CLINICAL LABORATORY

PROCEDURES
VT 112
Spring Term 2020
Christine M. Fletcher, DVM

Lab 1: Hematology Basics: Packed Cell Volume and

Total Protein
HEMATOCRIT
Hematocrit
• What is the hematocrit?
Hematocrit
• What is the hematocrit? % RBCs in whole blood

• What is another term for the hematocrit?

Hematocrit
• What is the hematocrit? % RBCs in whole blood

• What is another term for the hematocrit? Packed cell

volume (PCV)
Hematocrit
• What is the hematocrit? % RBCs in whole blood

• What is another term for the hematocrit? Packed cell

volume (PCV)

• A hematocrit decreased below normal indicates…?

Hematocrit
• What is the hematocrit? % RBCs in whole blood

• What is another term for the hematocrit? Packed cell

volume (PCV)

• A hematocrit decreased below normal indicates…? Anemia

Hematocrit
• What is the hematocrit? % RBCs in whole blood

• What is another term for the hematocrit? Packed cell

volume (PCV)

• A hematocrit decreased below normal indicates…? Anemia

• A hematocrit increased above normal indicates…?

Hematocrit
• What is the hematocrit? % RBCs in whole blood

• What is another term for the hematocrit? Packed cell

volume (PCV)

• A hematocrit decreased below normal indicates…? Anemia

• A hematocrit increased above normal indicates…?

Polycythemia
Determining Hematocrit
• Packed cell volume (PCV aka microhematocrit)

• Macrohematocrit procedure

• Mathematical calculation
Packed Cell Volume (PCV)
• Precise test: Multiple tests on the same sample are within
+/- 1.0% of each other

• Sample: 1-2 drops anticoagulated blood

Equipment
• Microhematocrit tubes
(aka Capillary tubes)
• Plain (no anticoagulant):
Used if blood sample
already mixed with an
anticoagulant (i.e., EDTA,
heparin, etc.)

• Heparinized: Used when

blood has not yet been
anticoagulated
Equipment
• Microhematocrit tubes
(aka Capillary tubes)
• One way or another, blood
MUST be anticoagulated!

• This is necessary to get the

RBCs packed as tightly as
possible when centrifuged

• Maximal packing of the

RBCs is essential for test
accuracy & precision
Equipment
• Microhematocrit tubes
(aka Capillary tubes)
• How come you shouldn’t
use a heparinized tube if
the blood sample is already
anticoagulated?
• This can cause excessive
dilution of the blood sample,
which will make the result
falsely decreased
Packed Cell Volume (PCV): Equipment
• Sealing clay
• Used to seal one end of tube

• Keeps blood from leaking out

Packed Cell Volume (PCV): Equipment
• Centrifuge
• Must be able to accommodate microhematocrit tubes
Packed Cell Volume (PCV): Equipment
• Microhematocrit chart: To read results
Packed Cell Volume: Procedure

• Centrifuge 3 – 5 min at
>10,000 rpm

• This creates maximal

packing of RBCs 🡪 Accurate
& precise results!

• https://www.youtube.com/wa
tch?v=WywP3JC1QwY
Packed Cell Volume: Procedure
Packed Cell Volume: Procedure
ALWAYS make
and read 2 tubes!
🡪 QUALITY
CONTROL

Average the
results & report as
a whole number

If results are not

within 2% of each
other: 🡪
REPEAT PCV
Packed Cell Volume

Normal 🡪 🡪 Anemic
PCV: Plasma Color
• Normal: Colorless to pale
yellow

• Icteric: Medium / deep yellow

or gold
• Small animals: ABNORMAL 🡪
Indicates disease

• Adult ruminants & horses: May

be yellow or gold from carotenes
in diet. This is normal but must
be differentiated from icterus.
PCV: Plasma Color
• Hemolysis: Red
• Handling error?

• Disease process?
PCV: Plasma Turbidity
• Normal: Clear

• Opacity: Lipemia
• Increased fats in plasma

• May be normal or abnormal

PCV: Buffy Coat
• What makes up the buffy coat?
PCV: Buffy Coat
• What makes up the buffy coat?
• WBC

• Platelets
PCV: Buffy Coat
• What makes up the buffy coat?
• WBC

• Platelets

• Normal: Whitish-gray, < 2%

• Abnormal:
• Any other color, or > 2%

• Microfilariae (D. immitis or A. reconditum)

TOTAL PROTEIN BY
REFRACTOMETRY
Refractometry
• Refractive index: The tendency of light to “bend” when
passing through a material

• Refractometer: Device that measures the refractive index

of a solution
Refractometry
• Urine specific gravity
• Urine

• Read directly from scale

• Total protein
• Plasma or serum

• Read directly from scale

Refractometry
• Total solids
• Plasma or serum

• Must use a conversion table to determine total protein

• Uncommon these days

Total Protein: Procedure
Total Protein: Procedure

TP 8.9
Report result to the
nearest tenth
Total Protein: Procedure
• Always clean the refractometer after every use!
• Working surface and cover plate

• Distilled water, lens paper

• Dry all surfaces & close cover plate before putting away
Calibrating the Refractometer
• Refractometers need to
be calibrated periodically
(this is often overlooked!)

• Water = 1.000 on the

urine specific gravity
scale (U.G. or U.S.G.)

• If the refractometer gives

a different reading, it
needs to be manually
adjusted
Calibrating the Refractometer
• Refractometers need to
be calibrated periodically
(this is often overlooked!)

• Water = 1.000 on the

urine specific gravity
scale (U.G. or U.S.G.)

• If the refractometer gives

a different reading, it
needs to be manually
adjusted
Total Protein: Sources of Technical
Error
• Blurred or faint light/shadow interface
• Not enough sample 🡪 Add additional sample

• If sample is adequate (covers working surface) 🡪 Sample may be

hemolyzed

• No light/shadow interface
• Cover plate not completely closed 🡪 Check carefully for broken
glass!

• Sources of biological error

Discussed in
• Common TP abnormalities/Relation to PCV
VT112