Clin Path 2 - Section 1 - Hematology

Name the types of blood cells

Erythrocytes (RBC's) Leukocytes (WBC's) - Neutrophils - Eosinophils - Basophils - Monocytes - Lymphocytes Thrombocytes (Platelets)

Describe the procedure for performing a platelet estimate.

Platelet eval done by looking at blood film - #'s of platelets in minimum of 10 microscopic fields is counted - 7-10 platelets per oil-immersion field is normal - Indirect measurement: multiply estimated platelet # (as averaged over 10 fields) by 15,000 or 20,000 is also used

List and describe the terms used to describe abnormal changes in red blood cell shape

Poikilocytes Schistocytes Acanthocytes Echinocytes Drepanocytes Karatocytes Spherocytes Leptocytes Elliptocytes Eccentrocytes Dacrocytes

List and describe the terms used to describe abnormal changes in red blood cell color

Polychromasia Hypochromasia Hyperchromasia

Differentiate between hematopoiesis in prenatal and adult animals.

Prenatal: blood cells are formed at multiple organ sites (liver & spleen) Adults: blood cells formed in bone marrow

Prussian blue stain is used to evaluate

bone marrow samples for the presence of hemosiderin.

Erythrocyte indices are

calculated values that provide a measure of the size of the RBCs and their average hemoglobin concentration

The microhematocrit test uses

capillary tubes that are filled with blood and centrifuged

Changes in erythrocyte morphologic features affect

cells' size, shape, color, and arrangement.

A variety of techniques can be used to prepare bone marrow samples, with the ______________________ being the most common method.

compression smear

Bone marrow samples may be collected via ______________________ techniques.

core sampling or aspiration

pancytopenia

decrease in the number of ALL blood cell types

-penia

decreased number of cells in the blood

lymphopenia

decreased number of lymphocytes in the blood

Blood cell films are used to determine

estimated platelet #'s

All blood cells are derived from

hematopoietic stem cells (HSC's)

-philia or -cytosis

increased number of cells in the blood

leukocytosis

increased numbers of leukocytes in the blood

neutrophilia

increased numbers of neutrophils present in the blood

List the organs involved in hematopoiesis.

liver, spleen, thymus, and red bone marrow

Inclusions that are seen in leukocytes include

lysosomes, a variety of abnormal granules, and blood parasites.

Changes in erythrocyte size can involve

microcytes, macrocytes, or both

Mature* segmented granulocytes are produced through the

myeloblast, promyelocyte, myelocyte, metamyelocyte, & band cell precursors

leukemia

neoplastic cells in the blood or bone marrow

The results of bone marrow evaluation include, at a minimum, the

overall cellularity and the M : E ratio, the maturation index, or the left shift index.

Platelet production proceeds through the

progenitor cell develops into megakaryoblast, then promegakaryocyte, & then megakaryocyte stage *platelets are fragments of the cytoplasm of megakaryocytes

Define histogram and explain the use of histograms.

provide a visual representation of the numbers and sizes of the cells present in a sample

List the tests that comprise the complete blood count.

red and white blood cell counts, hemoglobin concentration, packed cell volume (PCV), a differential white blood film examination, and calculation of absolute values and erythrocyte indices Additional tests: Reticulocyte counts, measurement of total solids, thrombocyte estimates, & bone marrow evaluation

Differential WBC count provides the

relative percentage of each WBC type present in a sample

When examining bone marrow samples, evaluate cellular morphologic characteristics to determine the

relative percentages of nucleated cells and adipocytes, the relative percentages of erythroid and myeloid cells, and the M : E ratio.

Alterations in erythrocyte behavior include

rouleaux formation and autoagglutination

List the cells in the erythrocyte maturation series.***

rubriblast, prorubricyte, basophilic rubricyte, polychromatophilic rubricyte, metarubricyte, reticulocyte

Morphologic changes that are seen on a blood film must be _______________.

semiquantified

Reticulocytes are immature RBCs that require staining with a _______________ for identification

supravital stain

T-lymphocytes, B-lymphocytes, and NK cells develop through

the lymphoblast and prolymphocyte stages

Changes in leukocyte morphologic features may affect

the nucleus or the cytoplasm or involve inclusions within the cell.

The ESR is a measure of...

the speed at which erythrocytes fall in their own plasma under controlled conditions.

Hematology

the study of blood cells and their formation

Describe the laser flow cytometry test principles

uses focused laser beams to evaluate the size & density of solid components - Cells scatter light depending on shape & volume of the cell & the presence of absence of granules & nuclei - Degree & direction of light scatter from cells allows for enumeration of monocytes, lymphocytes, granulocytes, & erythrocytes

Describe the principle of the electrical impedance analyzer.

work by measuring the change in current as cells in electrolyte solution pass through an aperture

The preferred method of blood collection is the

Vacutainer system.

Describe the types of morphologic changes seen in white blood cells

WBC's - Nuclear Hypo-segmenatation: neutrophils with fewer than 3 nuclear lobes - Nuclear Hyper-segmentation: neutrophils with 5 or more nuclear lobes - Toxic change: Cytoplasmic changes in neutrophils = Giantism, cytoplasmic basophilia, Dohle bodies, & vacuoles or "foaminess" - Atypical (may have basophilic cytoplasm & cleaved nuclei) & Reactive Lymphocytes (increased basophilia in cytoplasm; may have more abundant cytoplasm, sometimes contain larger & more convoluted nucleus) - Lysosomal Storage Disorders (substances are abnormally stored w/in cells - appearance of leukocytes varies depending on type of lysosomal storage disease - Birman Cat Neutrophil Granulation Anomaly: neutrophils contain fine eosinophilic to magenta granules; must be distinguished from toxic change - Chediak-Higashi Syndrome: neutrophils have large, fused lysosomes in cytoplasm; stain lightly pink or eosinophilic - Siderotic Granules: appear similar to Dohle bodies; differentiated with use of Prussian blue stain - Smudge Cells: degenerative leukocytes that have ruptured - Karyolysis: degenerative change to nucleus - Pyknosis: condensing of nucleus - Karyorrhexis: fragmentation of nucleus

Describe the procedures for preparing blood samples for evaluation.

Wedge smear: 1) Remove drop of blood from EDTA tube 2) Place drop toward frosted end of microscope slide 3) End of 2nd slide is placed against surface of 1st slide at 30-degree angle & drawn back into drop of blood 4) Once blood spreads along most of width of spreader slide, it is pushed forward with steady, even, rapid motion 5) Slide is gently waved to allow it to dry quickly Coverslip smear: 1) 1) Place 1 drop of blood in the center of a clean square coverslip 2) Add a 2nd coverslip diagonally on top of the 1st 3) Allow blood to spread evenly between 2 surfaces until the blood almost fills the area between the coverslips 4) Pull coverslips apart in single smooth motion 5) Wave smears gently in air to promote drying

leukemoid response

a condition that can be mistaken for leukemia; characterized by marked leukocytosis (>50,000/mL), and usually the result of inflammatory disease

What is neutropenia?

a decreased number of neutrophils

What is a reticulocyte?

an immature RBC that contains organelles (ribosomes) that are lost as cells mature; lack a nucleus

What is neutrophilia?

an increase in number of neutrophils

Decreased PCV may indicate

anemia

Hemoglobin testing can be performed with

automated analyzers or with handheld meters

Differentiate between aggregate and punctate reticulocytes.

*These are the 2 morphologic forms of reticulocytes in cats A: contains large clumps of reticulum; similar to reticulocytes in other species; represents same cell that stains polychromatophilic with Wright's stain P: contains 2-8 small, singular, basophilic granules (unique to cats) & do NOT stain polychromatophilic with Wright's stain

Describe procedures for the collection of blood samples from small and large animals.

1) Collect appropriate supplies: needle & syringe of appropriate size; tubes with proper anticoagulants; isopropyl alcohol 2) Restrain patient 3) Select appropriate site depending on species 4) Occlude the vein 5) Clean the area with the alcohol 6) Insert needle bevel up 7) Collect sample

List and describe the equipment needed to perform a complete blood count.

1) blood sample - collected via needle & syringe; preferred method = Vacutainer system 2) Proper anticoagulant 3) Automated analyzers - Impedance analyzer: classifies cells based on their sizes - Laser-based flow cytometer: counts & classifies cells based on size and density - Quantitative buffy coat system: estimates cell counts

Describe the procedure for calibrating the centrifuge for optimum microhematocrit spin time.

1. Use a stopwatch to verify the centrifuge timer operation. Run several tests at different time intervals, and repeat each at least twice to verify reproducibility. 2. Use a tachometer to check the centrifuge speed. 3. Verify the minimum time required to obtain an accurate packed cell volume. 4. The minimum time needed to achieve optimal packing of the red cells should be checked with the following procedure: a. Choose two fresh EDTA blood samples; one sample should have a hematocrit level of more than 50%. b. Fill 10 to 12 microhematocrit tubes for each sample. c. Perform duplicate microhematocrit determinations at increasing times, beginning at 2 minutes. Centrifuge times should be increased by 30-second intervals. Record duplicate values at each time interval. d. Continue to increase the centrifuge time until the value remains the same for two consecutive time intervals. e. Centrifuge two more samples for an additional 30 and 60 seconds beyond that interval. f. Plot the results on a graph. The plateau point is the first point on the curve after the curve flattens out. This is the optimum spin time. g. Repeat the procedure periodically, because brushes and motors can wear, thereby reducing the speed of the centrifuge.

Describe methods for semiquantifying morphologic changes.

2 methods used to assess degree of morphologic changes One method: uses scale of 1+, 2+, 3+, & 4+ to indicate relative % of cells w/ morphologic change —> 1+ usually equates to 5-10% of cells being affected; 2+ = 10-25%; 3+ = 50%; 4+ = more than 75% 2nd method: uses "slight" = 10%, "moderate" = 25%, or "marked" = 50%

Describe the care and maintenance of automated hematology analyzers.

A daily "background count" is generally required - involves running the electrolyte solution through the analyzer so that any small particles may be identified and subsequently not counted by the analyzer The aperture may become partially or totally obstructed and require cleaning. The threshold setting may be improperly set on a counter with a variable threshold control Cold agglutinins may cause a decreased RBC count as a result of RBC clumping. Before processing, refrigerated blood samples must be warmed to room temperature

How many WBCs are counted and classified when performing the differential WBC count?

A minimum of 100

Describe the procedure for the proper staining of differential blood cell film.

After the slides air dry, they must be stained to clearly distinguish individual cells & identify any abnormal cellular characteristics Smears typically must be immersed in each solution for 5 to 10 seconds. 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 of the slide and away from the smear. Usually use Romanowsky-type stain Most common: Wright's stain & Wright-Giemsa stain Available in 1 step or 3 step formulations - Usually include fixative & buffered solutions of eosin & methylene blue

List and describe the terms used to describe abnormal changes in red blood cell size

Anisocytosis: RBC's that are unequal in size - Microcytic RBC: smaller cell, but central pallor still present - Macrocytic RBC: larger cell, but central pallor still present

How can PCV increase?

As a result of dehydration or polycythemia

Describe methods for preparing bone marrow samples for evaluation.

BM samples made asap if not mixed with EDTA at collection time - If EDTA is used, smears must be made w/in 1 hr of collection Smears of aspirate samples made similarly to smears of peripheral blood - 2-12 slides are prepared so samples are available for additional testing - Aspiration samples are pushed out of the hub of the needle using pressure from the syringe - BM samples are thicker than blood & should contain particles or spicules - Excess blood or EDTA in sample can be removed by tilting slide to allow it to run off —> Or, sample can be expelled in Petri dish & BM spicules removed w/ small plastic pipette or capillary tube Line, starfish, & compression smears can be used for BM samples - Modified comp technique may be most useful - Compression is similar to technique used for coverslip preparation of peripheral blood films BM smears rapidly air dried & stained, usually w/ Romanowsky-type stain - Staining time increases based on cellularity & thickness of sample Smears sent to reference labs usually stained with Prussian blue to identify iron particles in marrow sample - Hematoxylin-eosin stain often used for core biopsy samples, tho Giemsa stain may be used

________ are not commonly seen on the differential blood cell film

Basophils

Describe the processes and pathways that lead to the clotting of blood.

Coagulation proceeds through a mechanical & a chemical phase *which are interrelated* Mechanical: initiated when a blood vessel is ruptured or torn; platelets are attracted to this exposed, charged surface. Platelets will undergo changes to adhere to each other as well as to the blood vessel endothelium; causes aggregation; adhesion requires von Willebrand factor - serves to stabilize the platelet plug. This causes platelets to release the initiating factor for the chemical phase of hemostasis Chemical phase = coagulation cascade; involves # of coagulation factors that activate each other (Factors I - XIII) - Intrinsic Pathway: activated by trauma inside the vascular system, & is activated by platelets, exposed endothelium, chemicals, or collagen. Slower than extrinsic. Involves factors XII, XI, IX, VIII - Extrinsic Pathway: activated by external trauma that causes blood to escape from the vascular system; quicker than intrinsic; involves factor VII - Common Pathway: where both pathways meet & finish the pathway of clot production; involves factors I, II, V, & X End result of coagulation cascade = formation of a mesh of fibrin strands that forms the clot Final phase of hemostasis = degradation of fibrin clot

Describe the collection and handling of bone marrow aspiration and core samples.

Collected via aspiration or removal of bone marrow core - Proper restraint is crucial & sedation or local anesthesia is needed - Aseptic technique is vital Procedure: 1) Prepare syringes with EDTA solution by drawing 0.5mL of saline into syringe & dispensing it into empty small EDTA tube 2) Withdraw mixture & repeat procedure; creates a diluted EDTA flush to use on syringe & bone marrow needle 3) Gather slides - set them on an incline (usually about 12 slides) - Other equipment: no.11 scalpel blade, sterile skin preparation supplies, & suture material, if needed - Special bone marrow kit is preferred, altho an 18-gauge needle may be used for collection from puppies & kittens - BM needles have a stylet - prevents occlusion of the needle with bone and surrounding tissue as it's inserted into marrow cavity - Needle types used for BM collection = Rosenthal, Illinois sternal, and Jamshidi For aspiration biopsy: 1) Site must be aseptically prepared & draped 2) Stab incision is made at the site w/ sterile scalpel blade & needle is inserted w/ stylet in place 3) Slight pressure applied on side of needle against stylet to avoid blockage of needle w/ bony material 4) Slight caudo-ventral angle introduces needle into head of humerus 5) Needle & stylet are advanced until cortex of humerus is reached 6) Needle is rotated while slight forward pressure is applied; allows needle to penetrate cortical bone, & keeps needle in place 7) Stylet is removed, & syringe is attached 8) Large syringe (10-20mL) usually preferred to allow greater negative pressure 9) Syringe plunger is rapidly & vigorously withdrawn until a few drops of blood enter the hub of the needle 10) Once few drops of material are present in hub of needle, pressure is released to minimize hemodilution of the sample 11) Stylet should be replaced & needle left in place until smears are prepared to ensure adequate sample was obtained 12) Small suture may be needed to close the site For Core Biopsy: - Procedure is similar to aspiration biopsy: 1) After the needle is introduced into the cortical bone, the stylet is removed, and the needle is advanced ~1" and rotated back and forth to cut the piece of bone from the cortex. 2) Needle is removed and the stylet used to expel the core sample through the proximal (hub) end of the needle 3) Forcing the sample back through the narrow distal end of the needle introduces pressure artifact into the sample 4) An imprint should be made from the sample before it is placed in formalin *Usually, better-quality samples & greater diagnostic info is obtained if core sample of marrow is also collected with aspiration biopsy - Use different sites when collecting both to ensure procedures do NOT introduce artifact into next sample

Describe the criteria used to characterize a bone marrow sample as acellular, hypercellular, or hypocellular.

Determined by the proportion of nucleated cells vs amount of fat present Acellular: normal bone marrow that is 50% nucleated cells & 50% fat Hypercellular: marrow samples that have over 70% cellularity Hypocellular: marrow samples that have less than 30% cellularity

List the commonly used blood collection sites for various species.

Dogs/Cats: ***jugular vein is the vessel of choice for blood collection from most mammals; cephalic vein, saphenous vein Horse: jugular vein Cattle: coccygeal vein & jugular vein Bird: jugular vein & medial metatarsal vein Rabbit: ear vein Rodent: tail vein

Discuss aspects of quality control related to hematology testing.

Doing quality control regularly assures accuracy of test results

List the commonly used anticoagulants and the purpose and mode of action for each.

EDTA: prevents clotting by forming an insoluble complex (chelates) with calcium - which is needed for clot formation; preferred for hematologic tests Heparin: prevents conversion of prothrombin to thrombin; used for tests that require plasma samples Oxalates & Citrates: prevents clotting by forming insoluble complexes with calcium (chelates calcium) Sodium Fluoride: preserves glucose + has anticoagulant properties; chelates calcium

The preferred anticoagulant for hematology testing is_____; the preferred anticoagulant for coagulation testing is ______.

EDTA; citrate

List and describe the parasites that may be seen on a blood smear

Ehrlichia: can infect any of the leukocytes; appear as small clusters Mycoplasma (M. haemofelis = common parasite of feline RBC's); causes hemobartonellosis or feline infectious anemia; appear small, coccoid, rod-shaped, or ringlike structures that stain dark purple w/ Wright's stain; often appear as short rods on the periphery of RBCs in cats Microfilaria or D. immitis Eperythrozoa: swine, cattle, llamas; appear as small cocci, rods, or rings on RBC surface or free in plasma - ring form is most common Anaplasma: affects only platelets; causes infectious cyclic thrombocytopenia Cytauxzoon: rare cause of hemolytic anemia in cats; org's appear as small, irregular ring forms w/in RBC's, WBC's, & macrophages Babesia (Texas fever in cattle): appear as large, pleomorphic, teardrop-shaped intracellular org's often seen in pairs

List the cells in the leukocyte maturation series.

Eosinophils: eosinophilic myelocyte, eosinophilic metamyelocyte, eosinophilic band, eosinophil Neutrophils: myeloblast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophilic band, neutrophil Basophils: basophilic myelocyte, basophilic metamyelocyte, basophilic band, basophil Monocytes: monoblast, promonocyte, & monocyte Lymphocytes: lymphoblast, prolymphocyte - differentiates into small lymphocyte OR natural killer cell (NK cell); small lymphocyte differentiates into B lymphocyte or T lymphocyte

List and describe the cells that are commonly seen in bone marrow samples.

Erythroid cells: - rubricytes: smaller than rubriblast and prorubricyte with clumpier chromatin - metarubricytes: smaller than rubricyte; has pyknotic smooth nucleus that is shruken Myeloid cells: - metamyelocytes: kidney bean-shaped nucleus - bands: horseshoe-shaped nucleus - segmented myeloid cells Mature segmented neutrophils, eosinophils, & basophils present in small numbers Other cell types that are present in bone marrow samples include - macrophages: often contain phagocytized material that may aid in diagnosis - lymphocytes: usually present in low #'s —> Immature stages (i.e., lymphoblasts and prolymphocytes) are difficult to distinguish from rubriblasts and prorubricytes —> Reactive lymphocytes and normal mature lymphocytes may also be present and appear as they would if they were seen in peripheral blood samples - plasma cells: slightly larger than lymphocytes, with a greater nucleus-to-cytoplasm ratio; often have a perinuclear clear area around the eccentrically located nucleus, and they have distinctly basophilic cytoplasm - mast cells: characterized by the presence of abundant, small, metachromatic cytoplasmic granules - osteoblasts: appear similar to plasma cells except that they are much larger and contain paler-appearing nuclear material; tend to be found in clusters when they are seen in samples collected by aspirate techniques - osteoclasts: contain multiple nuclei and may appear somewhat fused and similar in appearance to megakaryocytes, except that the megakaryocyte nucleus is multilobed

Troubleshoot stain quality on a differential blood cell film.

Excessive Blue Staining - Inadequate wash = wash longer - Specimen too think = make thinner smears, if possible - Delayed fixation: fix smears sooner, if possible Excessive Pink Staining - Insufficient staining time = increase staining time - Excessive time in red stain solution = decrease time in solution - Mounting coverslip before preparation is dry = allow preparation to dry completely before mounting coverslip Weak Staining - Fatigued (old) stains = change stains - Insufficient contact with 1 or more stain solutions = increase staining time Uneven Staining - Water allowed to stand on some areas of the slide after staining and washing = Tilt slides close to vertical to drain water from the surface or dry with a fan - Inadequate mixing of stain & buffer = Mix stain and buffer thoroughly

Define hematopoiesis, leukopoiesis, erythropoiesis, and thrombopoiesis.

H: production of blood cells & platelets L: production of leukocytes E: production of erythrocytes T: production of platelets; involve specific cytokines

Describe the formation of the formed elements in blood.

Hematopoiesis; the formed elements of blood are continually produced, replacing the relatively short-lived erythrocytes, leukocytes, and platelets. Hemopoiesis begins in the red bone marrow, with hemopoietic stem cells that differentiate into myeloid and lymphoid lineages

Inclusions seen in erythrocytes include

Howell-Jolly bodies, Heinz bodies, basophilic stippling, and a variety of blood parasites

How do the different types of analyzers classify cells? Impedance, Laser, & Buffy coat?

I: classify cells according to their sizes L: classify cells on basis of size/density as they pass through focused laser beam BC: provide estimates of cell counts

left shift

Increased numbers of immature neutrophils in the blood

Explain the role of erythropoietin in hematopoiesis.

It is the primary cytokine responsible for the production of RBC's

List the calculations used to obtain the mean corpuscular volume, the mean corpuscular hemoglobin, and the mean corpuscular hemoglobin concentration.

MCV = PCV/RBC count x 10 MCH = Hb (g/dL) / RBC concentration x 10 MCHC: Hb (g/dL) / PCV (%) x 100

Describe the procedure for performing a packed cell volume test with the microhematocrit method.

Microhematocrit (mHct) method = most commonly performed PCV test - Requires anticoagulated blood sample be placed in 75-mm capillary tube - mHct tubes filled ~3/4 full - Usually 2 tubes are filled from one sample at same time so they can be centrifuged together - After tube is filled to appropriate volume, one end is plugged with clay sealant - Tube is placed in centrifuge with plugged end facing outward& spun for 2-5 minutes, depending on centrifuge used Blood components separate into different layers according to relate weights

________ tend to have an amoeboid nucleus, and they are the largest of the WBCs in circulation

Monocytes

Describe the procedure for performing a leukocyte absolute value calculation.

Multiply the relative percentage of each cell type by the total WBC count.

________ are the largest of the granulocytes, and they contain a nucleus with three to five lobes

Neutrophils

Differentiate among oxyhemoglobin, methemoglobin, and sulfhemoglobin.

O: occurs when oxygen binds to the heme component of hemoglobin in RBC's M: occurs naturally both in plasma and within RBCs; cannot bind to oxygen, which means it cannot carry oxygen to tissues; but it can be converted to hemoglobin and used for oxygen delivery S: not normally present in the body; made by the oxidation of iron in hemoglobin to a ferric state by drugs & chemicals that contain sulfur; formed by sulfur binding to the hemoglobin molecule's prphyrin ring

Describe the components of blood.

Plasma (consists of 90% water and the remainder = proteins, hormones, vitamins, & other molecules) Cells contained in plasma = RBC's, WBC's, & platelets

Plasma vs serum

Plasma is whole blood minus the cells; serum is whole blood minus the cells and the clotting elements.

Describe the types of morphologic changes seen in red blood cells

RBC's 1) Variations in Cell Arrangement: - Rouleaux: grouping of RBC's in stacks - Autoagglutination: bridging/clumping of RBC's 2) Variations in Cell Size - Anisocytosis: may indicate presence of macrocytes & microcytes 3) Variations in Cell Color: - Polychromasia: exhibit bluish tint when stained with Romankowsky-type stain - Hyperchromasia: decreased staining intensity caused by insufficient amount of hemoglobin w/in the cell - Hyperchromatophilc: cells appear more dearly stained than normal cells 4) Variations in Cell Shape - Poikilocytes: abnormally shaped RBC's; not helpful term - Schistocytes: RBC fragments appearing in a variety of morphologic forms such as small triangular erythrocytes, helmet cells, and normal-size erythrocytes with 2 to 3 pointed surface projections - Acanthocytes: have several (usually 3 to 7) irregularly spaced blunted projections from the margin of the cells - Echinocytes: are also cells with cytoplasmic projections, but in contrast to acanthocytes, the projections are typically evenly spaced on the cell surface, more numerous (often 10 to 15), and frequently have sharper points - Drepanocytes: elongated, sometimes crescent-shaped, erythrocytes with pointed ends - Keratocytes: (helmet cells); cells appear to contain a vacuole - Spherocytes: round, densely staining red cells that lack central pallor and have a smaller than normal diameter - Leptocytes: thin, flat cells with the hemoglobin at the periphery of the cell - Elliptocytes: range from slightly oval to elongated cigar-shaped forms - Eccentrocytes: hemoglobin is primarily pushed to one side - Dacrocytes: red cells with one end round and the other end more pointed (teardrop) 5) Inclusions - Basophilic Stippling: presence of small, dark-blue bodies /win RBC's - Howell-Jolly Bodies: basophilic nuclear remnants in young RBCs - Heinz Bodies: round structures that represent denatured hemoglobin

Describe the appearance of normal blood cells and platelets.

RBCs = small, round, biconcave discs; NO nuclei present in mammals; normal canine RBCs have central pallor (lightness) to them WBC's = - Neutrophils: irregular/elongated nucleus + 3-5 nuclear lobes - Eosinophils: shapes vary among species; nucleus is similar to neutrophils, but chromatin usually not as coarsely clumped - Basophils: nuclei similar to monocytes (sausage-shaped); granules in dogs/cats stain purple to blue-black; in equine/bovine stain blue-black & may completely pack cytoplasm; feline are round/stain light lavender - Lymphocytes: variety of sizes in peripheral blood; small lymphocytes in dogs & cats have slightly indented nuclei + chromatin is coarsely clumped & cytoplasm is light blue & scanty; medium-large lymphocytes have more abundant cytoplasm that may contain pink-purple granules - Monocytes = largest of WBCs in peripheral blood; contain variably shaped nuclei - occasional shape of kidney bean, but often elongated, lobulated, or amoeboid; nuclear chromatin = more diffuse than neutrophils; cytoplasm is blue-gray color; may contain vacuoles & small, fine, pink granules Platelets (thrombocytes) = shape varies greatly, but usually round, oval, or rod-shaped; stain light blue to purple; very granular

Erythrocyte OF refers to the ability of

RBCs to withstand hemolysis in varying concentrations of saline solution.

Describe the procedure for performing the reticulocyte count.

RC = expression of % of RBCs that are reticulocytes Percentage of R per 1000 RBC's determined using oil-immersion lens Perform on blood of all anemic domestic animals, except horses who do NOT release reticulocytes from the bone marrow Reticulocyte concentration is useful for assessing bone marrow's response to anemia

Explain the significance of reddish, yellow, and cloudy plasma colors in the centrifuged microhematocrit tube

Reddish tinge = hemolyzed = can be artifact if not collected/handled properly, or may be evidence of pathologic condition Deep yellow = icteric = seen in animals with liver disease or hemolytic anemia Cloudy serum = lipemia = can be pathologic condition or may be artifact if P was not properly fasted before blood collection

List and describe the terms used to describe abnormal changes in red blood cell arrangement

Rouleaux Autoagglutination

Describe the formation of platelets

Starts as a hematopoietic stem cell; produced from very large bone marrow cells called megakaryocytes. As megakaryocytes develop into giant cells, they undergo a process of fragmentation that results in the release of over 1,000 platelets per megakaryocyte

List and describe the hematology evaluations that are commonly performed in veterinary practice.

The complete blood count includes red and white blood cell counts, hemoglobin concentration, packed cell volume (PCV), a differential white blood film examination, and calculation of absolute values and erythrocyte indices. Additional tests that may be needed include reticulocyte counts, measurement of total solids, and thrombocyte (platelet) estimates

List the layers in the centrifuged microhematocrit tube in order from bottom to top.

The layers in the microhematocrit tube are the packed RBCs, the buffy coat, and the plasma

Describe toxic change

These are cytoplasmic morphologic features seen in neutrophils as a result of accelerated maturation through the bone marrow, in response to inflammatory cytokines

Describe the procedures for the manual counting of cells.

Use a hemocytometer or Leukopet system (exotic animals) 1) Clean hemocytometer & glass coverslip 2) Place glass coverslip over counting chambers 3) Pipette 10 microliters of cell sample into hemocytometer 4) Allow sample to stand for 10 mins so cells can settle 5) Place hemocytometer under microscope with 100x magnification 5) Calculate total leukocyte count as: Total heterophil + eosinophil (both chambers) x 1.1 x 16 x 100 / % Heterophils + % Eosinophils (from differential count)

Describe the principle of quantitative buffy coat analysis.

Uses differential centrifugation & staining to provide an estimation of cellular elements Measurements made using expanded buffy coat layer in specialized microhematocrit tube Partial differential count divides cells into granulocytes & lymphocytes and monocyte categories Best used as a screening tool b/c it only provides an estimation of cell #'s

Discuss indications for bone marrow evaluation.

Usually needed to provide sufficient detail for diagnosis when the differential leukocyte count from the peripheral blood film is ambiguous or has unexplained abnormal results Specific indications: persistent unexplained pancytopenia, neutropenia or thrombocytopenia, & nonregenerative anemia