c y t o p e n i a w i t h A s s o c i a t e d D r u g s 38

acetaminophen antiarrhythmics anticonvulsants anti-inflammator drugs (nonsteroidal) barbituates benzocaine cephalosporins chemotherapeutic agents chloramphenicol cimetidine estrogen gold salts griseofulvin immunosuppressive agents levamisole methimazole methionine methylene blue metronidazole penicillins phenobarbital phenothiazines phenylbutazone propylene glycol propylthiouracil sulfa derivatives sulfamethoxazole/trimethoprim zinc

C o a g u l a t i o n I n t r a v a s c u l a r D i s s e m i n a t e d

i n R e s u l t M a y T h a t C o n d i t i o n s

• septicemia (various gram-negative and gram-positive bacteria) • viremia (infectious canine hepatitis, feline infectious peritonitis, african swine fever, hog cholera, african horse sickness) • protozoal parasites (babesiosis, trypanosomiasis, sarcocystosis, leishmaniasis, cytauxzoonosis) • metazoal parasites (heartworms, lungworms) • marked tissue injury (heatstroke, trauma, surgical procedures) • intravascular hemolysis • obstetric complications • malignancy (hemangiosarcoma, disseminated carcinomas, leukemia, lymphoma) • traumatic shock • liver disease • pancreatitis • gastric-dilatation volvulus and abdominal displacement • toxins (snake and insect venoms, aflatoxin, insecticides)

T e r m s K e y I m m u n o l o g y 4 U n i t 40

A B C

active immunity : an animal’s production of antibody as a result of infection with an antigen or immunization allergen : a type of antigen that produces an abnormally vigorous immune response in which the immune system fights off a perceived threat that would otherwise be harmless to the body

alloantibodies : a naturally occurring antibody that is produced by an individual and that reacts with antigens of another individual of the same species anaphylactic shock : an extreme, often life-threatening allergic reaction to an antigen to which the body has become hypersensitive angioedema : Painless swelling under the skin, triggered by an allergy to animal dander, pollen, drugs, venom, food, or medication

antibody titer : the level of a specific antibody that is present in serum; this is calculated as the reciprocal of the highest dilution at which a sample no longer exhibits a positive reaction for the presence of the antibody; it is often used to help differentiate active infection from prior exposure to an antigen antigen : Any substance that causes the body to make an immune response against that substance; antigens include toxins, chemicals, bacteria, viruses, or other substances that come from outside the body atopy : refers to the genetic tendency to develop allergic diseases such as allergic rhinitis, asthma and atopic dermatitis (eczema); atopy is typically associated with heightened immune responses to common allergens, especially inhaled allergens and food allergens avidity : refers to the strength of the binding of antigen and antibody

blood group antigens : a test that uses a standardized shallow incision into the buccal mucosa of the upper lip to evaluate primary hemostasis

cell-mediated immune system : an immune system mechanism that involves actions of the cells of the immune system rather than antibodies chemiluminescence : describes a chemical reaction that results in the emission of light

competitive ELISA : an immunoassay; patient antigen, if present, competes with enzyme-labeled antigens for the antibodies that are coating the test wells complement system : a group of plasma proteins that function to enhance the activities of the immune system

FI DH L E

immunologic tolerance : a state of nonresponsiveness to antigens, whether self or foreign

coombs test : an immunologic test designed to detect antibodies on the surface erythrocytes (direct coombs test) or antibodies against erythrocytes in plasma ( indirect coombs test)

crossmatching : a blood test designed to indentify compatibility between donor and recipient samples before transfusion

inflammatory response : the defensive response of body tissues that is initiated by the release of histamine from damaged cells

fluorescent antibody : a specific antibody that has been labeled with a fluorochrome and that is used in immunoassasys

interferons : small soluble proteins that enhance the function of the immune system

immune-complex disease : a diverse group of inflammatory conditions characterized by antigen–antibody deposition and attendant activation of complement.

dog erythrocyte antigen (DEA) : a naming convention for canine blood types

histamine : a compound which is released by cells in response to injury and in allergic and inflammatory reactions, causing contraction of smooth muscle and dilation of capillaries humoral immunity : an immune response that involves the production of specific antibody

enzyme-linked immunosorbent assay (ELISA) : an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples

hypersensitivity : undesirable reactions produced by the normal immune system, including allergies and autoimmunity; an overreaction of the immune system; reactions may be damaging and uncomfortable

immune-mediated hemolytic anemia : an autoimmune disease in dogs in which the body attacks its own red blood cells. This can lead to very low red blood cell counts (anemia) that require multiple blood transfusions. It can also predispose dogs to forming blood clots, mostly in the lungs or in the brain. immunodiffusion : an immunologic test that is performed by placing reactants in an agar plate and allowing them to migrate through the gel toward each other immunoglobulin : antibodies; plasma proteins produced against specific antigens

lateral flow immunoassay : simple devices intended to detect the presence of a target substance in a liquid sample without the need for specialized and costly equipment latex agglutination : uses the patient’s antigen-antibody immune response; occurs when the body detects a pathogen and forms an antibody specific to an identified antigen present on the surface of the pathogen lymphoma : a neoplastic disorder of the lymphoid tissue

N RT P O SU specificity : the specificity of a test is its ability to designate an individual who does not have a disease as negative

radioimmunoassay : an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes

natural killer (NK) cells : a lymphocyte able to bind to certain tumor cells and virus-infected cells without the stimulation of antigens, and kill them by the insertion of granules containing perforin

neonatal erythrolysis : hemolytic anemia of the newborn

passive immunity : a condition that involves receiving antibodies from colostrum or synthesized antibodies

opsonization : the complementmediated adherence of phagocytes to antigens that enhances the phagocytosis of the antigen

phagocytosis : the process by which a phagocyte (a type of white blood cell) surrounds and destroys foreign substances (such as bacteria) and removes dead cells

polymerase chain reaction : a method that is used to replicate and amplify DNA molecules in a sample

rapid immunomigration : also known as lateral flow immunochromatograhic or rapid tests; simple devices intended to detect the presence of a target substance in a liquid sample without the need for specialized and costly equipment

sensitivity : refers to a test’s ability to designate an individual with disease as positive; a highly sensitive test means that there are few false negative results, and thus fewer cases of disease are missed

tuberculin skin test : the PPD test determines if a patient has developed an immune response to the bacterium that causes tuberculosis (TB)

urticaria : wheals; a rash of round, red welts on the skin that itch intensely, sometimes with dangerous swelling, caused by an allergic reaction, typically to specific food

V vaccination : any injection of attenuated microorganisms (e.g., bacteria, viruses, rickettsiae) that is administered to induce immunity or to reduce the effects of associated infectious diseases

W wheals : uticaria; Swelling of the surface of the skin into red- or skin-colored welts with clearly defined edges

S y s t e m C o m p l e m e n t t h e o f S u m m a r y 44

The complement system is one of the major mechanisms by which pathogen recognition is converted into an effective host defense against initial infection. Complement is a system of plasma proteins that can be activated directly by pathogens or indirectly by pathogen-bound antibody, leading to a cascade of reactions that occurs on the surface of pathogens and generates active components with various effector functions. There are three pathways of complement activation: the classical pathway, which is triggered directly by pathogen or indirectly by antibody binding to the pathogen surface; the MB-lectin pathway; and the alternative pathway, which also provides an amplification loop for the other two pathways. All three pathways can be initiated independently of antibody as part of innate immunity. The early events in all pathways consist of a sequence of cleavage reactions in which the larger cleavage product binds covalently to the pathogen surface and contributes to the activation of the next component. The pathways converge with the formation of a C3 convertase enzyme, which cleaves C3 to produce the active complement component C3b. The binding of large numbers of C3b molecules to the pathogen is the central event in complement activation. Bound complement components, especially bound C3b and its inactive fragments, are recognized by specific complement receptors on phagocytic cells, which engulf pathogens opsonized by C3b and its inactive fragments. The small cleavage fragments of C3, C4, and especially C5, recruit phagocytes to sites of infection and activate them by binding to specific trimeric G protein-coupled receptors. Together, these activities promote the uptake and destruction of pathogens by phagocytes. The molecules of C3b that bind the C3 convertase itself initiate the late events, binding C5 to make it susceptible to cleavage by C2b or Bb. The larger C5b fragment triggers the assembly of a membrane-attack complex, which can result in the lysis of certain pathogens. The activity of complement components is modulated by a system of regulatory proteins that prevent tissue damage as a result of inadvertent binding of activated complement components to host cells or spontaneous activation of complement components in plasma.

s e p y T d o o

Type A

DEA 3

Type B

DEA 4

Type AB

DEA 5 DEA 7

DEA 1

• all canine and feline blood donors should be blood-typed. • most serious transfusion reactions in cats are caused by alloantibodies in type B cats directed against type A red cell antigens. • transfuse type B cats with type B blood, transfuse type A cats with type A blood. • type A kittens born to type B queens are at risk for neonatal hemolysis. • canine typing is most important for the DEA (dog erythrocyte antigen) 1 blood group; the DEA 1 group consists of two alternate alleles: 1.1 and 1.2. • naturally occurring antibodies against DEA 1.1 and 1.2 are uncommon in dogs, but sensitization to these antigens occurs after transfusion of positive cells into a DEA 1 negative recipient; once formed, these antibodies may cause: • delayed transfusion reactions-shortened red cell survival • acute transfusion reactions-hemolysis or anaphylaxis (especially DEA 1.1 incompatibility) • dogs previously transfused with incompatible or unknown type red cells should be cross-matched before receiving a second transfusion.

Possible Causes of Urticaria & Angioedema in Dogs & Cats

• foods • drugs: penicillan, cephalexn, ampicillin, tetracycline, vitamin K, propylthiouracil, amitraz, ivermectin, moxidectin, radiocontrast agents, HyLyt efa shampoo, vincristine, azathioprine • antisera, bacterins, and vaccines: panleukopenia, lepotspirosis, distemper-hepatitis, rabies, feline leukemia • stining and biting insects: bees, hornets, mosquitos, black flies, ants • hairs from processionary caterpillars • allergenic extracts • blood transfusions • plants: nettles, buttercups • intestinal parasites: ascarids, hookworms, tapeworms • infections: staphlococcal pyoderma, canine distemper* • sunlight* • excessive heat or cold* • estrus* • dermatographism* • atopy* • phychogentic factors* • vasculitits, food allergy induced*

* reported in dogs only

Reasons for False-Negative Intradermal Skin Test Reactions 48

• subcutaneous injections • too little allergen • testing with mixes • outdated allergens • allergens too dilute (1000 PNU/mL recommended) • too small volume of allergen injected • drug interferance glucocorticoids antihistamines tranquilizers progestational compounds any drugs that significantly lower blood pressure • anergy (testing during peak of hypersensitivity reaction) • inherent host factors • estrus, pseudopregnancy • severe stress (systemic diseases, fright, struggling) • endoparatsitism or ectoparasitism ("blocking" mast cells with antiparasitic immunogloblin E?) • Off-season testing (tesing more than 1 to 2 months after clinical signs have disappeared) • histamine "hyporeactitivity"

Reasons for False-Positive Intradermal Skin Test Reactions

• irritant test allergens (especially those that conatain glycerin; also, some house dust, feathers, wool, mold, and all food preparations) • contaminated test allergens (bacteria, fungi) • skin-sensitizing antibody only (prior clinical or present subclinical sensitivity) • poor technique (traumatic placement of needle, dull or burred needle, too large a volume injected, air injected) • substance that cause nonimmunologic histamine release (narcotics) • "irritable" skin (large reaction seen to all injected substances, including saline control_ • dermatographism • mitogenic allergen

Molecular Diagnostic Tests for Veterinary Pathogens 50

organism

suggested samples

Bacillus anthracis

blood

bovine viral diarrhea virus 1 and 2

lymph nodes, spleen, serum

Clamydia spp.

placenta, liver

Colstridium perfringens

isolated colony from bacteria culture

Escherichia coli virulence typing panel

isolated colony from bacteria culture

Leptospira spp.

urine, liver, kidney

Mycobacterium paratuberculosis

intestinal mucosa, lymph nodes

porcine repro/respiratory syndrome virus

serum, spleen, lymph

Salmonella spp.

intestinal mucosa, feces, other tissues

West Nile virus

kidney, heart, brain, liver, spleen

T y p e s

o f

H y p e r s e n s i t i v y

R e a c t i o n s

T e r m s K e y U r i n a l y s i s 5 U n i t 52

A C B F

crystalluria : the presence of crystals in the urine indicating renal irritation; as that is caused by sulfa drugs

ammonium biurate : brownish crystals seen in the urine of animals with severe liver disease

crystine : an amino acid that may be present in the form of hexagonal crystals in the urine

anuria : absence of urine

cystocentesis : the aspiration of fluid from the urinary bladder

calcium carbonate : a type of crystal that is commonly seen in the urine of rabbits and horses

Bence Jones protein : a light chain protein of immunoglobin molecules that readily passes through the glomerulus and into the urine bilirubinuria : an abnormal increase in the concentration of bilirubin in the urine

bladder expression : the manual compression of the urinary bladder to cause the release of urine through the urethra

calcium oxalate : a crystal that is found in acidic and neutral urine; commonly seen in small amounts in dogs and horses

casts : structures that are formed from the protein precipitates of degenerating kidney tubule cells; may contain embedded materials catherization : the placement of a catheter in the urethra or the placement of an indwelling catheter in a blood vessel cellular casts : a formed element in urine that consists of a hyaline cast that contains blood cells or epithelial cells

fatty casts : formed elements that may be found in urine and that consist of a hyaline cast with embedded globules of fat flocculent : urine containing shreds of fluffy particles of graywhite or white mucus or other material free catche : a method of collecting a urine sample b collecting the sample as the animal voids naturally

G KM N L I H hyaline casts : the structures that are formed from protein precipitates of degenerating kidney tubule cells with no embedded materials

glomerulus : a tuft of capillaries located in the renal cortex

glucosuria : the presence of glucose in the urine

granular casts : a structure that is formed from the protein precipitate of degenerating kidney tubule cells that contain granular material derived from the breakdown of cells incorporated into the cast

hematuria : the presence of intact erythrocytes in the urine hemoglobinuria : the presence of free hemoglobin in urine

hypersthenuria : urine of unusually high specific gravity and concentration of solutes, resulting usually from a loss or deprivation of water hyposthenuria : urine of low specific gravity due to inability of the kidney to concentrate the urine normally

isosthenuria : inability of the kidneys to produce either a concentrated or a dilute urine; refers to a state of chronic renal disease in which the kidney cannot form urine with a higher or a lower specific gravity than that of protein-free plasma; the specific gravity of the urine becomes fixed

ketones : any of a class of organic compounds (such as acetone) characterized by a carbonyl group attached to two carbon atoms ketonuria : the presence of detectable ketone bodies in urine

leucine : a hydrophobic amino acid which is a constituent of most proteins; an essential nutrient in the diet of vertebrates

myoglobinuria : presence of an excess amount of myoglobin in the urine

nephron : a structured and functional unit of the kidney that resembles a microscopic funnel with a long stem and two convoluted tubular sections

OR T P W S U tom cat catheter : a feline catheter intended for assisting the relief of urethral obstruction in male cats

oliguria : the production of abnormally small amounts of urine

renal epithelial cells : the single layer of cells lining the nephron

pH : a measure of the hydrogen ion concentration of a solution

renal threshold : the concentration level up to which a substance, such as glucose, in the blood is prevented from passing through the kidneys into the urine

pollakiuria : frequent urination

polydipsia : abnormally great thirst as a symptom of disease, such as disease or psychological disturbance polyuria : an increase in the total volume of urine produced

proteinuria : the abnormal presence of protein in the urine

transitional epithelial cells : a type of stratified epithelium consisting of multiple layers of cells where the shape of the cell changes according to the function of the organ tyrosine : an amino acid that is synthesized in the body from the essential amino acid phenylalanine; it is found in most proteins and is a precursor of melanin and several hormones, including epinephrine and thyroxine

urinometer : a small hydrometer for determining the specific gravity of urine

urochromes : a yellow pigment to which the color of normal urine is principally due uroliths : a calculus in the urinary tract

waxy casts : likely represent an advanced stage of the disintegrative process that results in coarsely and finely granular casts and are usually indicative of advanced renal disease

specific gravity : measurement of the concentration of particles in urine and the density of urine compared with the density of water

struvite : a common crystal that is seen in alkaline to slightly acidic urine; also referred to as triple phosphate crystals or magnesium ammonium phosphate crystals

uric acid : a metabolic by-product of nitrogen catabolism

urease : a naturally occurring enzyme that hydrolyzes urea into ammonium carbonate

P r o c e d u r e s U r i n a l y s i s R o u t i n e 56

1. prepare a laboratory sheet with the patient's information and the date, time, and method of urine collection 2. if the sample was refrigerated, make note on the record and allow the sample to warm to room temperature 3. properly mix the sample by gentle inversion 4. record the physical characteristics: color, clarity, volume, and odor of sample 5. calibrate the refractometer with distilled water to 1.000 6. determine and record the specific gravity of the sample 7. dip a reagent test strip into the urine sample then remove it promptly, making sure to lightly tap the edge of the strip onto a paper towel to remove excess urine 8. read the pad's color at the appropriate time intervals as stated by the manufacturer's directions, and record the results 9. properly label a 15-mL conical centrifuge tube 10. pour approximately 5 to 10 mL of the urine sample into the centrifuge tube 11. centrifuge the sample for 5 to 6 minutes at 1000 to 2000 rpm 12. make a note of the amount of sediment 13. pour off the supernatant, leaving approximately 0.5 to 1 mL in the tube 14. resuspend the sediment by gently mixing it with a pipette or flicking the tube with the fingers 15. transfer a drop of reconstituted sediment to a microscope slide with a transfer pipette, and place a coverslip over it 16. subdue the light of the microscope by partially closing the iris diaphragm 17. examine the entire specimen under the coverslip with the high-power (40x) objective to identify cells, casts, crystals, and bacteria 18. to help with the detection of these elements, the fine adjustment knob should be continuously focused 19. record the results

t r a h C r o l o C

significance

pale yellow

low specific gravity; decreased concentration of urine

very yellow

high specific gravity; oliguria (yellow-brown)

brown-green

bile pigment

red-brown

hematuria; hemoglobin

brown

myoglobin

orange

drugs (e.g., tetracycline

color

s a t C & s

specific gravity

1.025 (1.001 - 1.065)

1.030 (1.001 - 1.080)

6-7

glucose

none

protein

none/trace

bilirubin

none/trace

cat

ketone

none

occult blood

none

V a

dog

s t c a f i t

debris

fungus • pollen

fungus • pollen • bacilli

fungus • pollen • grain

fungus • pollen • epithelial cells

cast mimic

s l l

crenated RBCs

epithelial cells • lipids • crystals

hemoglobin globules

inflammation • bacteria

neutrophil • epithelial • aggregate

neutrophils activated

pyuria • hematuria

squamous epithelial cells

transitional cell carcinoma

transitional epithelial cells

urothelial carcinoma

columnar epithelial cells

s l a t

amorphous

bilirubin • granular crystal

calcium carbonate

calcium oxalate dihydrate

calcium oxalate monohydrate

cystine

struvite

uric acid

unknown blocks

unknown needles

unknown triangles

ammonium biurate

s t s a

granular cast

hemoglobin cast

hemosiderin

hyaline cast

mixed cast

mixture of casts

waxy cast • epithelial

waxy cast

granular cast • hyaline cast

s t n e g A s u o i

cocci

cryptococcus

giardia

yeast • bacteria

yeast

capillaria

T e r m s K e y C h e m i s t r y C l i n i c a l 6 U n i t 64

A acid-base balance : a state of equilibrium between the acidity and alkalinity of the body fluids; also call the hydrogen ion balance acidosis : a pathologic decrease in the pH of blood or body tissues as a result of the accumulation of acids or a decrease in bicarbonate acinar : pertaining to or affecting an acinus or acini; this term refers specifically to glandular tissue with structure that is often described as grapelike clusters ACTH stimulation test : a test designed to test the response of the hormone that stimulates adrenocortical growth and secretion acute-phase proteins : proteins, including serum amyloid A and C-reactive protein, that are produced by hepatocytes immediately following injury or inflammation Addison’s disease : see hypoadrenocorticism

arenocorticotropic hormone : a hormone secreted by the anterior pituitary gland that has a stimulating effect on the adrenal cortex; also referred to a corticotropin and abbreviated ACTH alanine transaminase : cytoplasmic enzyme of hepatocytes released when hepatocytes are damaged albumin : a group of plasma proteins that comprises the majority of protein in plasma alkaline phosphate : a group of enzymes that functions at an alkaline pH and that catalyzes the reaction of organic phosphates alkaline phosphate : a group of enzymes that functions at an alkaline pH and that catalyzes the reaction of organic phosphates alkalosis : a condition in which the blood pH is higher than 7.45 allatoin : a crystalline substance produced by the oxidation of uric acid by uricase and present in the urine of most mammals, except primates and Dalmatian dogs (which lack uricase) amylase : an enzyme derived primarily from the pancreas that functions in the breakdown of starch

amyloclastic : a method of measuring serum amylase by evaluating the disappearance of starch substrate anion : a negatively charged ion anion gap : a method that is used to evaluate a patient’s acid-base status; the calculation is based on subtracting the sum of measured major serum anions form the sum of measured majored serum cations aspartate transaminase : an enzyme that is present in body serum and in certain body tissues that catalyzes the transfer of an amino group from aspartic acid to alpha-ketoglutaric acid, thereby forming glutamic acid and oxaloacetic acid; also referred to as aspartate aminotransferase azotemia : the increased retention of urea in the blood

base excess : the amount of acid or base required to titrate a sample of whole arterial blood to the normal pH of 7.4

D F E G C

Beer’s law : a principle that describes the relationship between light absorbance, transmission and the concentration of a substance in solution

bicarbonate : an electrolyte in plasma; part of the bicarbonate-carbonic acid buffer system that maintains the blood pH in equilibrium

bile acids : a group of compounds that are synthesized by hepatocytes from cholesterol that help with fat absorption bilirubin : an insoluble pigment derived from the breakdown of hemoglobin, which is processed by hepatocytes

blood urea nitrogen : the principal end product of amino acid breakdown in mammals

buffers : a substance that increases the amount of acid or alkali necessary to produce a unit change in pH

calcium : the most abundant mineral in the body; calcium is an important cation in intracellular and extracellular fluid; it is essential to the normal clotting of blood, the maintenance of a normal heartbeat, and the initiation of neuromuscular and metabolic activities chloride : the principal anion in extracellular fluid and gastric juice

dexamethasone suppression test : an endocrine system test designed to detect hyperadrenocorticism

cholesterol : a plasma lipoprotein that is produced primarily in the liver as well as ingested in food; used in the synthesis of bile acids conjugated bilirubin : bilirubin that has been taken up by the liver cells and conjugated to form the water-soluble compound bilirubin diglucuronide

cortisol : a steroid hormone produced by the adrenal glands

creatine kinase : an enzyme that is found predominantly in cells of the heart, brain, and skeletal muscle; released when cells are damaged creatinine : a waste product that is formed during normal muscle cell metabolism Cushing’s disease : see hyperadrenocorticism

cation : a positively charged ion

ethylene glycol : a solvent with a sweet, acrid taste that is found in many products, such as antifreeze, drying agents, and inks; ingestion or excessive skin exposure can be toxic

effective renal plasma flow : the effective rate of blood flow through the kidneys; the determining factor relative to the rate of glomerular filtration

fractional excretion of electrolytes : a mathematical manipulation that describes the excretion of specific electrolytes relative to the glomerular filtration rate fructosamine : a molecule formed as a result of the irreversible reaction of glucose bound to protein

electrolytes : any substance that dissociates into ions when in solution

end point assay : a chemical reaction that proceeds to a stable end point endocrine : a term that refers to the system of glands and other structures that secrete hormones directly into the circulatory system

enzymuria : the presence of specific enzymes in urine

gamma glutamyl transferase : an intracellular enzyme found in high concentration in liver; pancreatic, and renal tubular cells

globulins : a complex group of plasm proteins that have been designed as alpha, beta, or gamma; this group includes immunoglobulins, complement, and transferrin glomerular flitration rate : the rate at which substances are filtered through the glomerulus and excreted in the urine glucagon : a hormone secreted by the alpha cells of the islets of Langerhans in response to hypoglycemia glucose : a monosaccharide that represents the end product of carbohydrate metabolism glucose tolerance test : a metabolic test of carbohydrate tolerance glutamate dehydrogenase : a mitochondrial-bound enzyme that is found in high concentration in the hepatocytes of cattle, sheep, and goats glycosylated hemoglobin : the irreversible reaction of hemoglobin bound to glucose

hypophospatemia : a decreased number of phosphates in the blood

hyperlipoproteinemia : a condition characterized by excess lipids in the blood; also referred to as hyperlipidemia and hyperlipemia

hypoproteinemia : a condition characterized by an abnormally low level of protein in the blood

hematachezia : the presence of blood in the feces

hypernatremia : an increased plasma sodium level

hemolysis : the destruction of erythrocytes

hyperphosphatemia : an excessive number of phosphates in the blood

hepatoencephalopathy : severe hepatic insufficiency that may induce a syndrome of excitability, tremor, compulsive walking, head pressing, and apparent blindness, followed by coma and convulsions

hyperproteinemia : an increased protein level in the blood

hyperadrenocorticism : the abnormally increased secretion of adrenocortical hormones, as with conditions such as Cushing’s syndrome hypercalcemia : an increased plasma calcium level hypercapnia : an excess of carbon dioxide in the blood that is indicated by an elevated PCO2 level as determined by blood gas analysis and that results in respiratory acidosis; also known as hypercarbia or hypercarbemia hyperglycemia : an abnormally increased glucose level in the blood

hyperkalemia : in increased plasma potassium level

hyperthyroidism : a condition that is caused by the excessive production of iodinated thyroid hormones hypoadrenocorticism : a deficiency in the production of mineralocorticoid or glucocorticoid steroid hormones hypoalbuminemia : a decrease in the circulating levels of albumin in the blood hypocalcemia : a decreased plasma calcium level hypocapnia : a deficiency of carbon dioxide in the blood; also called hypocarbia hypoglycemia : a decreased plasma glucose level hypokalemia : a decreased plasma potassium level hyponatremia : a decreased plasma sodium level

icterus : abnormal yellowish discoloration of skin, mucous membranes, or plasma as a result of an increased concentration of bile pigments iditol dehydrogenase : an enzyme of the oxidoreductase class that catalyzes the oxidation of I-iditol to I-fructose; it occurs in significant quantities only in the liver and its increased activity in serum is used as an indicator of parenchymal liver damage; also referred to as sorbitol dehydrogenase inorganic phosphorus : essential for every known form of life; forms many compounds such as metal phosphates, phosphides, oxides, halides, and oxyacids insulin : a protein hormone that is secreted by the beta cells of the pancreatic islets in response to elevated blood levels of glucose and amino acids

O JM R P K S L

ion-selective electrode : a transducer (or sensor) that converts the activity of a specific ion dissolved in a solution into an electrical potential

lipase : a pancreatic enzyme that functions in the breakdown of fats lipemia : the presence of fatty material in plasma or serum

optical density : the degree to which light is transmitted through a medium

jaundice : a condition that is characterized by hyperbilirubinemia and the disposition of bile pigments in the skin, mucous membranes, and sclera

magnesium : a mineral involved in many processes in the body including nerve signaling, the building of healthy bones and normal muscle contraction

melena : dark sticky feces containing partly digested blood

kinetic assay : a chemical test that measures the rate of change of a substance in the test system

lactate : the anionic form of lactic acid

mucin clot test : the adding of acetic acid to normal synovial fluid, which cause clot formation; the compactness of the clot and clarity of the supernatant fluid are the criteria on which the result is based

reference range : a set of values that a veterinarian uses to interpret a patient’s test results

pancreatic lipase immunoreactivity : an assay test to measure pancreasspecific lipase

plasma : the fluid portion of the blood

reflectometer : an instrument for measuring the refractive index of transparent substances such as urine and plasma or serum

plumbism : a chronic form of lead poisoning that is caused by the absorption of lead or lead salts

potassium : major positive ion (cation) found inside cell; essential for normal cell function

protein : large molecules composed of one or more chains of amino acids in a specific order determined by the base sequence of nucleotides in the DNA coding for the protein

serum : the fluid portion of blood after it has clotted; it does not contain cells or coagulation proteins sodium : a major positive ion (cation) in the fluid surrounding cells in the body 67

spectrophotometer : a piece of equipment designed to measure the amount of light that is transmitted through a solution

thyroxine : a hormone of the thyroid gland that is derived from tyrosine and that influences the metabolic rate

T U

thyroid-stimulating hormone : a substance secreted by the anterior lobe of the pituitary gland that controls the release of thyroid hormone and that is necessary for the growth and function of the thyroid gland

trypsinogen : the inactive precursor form of trypsin; it is secreted in pancreatic juice and converted into active trypsin through the action of enterokinase in the intestine

uric acid : the metabolic byproduct of nitrogen catabolism

n o i t a r a p e r P e l p m a S a m s a l P 70

1. collect a blood sample in a container with the appropriate anticoagulant 2. mix the blood-filled container with a gentle rocking motion 12 times 3. make sure that the container is covered to prevent evaporation during centrifugation 4. centrifuge (within 1 hour of collection) at 2000 to 3000 rpm for 10 minutes 5. with a capillary pipette, carefully remove the fluid plasma layer from the bottom layer of cells 6. transfer the plasma to a container that has been labeled with the date, the time of collection, the patient's name, and the case or clinic number 7. process the sample immediately, or refrigerate or freeze it, as appropriate

n o i t a r a p e r P e l S

1. collect a whole blood sample in a container that contains no anticoagulant 2. all the blood to clot in its original container at room temperature for 20 to 30 minutes 3. gently separate the clot from the container by running a wooden applicator stick around the wall of the container between the clot and the wall 4. cover the sample and centrifuge at a 2000 to 3000 rpm for 10 minutes 5. with a capillary pipette, remove the serum from the clot 6. transfer the serum to a container that has been labeled with the date, time of collection, the patient's name, and the case or clinic number 7. refrigerate or freeze the sample, as appropriate

s n o i t c n u F n i e t o r P a m s a l P 72

• • • • • • • •

help form the structural matrix of cells, organs and tissues maintain osmotic pressure serve as enzymes for biochemical reactions act as buffers in acid-base balance serve as hormones function in blood coagulation defend the body against pathogenic microorganisms serve as transport/carrier molecules for most constituents of plasma

Co n d i t i o n s A s s o c i at e d w i t h A lt e r e d S e r u m P r o t e i n

• • • • • • • • • • • •

hyperproteinemia and/or hyperalbuminemia hemoconcentration (dehydration) inflammatory disease plasmacytoma lymphoma hypoproteinemia and/or hypoalbuminemia hemodilution (overhydration) blood loss glomerulonephritis hepatic insufficiency malabsorption malnutrition

e c n a l A B e s a B d i c A 74

acid-base balance refers to the steady state of the pH of the body; pH is used to describe the hydrogen ion concentration; every change on the pH scale of one number indicates a power-of-10 difference in the hydrogen ion concentrations; normal pH has a fairly narrow range of approximately 7.35 to 7.45; should pH values begin to fall outside of the ideal range, the functioning of many of the body's proteins is diminished or destroyed; when the pH value of body fluids is less than 7.3, the condition is called acidosis, which is characterized by excess hydrogen ions; when the pH is more that 7.4 the condition is called alkalosis, and it is characterized by a low hydrogen ion concentration

F l u i d s

anions

Na+

Cl-

K+

HCO3-

Ca++

PO4

Mg++

SO4

H+

cations and anions in body fluids

M a j o r

E l e c t r o l y t e s

i n

B o d y

cations

s m r e T y e K y g o l o t y C 9 t i n U 76

A D C B E

Curschmann’s spirals : the coiled mucinous fibrils that are sometimes found in cytology preparation of bronchial samples

abdominocentesis : paracentesis of the abdomen

anisokaryosis : variation in the size of the nuclei of cells in a sample

anisonucleoliosis : variation on the size of the nucleus arthrocentesis : removal of fluid from a joint

carcinoma : a term that describes tumors of epithelial cell origin

centesis : the act of puncturing a body cavity or organ with a hollow needle to draw out fluid

benign : a term used t describe a tumor or growth that is not malignant; this word can refer to any condition that is not life threatening

compression smears : the act of puncturing a body cavity or organ with a hollow needle to draw out fluid compression smears gently place a second slide at right angle to the first slide; the spreader slide is quickly and smoothly slid across the prep slide downward pressure should not be placed on the spreader slide

cornified : keratinized; used to describe vaginal epithelial cells as seen in a vaginal cytology smear from a patient in estrus

discrete round cell tumors : a neoplasia that is characterized by cells with discrete round shapes; examples of round cell neoplasms include mast cell tumors, histiocytomas, lymphomas, plasmacytomas, and transmissible venereal tumors

eosinophilic : a term that refers to an increase in circulating eosinophils or a reddish appearance of cells or components of cells that have a high affinity for stain with acid pH

GK M H F L I lymphoma : a neoplastic disorder of the lymphoid tissue

epithelial cell tumors : a type of neoplasm associated with a clustered arrangement of cells into ball shapes or monolayer sheets; examples include lung adenocarcinoma, perianal adenoma, basal cell tumor, sebaceous adenoma, transitional cell carcinoma, and mesothelioma exudate : a fluid accumulation that results from inflammatory processes; characterized by increased cellularity and protein concentration

lymphopenia : the presence of decreased numbers of leukocytes in a peripheral blood sample

granulomatous : granulomatous – a term that refers to an inflammatory condition that is characterized by high numbers (more than 70%) of macrophages

karyolysis : the degeneration or dissolution of a cell nucleus

karyorrhexis : the fragmentation of a cell nucleus

mast cell tumors : a benign local aggregation of mast cells that forms a nodular tumor that occurs in the skin of most species (most commonly dogs)

histiocytoma : a tumor that contains histiocytes (macrophages)

fine-needle biopsy : a sample collection method in which tissue is obtained by puncture of a lesion fixative : a chemical substance used to preserve or stabilize biological material prior to microscopy or other examination

impression smears : used to collect the surface material of a draining or oozing sore so that it can be examined under a microscope

malignant : a term for diseases in which abnormal cells divide without control and can invade nearby tissues

line smears : a drop of fluid is placed on the slide and a blood smear technique is used except the spreader slide is raised directly upward approximately three-quarter of the way through the smear, creating a line containing a higher concentration of cells; used for low cellularity

melanoma : a tumor that arises from melanocytes of the skin or other organs mesenchymal cell tumors : tumors of mixed mesenchymal tissues with two or more cellular elements that are not commonly associated (not counting fibrous tissue as one of the elements) modified transudate : a transudate with additional protein, cells, or both; it may be a transitional stage that ultimately progresses into an exudate

N R ST P

suppurative : containing, discharging, or causing the production of pus; cytology sample characterized by the presence of neutrophils representing more than 85% of the total nucleated cell in the sample; may also be described as purulent

peritoneal fluid : a naturally produced fluid in the abdominal cavity that lubricates surface, thereby preventing friction between the peritoneal membrane and the internal organs

neoplasia : a generic term that is used to describe any growth; often used to describe a tumor, which may be malignant or benign new methylene blue : an organic compound of the thiazine class of heterocycles; it is used as a stain and as an antimicrobial agent

nuclear molding : a deformation of nuclei by other nuclei within the same cell or adjacent cells

parabasal : an abnormal but not malignant cell seen in some cytologic specimens paracentesis : the removal of fluid from a body cavity

pleomorphism : a term that refers to something that takes a variety of shapes and forms or that has multiple morphologies

punch biopsy : the removal of living tissue for microscopic examination with the use of a punch

Romanowsky stains : a stain made from water-soluble eosin, methylene blue, and absolute methanol and used in parasitology, hematology and cytology

synovial fluid : a transparent, viscous fluid that is secreted by synovial membranes and that acts as a lubricant for many joints, bursae, and tendons; it contains mucin, albumin, fat and mineral salts

pykonsis : the presence of condensed nuclear chromatin in a degenerating cell

pyogranulomatous : a term used to describe a cytology sample that is characterized by the presence of macrophages representing more than 15% of total nucleated cells in the sample reactive lymph node – the build up of lymph fluid in an effort to trap bacteria, viruses, and other harmful pathogens; helps keep the infection from spreading to other parts of the body

sarcoma : a generic term that is used to describe any cancer that arises from cells of the connective tissue

starfish smears : drags the aspirate peripherally in several directions with point of a needle; tends to not damage cells but leaves a thick layer; ideal for viscous samples

thoracocentesis : the removal of fluid from the thoracic cavity transmissible venereal tumors : is a histiocytic tumor of the external genitalia of the dog and other canines; and is transmitted from animal to animal during mating; it is one of only three know transmissible cancers in mammals transtracheal wash : also known as bronchial lavage; performed to diagnose respiratory tract problem, most commonly a chronic and/or recurring cough

transudate : an effusion that is characterized by a low protein concentration and a low total nucleated cell count Tzanck preparation : a rapid test done to diagnose infections caused by herpesvirus; cells are examined under a microscope for signs of infection

W wave motion : the transfer of energy and momentum from one point of the medium to another point of the medium without actual transport of matter between two points wedge biopsy : a biopsy in which a wedge-shaped sample of tissue is obtained

s n r e

Non-degenerate neutrophils (resemble those in blood with condensed clumped chromatin): Causes include immune-mediated conditions, sterile irritants (bile, urine), bacterial infection, protozoal or fungal infection. The lack of degenerative change in neutrophils does not mean that the inflammation is not due to bacterial infection. Some species of bacteria will not cause this morphologic change in neutrophils.

Degenerate neutrophils: These are undergoing karyolysis and taking up water, resulting in cell and nuclear swelling. In these cases, suspect bacterial sepsis and look carefully for phagocytized organisms. However, such swelling can also be an artifact of sample collection into fluids and storage. Degenerate neutrophils do not always mean bacterial sepsis and can be seen with fungal infections and chemical irritants.

degenerate neutrophils

Erythrophages: This indicates recent hemorrhage (<24 hours). Note that erythrophagia can also occur as an artifact during storage of fluid specimens (e.g. body cavity fluids). Macrophages retain phagocytic activity in vitro.

neutrophilic inflammation

granulomatous inflammation

Hemosiderophages: These are macrophages that contain hemosiderin in their cytoplasm. The hemosiderin is from the iron component of the porphyrin ring of hemoglobin (from phagocytized erythrocytes) and represents the storage form of iron. Hemosiderophages supports prior hemorrhage (>24 hours duration as it takes time for hemosiderophages to form).

hemosiderophages

mixed inflammation

This is comprised of a mixture of neutrophils and macrophages, with neutrophils being typically nondegenerate, unless there is concurrent bacterial sepsis. Neutrophils usually dominate, but may not if the outer edges of the lesion are aspirated (the latter areas may be more macrophage-rich). This type of mixed inflammation is also called pyogranulomatous, particularly if there are multinucleated macrophages. Lymphocytes and plasma cells can be seen in low numbers well.

This is generally defined as inflammation consisting of more than 10-20% eosinophils, although cutoffs vary between clinical pathologists, with some pathologists indicating an eosinophilic component to the inflammation if <20%.

eosinophilic inflammation

dysplasia

Dysplasia, or disordered growth, is most often seen in epithelial tissue secondary to inflammation or irritation. Dysplasia results in loss of uniformity of the individual cells and disordered architectural arrangement of the cells. It also results in atypical cytologic features in the aspirates cells, including nuclear to cytoplasmic asynchrony, increased cytoplasmic basophilia, anisokaryosis and anisocytosis. Dysplasia can be cytologically difficult to distinguish from neoplasia as dysplastic lesions often contain more criteria of malignancy than strictly hyperplastic lesions.

Epithelial tumors are cohesive and form clusters or sheets. They can show trabecular, circular to papilliform arrangements. Acini may be seen in cells that produce secretory product. Examples of epithelial tumors include perianal gland adenoma, transitional cell carcinoma, biliary carcinoma, squamous cell carcinoma.

epithelial cells

Mesenchymal neoplasms carry features of their embryonic tissue of origin, the mesenchyme. The cells are generally individualized and spindled in shape. They can be seen in aggregates (not clusters), often held together by extracellular matrix. They do not typically demonstrate cell-to-cell adhesion. Due to increased matrix production, there are some mesenchymal tumors (e.g. fibroma) that do not exfoliate well and aspirates may be of low cellularity making a definitive cytologic diagnosis difficult.

mesenchymal cells

Mast cell tumor: These are readily recognized by the presence of purple cytoplasmic granules. They also have round eccentric nuclei with smooth chromatin. The nuclei can be hard to see as the granules soak up the stain.The degree of granularity varies between tumors. Granules may be harder to discern with waterbased stains, such as Rapid stains, particularly in the less well-granulated tumors. mast cell tumor

Histiocytoma: Cell of origin is the epidermal Langerhans cell. These may not be tumors per se as they regress without treatment due to a cytotoxic T cell immune response. They can be single (usually are) or multiple.

histiocytoma

Plasmacytoma: These arise from plasma cells, which form tumors (usually solitary) in extramedullary sites, such as the skin (digit, ears, mouth) in dogs.

plasmacytoma

cutaneous lymphoma

Lymphoma: There are multiple forms of cutaneous lymphoma, including inflammatory, epitheliotropic and non-epitheliotropic forms. In the skin, lymphoma can consist of solitary to multiple nodules, plaques or ulcerative/ exfoliative lesions. It can be pruritic. Lymphoma, as we know, also arises in other sites. They are usually of T or B cell origin. Lymphomas of natural killer cell origin are quite rare. Lymphoma is most easily recognized when it consists of large cells or cells that are not expected in inflammatory lesions, such as many granular lymphocytes.

Transmissible venereal tumor: This sexually transmitted tumor is thought to be of histiocytic origin. It is mostly seen in warmer cell climates and is often around the mouth or genital region, but can be seen in other sites.

transmissable venereal tumor

These tumors have a characteristic appearance, forming packets of cells. Cells often exfoliate in large numbers but are fragile and aspirates contain many bare nuclei from ruptured cells, hence some people call them “naked nuclei” neoplasms. They are of secretory epithelial (producing hormones, e.g. thyroid tumors) or neuroectodermal origin, with the latter secreting neurotransmitters, such as epinephrine in phaechromocytomas. endocrine cells

MESENCHYMAL

DISCRETE "ROUND" CELL

ENDOCRINE NEUROENDOCRINE

TYPICAL CELLULARITY OF ASPIRATES

high

low to moderate (may be high in a few specific tumors, e.g., soft tissue sarcoma)

high

high, may have large numbers of individualized free nuclei from ruptured cells ("naked nuclei")

CELL ASSOCIATIONS

clusters (adherent); can see acinar formation if secretory

individual cells, or in some non-cohesive aggregates

individual cell; may form aggregate-like arrangements in thicker aspirates

clusters (adherent); can see acinar formations in endocrine tumors

CELL SHAPES

variable; often polygonal, columnar, or cuboidal

spindled to stellate to rarely round (hystiocytic)

round ( ish)

generally round or cuboidal

CELL SIZE

medium to large

medium

small to medium; large with large cell lymphoma

small to medium

CELL BORDERS

distinct in most tumors

often distinct

distinct

indistinct

NUCLEAR SHAPE

round to oval

oval to elongated to round (e.g., soft tissue sarcoma)

round, sometimes indented (histiocytic tumors)

round

OTHER CHARACTERISTICS

can see features of differentiation (e.g., hepatoid cells, squamous cells)

some tumor types may be associated with characteristic extracellular matrix (e.g., chondrosarcoma)

cytoplasmic purple granules in mast cells tumor may occasionally stain poorly with Diff-Quik

tend to lack cytologic features of malignancy regardless of biologic behavior

EXAMPLES OF TUMORS

benign: adenomas malignant: carcinomas

benign: "-omas", e.g., fibromas malignant or invasive: sarcomas

lymphoma, plasmacytoma, mast cell tumor, histiocytoma, transmissible venereal tumor

insulinoma, thyroid tumors, apocrine anal sac adenocarcinoma, phaechromocytoma, paraganglioma

EPITHELIAL

Student: Jennifer L. Smith Instructor: Kathy McLemore, CVT, VTS School: Apex College of Veterinary Technology Program: Veterinary Technology, A.A.S. Course: VET 501: Clinical Chemistry and Interpretive Cytology 85