LabD 4 WBC Flashcards
Def infection
Parasitic, bacterial (septic), rickettsial, fungal, viral (infectious agent)
Def inflammation
Infection, endotoxin mediated, sterile necrosis (ie. pancreatitis, tumour, trauma), chemical (injections, toxins, acids, alkalines), immune medisted (SLE)
Laboratory examinations to evaluate severity, locality and ethiology of inflammatory processes
- Haematology, (qualitative, and quantitative = complete blood count, CBC)
- Erythrocyte sedimentation rate (ESR)
- Cytology
- serum biochemistry; routine ebiochem parameters(substrate, enzymes) and acute phase proteins
- Microbiology
- parasitology
- immunology (ELISA, RIA etc. methods)
- molecular biology
- morphological pathology, histology
WBC counting; blood, blood birds, reptiles, fish
Anticoagulated blood; Na2EDTA, K2EDTA or Na-citrate. In case of birds, reptile, fish; Li- or Ca Heparin is used.
WBC counting; techniques
- Bürker chamber
- Hematology analyzer
- Automatic cell counters, impedance and laser cell counters
WBC counting methods; Bürker chamber
- pipette homogenized whole blood sample
- wash with Türk solution (acetic acid hemolyser, gentiana violet blue - stains nuclei)
- incubate room temp.
- count cells with whole nuclei 25 squares, then divide number by 10 —> number of wbc in No x10 giga/L
WBC counting methods; hematology analyzer
- Some machines analyze diff cell types according to light absorbancy of diff cell layers - accurate
- Laser - very accurate and diff by poorly diff cells also
- Reagents (to measure peroxidase activity of the phagocytic cells - very accurate and diff by poorly diff cells also
- Flow cytometric method - very accurate
WBC counting methods; impendance method
- electrical impendance of cell entering between electrodes
- first dilution is made for wbc counting, then RBCs are hemolysed
- diff between wbc types
- regants decrease size of lymphocytes and increase size of neutrophils and macrophages
WBC counting methods; laser cell counters
- diff cells from size and inner structure
- cells flow through tube and are illuminated by laser light -> light beam scattered to a low angular and then to high angular detecting light energy
(Size is low angular scatter, inner structure side scatters) - two numerical values -> graph point, forms cloud of points with collection of similar cell types (process called gating)
- cloud diagrams called scatter diagram
- some reagents can detect biochem properties, ex. Myeloperoxidase activity of cell
Very accurate
Qualitative blood count; after preparing smears several staining methods can be used
May-Grünwald, Romanowsky, Giemsa, Diff-quick
Qualitative blood count using smears, how
- Immersion lens and immersion oil
- First low power, then high
- See the smear at edges and middle
- Count at least 50-200 cells and diff according to morphological pattern of cell types and make % ratio of cell types
Cell types in qualitative blood count smear; Neutrophil granulocyte cell line
- Myeloblast
- Promyelocyte
- Myelocyte
- Metamyelocyte (jugend)
- Band forms (stab)
- Segmented forms
Cell types in qualitative blood count smear; Lymphoid cell line
- Lymphoblast
- Small lymphocyte
- Middle sized lymphocyte
- Reactive T lymphocyte
Cell types in qualitative blood count smear; Eosinophil cell line
- Young form (band nucleated) eosinophil granulocyte
- Segmented form eosinophil granulocyte
Cell types in qualitative blood count smear; Basophil cell line
- Young form (band nucleated) basophil granulocyte
- Segmented form basophil granulocyte
Cell types in qualitative blood count smear; Monocyte cell line
- Monocytic (young) form
- Reactive macrophage form
Absolute WBC numbers equation
relative % of the cell type (no. x 10giga/L) = relative % of cell type/100 x WBC count (No. x10giga/L)
General normal values for dog, cat, horse, human absolute WBC count and % ; Neutrophil granulocyte
% 0-3 and 60-77
No. x10giga/L 3-11,8
General normal values for dog, cat, horse, human absolute WBC count and % ; Metamyelocyte (jugend)
% 0-3
No. x10giga/L 0-0,3
General normal values for dog, cat, horse, human absolute WBC count and % ; Neutrophil granulocyte, segmented form
% 60-77
No. x10giga/L 3-11,5
General normal values for dog, cat, horse, human absolute WBC count and % ; Lymphocyte
% 12-30
No. x10giga/L 1-4,8
General normal values for dog, cat, horse, human absolute WBC count and % ; Eosinophil granulocyte
% 2-10
No. x10giga/L 0,1-1,35
General normal values for dog, cat, horse, human absolute WBC count and % ; Basophil granulocyte
% <1
No. x10giga/L
General normal values for dog, cat, horse, human absolute WBC count and % ; Monocyte
% 1-5
No. x10giga/L 0,1-0,5
WBC pools in the body; bone marrow
Mitotic- (very young cells, proliferating), maturation- (under differentation) and storage pools (mature, differentiated WBCs)
WBC pools in the body; blood vessels
masrginal pool, cells attached to the inner surface of the blood vessels (mostly neutrophil granulocytes)
WBC pools in the body; circulating blood
where we take our sample from
WBC pools in the body; in the tissue of diff organs
tissue pool
What pool can the cells be mobilized very quickly from?
Blood vessel pool
Physiological
leukocytosis
- develops due to acute or chronic stress (epinephrine, norepinephrine, glucocorticoids)
Physiological
leukocytosis, effects of catecholamines WHYY an examples??
Visible within seconds, neutrophilia and lymphocytosis as the cells are mobilized from the marginal pool/cells attached to the vessels, lymphatic vessels and organs
Physiological
leukocytosis, effects of ACTH or glucocorticoids
Visible after hours - neutrophilia, lymphopenia, eosinopenia
Neutrophilia def
Neutrophilia is a medical condition that involves the increase of neutrophil in the blood or serum
Lymphocytosis def
Lymphocytosis is an increase in the number or proportion of lymphocytes in the blood
Lymphopenia def
Lymphocytopenia is the condition of having an abnormally low level of lymphocytes in the blood
Eosinopenia def
Eosinopenia is a form of agranulocytosis where the number of eosinophil granulocytes is lower than expected
In cats, stress can cause extreme neutrophilia due to?
the marginal pools are bigger in cats. (two third of the total neutrophilic count
Leukocytosis
a condition that involves the increase of leukocytes (white blood cells)
Pathologic leukocytosis; acute inflammation - what are the very first laboratory signs of inflammations
increase of positive acute phase proteins (APP) in the blood and the decrease of negative acute phase proteins
Stages of inflammation, blood
- First period or widespread and severe: neutropenia
- Later phases: neutrophilia –> increased WBC count
What causes neutropenia?
- Early phase inflammation
- Caused by the migrating factors: ex. leukotrienes, interleukins produced by tissue cells, macrophages.
- Neutrophils are migrating out of the blood vessels to the site of inflammation (tissues), and this move can lead to decrease in total WBC count.
What causes neutrophilia?
- Later phase inflammation
- Occurs due to granulocyte colony stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factors (GM-CSF) produced by the macrophages.
- These factors stimulate WBC production in bone marrow –> leads to increased WBC count
Left shift, def
- More young WBCs appear in the circulating blood
- Young metamyelocytes (jugend) and band (stab) forsm are visible in greater proportion and absolute no. in blood smear
Left shift, steps
- Cells ar mobilized from the marginal pool, then differentiated forms are migrating from the storage pool of the bone marrow
- Later younger cells enter the blood stream from maturation pool of bone marrow
- In case of severely increased tissue need (or in neoplastic disease ex. acute myelogenous leukemia), very young cells (myelocytes, promyelocytes) from the mitotic pool are going out.
Neoplastic disease, def
Abnormal growth of cells
Two different types of left shift
- Regenerative left shift
2. Degenerative left shift
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.
Prognosis when regenerative left shift is seen?
Favourable prognosis
Most common cause of appearance of young neutrophils?
Regenerative left shift
Degenerative left shift
- Low or normal WBC and neutrophil count and left shift (younger forms).
- If there is a great neutrophil utilization in the tissue (widespread or severe inflammation, big abcesses, periotonitis, pleuritis, phlegmone, pyometra), and the rate of utilization is bigger than the regenerative capacity of bone marrow, increased outflow of young neutrophils is not enough to increase WBC count (or even the maintenance of normal WBC count)
Prognosis when degenerative left shift is seen
Poor prognosis, very serious disease that needs immediate treatment
Phlegmone, def
A phlegmon is a localized area of acute inflammation of the soft tissues. It is a descriptive term which may be used for inflammation related to a bacterial infection or non-infectious causes. Usually with pus
Leukemoid reaction
- When there is a great stimulus for producing neutrophil granulocytes, an enormous number of neutrophils can be present in the peripheral blood due to increased effects of G-CSF and GM-CSF. (WBC count can be >70x10giga/L)
- Typical reaction in case of big abscesses, pyometra and some neoplastic diseases (but not leukemia).
- During blood smear analysis, leukemoid reaction can easily be confused with chronic myelogenous leukemia
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 basophilic cytoplasm.
This process is the toxic granulation of neutrophils
Granulocytopoesis def
Granulopoiesis is production of granulocytes
Döhle-bodies
- Due to the toxic effects some angular, basophilic inclusion bodies can be seen in some neutrophil granulocytes.
- These are the remnants of the endoplasmatic reticulum.
- Apperance of Döhle-bodies is more common in cats
Right shift
- Many segmented and hypersegmented, old neutrophils (3-4 segments are on one nucleus) are seen in smear.
- ## Apperance of these older forms and increased WBC count is typical for chronic inflammatory process
Right shift is typical for what substances
- Glucocorticoids (therapeutic drugs for diff diseases or increased production of endogenous glucocorticoids in the adrenal glands, Cushing´s disease)
Effect of Glucocorticoids on WBC
- Inhibit cellular proliferation (disappearance of young cells)
- membrane stabilizing effect (lets the neutrophils grow old and become hypersegmented)
- They are lympholytic and inhibit the outflow of eosinophils from the bone marrow (can cause lymphopenia and eosinopenia) Found is ex. macrocytosis in poodles.
Stress leukogram
Right shift, leukocytosis, neutrophilia, lymphopenia and eosinopenia together is called stress leukogram
Typical signs of chronic inflammation is similar to glucocorticoid effect, whats the similarities and differences
Similar: Leukocytosis, neutrophilia, right shift
Diff: in case of chronic inflammation, leukocytosis is sometimes associated with lymphocytosis, monocytosis and or eosinophilia
Other typical changes; Haematology of Addisons disease (hypoadrenocorticism)
- There is no inhibitory effect of glucocorticoids, as there is hypoplasia or necrosis of adrenal gland.
- The typical changes: increase in WBC (due to the polyuria caused increased hematocrit), increase in young neutrophils, left shift (no inhibition of cell proliferation), lymphocytosis and eosinophilia
Other typical changes; Pelger-Huet anomaly
- Normocytaemia and left shift (metamyelocytes, band forms of neutrophils)
- Inheritable problem causes diagnostic problem
Other typical changes; Cyclic neutropenia
- Inheritable disease of grey collies.
- Due to cyclic bone marrow activity, neutropenia occurs in weekly, monthly intervals.
- During these periods, animal is sensitive to infections
Other typical changes; Bone marrow damage
- Leukopenia and neutropenia occurs in case of bone marrow damage or decreased bone marrow function.
- Thrombocytopenia and aplastic anemia is often accompanied.
- When other cells suppress the hemopoetic cells in bone marrow, it is called Myelophtysis
Myelophtysis def
When other cells suppress the hemopoetic cells in bone marrow, it is called Myelophtysis
Other tests to examine inflammatory process
- Glutaric aldehyde test
- Erythrocyte sedimentation rate
- CRP
Glutaric aldehyde test
- Examine the increase of fibrinogen and globulin conc. in plasma
- Fibrinogen is an acute phase protein -> increase during inflammatory processes
- Used for adult cattle (more than 1 year old)
- Glutaric aldehyde solutions causes a rapid coagulation of fibrinogenand labile globulins, so blood mixed with this solution can show coagulation within seconds in case of acute inflammatory process (hig fibrinogen and globulin level in blood) even when blood contains anticoagulant
- The coagulation time is inversely correlated to the conc. of fibrinogen and globulins in the blood
Glutaric aldehyde test, blood used
- Heparinized blood
- Mix the sample with same amount of 1,25% glutaric aldehyde solution
- Tubes or syringes checked every 30 seconds for coagulations
Aid for evaluation of glutaric aldehyde test in cattle, breefly
Coagulation time, presence of the inflammatory proteins in the sample, character of the pathological process (see table)
- Severe: under 1 min coagulation
- Acute inflammation: 1-3 min
- Subacute: 3-8 min
- Not severe inflammation: 8-15 min
- No inflammatory process: over 15 min
If the symptoms and the results do not correlate, what then?
Repeat test after 24-48 hours
Glutaric aldehyde test mainly used for
To diagnose reticuloperitonitis, severe mastitis or endometritis of cattle.
Cattle show vague clinical signs, even when very sick
Erythrocyte sedimentation rate test (ESR)
- Increased sedimentation of RBCs due to inflammatory processes, as the acute phase proteins and other globulins tend to attach onto the surface of RBCs. Normally surface has negatively charged albumin molecules on their surface, this prevents RBC aggregation.
- In case of high globulin level in the blood (severe inflammation or some types neoplastic disease), the surface of RBC is covered by globulins instead of albumins. –> Globulins have no neg. charge, therefore RBCs attach to each other and form big aggregates and sediment quickly.
What can cause change of ESR?
- Increased globulin conc. causes increased ESR.
- Decreased albumin and increased globulin conc. (as a consequence), causes increased ESR.
- Chronic renal failure or glomerulonephropathy, when there is hypoalbuminaemia, ESR is increased
ESR test, equipment and test
- Specific tubes: glass Westergreen tubes. Tube contains Na-citrate, and has a mm scale. Tube has two openings, and another tube with rubber ending is used to measure.
- Should be checked after one hour
Physiological ESR should be
- 0,5-3 cm/hour
- ESR is inversely proportional with the Hematocrit
- Sedimentation is very fast in horses, must be evaluated within 20 min. In case of horses speed of sedimentation decreases in case of inflammatory processes! Contrary to other species
In ESR test, the sedimentation can be biphasic, the young RBCs sediment later than the older forms. When?
- Common in immunehemolytic anemia (regenerative anemia, many young RBCs, are produced.
ESR is proportional to
- Hematocrit (PCV) (Inversely proportional)
- serum viscosity
- Total protein and fibrinogen conc.
CRP, stands for?
C-reactive protein.
Origin of name of this acute phase protein: it can bind non-specifically to a protein of Pneumococcus bacteria, called protein-C and causes precipitation
In case of acute inflammatory processes, the conc. of some acute phase proteins increases in the plasma:
- CRP
- Haptoglobin
- Serum amyloid A (SAA) etc.
In case of acute inflammatory processes, the conc. of some ¨negative¨ acute phase proteins decrease:
- Transferrin
- Lactoferrin
Where is CRP produced
In the liver. Produced in the beginning of the inflammatory process, and stimulated by cytokins (interleukins)
Measurement of CRP, blood used and test
- Blood: serum sample
- Test: based on immunological ELISA method. Specific tests for diff. species are available
General physiological value
8 mg/L
Test during the beginning of acute inflammatory process, before clinical signs:
- CRP
- Can be extremely important for immunosuppressed animals, ex. during chemotherapy, or high dose glucocorticoid therapy, or other diseases where clinical and laboratory signs are absent
The most common leukemic hemopoietic tumor types in animals?
- Acute leukemic disease
- Chronic leukemic diseases
- Lymphomas
Acute leukemic disease
- Blast cells are typical cell types. Blast cells:
- Origin of these cells evaluated by (immune)cytochemical and bone marrow analysis.
- In some cases, neoplastic cells do not appear in peripheric blood, but anemia, leukopenia, thrombocytopenia. In these cases bone marrow analysis is important.
Chronic leukemic diseases
- Mature differentiated or well differentiated cellsm that appear in enormous number in peripheric blood.
- This presentation does not cause diagnostic difficulties in case of lymphoid, thrombodytes or erythroid forms, but it can be mixed with leukemoid reaction. Bone marrow analysis is essentially required for diagnosis
Lymphomas
- Are hemopoietic tumours too.
- Poorly differentiated lymphoid cells are overproduced in lymphatic organs (lymph nodes, spleen) and sometimes in other tissues (liver, skin, intestines etc.).
- In some cases tumorous lymphoid cells are overproduced in bone marrow too. The origin of this is proved in case of cats and cattle only! In these species: viral infection is causing the disease (Bovine Leukosis Virus-BVL, and Feline leukemia Virus-FeLV)
Blast cells in Acute leukemic disease:
- Coarse chromatin pattern, have nucleoli
Acute leukemic disease, examples
- Acute lymphoblastic leukemia
- Acute myeloblastic, promyelocytic leukemia
- Acute erythroblastic leukemia
- Lymphoma of stage V (metastasis to bone marrow)
Important to analyse bone marrow
Chronic leukemic disease, examples
- Chronic small lymphocytic anemia
- Chronic myeloid (neutrophil granulocytic, eosinophil granulocytic, basophil granulocytic, monocytic leukemia)
- Polycytaemia absoluta vera (overproduction of mature erythrocytes)
- Essential Thrombocytosis (overproduction of mature thrombocytes)
