6. WBC and inflammation Flashcards
Causes of infection
Parasitic, bacterial (septic), rickettsial, fungal, viral (infectious agent!!)
Causes of Inflammation
- infection
- endotoxin mediated
- sterile necrosis (i.e. pancreatitis, tumour, trauma),
- chemical (injections, toxins, acids, alkalines),
- immune mediated (SLE)
Laboratory examinations of 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
Samples used for WBC counting
- Anticoagulated blood. Na2EDTA, K2EDTA, or Nacitrate.
- In case of bird, reptile, fish samples: Li-, or Ca heparin
Ways to count WBC´s
- WBC counting by using Bürker-chamber
- WBC counting by hematology analyser
- Automatic cell counters can count WBC
- Laser cell counters
Qualitative blood count: staining methods
- May-Grünwald
- Romanowsky
- Giemsa
- Diff-Quick etc.
Qualitative blood count:
- Type of power used?
- Number of cells counted?
- Differentiate according to?
- First low power should be used, then high 1000x magnification can be provided by using immersion lens (and immersion oil).
- 50-200 cells
- Differentiate according to the morphological pattern of the cell types
Qualitative blood count: 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
Calculation of relative % of the cell type
relative % of the cell type = (relative % of the cell type / 100) x WBC count (x10^9/l)
Absolute WBC numbers in % and Absolute No x10^9/l
- Neutrophil granulocyte
b) metamyelocyte (jugend)
c) segmented form - Lymphocyte
- Eosinophil granulocyte
- Basophil granulocyte
- Monocyte
- Neutrophil granulocyte - - 3-11,8
b) metamyelocyte (jugend) 0-3 - 0-0,3
c) segmented form 60-77 - 3-11,5 - Lymphocyte 12-30 - 1-4,8
- Eosinophil granulocyte 2-10 - 0,1-1,35
- Basophil granulocyte < 1 - -
- Monocyte 1-5 - 0,1-0,5
White blood cell (WBC) pools in the body:
- In the bone marrow
- In the blood vessels
- In the circulating blood
- In the tissues of different organs
- In the bone marrow: mitotic- (very young cells, proliferating), maturation- (under differentiation) and storage- pools (mature, differentiated WBCs).
- 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!
- In the circulating blood: we take our sample from here, the circulating pool.
- In the tissues of different organs: tissue pool.
How does physiological leukocytosis develop?
Due to acute or chronic stress (epinephrine, norepinephrine, glucocorticoids).
The effects of catecholamines during physiological leukocytosis?
Visible within seconds (neutrophilia, lymphocytosis- as cells are mobilised from the marginal pool /cells attached to the vessels, lymphatic vessels, and organs/).
The effects of ACTH, or glucocorticoids during physiological leukocytosis?
Detectable after hours (or in some instances minutes) (neutrophilia, lymphopenia, eosinopenia).
What happens with cats during stress?
Can cause an extreme neutrophilia, as the marginal pool is bigger (two third of the total neutrophilic count) in cats.
Acute inflammation lab. signs
Increase of positive acute phase proteins (APP) in the blood and the decrease of negative acute phase proteins
Neutropenia
- When does neutropenia occur?
- What is the causes?
- What is the effect?
- What important lab. procedure in important due to this?
- During the first period of the inflammatory process, or widespread severe inflammation.
- Caused by the migrating factors (i.e. leukotriens, interleukins etc.) produced by tissue cells, macrophages. 3. Neutrophils are migrating out of the blood vessels to
the site of inflammation, and this can lead to a transient decrease in total WBC count. - Usually do not take blood samples in this early phase of inflam., that is why many people are convinced that inflammation always cause high WBC count
Neutrophilia:
- When does it occur?
- Causes?
- Effect?
- During later phases of inflammation
- 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 prod. in bone marrow, which leads to an increased total WBC count.
How can be observe a Left shift?
- More young WBCs appaear in the circulating blood.
- Young metamyelocytes (jugend), and band (stab) forms are visible in greater proportion and absolute number in the blood smear.
Types of left shift
- Regenerative left shift: Increased WBC count, neutrophilia, and left shift (younger neutrophils).
- Degenerative left shift: Low, or normal WBC, and neutrophil count and left shift (younger forms). It is a sign of poor prognosis, it means that the disease is very serious and needs immediate treatment.
Leukemoid reaction
- Occurance?
- Causes?
- What can it be confused with during blood smear analysis?
- In some cases, when there is a great stimulus for producing neutrophil granulocytes an enormous number (WBC count can be >70x109/l) of neutrophils can be present in the peripheral blood due to increased effects of G-CSF, GM-CSF.
- Big abscesses, endometritis (pyometra) and in case of some neoplastic (but not leukaemia) diseases.
- Chronic myelogenous leukemia.
Toxic neutrophils
- Occurance?
- How can it be observed?
- In very severe inflam. processes the granulocytopoesis can be disturbed.
- When the granule prod. is not physiological, some azurophilic (orange-red) granules can be seen in basophilic cytoplasm. These are “toxic” neutrophils, or we can say that this process is the toxic granulation of neutrophils.
Döhle-bodies:
- Occurance?
- When is it more common?
- Due to toxic effects some angular, basophilic inclusion bodies can be seen in some neutrophil granulocytes. These are the remnants of the endoplasmatic reticulum. 2. Appearance of Döhlebodies is more common in cats.
Chronic inflammation - Right shift: What is it?
Right shift means that many segmented and hypersegmented, old neutrophils (3-4 segments are on one nucleus) are seen in the smear.
Chronic inflammation - Right shift:
-Occurance?
The appearance of old neutrophils while there is
an increased WBC count is typical for chronic inflammatory process. This phenomenon is
also typical for the effects of glucocorticoids (given as therapeutic drug for different diseases, or increased production of endogenous glucocorticoids in the adrenal glands, Cushing’s disease).
What does Glucocorticoids do during a right shift?
- Inhibit cellular proliferation (disappearance of young cells), and have membrane stabilising 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 (can cause lymphopenia and eosinopenia).
This is a common finding of right shift?
In case of macrocytosis of poodles.
What is a stress leukogram?
Right shift, leukocytosis, neutrophilia, lymphopenia and eosinopenia together is called as stress leukogram.
Typical signs of chronic inflammation in the CBC?
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.
Haematology of Addison’s disease (hypoadrenocorticism)
- There is no inhibitory effect of glucocorticoids, as there is hypoplasia or necrosis of adrenal gland.
- The typical changes are increase in WBC (due to the polyuria caused increased PCV), increase in young neutrophils, left shift (no inhibition of cell proliferation), lymphocytosis 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.
What is myelophtysis?
During BM damage, when other cells suppress the hemopoetic cells in bone marrow
What does Glutaric aldehyde test examine?
- The increase of fibrinogen and globulin concentration in plasma.
- This method is used in clinical practice to indicate inflammatory processes in adult cattle (more than one year old).
What can fibrinogen in Glutaric aldehyde test show?
As fibrinogen is an acute phase protein, it can show a marked increase during acute inflammatory processes.
What does Glutaric aldehyde solution cause?
A rapid coagulation of fibrinogen and labile globulins, so
blood mixed with this solution can show coagulation within seconds in case of acute inflammatory process (high fibrinogen and globulin level in the blood), even if the blood sample contains heparin or EDTA (as an anticoagulant).
Glutaric aldehyde test: sample type?
We generally use heparinised blood and mix the sample (i.e. 1-2 ml) with the same amount of 1,25% glutaric aldehyde solution. (Tubes (or syringes) should be checked for coagulation every 30 seconds by turning them upside down)
Aid for the evaluation of glutaric aldehyde test in cattle:
Presence of inflammatory proteins in the sample and Character of the pathological process if Coagulation time is…
1. < 1 min
2. 1-3 min
3. 3-8 min
4. 8-15 min
5. > 15 min
- < 1 min. ++++ Severe, widespread acute inflam
- 1-3 min. +++ Acute inflam
- 3-8 min. ++ Subacute inflam
- 8-15 min. +/- Not severe inflam process
- > 15 min. - There is no inflam process
Usage of glutaric aldehyde test in cattle
To diagnose reticuloperitonitis, severe mastitis or endometritis of cattle
Erythrocyte sedimentation rate (ESR) - theory
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.
How is the surface of RBC normally?
How is it during high globulin level?
- Normally RBCs have negatively charged albumin molecules on their surface, this prevents RBC
aggregation. - In case of high globulin level in the blood, the surface of RBCs is covered by globulins, instead of albumins. Globulins have no negative charge, therefore RBCs are able to attach to each other, form big aggregates and sediment quickly.
Causes of increased globulin concentration?
Increased ESR, and this is generally due to inflammatory or neoplastic processes.
Causes of decreased albumin concentration
It causes as a consequence relatively increased globulin concentration, which causes increased ESR, too.
In case of chronic renal failure, or glomerulonephropathy when there is hypoalbuminaemia, ESR is?
Typically increased
Erythrocyte sedimentation rate (ESR) - sample type?
- Specific tubes (those made of glass: Westergreen tubes) are used to perform this test.
- The tube contains Na-citrate, and there is mm scale on it.
- 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.
Physiologically ESR is?
0,5-3 cm/hour.
ESR is inversely proportional with the Ht (PCV).
When is the sedimentation biphasic?
- When the young RBCs sediment later than the older forms.
- Common in case of immunhemolytic anemia (regenerative anaemia, many young RBCs are prod).
- In this case the older and even aggregated RBCs sediment much earlier then the younger forms.
ESR in dogs in case of different Ht (PCV)
- 10
- 15
- 20
- 25
- 30
- 35
- 40
- 45
- 50
- 10 79
- 15 64
- 20 49
- 25 36
- 30 26
- 35 16
- 40 10
- 45 5
- 50 0
(mm/hour)
ESR in horses in case of different Ht (PCV)
- 10
- 15
- 20
- 25
- 30
- 35
- 40
- 45
- 50
- 10 86 +/-1
- 15 80 +/-1
- 20 70 +/-2
- 25 60 +/-3
- 30 47 +/-5
- 35 28 +/-15
- 40 11 +/-8
- 45 2.5 +/-1
- 50 0 +/-1
(mm/20 min)
C-reactive protein, CRP
Origin of the name of this acute phase protein: it can bind nonspecifically to a protein of Pneumococcus bacteria, called protein-C, and cause precipitation
What happens with the conc. of APPs during acute inflammatory processes?
The conc becomes incr in the blood plasma. Such APPs is CRP, haptoglobin, serum amyloid A (SAA) etc. Some „negative” APPS (i.e. transferrin, lactoferrin) show a decr conc.
Where is CRP prod?
In the liver and in the beginning of the inflammatory process cytokins (interleukins) stimulate its production.
Samples used for the measurements of CRP?
Serum samples.
The determination of CRP is based upon which methods?
Immunological (ELISA or immunturbidimetric) method. Today species (dog, horse) specific ELISA methods are available.
General physiologial value of CRP
8 mg/l
What happens with the CRP during acute inflammatory processes?
2. In which species is early diagnostic detection of inflammatory processes important?
- In the beginning of acute inflammatory process CRP values are increased, before the appearance of the clinical signs. (In case of concomitant decreased liver function this increase might not be so marked.) Therefore it is a very useful diagnostic tool for the early detection of
inflammatory processes. - In immunosuppressed animals, for example during chemotherapy, or high dose glucocorticoid therapy. In these cases the well known clinical and laboratory signs of inflammation can be vague or absent
The most common leukemic types?
- Acute leukemic diseases
2. Chronic leukemic diseases
Acute leukemic diseases:
- What is the characteristics of the disease?
- Origin of these cells can be evaluated how?
- Types
- Typical cell types in these tumours are „blast” cells, that have coarse chromatin pattern, and have nucleoli.
- Origin of these cells: by (immune)cytochemical, and bone marrow analysis. In some cases neoplastic cells do not appear in peripheric blood, but anemia, a leukopenia, thrombocytopenia. In these cases bone marrow evaluation is important.
- Types:
Acute lymphoblastic leukemia
Acute myeloblastic, promyelocytic leukemia
Acute erythroblastic leukemia
Lymphoma of Stage V (metastatisis to bone marrow)
- Chronic leukemic diseases:
- What is the characteristics of the disease?
- Method to diagnose this disease?
- Types
- Typical cell types in these tumours are mature differentiated, or well differentiated cells, that appear in enormous number in the peripheric blood. This presentation does not cause diagnostic difficulties in case of lymphoid, thrombocytes, or erythroid forms, but it can be mixed with leukemoid reaction.
- Bone marrow evaluation is essentially required
- Types:
Chonic small lymphocytic leukemia
Chronic myeloid (neutrophil granulocytic, eosinophil granulocytic, basophil granulocytic, monocytic leukemia)
Polycythaemia absoluta vera (overprod of mature erythrocytes),
Essential thrombocytosis (overprod of mature thrombocytes).
Lymphomas
Are hemopoetic tumours, too. In this disease poorly differentiated lymphoid cells are overprod 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 this disease (Bovine Leukosis Virus/BVL/ a Feline Leukemia Virus/FeLV/).
