What is Clinical Pathology?
Hematology, clinical chemistry, exfoliative cytology, urinalysis, endocrinology, clinical immunology, toxicology
Reasons for testing
health screen, screen for disease, identify specific organ involvement, confirm presumptive diagnosis, confirm abnormal test, determine disease severity, formulate prognosis, monitor therapy or disease progression
Understanding reference intervals
2.5% of the healthy population will have values beyond either side of the median 95% and be deemed “abnormal” even though they are fine; is a compromise that increases the sensitivity of the test for recognizing sick animals because only a few healthy animals will be viewed as “abnormal”
What is the chance of an abnormal test result when testing a healthy animal?
5% chance when 1 analyze measured; 64% chance when 20 analytes measured
Factors affecting reference intervals
species, age, sex, time postprandial, time of day, emotional state, activity level, pregnancy/egg-laying, diet, region, time of year, generally collect overnight fasting samples from adult animals to increase chances of “normal”
Most common laboratory error
pre-analytical - ie. improper handling of samples (labeling), wrong anticoagulant/improper ratio of anticoagulant, traumatic blood draw or transfer of blood into tubes causing hemolysis
Blood samples getting old cause what cellular changes? and these changes affect what blood cell measurements.?
cell lysis
erythrocyte swelling -> affects MCV, MCHC, PCV
platelet activation -> affects MPV and platelet count
Platelet, leukocyte, and erythrocyte clumps could affect which blood cell measurements?
number/microliter blood decreased
platelets counted as leukocytes
MCV, MCHC, electronic HCT
MPV increased
What additive helps with platelet and leukocyte clumping?
citrate
Clot formation affects what blood cell measurements
all cell types decrease
Purple top tubes have what additive?
EDTA
Green top tubes have what additive?
Lithium heparin
Blue Top tubes have what additive?
citrate
What interfering substances cause analytical errors?
hemolysis, lipemia, hyperbilirubinemia
Falsely elevated potassium concentrations in horses and cattle can be due to what laboratory error/cellular process?
hemolysis - they have high K in RBCs
What three visual factors do you need to evaluate before examining blood samples?
color - tomato soup vs. syrump (methemoglobinemia)
Agglutination vs. rouleaux -if rouleaux you can use saline 10:1 to separate
is it mixed well?
Define rouleaux
“stack of coins” - due to non-specific binding of RBCs due to high protein content in the blood, will disperse with saline
Define agglutination
clumping of RBCs due to specific binding of RBCs by antibodies when IMHA is happening, will not disperse with saline test
What conditions cause lipemia in blood samples?
When animals not fasted before blood collection or with hyperlipidemic syndromes
How does lipemia affect blood samples?
causes turbidity
can interfere with anything that is measured by spectrophotometric assays
can dilute out normal substances like electrolytes in the aqueous component of the serum resulting in falsely decreased concentration (ion exclusion effect)
Give an example of a post-analytical laboratory error.
Error in data transcription
What are the four components of a microhematocrit tube after spinning?
plasma, buffy coat, packed red blood cells, wax plug
Three conditions that affect the appearance of the plasma in a microhematocrit tube?
hemolysis, lipemia, icterus
What types of cells are in the buffy coat?
leukocytes and platelets
What is fibrinogen and why is it important?
acute phase protein, precursor to fibrin in coagulation - important because it increases with inflammation so it can indicate infection in large animals that don’t have dramatic neutrophilic responses; present in plasma but not serum because it is used up in clotting process - causes optimal platelet aggregation
What are the functions of plasma proteins?
transport of nutrients, hormones, waste and drugs colloid osmotic effects acid-base immunity hemostasis
Where are most plasma proteins synthesized?
in the liver - big problem if liver failures
Why do adults tend to have higher concentrations of plasma proteins than neonates?
exposure to antigens
What is the composition of plasma?
92% water
8% solids - nutrients, proteins, hormones/enzymes, and electrolytes
Functions of plasma
transport of nutrients
transport of by-products and waste
transportation of cells
maintain homeostasis (pH, temp, etc.)
What important things do you evaluate on peripheral blood film?
red blood cell density - anemia?
RBC size, shape, color, and inclusions
What is a normal PCV?
Roughly 30-50% Canine - 37-54 Feline 30-47 Equine 32-47 Bovine 24-46
What is a normal plasma protein (TP)?
Roughly 6.0-8.0 g/dL
What is a normal plasma color?
dogs and cats: colorless
horses and bovines: colorless or pale yellow
What blood cell characteristics are analyzed by an automated hematology analyzer?
Quantity - RBC #/microliter, hemoglobin concentration/deciliter, hematocrit percentage
Quality - mean cell volume (size of RBCs), red cell distribution width (size variation of RBCs), mean cell hemoglobin concentration (hemoglobin within RBCs)
MCV
Describes average size of RBC
RDW
red cell distribution width - describes how much variation there is in RBC size, corresponds to the standard deviation of RBC mean cell volume; =SD/MCV, when RBC cell size is more variable than normal - RDW is increased
When calculated hematocrit and spun PCV are not within 3% of each other which do you trust?
spun PCV - certain diseases cause HCT to be false because it is a calculation not a direct measurement
MCHC
ratio of hemoglobin to the number of RBCs
decreased = regenerative anemia, iron deficiency anemia
increased = artifact! not real ( could be due to hemolysis, lipemia, heinz bodies, WBC elevation)
Heinz bodies cause what types of errors in blood cell measurement?
Hb and MCHC increased
sometimes total leukocyte counts increased
nucleated erythrocytes cause what error in blood cell measurement?
often counted as luekocytes
You should never trust an automated ______ count in cats
platelet - cats are very prone to platelet clumping
Name the four types of blood film stains and what you use them for.
Romanowsky type stains (Wright, Wright-Giemsa, Diff-Quik) - evaluation of RBCs
New Methylene Blue - reticulocytes, NMB wet prep
Prussian Blue - look for Iron
Cytochemical - ??
Describe ideal procedures for blood sample collection and handling
kept cool during storage and shipping - wrapped in paper towels to avoid direct contact with ice
blood smears made from freshly collected blood, not stored and transported blood
use DI water instead of tap
What does a water artifact look like?
moth eaten appearance of cells
refractile artifact - looks bright in one plane, dark in another
can be mistaken for RBC inclusion
Age related changes in RBCs
crenation - echinocyte formation
lysis - decrease RBC and HCT, falsely high MCHC
Hgb crystallization
cellular swelling - falsely high MCV ( and thus HCT), decreased MCHC
Age related changes in WBCs
swelling and smoothing of nuclear chromatin (looks like band neutrophil formation)
pyknosis and karyhorrhexis of nuclei
cell smudging
prominence of Dohle bodies
pyknotic leukocytes look like nucleated RBCs - can be misinterpreted
Age related changes in platelets
clumping- decreases platelet count and increases MPV (small clump seen as single large platelet), large platelet clumps are excluded from count altogether
degranulation (makes them difficult to see and enumerate)
The three parts of a blood smear
base, feathered edge, “counting area”
What do you analyze in the monolayer of a blood smear slide?
right behind feathered edge - cells spread, not overlapping and not disrupted so analyze size and shape of RBCs, WBCs uniform distributed in this section
What should you analyze in the feathered edge of a blood smear slide?
detect platelet clumps and microfilaria