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CBC tests should include:
1.PCV
2.TPP
3.TWBC
4.differential WBC count
5. erythrocyte morphology
6.PLT
7. Reticulocytes count if anemic
8. Hgb oncentratin
9, MCV
10. WBC differential
Methods for performing CBC
1. Electronic cell counters
2. quantitative buffy coat analysis
How does the electronic cell counter work
Cells are passed btwn 2 elerodes which interrupts the flow of electricity. Each interruption counts the cell as well assize & volume due to the interruption
Define MCV
mean corpuscular volume, determines the erythrocyte volume distribution
Why are WBC samples diluted with a lytic agent
It destroys the cell membrane leaving only the nuclei for counting
Define blood
cells embedded into a fluid matrix
What is the purpose of blood
1. Transport system for supplies (glucose, O2, H2O electrolytes, hormones)
2. Carry waste (Co2 & nitrogenous protein waste
What can a blood exam help to determine
1. Disease
2. View of overall health
3, Adjunct to evaluation & dz dx
4. assess ability to fight infection
5, evaluate trends - deviation from normal
6. ck for effects of tx
7. View in light of PE, Hx
Common veins for blood draws
Large veins like jugular, femoral, saphenous
Why use anticoagulants
Blood may clot too quickly for test to be completed
What anticoagulant is in a Purple Top
EDTA
Why is the ratio of anticoagulant to blood so important
The shape of the blood cells can be altered if the ratio isn't correct
What anticoagulant is in a green top
Heparin
Used for certain specie like reptiles ck for RBC
How is the proper ratio of blood added to the anticoagulant in the tubes
The tubeis a vaccuum so will suck out enough blood in the correct proportions
What is the best gauge needle to withdraw blood & why
Nothing less than 20 gauge b/c will damage cells and give a miss reading
What measures should you take to preserve cell shape if you use a vacutainer or syringe smaller than 20 gauge
After blood draw, remove the needle & let sample dribble down the side of the tube so that the blood is not forced 2x thru the narrow opening of the needle
Who has fragile blood
birds
What is quantitative data
Cell counts
Cell measurements - ex. volume of RBC
What does the volume of RBC help to determine
The volume of hte RBC helps to determine its capacity to carry O2. The hemoglobin in the RBC is the O2 carrier
Why collect qualitative data
1. morphology
2. Cell shape evaluation
3. Cytoplasm of WBC gets weird when fighting infection
4. Parasites change cells
What values do CBC determine
1. PCV
2. TPP
3.TWBC
4. Blood film
5. Reticulocyte count
What is PCv & how is it obtained
Packed Cell Volume
1. fill a hermatocrit tube 3/4 full using capillary action
2. may or may not have anticoagulant depending on test you want to run
3. Mix gently
4. Stab the end in clay
5. Centrifuge
6. Read against the hermatocrit reader
What does PCV help to determine
RBC mass. If you know the volume of the cylinder, you can compute the # of RBC
What 3 components are separated during centrifugation
1. RBC on the bottom (Hgb is heavy with iron)
2. Buffy coat holding the WBC & platelets & parasites
3. Top coat - serum (if blood is allowed to coagulate)
or plasma if blood is not allowed to coagulate
How do you determine the PCV
You know the total volume of the blood and reading the fine line at the top of the RBC layer
What test can be done with the buffy coat/
Plasma Protien concentration using a refractometer
What does specific gravity determine
extrapolate to determine the amount of protein in the blood. Thehigher the specific gravity, the higher the protein in the blood
What can be measured with the refractometer
1. Specific gravity (g/dl)
2. Urine specific gravity
What is the major protein in blood
albumin
Which tube top has no anticoagulate
Blue top
Why make a blood smear & what are important components
Trying to get a singel layer of cells to examine & to count & to enumerate RBC, WBC & stuff
What does Dif quik do
Differential stain that stains different parts of the cell differently
What are the 3 components of Dif Quik
1. Fix with alcohol
2. Eosin which stains the cytoplasm (red stuff)
3. methylene - stains the nuclei purple
How do you measure a WBC Count using the unopette
Unopette hematocytometer designed for TWBC
Dilute blood sample 1:100.Microhermatocrite designed to draw in specific volume of blood
Mix gently & wait 10 minutes
Fill hermatocytometer,
Cover with special slide
Look on 4x for grid
Count each of he sections on 10x
RBC lysed so easier to count
Formula for determining WBC
(Total cells counted +10% of those counted)x dilution factor (100) = #/ul
How do you calculate HCT
MCV x RBC count
How big is a RBC
7 microns
What is the function of RBC
1. Produce, protect & carry Hgb & O2
2. Produced in teh bone marrow
How long does a RBC last
90-120 days
Where do dead RBC go
Spleen gets rid of them. Onoly the young RBC can fold up & pass through the capillaries

Spleen recycles Hgb, iron and other parts & send to Bonne Marrow
What does pink urine indicate
You ate beets or you are losing RBC faster than the spleen can clean them up
Why is the nucleus of the RBC kicked out
Allows more room for Hgb
2. cell is more flexible to fold in splenic cappilaries
What are signs of anemia
Decreased circulation of RBC
2. Decreased PCV
3. Decreased Hgb

Decrease in bodies ability to oxygenate teh blood
Why do a Blood Film
1. Differential WBC count
2. Estimate platelet #
3. Evaluate morphology
What is the most clinically relevant classification of anemia
Bone Marrow Respones
What is regenerative anemia
Bone Marrow increases erythrocyte production & releases immature RBC (nRBC) or polychromatophilic RBC = reticulocytes.
What does the presence of reticulocytes indicate
regenerative anemia
What are the usual causes of regenerative anemia
1. blood loss - hemorrhage
2. blood destruction - hemolysis
What is non-regenerative anemia
Bone marrow malfunction requiring bone marrow biopsy
What are signs of non-generative anemia
Absence of reticulocytes in blood
Less erythropoesis
What are causes of non-generative anemia
1. endocrine
2. metabolic
3. bone marrow mal function
What does the blood look like in regenerative anemia
1. reticulocytes
2. polychromasia
3. anisocytosis
4. Purple nucleus when stained
5. nRBC have bluish cast to ribosomes & RBC & are a little bigger
Define normocytes
RBC of normal size 7micron
Define microcyte & possible cause
small cells

Iron Deficiency
macrocyte & possible cause
larger than normal size which are larger & stain blue

reticulocytosis
Vit B12 deficiency in humans
Hypochromic RBC may indicate -----
reticulocytes b/c full concentration of Hgb is not attained
What die macrocytic hypochromic RBC suggest
regenerative anemia
What are indications of ypochromic anemia
1. iron deficiency
2. microcytosis
Why is iron important in RBC formation
Iron helps make Hgb,
Cell division stops when full concentration of Hgb is met. If there is no signal to tell cell to stop dividing, this results in smaller cells
What does macrocyticm hypochromia anemia look like
& which kind is it
Big pale RBC

regenerative
What does micromyctic, hypochromic RBC indicate
iron deficiency
Macrocytic, normochromic RBC indicates ?
non-regenerative anemia
Normocytic, normochromic RBC
non-regenerative anema - cat in chronic renal failure
How is MCV measured
femtoliters
What is formulat to determine MCV
PVC (%) x 10/RBC ( millions/ul)
What is MCH
Mean corpusculat corpuscular hemoglobin is the mean weight of Hb

MCH (in picograms)= Hb (g.dl) x RBC (millions/ul)
MCHC
Mean CorpuscularHb concentration
Animals pack in as much Hb as they can

MCHC (g/dl) = Hb (g/dl) x 100/PVC (%)
What is normal MCHC range for mammels
30- 36 g/dl (wt/volume)
Why care about erothrocyte indices
Helps to classify type of anemia
Stain reticulocytes with----
New Metholine Blue
What are poikilocytes
abnormally shaped RBC
What is a reticulocyte
Immature RBC with organells (ribosomes) that look blue gray when Wright's stained
What does a reticulocyte look like
Clumps of blue in cytoplasm of cells
What forms of reticulocytes do cats have
1. Aggregate form -normal one of bluish chains
2. Punctate form - 2-8 small singlular granulosesbasophilic cells that dosn't stain
Why count reticulocytes
Indicates if animla is anemic and if BMis working (regenerative) or not
How do you count reticulocytes
equal amounts of whole blood & new methylen blue
2. wait 10 min
3. smear
4. count RBC and # of teticluocytes to determine %
What is a corrected Reticulocyte %
Observed reticulocyte % x PCV/normal mean PCV
What aspects of cell morphology are improtant
1. species
2. cell arrangement
3. Cell size
4. cell color
5. cell shape
What is a rouleaux
Stacke sof RBC
Common in horses
What causes rouleaux
Increases w' increased fibrinogen (serum protein of blood coaggulation whic increases coagulation)
Add saline & cells come apart
Define agglutination
Clumps of cells
Caused by immune mediated dissease
RBC coated w/ antibioties
If add saline cells do not come apart
What is the big test to determine rouleaux or agglutination RBC
add saline
1. Agglutination - cells stick together
2. Rouleaux - cells separate
define anisocytosis

& what do they indicate?
unequal cell size

Macrocytes -Large cells. MCV = reticulocytosis


Microcytes - small cells/MCV - iron deficiency
How is polychromasia determined
Bluish stain of organelles in cytoplasm indicating young RBC
How is hypochromia determined
Decreased staining intensity caused by insufficent Hb usually the result of iron deficiency in RBC
What are spiculated cells
spiky TBCcells
What do schistocytes indicate
RBC fragments indicating intravascular trauma where RBC get sheared by fibrin strands
DIC, neoplasia, iron deficiency
Spur cells are ---

& are caused by
acanthocytes

Change in membrane chlestrol
altered Lipid metabolism from hepatic lipidosis in a cat or liver dz in a dog (hemangiosarcoma
What are burr cells

& cause
echinocytes

artifact of slow drying -pH & fluids leak out which changes membrane shape
renal dz - dogs
lymphosarcoma - dogs
after excercise in horses
rattlesnake envenomization in dogs
in vitro artifact know as crenation
What do spherocytes look like
Dark staining RBC with little central pallor

Best detected in dogs

Or blood mismatch
What is a sign of lead poisoning
Basophilic stippling
small dark-blue dots in
When is it most likely to see Howell-Jolly bodies
after spelectomy b/c the spleen usually removes remnants in RBC

They look like a purple concentrated nuclear remnantdot
What are Heinz bodies
Denatured Hb caused by drug reaction, poisonings that harm the Hb

denatured Hb becomes detached & sticks to the cell wall
Increase in Heinz bodies may indicate what
1. lymphosarcoma
2. hyperthyroidism
3. diabetes mellitis in cats
How do you differentiate btwn an artifact & a parasite in teh blood
Artifact is refractile & form rings

Parasites will probably see more than 1
Define Hemobartonella
Common feline RBC parasite

Coccoid, rod shaped or ring-like structures that stain purple
In hemobartonella felis what doest hte blood lok like
Anisocytosis
polychromasia
nRBC with almost black nuclei
Black oval on the edge of the cytoplasm
Hemobartonella canis is distinguished by
chain of cocci across RBC
Who gets eperythrozoa
it is hemobartonella in large animals - ring form is most common.
Organismas are free all over theRBC
What 2 RBC dz cells look alike
1. Howell-Jolly bodies
2. anaplasma marginale in cattle with peripheral dark spot on cells
Descripe babesia
tear chaped protozoan in RBC

paired in dogs at the feathered edge. Look like 2 eyeballs in teh RBC
What does rhinicephalus canis cause
Ehrlichia - intercellular parasite of monocytes & neutrophils

Morulae or small clusters in cytoplasm
What is a rare cause of hemolytic anemia in cats
Cytauxzoon Felis

small irregular rings in erythrocytes, lymphocytes, & macrophages
What is the cause of feline infectious anemia
hemobartonella
What are the 5 types of leukocytes in teh blood
1. Neutrophils - infection fighters
2. Lymphocytes - nuerous in cows than neutrophils
3. Monocytes
4. Eosinophils - lots if parasite infestatin
5. Basophils - rare in mammals but common in reptiles
Define -penia
decreased # of cells in the blood
Define -philia or - cytosis
increased # of cells in th blood
Define left shift
Increased # of immature neutrophils in the blood

INDICATES SEVERE INFECTION
Define leukemia
neoplastic cells in blood or BM
Define leukemoid response
indicated by marked leukocytosis & usually the result of inflammatory dx

Often confuese tiwh leukemia
NOT CANCER
Define lymphoproliferative disorders
conditions in which lymphocytes and plasma cells proliferate abnormally
Myeloproliferative disorders
BM disorders usually neoplastic characterized by proliferation of 1, several or all BM cell lines

REALLY BAD CANCER
What is a differential WBC count
to determine dz & help with dx
What is the criteria for counting WBC
1. nice monolayer
2. near the feathered edge
3. perform each section consistently
4. coutn 100WBC
5. View under oil immersion
6. count with 5 clickers to enumerate each type
7. Report as % of each type
8. Determine absolute values
9. Consider TWBC to get relavent absolute value
Formulate for correcting WBC
Observed WBC x 100/100 + % of nucleated RBC
% of nRBC is determined by---
count in differential & just subtract from the total count

TWBC - |nRBC| = corrected amount
How do you differentiate a horse neutrophil from that of a D&C
Heavy clumped coarse chromatin with ligth blue to pink cytoplasm
Which cells get Dohle bodies
Toxic neutrophils
What are distinguishing characteristics of neurtrophils
1.granulated cytoplasm that doesn't take up a stain
2. obvious nucleus b/c granules don't stain
3.If pinched nucleus - mature
What distinguishes lymphocytes
1.Beautiful light blue stain with big dark nucleus
2. may have a slight dent
3. rim of bright blue cytoplasm with large nucleus
Describe a band neutrophil
Horseshoe shaped with rounded ends
No vacuoles in Cytotoplasm
Young neutrophil possibly indicating massive infection b/c didn't get a chance to mature before put to work
Describe a Seg
1.Segmented neutrophil looks like a clogged colon
describe a monocyte
1. diffuse muclear chromatin
2. lobulated nucleus
3. cytoplasm is blue-gray & vacuoles - lots of vacuoles
4. Absence of Left shift(aka increased immature neutrophils in the blood
Describe lymphocyte
1.small lymphocytes have slightly indented nucleus
2. coarsely clumped chromatin
3. Cytoplasm may contain pink-purple granules
4. similar in size to RBC
Describe eosinophils
1. Obvious granules b/c stain
2. Horses - grape like big granules
3. Roudn & oval red granules
How do you avoid smudge cells
Don't destroy the monolayer with too much turbulence
Dog esoinophils are distinguished by
Granules stain closer in color to Hg, horses stain intense red-orange
cats - granules are rod shaped
Basophil
1.Rarely seen,
2.stain dark purple to blue-black that over crowd the nucleus
3.Larger in size
4. Look like monocytes but very rare
5. poorly defined round lite purple granules
Define hyposegmentation

& cause
1.WBC with too few lobes
2. indicates early release of band neutrophils

1. normal inflammatory response
2.Drug reaction
3. Pelger-Huet anomaly - rare inherited
Define hypersegmentation
1. 5 or more lobes
2. aging neutrophil
3. Seen in poodles macrocytosis
4. prolonged blood storage before making films
5. in vivo - glucocorticoids
What doe toxic change cells look like
1. inflammation
2. infection
3. drug toxicity

Presence ot basophilia, Dohle bodu=ies, vacuoles or foamyness
What causes toxic change
decreased time of neutrophil maturation w/in the BM
What is another name for platelets
thrombocytes
Describe platelets
non-nucleated cells expelled by the cytoplasm of megakeryocytes in the BM.

Clotting agents
What is indicated if platelet count drops below 20-50K/ul
Increased bleeding

Normal is 2-5 platelets per oil immersion field
Where do you find platelets in a slide
in the feathered edge where they clump
What does the refractometer measure
3 major plasma proteins:
1. albumin - wworkhorse
2. Globulin - response to infection
3. Fibrinogen - soluble circulating plasma protein
Which plasma protein in important in determining inflammation in cows
Fibrinogen inceases with increases in inflammation
How is PP measured
mg/dl
What is normal fibrinogen measurement in a cow
200-800mg/dl
Why heat centrifuged blood
fibrinogen is sensitive to heat & will precipitate out.
1. get 2 samples
1.Centrifuge a 2nd time
2.Compare to other centrifuged sample
3. read both in refractometer
4.Difference in 2 values = amt of fibrinogen
Define hemostasis
Blood clotting or coagulation
Where are clotting factors made
Protein made by liver
What are the 2 methods of fibronogen production
1. intrinsic - intravascular
2. extrinsic - extravascular (tissue)
Why is Calcium important
Involved in activating proteins to vreat fibrinogen
How does blood clot
Fibrinogen form mesh which traps the RBC to clot the blood & stop bleeding
What are the components of clot
1. fibrinogen
2. platelets or thrombocytes
3. blood
What does Whole Blood Clotting Time test
intrinsic clotting mechanism
What is the normal Whole Blood clotting time for a dog

Horse

Cows
dog- 2 to 10 min

horse - 4 -15 min

cows- 10 - 15 min
What is the normal clotting time using a simple syringe tilt test
2-10 minutes
What activates clotting time
Diatomacious earth
What is the normal clotting time in an Activated Clotting time
60-90 seconds
What is importatnt to remember when performing Activated Clotting time
1. do not use an anti-coagulant
2. Keep warm for 60 seonds
3. Compare with normal donor blood for
Why is the activated clotting test good to use
1. Fast
2. Helps to determine if it is okay to proceed with a surgery
What dz indicate a bleeding disorder with inadequate platelets
1. petecchiae
2. epistaxis
3. hematuria
Why compute bleeding time
Increased bleeding time indicates
1.platelet defects
2. Increased capillary fragility
3. von Willebrands disease
What are the tests for intrinsic clotting mechanism
1.whole blood clotting time
2. activated clotting time (
ACT)
3. bleeding time
4. activated partial thromboplastin time (APTT)
What are tests for extrinsic clotting mechanisms
1. One stage Prothrombin Time (OSPT)

Citrated plasma is added to thrombo-calcium mix
Time to form fibrin
What does prolonged OSPT indicate
1.Severe liver dz
2. DIC (disseminated intravascular coagulation)
3. warfarin toxicity
4. Vit K deficiency
5. hereditary or acquired defeciencies
What is normal in a OSPT
7-10 seconds for dogs
If TPP is elevated, why it imporant to determine which factor is elevated
1. Albumin increase = dehydration
2. globulin increase= active infection
In the fibrinogen test what temperature do you use & for how long
1. 56C for 5 min
Why do a BM biopsy
to determine if there is BM failure or neoplasia when unable to dtermine through peripheral blood
What cells have granulocytes
WBC
1. Neutrophil
2. eosinophils
3.basophils
What are platelet precusors
1. Megakaryoblast -
2. Promegakaryocytes
3. Mwegakaryocyte
4. Platelets
What do megakaryoblast look like
1. large
2. 2 reddish nuclei
3. basophilic
4. scanty cytoplasm
What do promegakaryocytes look
1. Partially divided nuclei in 4,8,16,32 numbers
2. nuclear lobes remain connected
3. cytoplasm does NOT divide
4. cytoplasm rim around nuclear mass
Describe megakaryocytes
1. abundant cytoplasm
2. Pale blue cytoplasm with azurophilic granules
3. Really large
Where do platelets come from
Cytoplasmic structures of megakaryocytes
2. vary in size
3. azurophilic granules in pale blue background
4. Giant cigar shape - esp in cats

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