Lecture 1-2

How do purple top tubes prevent coagulation?

Chelating calcium

How do green top tubes prevent coagulation?

Potentiation antithrombin

What do you get from a spun green top?

Plasma

How do blue top tubes prevent coagulation?

Reversibly prevents coagulation by weakly chelating calcium

Why shouldn’t you use a red top tube for phenobarbital measurement?

Decreases measurement b/c fluid fraction contact with gel separator

What does traumatic venipuncture cause?

Hemolysis, reduce platelet counts

Can you freeze plasma/serum samples?

Yes, usually long-term storage

Can you freeze CBC samples?

No

Why might plasma/serum have red coloration?

Hemolysis → free hemoglobin from the destruction of RBCs

Why might plasma/serum have lactescent/opaque coloration?

Lipemia → post-prandial samples

Why might plasma/serum have yellow coloration?

Icterus → elevated bilirubin, LA usually have yellow appearance

What interference does plasma/serum hemolysis have on bloodwork?

CBC → decreased PCV, HCT and RBC count

• MCHC falsely increased (MCV unaffected)

• Incrase in PLT due to erythrocytes falsely counted as platelets

• Difficult to read TP on refractometer

Chemistry → Increased K, Phosphorous, ALT, LDH, Mg

• Electrophoresis: beta globulin spikes

What interference does plasma/serum lipemia have on bloodwork?

CBC → increases HGB, MCHC and platelets

• Increases refractometer TP

Chemistry → decrease in Na and Cl, slight decrease in K

Lipemia also promotes in vitro _________.

hemolysis

What can be done to precipitate out lipemia lipids?

Refridgerate samples

What interference does plasma/serum icterus have on bloodwork?

CBC → little to no effect

Chemistry → hyperbilirubinemia falsely decreases biuret test TP and CREA

What do CBCs look at?

Erythrocyte parameters, leukocyte counts and platelets (“red, white and platelets”)

What erythrocyte parameters are measured on CBC?

Hematocrit, hemoglobin, RBC count

Indices like MCV, MCH, MCHC and sometimes RDW

What leukocyte counts are measured on CBC?

Total and differential

What platelet calculations are measured on CBC?

Platelet count and MPV

Electrical Impedance Method

Counts platelets by drawing diluted blood through a small aperture. As each cell passes through, it changes the electrical resistance. This creates a voltage pulse proportional to the cell's volume, allowing the analyzer to size and count the platelets

Which is smaller: platelets or RBCs?

Platelets

How do impedance instruments measure hemoglobin?

Spectrophotometrically

What can impedance instruments measure vs. not?

Can: measures count and size of cells → provides cell volume distributions

Cannot: determine intracellular cell characteristics, count reticulocytes or do accurate platelet counts in cats

Because impedence instruments base their differential on ______ ______, it makes them _____ ______.

cell size, less accurate

In Laser Flow Cytometry, what does forward scatter indicate?

Cell’s shadow → cell size

What size of forward scatter would a neutrophil make?

Large shadow because it’s a large cell

What size of forward scatter would a lymphocyte make?

Small shadow because it’s a small cell

In Laser Flow Cytometry, what does side scatter indicate?

Cell complexity/granularity → more granularity = more side scatter

What size of side scatter would a lymphocyte make?

Lower = less granular

What size of side scatter would a neutrophil make?

Higher = more granular/complex

What does Laser Flow Cytometry measure?

Determines number, size and internal complexity of cells based on extinction of light and scattered light

Impedance Instruments have a ___-part differential.

3

Laser Flow Cytometry have a ___-part differential

5

What can laser flow cytometry measure vs. not?

Can: Total WBC, “5-part” differential counts, Platelets, Reticulocytes

Cannot: accurately count basophils in dogs

Which instrument can count reticulocytes?

Laser Flow Cytometry

What are the RBC counts used for?

Little value by itself, but used to calculate Hct (electronically), MCV and MCH (manually)

What reagent is used to measure hemoglobin? How?

Sodium lauryl sulfate (SLS) → lyses RBC and oxidizes Hb to MetHb to make more stable product

What causes false increases in hemoglobin?

What is MCV, MCH, MCHC and RDW?

MCV - Mean Cell Volume

MCH - Mean Cell Hemoglobin

MCHC - Mean Cell Hemoglobin Concentration

RDW - Red Cell Distribution Width

Do cats or dogs have less erythrocytes?

Cats

What measures MCV?

Hematology analyzers

What causes low MCH?

Severe iron deficiency anemia → low MCV and low internal hemoglobin concentration

What is MCH and what does it correlate with?

Average amount of hemoglobin in a single erythrocyte, usually based on erythrocyte size (MCV)

High MCV means you will likely have a _____ MCH.

High

What is the measurement of MCHC?

g of Hb/dL of packed erythrocytes

What is the measurement of MCV?

femtoliters

How is MCHC calculated?

Hb/HCT x 100

Red Cell Distribution Width is an electronic measure of ______.

anisocytosis (different RBC cell sizes)

What might cause a false increase in RDW?

• RBC agglutination (makes RBCs look bigger than they are)

• Platelets counted in erythrocyte count of severely anemic patients (which are smaller than RBCs)

Aboslute ______ counts should be validated using manual counts.

reticulocyte

What kind of reticulocytes are counted in cats?

aggregate

What cell types show up as red, pink and blue on a ProCyte Dot Plot?

What is reticulocytosis?

High # of reticulocytes

What can cause false reticulocytosis?

• Erythrocyte parasites that have RNA and DNA (mycoplasmas, Babesia)

• Howell-Jolly Bodies

• Nucleated erythrocytes

• Large, immature platelets or platelet clumps

• Heinz bodies

• Leukocytosis (uncommon)

What can cause false platelet counts?

Platelet aggregates → false thrombocytopenia (low platelets)

• Collection and handling error

In what species is automated platelet count often unreliable?

Cats

_______ should be verified by stained blood film examination.

Thrombocytopenia

How are platelet clumps seen on a RBC-PLT Dot Plot?

What is MPV?

The average volume of a single platelet

What is the reference interval of a MPV of cats vs dogs?

Cats: 11.0-18.1

Dogs: 6.7-11.1

What can cause false increased MPV?

Platelet aggregates or storing blood at 5 C (fridge temp)

What should you do before calculating the absolute differential leukocyte counts if there’s nucleated erythrocytrs in the blood? Why?

Calculate the corrected total leukocyte count because they can be counted as leukocytes!

What errors do old blood samples show on bloodwork?

• Erythrocyte swelling (increase in MCV and HCT, lower MCHC)

• Platelet aggregation (increase MPV and decreased platelet count)

• Decrease of all cell types due to lysis

What errors do platelet, leukocyte and erythrocyte aggregates show on bloodwork?

• Decrease in cell concentration

• Erythrocyte → increase MCV and MCHC, decrease in HCT on instrument

• Platelet → may be counted as leukocytes and increased MPV

What errors do blood clots show on bloodwork?

Decrease in all cell types

What errors do cryoglobulins (precipitate below body temp) show on bloodwork?

May be counted as leukocytes or platelets

What errors do Heinz Bodies show on bloodwork?

• Increase in Hb and MCHC

• Sometimes increase in total leukocytes and/or reticulocytes