Lab 4 - WBC, inflammation Flashcards
What can inflammations be caused by
infection endotoxin mediated sterile necrosis (i.e. pancreatitis, tumour, trauma), chemical (injections, toxins, acids, alkalines), immune mediated (SLE)
Name the laboratory examinations used to examine inflammatory processes
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 samplentupe is used for wbc counting
anticoagulated blood. Na2EDTA, K2EDTA, or Na- citrate can be used as anticoagulant
In case of bird, reptile, fish samples Li-, or Ca heparin is used.
wbc counting by hematology analysers - Give the different types
- automatic cell counters: the impedance is measured to give info about number and size of cells different cell layers.
- the laser beam method: the cells will scatter the laser beam and give two measures giving information about size, shape and number
How does automatic cell counters aka volumetric impedance method work?
SIZE AND NUMBER
the impedance gives A jump indicates a cell eg counted - small jump means small cell and vice versa. Its a very reliable methods as all cells are counted. The result is given in a histogram. The wbc and rbc are differentiated by heamolysing the rbc`s after making a dilution for the wbc
Differentiated wbc by size. Reagents are used to decr the size of lymphocytes and incr size of ng and macrophages
How does laser cell counters work
SIZE, NUMBER and SHAPE/INNER STRUCTURE, better that ACC
- Very reliable as all cells will scatter the light from the laser. cells flow through tube and are illuminated abd they will scatter the light
- the mistakes in ACC are more or less fixed in this machine eg. It wont count clumps
- gives a scattergram and histogram
Describe the two types of scatter and what they respresent (laser beam method)
x: Low angular scatter - size
Y: High angular side scatter - inner structure
X and y values create point on graph making the cloud
What is gating
The process of counting the points in the „cloud” giving the cell count of the similar cell types
How do you perform a qualitative blood count
Smears are examined using different stainings to enhance different traits. The smears are analysed at the middle of the slides and at the edges aswell.
First low power should be used, then high 1000x magnification can be provided by using 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
Neutrophil granulocyte cell line
myeloblast
promyelocyte myelocyte metamyelocyte (jugend) band forms (stab) segmented forms
Lymphoid cell line:
lymphoblast
small lymphocyte middle sized lymphocyte reactive T lymphocyte
Eosinophil cell line
young form (band nucleated) eosinophil granulocyte segmented form eosinophil granulocyte
Basophil cell line:
young form (band nucleated) basophil granulocyte segmented form basophil granulocyte
Monocyte cell line:
monocytic (young) form
reactive macrophage form
How do you determine the absolute wbc numbers
(relative % of the cell type/100) * WBC count
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).
White blood cell (WBC) pools in the body:
In the blood vessels
In the blood vessels: marginal pool, cells attached to the inner surface of the blood vessels (mostly neutrophil granulocytes), cells can be mobilised very quickly from this pool!
White blood cell (WBC) pools in the body: In the circulating blood:
we take our sample from here, the circulating pool.
White blood cell (WBC) pools in the body: In the tissues of different organs
tissue pool
Physiological leukocytosis: acute
develops due to acute or chronic stress (epinephrine, norepinephrine, glucocorticoids) give their effects
catecholamines are visible within seconds (neutrophilia, lymphocytosisu- as cells are mobilised from the marginal pool /cells attached to the vessels, lymphatic vessels, and organs/
The effects of catecholamines during Physiological leukocytosis/inflammation
catecholamines are visible within seconds (neutrophilia, lymphocytosis- as cells are mobilised from the marginal pool /cells attached to the vessels, lymphatic vessels, and organs/
Physiological leukocytosis: chronic inflammation
can be detectable after hours (or in some instances minutes) (neutrophilia, lymphopenia, eosinopenia). The older neutrophils are mobilised from the bone marrow and marginal pools, they will age faster leading to hypersegmentation, the cellular proliferation is inhibited, lympholysis, and the eosinophils are inhibited from from leaving the BM
Physiological inflammation/ leukocytosis in cats
In cats stress can cause an extreme neutrophilia, as the marginal pool is bigger (two third of the total neutrophilic count) in cats.
Pathologic inflammation: Characteristics of acute and chronic
acute (increase of positive acute phase proteins (APP) in the blood and the decrease of negative acute phase proteins) inflammation
Chronic (is not part of the body’s natural healing. dilated blood vessels and a hyped up immune system) incr GCs by e.g. cushings or GC therapy
Stress leukogram/Chronic: right shift, Neutrophilia, leukocytosis, lymphopenia, eosinopenia
Acute: left shift, neutropenia, neutrophilia
Neutropenia in pathological acute inflammation
First stage of scute inflammaton bc the ng will travel to the tissues to work by the fsctors released by macrophages, the total wbc count will therefore initially decrease - we dont do blood samples in this part!
Neutrophilia in pathological acute inflammation
During later phases of acute inflammation neutrophilia occurs due to the granulocyte colony stimulating factor (G-CSF), or granulocyte macrophage colony stimulating factors GM-CSF produced by the macrophages. These factors stimulate WBC production in the bone marrow, which leads to an increased total WBC count.
Left shift: types, examples and how the cells travel
Acute inflammation
- regen, degen, leukemoid or leukemic L shift
- neoplastic disease, addisons disease, Pelger-Huet anomaly(band form NG)
- cells are mobilised form the marginal pool, then differentiated forms are migrating from the storage pool of the bone marrow. Later younger cells enter the blood stream from the maturation pool of bone marrow.
Regenerative left shift
ng will migrate from the circulation to the tissues causing Leucopenia to signal to bm to replace wbc loss - younger cell population from the storage, maturation, and/or the mitotic pool (from bm and circulation to the tissues)
INCR. Wbc and ng
Degenerative left shift
the 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.
DECR wbc, ng
Due to severe inflammation, big abcesses, peritoneitis, pleuritis
Leukemoid left shift (leukemoid reaction)
Wbc and ng increase a lot, due to G- and GM-CSF. “Moid”=like leukemia but other unknown cause such as big abcesses, endometritis. Normal reaction woukd be degr L shift - dont really know difference other than that the incr wbc and ng is much higher in leukemoid reaction
typical reaction in case of big abscesses, endometritis
Toxic neutrophils
Left shift
During inflammations its normal for the granulocytopoesis to be disturbed causong some toxic neutrophils. The situation becomes pathological when these toxic np get into the blood during eg. Septicemia
Döhle-bodies, pathological granules
Toxic neutrophil:
Due to toxic effects some angular, basophilic inclusion bodies can be seen in some neutrophil granulocytes. These are the remnants of the endoplasmatic reticulum. more common in cats.
Pathological (toxic) granules
Right shift: types, examples
Incr wbc and ng, chronic
- appearance of the older forms (segmented, hypersegmented forms)
- always glucocorticoid effects: 1️⃣Chronic inflammations
2️⃣GC as theraputic drug
3️⃣incr. Production in adrenal gland: Cushings disease.
What is a stress leukogram
Right shift, leukocytosis, neutrophilia, lymphopenia and eosinopenia
Glucocorticoids effect on wbc’s
- Glucocorticoids inhibit cellular proliferation
- have membrane stabilising effect (which let the neutrophils to grow old and become hypersegmented
- glucocorticoids are lympholytic
- inhibit the outflow of eosinophils from the bone marrow (it can cause lymphopenia and eosinopenia
What differs glucocorticoid effect from chronic inflammation?
similar to glucocorticoid effect (leukocytosis, neutrophilia, right shift), the difference is that in this case leukocytosis is sometimes associated with lymphocytosis, monocytosis, and/or eosinophylia.
Addison’s disease (hypoadrenocorticism)
Hypoplasia or necrosis of adrenal gland cause ⬇️GC. no inhib effect of glucocorticoids -> the wbc count will increase; LEFT SHIFT (young ng) as there id no inhibition of cell proliferation, lympocytosis and eosinophilia.
Pelger-Huet anomaly:
Normocytaemia, and left shift (metamyelocytes, band forms of neutrophils) Inheritable problem causes diagnostic problem.
Cyclic neutropenia:
This is an inhertitable disease of Grey Collies. Due to cyclic bone marrow activity, neutropenia occurs in weekly, monthly intervals. During these periods the animals are sensitive to infections.
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.
What is the clinical approach to give R/L shift
Only smear analysis!
The process of inflammation
1️⃣ neutropenia 2️⃣ use the ng from pools 3️⃣ incr bm wbc production, mobilisation 4️⃣ L shift (if it reaches here its getting bad)(= acute and big inflammation at this stage) 5️⃣ degen(worst) or regen L shift 6️⃣ R shift
What is the mechanism of the glutaric aldehyde test?
- causes a rapid coagulation of fibrinogen and labile globulins
- show coagulation within seconds in case of acute inflammatory process (high fibrinogen and globulin level in the blood), even with heparin or EDTA
- faster coag means more severe inflamm, more globulins and fibrinogens!!!!
- Tubes should be checked for coagulation every 30 seconds by turning them upside down
What is the glutaric aldehyde test primarily used to
diagnose? In what species?
- examine the increase of fibrinogen and globulin concentration in plasma.
- fibrinogen is an APP, incr during acute inflammation
- in adult cattle (more than one year old) best clottimg in cattle, not really used in other spp
- reticuloperitonitis, severe mastitis, endometritis
Important test as they show vague clinical signs
What percentage glutaric aldehyde solution is used?
We generally use heparinised blood and mix the sample (i.e. 1-2 ml) with the same amount of 1,25% glutaric aldehyde solution.
glutaric aldehyde test, used for what type of diseases
The test is primarily used to diagnose reticuloperitonitis, severe mastitis or endometritis of cattle. These animals can be extremely indolent, sometimes they show just vague clinical signs, (or no signs at all!!) even if they are suffering from very severe diseases
What is the mechanism of the erythrocyte
sedimentation rate?
the increased sedimentation of RBCs due to inflammatory processes, as the acute phase proteins and other globulins tend to attach onto the surface of RBCs (no neg charge so aggregation is possible apposed to albumins who do have neg charge so no agg occur in normal circumstances)
Erythrocyte sedimentation rate method
Westergreen tubes are used to perform this test. The tube contains Na-citrate to prevent coag by binding ca ions.
The tube has a hole on the bottom (and on the top), so when it is driven through the rubber plug of another tube containing the blood sample, blood will flow up in the tube with the scale until the zero point.
- Check sedimentation rate of RBCs after one hour.
How is the ESR of horses different to other species?
Sedimentation is very fast in horses, so ESR must be evaluated after 20 minutes.
- the speed of the sedimentation DECREASE due to inflammatory processes contrary to other species.
Name some positive and negative acute phase proteins.
Pos:
- CRP(dog)
- serum amyloid A(cat, horse)
- haptoglobin(cattle)
- fibrinogen
negative: transferrin, lactoferrin
(The major app That incr the most are different for diff spp, diff are used to measure)
When is ESR increased
During initial stage of acute inflammation before clinical sings! Diagnostics in eg immunosupressed animals where known clinical and laboratory signs of inflammation can be vague or absent!
- Increased globulin concentration
- Decreased albumin concentration and as a consequence relatively increased globulin concentration causes increased ESR, too
(Neutropenia)
Esr mechanism
Decreased albumin concentration and as a consequence relatively increased globulin concentration causes increased ESR, too
sedimentstion rate interpretation
- ⬆️ ESR means if:
⬇️ ht, serum viscosity, total protein
⬆️ fibrinogen concentration (APP - cells stick together and sink faster) - The sedimentation can be biphasic, when the young RBCs sediment later than the older forms. This is common in case of immunhemolytic anemia (regenerative anaemia, many young RBCs are produced). In this case the older and even aggregated RBCs sediment much earlier then the younger forms.
C-reactive protein: what is it, used for, produced where
An app which incr during acute inflammations, esp in dog.
- prod in liver
- serum sample for measurement - elisa: dog and horse specific methods
- measurement before clinical signs appear. Imp for immunosuppressed animals or high dose glucocorticoid therapy(cant see difference?)
What are Haemopoetic (leukemic) tumours of animals
Hematopoietic cancers (HCs) are malignancies of immune system cells. Leukemias are “liquid tumors” in the blood and are derived from the transformation of either a hematopoietic precursor in the bone marrow or a mature hematopoietic cell in the blood
What differentiates the different Haemopoetic (leukemic) tumours of animals
Leukemias can be lymphoid or myeloid, and acute(blast cells) or chronic(differentiated)
myelomas: . In blood or BM. Fully differentiated cell. Leucopenia and thrombocytopenia indicates that BM
lymphomas: a transformed lymphocyte in a secondary lymphoid tissue(liver, skin, int) generates a solid mass. May occur in bone marrow too.
Lymphomas
Overprod of poorly differentiated lymphocytes in lymphayic organs mostly: lymphnodes, spleen
(Sometimes in other tissues too)
Origin of lymphomas are only proved in the case of cats snd cattle! - viral infections
What are the Acute leukemic diseases:
Acute lymphoblastic leukemia
Acute myeloblastic, promyelocytic leukemia
Acute erythroblastic leukemia
Lymphoma of Stage V (metastatisis to bone marrow)
What are the Chronic leukemic diseases:
Chonic small lymphocytic leukemia, Problem with mature/ differentiated cells
Chronic myeloid (neutrophil granulocytic, eosinophil granulocytic, basophil granulocytic,
monocytic leukemia)
Polycythaemia absoluta vera (overproduction of mature erythrocytes)
Essential thrombocytosis (overproduction of mature thrombocytes).
Name 8 causes of high WBC count
- overproduction in bone marrow - neoplasias: SOME myeloid leukemias eg chronic neutrophilic leukemia
- Regenerative L shift
- R shift - neutrophilia
- Later phase of physiological inflammation: neutrophilia
- Leukemoid reaction, neutrophilia
- Glucocorticoid therapy
- R: Cushings disease: incr glucocorticoid production
- L: Addisons disease: decr GC prod so incr eosins, lymphocytes
Name 6 causes of low WBC count
- Bone marrow suppression: chemo, haemotoxins, mycotoxins, drug side effects
- Degenerative L shift
- First phase of imflammation
- Some leukemias: Lymphomas - accumulation in lymphatic organs
- Cyclic neutropenia in grey collies: cyclic BM activity, neutropenia in monthky/weekly intervals
- Autoimmune neutropenia
Myelophtysis
When other cells suppress the hemopoetic cells of the BM - a cause of low WBC count (see last FC)
Neutrophilic animals and their ratio of wbc
Dog, cat, human
NG : LC
60 : 35
Lymphophilic animals and their ratio of wbc
The rest (swine, cattle, domestic fowl etc.)
NG : LC
35 : 60
