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Lab diagnostics > 4. White blood cells and analysis of the inflammatory process > Flashcards

4. White blood cells and analysis of the inflammatory process Flashcards

1
Q

What do we use for WBC counting?

A
  • It is necessary to use anticoagulated blood.
  • Na2EDTA, K2EDTA of Na-citrate can be used as anticoagulant.
  • In case of birds, reptile, fish samples Li,or Ca heparin is used.
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2
Q

what can be the causes of infection:

A
  • Parasitic
  • Bacterial (septic)
  • Rickettsial
  • fungal
  • viral (infectious agent!!)
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3
Q

What can cause inflammation?

A
  • Infection
  • Endotoxin mediated (Endotoxins are bacterial toxins)
  • Sterile necrosis (pancreatitis, tumour, trauma)
  • Chemical (injections, toxins, acids, alkalines)
  • Immune mediated (SLE = Systemic lupus erythematosus, also known simply as lupus, is an autoimmune disease in which the body’s immune system mistakenly attacks healthy tissue in many parts of the body.)
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4
Q

To evaluate the sverity. locality and ethiology of inflammatory processes, labatory examinations are needed, WHICH ONES?

A

HECSPIMMM

  • Haematology (complete blood count)
  • 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
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5
Q

Why do we use Li or Ca heparin on fish, birds and reptile cells instead of Na?

A

Because cells swell when we use Na, they become hypotonic.

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6
Q

Explain the WBC counting using the Bürker-chamber:

A
  • Aspirate 0.1 ml homogenised whole blood sample.
  • Wash the sample into 0.9ml Türk-solution.
  • Incubate for 1-2min in room temp
  • Drip one drop onto Bürker-chamber
  • Count number of blood cells above 25 large squares.
  • The number must be divided by 10, that gives the number of WBCs in N (giga)
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7
Q

Explain WBC counting by hematology analyser:

A
  • Some machines make a centrifugation and analyse different cell types according to the light absorbancy of the different cell layers.
  • These are more accurate.
  • Some machines use laser technique and use reagents to measure peroxidase activity of the phagocytic cells.
  • These are very accurate and can make difference among the poorly differentiated cells too.
  • Even more accurate diagnosis can be performed by using the flow cytometric method.
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8
Q

Which species are neutrophilic?

A

Horse, cat, dog.
60% neutrophil, 40% lymphocytic

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9
Q

Which species are lymphocytic?

A

Sheep, cattle, swine, poultry

60%lymphocytic, 40%neutrophilic

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10
Q

WBCs count in mammals and birds:

A
  • 5-15 giga in mammals
  • 15-30 giga in birds
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11
Q

Explain the automatic cell counters:

A
  • It can count WBC, as they make a distribution according to the electrical impedance of a particle (cell) entering in between two electrodes.
  • Analyser first makes a dilution for WBC counting, then hemolysys RBCs.
  • These analysers can differentiate among the WNC types.
  • They distribute the cells according to the greatnes of the electrical impedance of the cell particle which is corresponding with the size of the neutrophils and macrophages.
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12
Q

Histogram of a feline blood sample: Laser cell counters

A
  • It can see the size of the cells AND their inner structure.
  • The cells flow through a tube and are iluminated by the laser light, which will be scattered by the cell.
  • The scatters are detected by two light detectors; one low angular/forward scatter detector and one high angular/side scatter detector- detects the greatness of light energy(lux).
  • The low angular = size
  • The high anuglar = inner structure
  • The numerical value of the two detectors can be extrapolated into a X and Y axis.
  • The similar cells will have almost same points, so they will form a cloud and we can count the similar cells. This is called THE GATING.
  • The cloud diagram are called scatter grams. Some reagents of the instrument can determind the biochemical properties of the cells. The cell types can easily be differentiated by this method.
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13
Q
A
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14
Q

Which staining methods can be used after preparing smears?

A
  • May- grunwald
  • Romanowsky
  • Diff-quick
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15
Q

How should smears be analyzed? (qualitative blood count)

A
  • The smears should be analyzed at the edge of the slide and in the middle.
  • First low power should be used, then 1000x magnification can be provided by using immersion lens (and 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.
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16
Q

Neutrophil granulocyte cell line:

A
  • Myeloblast
  • promyeloblast
  • myelocyte
  • metmyelocyte (jugend)
  • band forms (stand)
  • segmented forms
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17
Q

Lymphoid cell line:

A
  • Lymphoblast
  • small lymphocyte
  • middle size lymphocyte
  • reactive T lymphocyte
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18
Q

Eosinophil cell line:

A
  • Young form (band nucleated) eosinophil granulocyte
  • segmented form eosinophil granulocyte
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19
Q

Basophil cell line:

A
  • Young form (band nucleated) basophilgranulocyte
  • segmented form basophil granulocyte
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20
Q

Monocyte cell line:

A
  • Monocyte young form
  • Reactive macrophage form
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21
Q

Function of Neutrophil cells:

A
  • Phagocytosis
  • Eliminate antigens with lysosomal enzymes
  • Lifespan: hours
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22
Q

Function of Eosinophil cells

A
  • Phagocytosis
  • Inactivate histamin with lysosomal enzymes
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23
Q

Function of Basophil cells?

A
  • Stimulate the elimination of antigens with lysosomal enzymes.
  • Histamine, heparin and seotonin
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24
Q

Function of monocytes:

A
  • Phagocytosis
  • Presentation of antigens

-Life: intravascular : hours to days
Extravascular: weeks to months

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25
Q

How to calsulate absolute WBC numbers

A

-Relative% of the cell type/100 x WBC count N giga/L

26
Q

General normal values of dog,cat,horse and humans: in %

A

Neu: -

  • Metamyelocyte: 0-3
  • Segmented form: 60-77

Lymp: 12-30

Eosin: 2-10

Baso: <1

Mono: 1-5

27
Q

General normal values for dog, cat, horse, humans: Absolute

A

Neu: 3-11.8

  • Metamyelocyte: 0-0.3
  • Segmented form: 3-11.5

Lymp: 1-4.8

Eosin: 0.1 - 1.35

Baso: -

Mono: 0.1 - 0.5

28
Q

what’s it called if you have high numbers of absolute neturophil granulocytes or too low?

A

Too high - neutrophilia

Too low - neutropenia

29
Q

Name the WBC pools in the body and their characteristics:

A
  • In the bone marrow: mitotic (very young cells, proliferating), maturation- (under differentation) 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 form this pool.
  • In the circulating blood: we take our sample from here, the circulating pool.
  • In the tissues of different organs: tissue pool.
30
Q
A
31
Q

Why do physiological leukocytosis happen?

A
  • It develops due to acute or chronic stress (ephinephrine, norepinephrine, glucocorticoids).
  • The effect of catecholamines are visible within seconds (neutrophilia, lymphocytosis, as cells mobilised from the marginal pool/cells attached to the vessels, lymphatic vessels and organs.)
  • The effect of ACTH or glucocorticoids can be detectable after hours. (or in some instances minutes (neutrophilia, lymphopenia, eosinophilia).
32
Q

what about the older neutrophils in physiological leukocytosis?

A
  • The older neutrophils are mobilised from the bone marrow and marginal pools, they do not die, they stabilize the lysosomal membranes, meanwhile there is a severe lympholysis.
  • The removal of lymphocytes to the lymphatic organs, eosinophil granulocytes are not released to the circulation from the bone marrow.
33
Q

what can happen with cats in stress?

A

-can cause an extreme nautrophilia, as the marginal pool is bigger (two third of the total neutrophilic count)

34
Q

Signs of acute inflammation:

A

the very first sign of inflammation is the increase of positive acute phase proteins (APP) in the blood and the decrease of negative acute phase proteins.

35
Q

Explain neutropenia:

A
  • During the first period of inflammation - neutropenia occurs.
  • Caused by migrating factors produced by tissue cells, macrophages.
  • Neutrophils are migrating out of blood vessels to the site of inflammation, this can lead to transient decrease in the total WBC count.
  • Blood samples are not taken in this period.
36
Q

Explain Neutrophilia:

A

-During late phases of inflammation neutrophilia occurs due to the granulocyte colony stimulating factor (G-CSF), or granulocyte macrophage colony stimulating GM-CSF produced by the macrophages. These stimulate WBC production in the bone marrow, which leads to an increased total WBC count.

37
Q

what happens during a left shift?

A
  • 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 mobilised 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 the maturation pool of bone marrow.
  • In case of severly increased tissue requirement (or in neoplastic diseases i.e acute myelogenous leukemia) very young cells (myelocytes, promyelocytes) from the mitotic pool are going out.
38
Q

Explain regenerative left shift:

A
  • Increased WBC count, neutrophilia, and left shift (young neutrophils).
  • After the first neutropenic phase, bone marrow regenerates the utilised neutrophils in the circulation by the younger cell population form the storage,maturation, and/or the mitotic pool.
  • This is the most common cause of appearance of the young neutrophils.
  • Regenerative left shift is a sign of favorable prognosis.
39
Q

Explain degenerative left shift:

A
  • Low or normal WBC, and neutrophil count and left shift (younger forms). If there is a great neutrophil utilisation in the tissues (widespread or very severe inflammation, big abscess,peritonitis,pleuritis, phlegmone,puometra) and the rate of utilisation is bigger than regenerative capacity of bone marrow, increased outflow of the young neutrophils is not enough to increase WBC count.
  • Degenerative left shift is a sign of poor prognosis, it means that the disease is very serious and needs immediate treatment.
40
Q

What is the leukemoid reaction?

A
  • When there is a great stimulus for producing neutrophil granulocytes an enormous number of neutrophils can be present in peripheral blood due to increased effects of GCSF,GM-CSF.
  • Typical reaction in case of big adscesses, endometritis (pyometra) and in case of some neoplastic (but not leukemia) diseases.
  • can be confused with chronic myelogenous leukemia.
41
Q

What is toxic neutrophils?

A
  • The granulocytopoesis can be disturbed.
  • Some orange-red ganules can be seen in basophilic cytoplasm. These are Toxic neutrophils.
  • Toxic granulation.
42
Q

what is Döhle-bodies?

A
  • Due to toxic effects some angular, basophilic inclusion bodies can be seen in some neutrophil granulocytes.
  • These are the remants of the endoplasmatic reticulum. Apperance of Döhle-bodies is more common in cats.
43
Q

what is right shift?

A
  • Many old, segmentes and hypersegmented neutrophils are in the same smear.
  • If this is visable at the same time as an increased WBC count = chronic inflammatory process.
  • Also typical for the effects of glucocorticoids. This inhibits cellular proliferation and have membran stabilising effect (which makes the cells more segmented and old.)
  • They are also lympholytic and inhobit the outflow of eosinophils for the bone marrow (lymphopenia/eosinopenia).Often in poodles.
  • Right shift, leukocytosis,neutrophilia, lymphopenia and eosinopenia is called stress leukogram.
44
Q

what are typical signs of chronic inflammation in the CBC ?

A
  • 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.
45
Q

Haematology of Addison’s disease :

A
  • 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.
46
Q

What is Pelger-Huet anomaly?

A

-Normocytaemia, and left shift (metamyelocytes, band forms of neutrophils) Inheritable problem causes diagnostic problem.

47
Q

What is cyclic neutropenia?

A
  • 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.
48
Q

what is bone marrow damage?

A
  • Leukopenia and neutropenia occurs in case of bone marrow damge or decreased bone marrow function.
  • Thrombocytopenia and aplastic anemia is often accompanied.
  • When other cells supress the hemopoetic cells in bone marro, it is called: myelophtysis.
49
Q

Describe the schilling-phases:

A
  • Struggle against microbes.
    1. Premonitory phase - Leukopenia, lymphocytosis - loose neutrophils
    2. Phase of neutrophils - granulose cytosis - shift to left
    3. Phase of monocytes- monocytosis - nr of granulocyte is normalised
    4. Phase of lymphocytes - lymphocytosis, eosinophilia
50
Q

Explain the glutaric aldehyde test:

A
  • Examines the increase of fibrinogen and globulin conc in plasma. (in adult cattle)
  • Fibrinogen is an acute phase protein, increase if there is an acute inflammation.
  • Glutaric aldehyde solution causes a rapid coagulation of fibrinogen and labile globulins, so blood mixed with this solution coagulants in seconds if there is an acute inflam. (even blood with anticoagulants)
  • We use heparinised blood and mix the sample with the same amount of 1,25% glutaric aldehyde solution.
  • Check for coagulation every 30 seconds.
  • Test is done to diagnose reticuloperitonitis, severe mastitis or endometeritis of cattle.
51
Q

Erythrocyte sedimentation rate: ESR

A
  • It is the increased sedimentation of RBC due to inflammatory processes. as the acute phase proteins and ther glubulins tend to attach onto the surface of RBCs.
  • Normally RBCs are covered by albumin (-), but bc of inflammation or neoplastic disease, globulins (no charge) cover the surface = form big aggregates and sediment quick.
  • Westergreen tubes are used to perform a test on this. They contain Na-citrate. check after an hour.
52
Q

What is special about the horse ? (ESR)

A

Sedimentation is very fast, so ESR are checked after 20 min, The speed decreases after a while. Inversely proportional to the Ht(PCV), proportional with the serum viscosity, total protein and fibrinogen conc.

53
Q

How can the sedimentation be biphasic?

A
  • When young RBCs sediment later than the older.
  • In a commen case of immunhemolytic anemia, the older and even aggregated RBCs sediment much earlier then the younger.
54
Q

Physiologically ESR :

A

0.5-3 cm/hour , inversely proportional with Ht (PCV).

55
Q

what is CRP?

A
  • C-Reactive Protein (acute phase protein, it can bind non-specifically to a protein of Pneumococcus bacteria, called protein-C and cause presipitation.
  • Acute inflam = increased acute phase proteins (CRP, haptoglobin, serum amyloid A)
  • Some negative acute phase proteins decrease (lactoferrin,transferrin)
  • CRP is produced in the liver and in the beginning of the inflam process cytokins (interleukins) stimulate this production.
56
Q

How do we measure CRP?

A

we use serum samples, the determination is based upon immunological (ELISA or immunturbidimetric) method.

-General physiological value is 8 mg/L

57
Q

why can CRP be so important?

A

The values might increase before the clinical signs appear, so in immunosuppressed animals it is very important for early detection of inflammatory processes.

  • Especially during chemoterapy or high dose glucocorticoid therapy, in these cases the well known clinical and laboratory signs of inflammation can be vague and absent.
58
Q

Describe the acute leukemic diseases:

A

Cell type: blast cells, they have coarse chromatin pattern and nucleoli.

Origin of cell: evaluated by immunocytochemical 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.

  • Acute lymphoblastic leukemia.
  • Acute myeloblastic, promyelocytic leukemia
  • Acute erythroblastic leukemia
  • Lymphoma of stage V (metastatisis to bone marrow)
59
Q

Describe leukemic diseases:

A

Cell type: mature differentiated or well differentiated cells, that appear in enormous number in peripheric blood.

  • This presentation does not cause diagnostic difficulties in case of lymphoid, thrombocytes or erythroid forms, but it can be mixed with leukemod reaction.
  • In order to diagnose these diseases, bone marrow evaluation is essentially required.
  • Chronic small lymphocytic leukemia
  • Chronic myeloid
  • Polycythaemia absolute vera
  • Essential thromobocytosis
60
Q

Describe lymphomas:

A
  • They are hemopoetic tumours too.
  • In this disease poorly differentiated lymphoid cells are overproduced in lymphatic organs (lymph nodes, spleen) and sometimes in other tissues (liver, skin, intestines)

-In some cases tumours lymphoid cells are overproduced in bone marrow too. The origin of this is proved in case of cats and cattle only. Caused by viral infections.

Lab diagnostics (6 decks)

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