6. Leukocyte Identification and Evaluation

Question 1

What is leukocyte identification? What is a leukogram

Answer 1

id of WBC’s found in the perpheral blood
A leukogram - is the quantification of the diff classes of leukocytes as well as any morphologic abnormalities of the cells - calculated by evaluating a peripheral blood smear, species differences

Question 2

How do we examine a blood smear

Answer 2

1. start by examining on 40x objective for platelet clumps, enlarged cells and microfilariae
2. go to feathered edge of slide for platelet or cell clumping
3. Move 3-4 fields inwards to the monolayer where you’ll fine approx 200 cells/field, cells are single layer, they are not overlapping and they are not touching
4. Go to 100x objective and perform differential cell count, 100 WBC will be counted to give % for each cell type observed

Question 3

What are absolute values

Answer 3

once your differential cell count is performed and your total WBC count is known, absolute values of the wbc’s can be calculated

Question 4

For absolute values, if the total WBC count is 4.4x10^9/L and differential count is as follows

Answer 4

neutrophils: 49% x4.4x10^9/L = 2.2x10^9/L
Lymphocytes: 44% x 4.4x10^9/L = 1.9x10^9/L
Monocytes: 2% = 0.1x10^9/L
Eosinophils: 5% = 0.2x10^9/L
Basophils: 0% = 0.0x10^9/L

Question 5

What do neutrophils look like

Answer 5

aka “segs”
Nucleus: irregular, elongted and lobed
Segmented neutrophil - mature neutrophil is the most abundant leukocyte in peripheral blood in SA, granulocytic cell
Small, very poorly stained granules
may contain small vacuoles, cells will be larger if actively phagocytic

Question 6

What is the neutrophils job?

Answer 6

Participate in the inflam response with an inc # generally indicate inflam or infection
Phagocytosis of foreign invaders or diseased self

Question 7

Do all neutrophils across the species look the same? If not, what are the differences

Answer 7

Cattle - granules stain faintly pink, giving cytoplasm an orange-pink tint overall
Horses - fine diffuse granules
Reptiles, birds, some fish and some small mammals like rabbits and guinea pigs have heterophils in the place of neutrophils (granules are eosinophilic)

Question 8

What are band neutrophils

Answer 8

immature stage of neutrophils
has a smooth, parallel sided, horseshoe-shaped nucleus with clubbed ends and no constrictions, the chromatin is visible
if the narrowest part is 2/3 the sides of the widest part of the nucleus = band beutro
When unsure, always class as more mature stage
Normal values for all species 0-3%

Question 9

What are eosinophils?

Answer 9

granulocytic cell, nucleus similar to neutrophils
primary func is in immune system modulation - capable of phagocytosis, #’s inc w/ allergic reactions and parasitic infections
Found in sm #’s in peripheral blood in healthy animals
Contain granules that are large and stain red - vary considerably btw species

Question 10

How do the granules of k9, fel, equine and bovine differ?

Answer 10

k(: granules light reddish color and will range from sm-lg within same cell 2-10%
Feline : granules are rod-shapped, sm, numerous and stain a reddish color 2-12%
Equine : bright orange-red granules are very lg, often filling the cell and occluding the nucleus 0-4%
Bovine: granules are relatively sm, round and red 2-20%

Question 11

What are basophils and what do they do?

Answer 11

involved in the mediation of the immune system - will see inc #’s w/ inflam or infection
not often found in peripheral blood smears - larger than neutrophils, have a nucleus similar to monocytes

Question 12

What are the differences of basophils btw k9, feel, eq and bovine

Answer 12

K9 - small # of dark - purple-blue/black granules
Feline: Large, round, lavender granules
Equine/Bovine: large number of dark blue/granules that may occlude the nucleus (0-3%)

Question 13

What are lymphocytes and what do they do?

Answer 13

Agranulocytic cell, func in the humoral immune response (fever) & cell mediated responses (antibody production)
Most abundant cell in blood smear of ruminants
an inc in lymphocytes usually indicates viral infection

Question 14

What do lymphocytes look like?

Answer 14

dark purple/blue round to oval or slightly indented nucleus (bean)
nucleus will have variations of color due to clumping of chromatin
sm amount of clear to sky blue cytoplasm, may appear to have none
k9/cats have smler lymphocyte with scant cytoplasm
cattle have med to large (neutrophil size) lymphocytes with more abundant cytoplasm - pink/purple granules)

Question 15

What are monocytes?

Answer 15

Classed as agranulocytic cells
largest of the WBC’s, participate in the inflame response - numbers are often inc w/ chronic infections
capable of migrating into tissues(become macrophages)
then phagocytize foreign material and dead, dying or dz’d cells
responsible for normal erythrocyte destruction and associated metabolic iron recycling

Question 16

What is the most misidentified cell on blood smears and why?

Answer 16

nucleus varies in shape, generally bean shaped with occasional lobes
a soft cell, so can fold over itself
nucleus does not stain as dark as other cells and appears to have a lacy appearance
cytoplasm is grey-blue and may contain some small, pink granules or vacuoles

Question 17

How can you tell a monocyte from a lymphocyte in LA?

Answer 17

Monocyte - cytoplasm grey/blue, ground glass appearance, tiny pink granules
Nucleus deeply indented, maybe amoeboid traits
Large/sm vacuoles maybe present
chromatic is reticulated (lacy)
LYMPHO - cytoplasm trye or sky-blue, no granules except occasional large one. Nucleus round to slightly indented, vacuoles are extremely rare and are tiny if present, chromatin is clumped

Question 18

How do we quantify abnormal morphology? What is the system we will be using in lab?

Answer 18

Subjective assessment, 2 systems
1. Slight: ~10% of cells affected
Moderate: ~25% of cells affected
Marked: >50% of cells affected
2. 1+: 5-10% of cells affected
2+: 10-25% of cells affected
3+: ~50% of cells affected
4+: >75% of cells affected

Question 19

What might happen with abnormal leukocyte morphology?

Answer 19

nuclear hypersegmentation
toxic change
intracytoplasmic inclusions
atypical and reactive lymphocytes
smudge cells/basket cells
neoplasia

Question 20

What is nuclear hypersegmentation?

Answer 20

canine or feline neutrophils with >5 lobes
usually due to aging of cells either in the patient or in the blood tube

Question 21

What is toxic change in cells?

Answer 21

most common dz-induced cytoplasmic change in neutrophils
found in association w/ inflam, infection and drug toxicity - if severe, often associated w/ bact infection
Significant in dogs, common in cats

Question 22

What happens with toxic change?

Answer 22

cytoplasmic basophilia
Dohle doies - blue staining cytoplasm inclusions, single or multiple
Vacuoles or “foamy” appearance to the cytoplasm
Toxic granulation (rare) -intensly stained granules
may be significantly lger than normal neutrophils

Question 23

How do we quantify toxic change?

Answer 23

slightly diff than quantification of other abnormal WBC morphology
May be based on #’s of cells affected OR observations noted

Question 24

Through quantifying toxic change, what system do you use when noting percentage of neutrophils with toxic change?

Answer 24

Mild: 5-10%
Moderate: 11-30%
Marked: >30%

Question 25

Through quantifying toxic change, what system do you use when noting severity of toxic change within the cell

Answer 25

Dohle bodies 1+
Mildly basophilic 1+
Moderatelty basophilic w/ dohle bodies 2+
Moderately basophilic and foamy 3+
basophilic with toxic granules 3+

Question 26

What are examples of intracytoplasmic inclusions?

Answer 26

canine distemper inclusions - RBC’s and neutrophils
Ehrlichia and anaplasm
Others including histoplasma capsulatum, francisella philomiragia, mycobacterium, hepatozoon canis, leishmania infantum

Question 27

What do reactive lympocytes look like?

Answer 27

basophilic cytoplasm, more abundant cytoplasm, inc cell size, lger and more convoluted nucleus, also called immunocytes, seen w/ antigenic stimulation - this will occur w/ Ag exposure thru infection or vx

Question 28

What are immunocytes?

Answer 28

reactive lymphocytes

Question 29

What are atypical lymphocytes?

Answer 29

basophilic cytoplasm
inc cell size
cleaved nuclei
asynchronous maturation of the nucleus and cytoplasm
Azurophilic(pink) granules - chronic antigenic stimulation (ex. k9 ehrlichiosis), may be present in normal bovine lymphocytes

Question 30

What are smudge/basket cells?

Answer 30

ruptured, degernerative leukocytes
significant when lg #’s are present - leukemia
Can be an artifact

Question 31

How might neoplasia and blood cells be related

Answer 31

neoplastic profliferation of hematopoietic cells
AKA leukemia - there are neoplastic cells in the blood and/or bone marrow
Neoplastic blood cells in peripheral blood AKA type cells
Include: blast, atypical, bizarre cells, mitotic figures, unclassified neoplastic cells - nucleus appears to belong to one cell line, while the cytoplasm appears to belong to another cell line

Question 32

What three types of neoplastic/type cells are there?

Answer 32

1. mitotic figures - when seen on the blood smear are indicative of neoplasia(cells that are splitting in the peripheral blood= BAD)
2. blast cells - confined to the bone marrow In relatively sm #’s - w/ neoplasia, blast cell #’s will inc, or they will be present in the blood smear
3. Abnormal lymphocytes - will be vacuolated, clumped together or appear more basiphilic - usually seen @feathered edge of a smear and are rare

Question 33

What should you consider if you are seeing bizarre cells or if an unexpected cell type dominates on blood smear

Answer 33

Neoplasia
only exception being that of the neutrophil series - toxic change is common w/ significant inflam, immature neutrophils may be present in peripheral blood w/ severe infections, clin hx must be considered

Question 34

what is myeloproliferative dz

Answer 34

characterized by bone marrow - that has hypercellularity, loss of orderliness in maturation, abnormal (neoplastic) cells
Not uncommon in cats (often infected with FeLV

Question 35

What will a myeloproliferative dz show on a CBC?

Answer 35

inc WBC count, inc nucleated RBC count, dec PCV, abnormal cells in the blood smear, nonregenerative anemia, thrombocytopenia

Question 36

What are lymphoproliferative disorders?

Answer 36

can be used to describe any abnormal proliferation of lymphoid cells
more commonly used to describe tumors that are derived from lymphocytes or plasma cells
more common than myeloproliferative disorders
common in cats - often associated with FeLV and FIV infection
also seen in cattle with bovine leukosis virus (BLV)

Question 37

How is lymphoproliferative disorders categorized?

Answer 37

primary lymphoid leukemia, lymphoma or plasma cell tumors
we can use PCR testing to distinguish btw neoplastic and non-neoplastic lymphoproliferative disorders
main difference btw lymphoid leukemia and lymphoma is the distribution of dz
Lymphoid - neoplastic cells are in the bone marrow +/- blood
Lymphoma - neoplastic cells are in solid tissue in the form of tumors (ex lymph nodes)