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what is involoved in a manual CBC?
- packed cell volume (PCV)
- total protein (TP)
- WBC count
- WBC differential
- blood cell morphology
- platelet estimate
what is WBC differential?
count at least 100 cells and get a % of WBC from those 100 cells
what is the qualitative analysis for a CBC?
what is the quantitative analysis for a CBC?
- WBC count
- WBC differential
- platelet estimate
why do we do a CBC?
provides a borad overview of general health status
what is the hematopoietic system?
what is blood?
cells and plasma
what are cells
- RBC and WBC
- we count platelets as cells but they are really just cell fragments
what is plasma?
- 90% water
- 10% protein (albumins and clotting factors)
why do we do hematology?
- sick patients
- preanesthetic evaluations
- geriatric profile
- recheck after/during treatment
why do we do hematology for sick patients?
- animals can have the same clinical signs for a variety of reasons
- hematology helps with the diagnosis
why do we do hematology as a preanesthetic evaluation?
to count RBCs, WBCs, platelets, and TP
what would causes an increase in WBC above the normal range?
Leukocytosis - inflammation, infections, stress, excitement
what would cause a decrease in WBCs below the normal range?
leukopenia - can cause poor inflammatory response post op
what is thrombocytopenia?
not enough platelets
what are albumins?
proteins made by the liver and too big to leave the blood stream
what are protein-bound drugs?
binds to albumins and the rest are free floating. only free floating drug can get out of the blood stream. when some of the drugs leave, some of the drugs will unbind from albumin to become free floating to keep the equilbrium.
when are annual hematology screenings recommended?
geriatric patients over 7 years old
what are some problems that can effect the testing?
- effects from diet (just ate)
- keeping blood room temperature
- anticoagulant (using the wrong one)
what color should plasma be?
what is hemolysis?
breaking of red blood cells and the plasma will look red
what causes hemolysis?
- excess pressure drawing the blood
- rough handling (shaking the sample)
- injecting blood too fast into tube
- moisture in syringe or tube
what is lipemia?
fat in the blood, causes the plasma to look white
what causes lipemia?
- postprandial sample (getting sample after eating)
- diabetes mellitus
- liver disease
what is icterus?
plasma is yellow or brownish color
what causes icterus?
- caused by bilirubin
- breakdown product of RBCs - hemolytic anemia (RBC are breaking up in body and hemaglobin is changed to bilirubin in the liver), liver problems, cholestasis (blockage of bile)
what does RES stand for?
reticular endothelial system
what is RES?
- macrophages that live in the liver and spleen and they check RBC to make sure they are okay
- converts heme into bilirubin and will travel to a hepatocyte in the liver and will make bilirubin water soluble - changes to conjugated bilirubin which changes to bile
what happens to a blood test when an animal is excited?
increase in RBCs, WBCs, platelets, and glucose
what happens to a blood test when an animal just ate (post-prandial)?
increase in glucose and lipemia
which blood sample should never be frozen?
when should platelets be counted?
- after 4-6 hours they are not reliable
can a normal lab result help you? why?
- it will help rule-out as well as rule-in diseases
what are the steps to doing a manual CBC?
- take anticoagulated blood
- make a smear
- run PCV and TP
- perform WBC count (we will do this with a machine)
- examine smear (WBC differential, cell morphologhy, platelet estimate)
what is PCV?
packed cell volume - ratio of volume of erythrocytes to volume of whole blood
what is the normal range for total protein?
5.5 - 7.5 g/dl
what causes TP to be elevated?
- hemoconcentration due to dehydration
- nemolysis, lipemia, high glucose
- increased globulin production
what angle do we hold the slide cover?
what are the steps to using Diff Quik?
- dip slide in fixative about 10 times
- dip slide in red stain about 10 times (eosin stain)
- dip slide in blue stain about 10 times (methylene blue stain)
- rinse slide well in jar or under slow running water
what color are RBC when they pick up the stain?
pink (they pick up the eosinophilic stain)
what color are the nuclei of WBC?
blue/purple (they pick up the basophilic stain)
what color are granules when stained?
can be basophilic, eosinophilc, or neutrophilic
what color are platelets when stained?
what are the common problems when preparing a slide?
- stain precipitates
- water artifacts
what happens to RBC and WBC when they are over-stained?
- RBC's become more basophilic than normal
- WBC cytoplasm that should be clear takes up stain instead
what happens to cells when they are under-stained?
all cells are pale, cellular details of WBC are barely distinguishable
what causes stain precipitates?
- old stain
- stain left open
- stain refilled
- inadequate washing
what do water artifacts look like?
what is crenation?
round blood cell edges become distorted
what causes crenation?
- slow drying of smear
- old cells (not make smear fast enough)
what are skipocytes?
WBC that has lost its cell membrane and has ozed everywhere. we will skip these when counting.
how do you evaluate the quality of a slide and what are you looking for?
- look at slide with 10x and 40x objective.
- look for staining quality, cell distribution, check feathered edges, find the monolayer
what makes a slide insufficient?
- inadequate monolayer
- lots of WBC at feathered edges
- lots of smudge cells
- under or over stained
- stain precipitate
- water artifact
what do we examine under oil immersion?
- RBC morphology
- platelet morphology and distribution
- leukocyte morphology and then count them
- perform differential
how many cells do we count for a WBC differential?
100 - 200
what do we look for in erythrocytes?
- extracellular things
what are the 2 types of leukocytes?
- granulocytes (neutrophils, eosinophils, basophils)
- mononuclear cells (monocytes and lymphocytes)
what does the nucleus of a granulocyte look like?
has a segmented nucleus
what does the nucleus of mononuclear cells look like?
round nucleus with no segments
what is another name for platelets?
what color stain do thrombocytes pick up?
where do you usually find parasites on a blood smear?
at the feathered edge because they are heavy
Name the 6 components of the CBC?
- WBC count
- WBC differential
- Cell morphology
- Platelet estimate
Why is a CBC a good idea prior to anesthesia?
to check for RBC, WBC, and platelet levels and to make sure the patient is well hydrated by testing the TP
Why is anemia dangerous to anesthestized patients?
because they're blood would not be able to carry enough oxygen throughout their body
What is polycythemia?
What is meant by "relative polycythemia"?
decrease in plasma which makes it look like there is a high PCV so it shows a false high PCV
What things besides physical exam can indicate a patient's dehydration
- high PCV
- high TP
- low plasma volume
What annual tests should be routinely performed in geriatric animals?
The number one cause of bloodwork errors is ____.
not letting it spin down for 10 minutes
Blood smears should be made within ___ minutes of the blood collection. Why?
- 30 minutes
- after 30 minutes WBC start to distort, platelets clump together, and after 24 hours RBC lyse
What happens to RBC's stored in EDTA?
they lyse after 24 hours
Why should heparin not be used as an anticoagulant when making mammmalian blood smears?
it causes the WBC to distort and the platelets to clump together
What happens to platelts in a blood sample over time?
Describe in detail two methods of making a blood smear.
- put the drop of blood on the etched/cloudy/white side, hold the other slide at 30 degrees, move the edge of the slide to the blood drop until the blood drop spreads 3/4th the width of the slide, then push forwards
- make the two slide covers in a star then pull them apart
At what angle should you hold the spreader slide?
Describe where on the slide to read a blood smear
the monolayer, away from the feathered edge
What are causes of too thin of a smear?
- too small of a drop
- pushing the spreader slide too fast
- angle of spreader slide is less than 30 degrees
What are causes of too thick of a smear?
- too big of a drop
- pushing the spreader slide too slow
- angle of the spreader slide is more than 30 degrees
What happens if a blood smear dries too slowly?
it will cause crenation of the RBC
How should you dry a blood smear?
waving it in the air
How should smears be stored?
at room temperature and away from formalin
The gross pattern of a good smear should resemble a _____.
If you see a bunch of monocytes and segmented neutrophils at your feathered edge, what did you do wrong in preparation of the slide?
pushed too slow
What the heck is a smudge cell (aka skipocyte) and should you count them?
they are WBC's that have lost their cell membrane and their contents have spilled out everywhere. you don't count them
Where on the blood smear should the differential be performed?
at the monolayer
What is a supravital stain? Name one example
when the stain is added to the vial and is absorbed by the live cells. New methylene blue - Zynostain
What should you do if your eosin and methylene blue are getting low in your Diff-QUik stains? Why?
rinse out the containers really really well then fill them up with new stain
Define eosinophilic, basophilic, and neutrophilic
- eosinophilic - red/pink
- basophilic - blue/purple
- neutrophilic - neutral (did not pick up any stain)
What do over-staining, under-staining, water artifact, stain precipitate, and crenation look like?
- over-staining: dark, cytoplasm of the WBC's pick up color
- under-staining: pale, hard to see
- water artifact: refractile bubbles (shiny)
- stain precipitate: stain on the slide that should not be there (can be confused with parasites)
- crenation: distorted edges of RBC
What should you look at on a blood smear while using the 10x objective?
- evaulate stainig
- look at cell distribution
- check feathered edge, edges of smear
- find the monolayer
What should you look at on a blood smear when performing a differential?
- Erythrocytes: size, shape, color, inclusions, extracellular things
- Leukocytes: granulocytes and mononuclear cells
- Platelets: estimate how many
- Parasites: identify them
What is a PCV?
Packed Cell Volume: the ratio of erythrocytes to the total cell volume
What happens to the PCV if an animal is dehydrated?
- it increases
- bc there is less plasma so it makes it look like the RBC are rising
Anticoagulated blood should be placed in what color hematocrit tube?
Whole blood that does not contain an anticoagulant should be placed in what color hematocrit tube?
What is the buffy coat composed of and where is it found in the hematocrit tube?
platelets and WBCs
Explain how dehydration can mask anemia
because of the low plasma volume gives a false high PCV
Where are microfilaria concentrated in the hematocrit?
just above the buffy coat
How can you adjust your refractometer for its zero point?
using distilled water and set it to 1.0
What happens to total protein if an animal is dehydrated? Why?
List five causes of hypoalbuminemia
- protein losing nephropathy
- protein enteropathy
- lose of lymph
- chronic or severe blood lose
- lack of hepatic production (albumin not being made)
What is the major plasma protein?
What other proteins are found in plasma?
globulins, hormones, and enzymes