Lab 6 - WBC Flashcards
Infection could be
Parasitic,
bacterial (septic),
rickettsial,
fungal,
viral (infectious agent!!)
Inflammation could be like:
- Infection:
- endotoxin mediated
- sterile necrosis (i.e.
- pancreatitis,
- tumour, trauma),
- chemical (injections, toxins, acids, alkalines),
- immune mediated (SLE)
To evaluate the severity, locality and ethiology of inflammatory processes what are needed?
Laboratory examinations are needed.
Laboratory examinations:
- Haematology (qualitative and quantitative=complete blood count, CBC)
- Erythrocyte sedimentation rate (ESR) cytology
- serum biochemistry
- routine biochemical parameters (substrates, enzymes)
- acute phase proteins
- microbiology
- parasitology
- immunology (ELISA, RIA etc. methods)
- molecular biology
- morphological pathology,
- histology
What is nessesary for White Blood Cell (WBC) counting
For WBC counting it is necessary to use anticoagulated blood
For WBC counting - what is used as a anticoagulant?
Na2EDTA, K2EDTA, or Na- citrate can be used as anticoagulant.
In case of bird, reptile, fish samples Li-, or Ca heparin is used.
Methods used for
White Blood Cell (WBC) counting
- WBC counting by using Bürker-chamber
- WBC counting by hematology analyser
- Automatic cell counters can count WBC (electrical impedance of a particle (cell))
- Laser cell counters
Qualitative blood count
Smear - Staining
After preparing smears several STAINING methods can be used, i.e.
- May-Grünwald,
- Romanowsky,
- Giemsa,
- Diff-Quick etc.
The smears should be analysed at the edge of the slide and in the middle, too.
How to perform Qualitative Blood cell count
- Prepare smare
- Stain
Low magnification - High magnification. First low power should be used, then high.
1000x magnification can be provided by using an immersion lens (and immersion oil).
- One should count at least 50-200 cells and differentiate according to the morphological pattern of the cell types and make the ratio in % among the different cell types:
Qualitative blood count
Neutrophil granulocyte cell line:
- Myeloblast
- Promyelocyte myelocyte
- Metamyelocyte (Jugend)
- Band forms (stab)
- Segmented forms
Qualitative blood count
Lymphoid cell line:
- Lymphoblast
- Small lymphocyte
- Middle sized lymphocyte
- Reactive T lymphocyte
Qualitative blood count
Eosinophil cell line:
- Young form (band nucleated) eosinophil granulocyte
- Segmented form eosinophil granulocyte
Qualitative blood count
Basophil cell line:
- Young form (band nucleated) basophil granulocyte
- Segmented form basophil granulocyte
Qualitative blood count
Monocyte cell line:
- Monocytic (young) form
- Reactive macrophage form
Function of different white blood cells
The function of different white blood cells is mentioned during the lectures.
Absolute WBC numbers
Relative % is defined routinely, but it is useful to know the absolute number of the different cell types.
Neutrophil granulocyte ratio is 70 % in a dog. This is physiological.
Example:
WBC count: 20 x109/l (high!)
Neutrophil granulocyte: 70 %
Lymhocyte: 29 %
Eosinophil granulocyte: 1 %
This value is pathological: 14 x10^9/l (normal: 3-11,8 x10^9/l).
Diagnosis: neutrophilia.
2.
Lymphocyte is 60 % in a cat, that seems to be increased.
WBC count is 3,5 x109/l.
WBC count: 3,5 x109/l (low!)
Neutrophil granulocyte: 40 %
Lymhocyte: 60 %
Absolute neutrophil granulocyte No is 1,4 x 10^9/l (normal: 3-11,8 x109/l).
Diagnosis: neutropenia.
WBC (x 109/l) in Swine, Sheep, Cow and Rabbit
Swine 7 – 20 (x 109/l)
Sheep 4 – 12 (x 109/l)
Cow 4 – 12 (x 109/l)
Rabbit 4.5 – 11 (x 109/l)
Lymphocytes (%) in Swine, Sheep, Cow and Rabit
Swine 40 – 60 (%)
Sheep 40 – 70 (%)
Cow 40 – 70 (%)
Rabbit 40 – 80 (%)
General normal values for dog, cat, horse, humans:
Neutrophil granulocyte
metamyelocyte (Jugend)
segmented form
% and Absolute No x10^9/l
1. Absolute No x10^9/l
Neutrophil granulocyte = 3-11,8 x10^9/l
- metamyelocyte (jugend)= 0-0,3 x10^9/l
- segmented form = 3-11,5 x10^9/l
2. %
Metamyelocyte (jugend) = 0-3%
segmented form = 60-77%
General normal values for dog, cat, horse, humans:
Lymphocyte
Absolute and %
% = 12-30 %
Absolute = 1-4,8 *10^9
General normal values for dog, cat, horse, humans:
Eosinophil granulocyte
% and Absolute
% = 2-10 %
Absolute = 0,1-1,35 *10^9
General normal values for dog, cat, horse, humans:
Basophil granulocyte
%
< 1 %
General normal values for dog, cat, horse, humans:
Monocyte
% and Absolute
% = 1-5%
Absolute = 0,1-0,5 *10^9
Haematological analysis of inflammatory processes
Where do you find the White blood cell (WBC) pools in the body:
- In the bone marrow:
- mitotic- (very young cells, proliferating),
- maturation- (under differentiation) and
-
storage- pools (mature, differentiated WBCs).
2. In the blood vessels: - marginal pool, cells attached to the inner surface of the blood vessels (mostly neutrophil granulocytes),
- Cells can be mobilized very quickly from this pool!
3. In the circulating blood: - we take our sample from here,
- the circulating pool.
- In the tissues of different organs: tissue pool.
What is Physiological leukocytosis and what happens?
Physiological leukocytosis develops due to acute or chronic stress (epinephrine, norepinephrine, glucocorticoids).
- The effects of catecholamines are visible within seconds (neutrophilia, lymphocytosis- as cells are mobilized from the marginal pool /cells attached to the vessels, lymphatic vessels, and organs/).
- The effects of ACTH or glucocorticoids can be detectable after hours (or in some instances minutes) (neutrophilia, lymphopenia, eosinopenia).
- The older neutrophils are mobilized from the bone marrow and marginal pools, they do not die too early due to the stabilisation of the lysosomal membranes, meanwhile, there is a severe lympholysis, and the sequestration of lymphocytes to the lymphatic organs, eosinophil granulocytes are NOT released to the circulation from the bone marrow.
In cats, stress can cause extreme neutrophilia, as the marginal pool is bigger (2/3 of the total neutrophilic count) in cats.
Cause of
Pathologic leukocytosis/leukopenia
Acute inflammation
The very first laboratory sign of inflammation is the:
1. increase of positive acute-phase proteins (APP) in the blood and the
2. decrease of negative acute-phase proteins. (see later in details)
Neutropenia
When does it happen
What is it caused by
- During the first period of the inflammatory process, or widespread severe inflammation = NEUTROPENIA.
- It is caused by the migrating factors (i.e. leukotriens, interleukins etc.) produced by tissue cells, macrophages.
Neutrophils are migrating out of the blood vessels to the site of inflammation (into the tissues), and this can lead to a transient decrease in the total WBC count.
We usually do not take blood samples in this very early phase of inflammation, that is why many people are convinced, that inflammation always causes a high WBC count!
Neutrophilia
When does it happen
What is it caused by
1. During later phases of inflammation = NEUTROPHILIA occurs due to the (G-CSF), or GM-CSF produced by the macrophages.
2. These factors stimulate WBC production in the bone marrow, which leads to an increased total WBC count.
What is “Left Shift”
When More young WBCs appear in the circulating blood.
- Young metamyelocytes (Jugend), and
- Band (stab) forms are visible in greater proportion and absolute number in the blood smear.
First, cells are mobilized from the marginal pool, then differentiated forms are migrating from the storage pool of the bone marrow.
Later younger cells enter the bloodstream from the maturation pool of bone marrow.
In case of severely increased tissue requirement (or in neoplastic diseases i.e. acute myelogenous leukemia) very young cells (myelocytes, promyelocytes) from the mitotic pool are going out.
Two different types of left shifts!
What is a regenerative left shift?
Increased WBC count, neutrophilia, and left shift (younger neutrophils).
After the first neutropenic phase, bone marrow regenerates the utilized neutrophils in the circulation by the younger cell population from the storage, maturation, and/or the mitotic pool.
=This is the most common cause of the appearance of young neutrophils.
Regenerative left shift is a sign of a favorable prognosis.
What is Degenerative left shift?
Low, or normal WBC, and neutrophil count, and left shift (younger forms).
1. If there is a great neutrophil utilization in the tissues
(widespread or very severe inflammation, big abscesses, peritonitis, pleuritis, phlegmone, pyometra), and the
2. Rate of utilisation is bigger than the regenerative capacity of bone marrow,
= increased outflow of young neutrophils is NOT enough to increase WBC count
(or even for the maintenance of normal WBC count!).
Degenerative left shift is a sign of poor prognosis, it means that the disease is very serious and needs immediate treatment.
What happens due to Leukemoid reactions
In some cases, when there is a great stimulus for producing neutrophil granulocytes in an enormous number (WBC count can be >70x10^9/l) of neutrophils can be present in the peripheral blood due to increased effects of G-CSF, GM-CSF.
This is a typical reaction in case of big abscesses, endometritis (pyometra), and in the case of some neoplastic (but not leukaemia) diseases. During blood smear analysis, a leukemoid reaction can be easily confused with chronic myelogenous leukemia.
What is Toxic Neutrophils
In very severe inflammatory processes the granulocytopoesis can be disturbed.
When the granule production is NOT physiological, some azurophilic (orange-red) granules can be seen in the basophilic cytoplasm.
These are “toxic” neutrophils, or we can say that this process is the toxic granulation of neutrophils.
What is Döhle-bodies?
Due to toxic effects some angular, basophilic inclusion bodies can be seen in some neutrophil granulocytes.
= These are the remnants of the endoplasmatic reticulum.
The appearance of Döhle- bodies is more common in cats.
Which shift is characteristics for Chronic inflammation
Right shift
Chronic Inflamation
What is characterizing the right shift?
Chronic inflammation
Right shift:
The right shift= many segmented and hypersegmented, old neutrophils (3-4 segments are on one nucleus) are seen in the smear.
The appearance of these older forms + increased WBC count = typical chronic inflammatory process.
This phenomenon is also typical for the effects of glucocorticoids (given as a therapeutic drug for different diseases, or increased production of endogenous glucocorticoids in the adrenal glands, Cushing’s disease).
Glucocorticoids inhibit cellular proliferation (disappearance of young cells), and have membrane stabilizing effect (which let the neutrophils to grow old and become hypersegmented).
Moreover, glucocorticoids are lympholytic and inhibit the outflow of eosinophils from the bone marrow (it can cause lymphopenia and eosinopenia). This is a common finding in the case of MACROCYTOSIS of POODLES..
Right shift, leukocytosis, neutrophilia, lymphopenia, and eosinopenia together is called a stress leukogram.
Typical signs of chronic inflammation in the CBC are similar to the glucocorticoid effect (leukocytosis, neutrophilia, right shift),
- the difference is that in this case leukocytosis is sometimes associated with lymphocytosis, monocytosis, and/or eosinophilia.
Other typical changes are
- Haematology of Addison’s disease (hypoadrenocorticism):
- Pelger-Huet anomaly:
3. Cyclic neutropenia:
- Bone marrow damage:
Lymphocytes
Morphology
● Size ~ RBC
● Round nucleus
● Smooth, dense chromatin
● Narrow rim of basophilic cytoplasm
● Vary in size
Monocytes
Morphology
Monocytes
● Larger than neutrophils
● Extreme variable shape, no consistent species differences
● Very variable shape of the nucleus:
- *● Ameboid**
- *● Bean-shaped**
● Band-like
● lobulated,
● U-shaped
● Cytoplasm:
- vacuoles,
- granules,
- slightly grainy
Eosinophils
Morphology
● Slightly larger than a neutrophil
● Segmented nucleus with 2-3 lobes
● Eosinophilic cytoplasmic granules
● Marked SPECIES VARIATION
1● Dog: granules are round, size, numbers are very variable
- Greyhounds: grey cytoplasm, and vacuolated appearance because their granules do not stain
2● Cat: small uniform, rod-shaped orange-pink granules
3● Horse: looks like a rasberry
Basophils
Morphology
● Mildly lobulated, ribbon-like nucleus
● Dark purplish cytoplasmic granules
● Canine: long, folded nucleus; grey, lavender cytoplasm
● Feline: round to oval granules, pale lavender, to deep purple
● Horse: deep purple granules
What is Acute phase proteins (APPs)
● Extremely sensitive markers of inflammation
● Extremely non-specific
● The decline in APP concentrations may indicate a response to treatment
● Triggered by proinflammatory cytokines:
- interleukin (IL)-1, IL-6 (produced by macrophages→ APPs production in hepatocytes
● Fibrinogen: in all species production is stimulated as part of the acute phase response
1● Major APPs: rapid increase (often within 24–48 hours) 5–50-fold
● Dog: C-reactive protein (CRP)
● Cat: Serum amyloid A (SAA)
2● Moderate APPs: slower increase (within 4–7 days) 2–10-fold
● Dog, cat: Haptoglobin (Hp)
3● Negative APPs: concentrations decline
- Albumin, transferrin
Characterize
Erythrocyte sedimentation rate (ESR)
● Non-specific measure of inflammation
1● In normal conditions:
- RBCs are negatively charged due to albumin attached to the surfaces
and repel each other
2● In inflammatory conditions albumin↓, fibrinogen↑, globulins (positively charged):
APPs, immunglobulins↑ → RBC aggregation, rouleaux formation, sedimentation↑
3. Method:
● Fresh Na-citrate (1:4)
● Westergren tubes
● Normal values Note: mm/h. = millimeters per hour.
● Eq: 20 min - ESR slower in inflammation
Characterize
Glutaric aldehyde test
● In Adult cattle
● Fibrinogen as APPs markedly increases during acute inflammatory processes
● “Point-of-care” test
● Fresh anti-coagulated (heparinized) whole blood mixed with 1-2 ml glutaric aldehyde solution
● Rapid coagulation of fibrinogen and labile globulins
● Coagulation within seconds in case of the acute inflammatory process
● Diagnose
- reticuloperitonitis,
- severe mastitis,
- endometritis
