Lab 4

Question 1

What type of sample are used for WBC count?

Answer 1

Anticoagulated blood, Na2EDTA, K2EDTA or Na-citrate. In case of bird, reptile fish: Li-, or Ca heparin is used.

Question 2

WBC counting by using Bürker-chamber:

Answer 2

• Use Hagedorn or other pipettes to aspirate 0.1 ml homogenised whole blood.
• wash the sample into 0.9 ml Türk-solution (acetic acid-hemolyser, gentiana-violet blue- stains nuclei) 10x dilution
• Incubate the solution for 1-2 min in room temp.
• drip one drop into the Bürker chamber and count the number of WBCs (total nucleated cell count) above 25 large swaures.
• the number must be divided by 10, that gives the number of WBC´s in Nô x 10^9/l

Question 3

WBC counting by hematology analyser:

Answer 3

• Some machines make a centrifugation and analyse the different cell type according to the ligh absorbancy of the different cell layers: these are more accurate
• Some machines use laser tecnnique and use som reagents, i.e to measure peroxidase activity of the phagocytiv cells. These are very accurate and can make difference among the poorly differentiated cells.
• Even more accurate: flow cytometric method.

Question 4

Impedance method -> haematology analyser:

Answer 4

• Automatic cell counter can count WBC as they make a distribution according to the electrical impedansce of a particle (cell) entering in between two electrodes.
• analyser first makes a dilution for WBC counting, then hemolyses RBC´s.
• these analysers can differentiate among the WBC types. they distribute the cells according to the greatness of the electrical impedance of the cell particle which is corresponding with the size of the cell types.
• these use reagents which decrease the size of lymphocytes and increase the size of the neutrophils and macrophages.

Question 5

Laser cell counters technique:

Answer 5

• they are contemporary cell analysers which can differentiate cell types not only according to their size but also according to their inner structure, which can be detected by the laser beam teqhnique.
• the cells flow thorugh a tube and they are illuminated by a laser light. The light beams are scattered (fractured) by the cells.
• The millions of scatters are detected by two light detectors. The low angular or forward scatter detector and a high angular, side scatter detector is detecting the greatness of the light energy (lux).
• the size of the cells is correlating with the low angular scatters and the inner structure complexity is correlating with the side scatters. The light energy is a numerical value which is detected by the detectors.
• The two numerical values gained by the two detectors can be extrapolated onto the x and y axis of a graph.

Question 6

Laser cell counters graph:

Answer 6

• The two numerical values gained by the two detectors can be extrapolated onto the x and y axis of a graph.
• The two numerical values on x and y axis will determinate a point. The similar cells have similar numerical values on the detectors, so the points gained by the similar cells will be located closer to each other on the graph.
• The closer point will form a “cloud”. The cells (points) in the cloud are the population of similar cells. If we frame the “cloud” and count the points in the “cloud” we can give he cell count of the similar cell types. This process is called the gating.
• The coud diagrams are called the “scatter grams”. some reagnets of the instrument can determine the biochemical property (i.e myeloperoxidase activity) of the cells. The cell types can be differentiated very accurately by this method.

Question 7

Qualitative blood count:

Answer 7

• After preparing smears several staining methods can be used. ie: May-Grunwald, Romanowsky, Giemsa, Diff-quick etc.
• the smears should be analysed at the edge of the slide and in the middle too.
• First low power should be used, then high 1000x magnification by using immersion lens (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.

Question 8

Neutrophil granulocyte cell line:

Answer 8

• myeloblast, promyelocyte, myelocyte, metamyelocyte (jugend), band forms (stab), segmented forms.

Question 9

Lymphoid cell line:

Answer 9

lymphoblast, small lymphocyte, middle sized lymphocyte, reactive T-lymphocyte.

Question 10

Eosinophil cell line:

Answer 10

young form (band nucleated) eosinophil granulocyte, segmented form eosinophil granulocyte

Question 11

basophil cell line:

Answer 11

young form (band nucleated) basophil granulocyte, segmented form basophil granulocyte.

Question 12

Monocyte cell line:

Answer 12

Monocytic (young) form, reactive macrophage form

Question 13

White blood cell (WBC) pools in the body:

Answer 13

• in the bone marrow: mitotic (very young cells, proliferating), maturation (under differentiation) and storage- pools (mature, differentaited, 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 tissue of different organs: tissue pool

Question 14

physiological leukocytosis:

Answer 14

• 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 mobilised 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 minutes) (neutrophilia, lymphphenia, eosinopenia).
• The older neutrophils are mobilised from the bone marrow and marginal pools, they do not die to 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.

Question 15

In cats, stress can cause?

Answer 15

an extreme neutrophilia, as the marginal pool is bigger (two third of the total neutrophilic count) in cats.

Question 16

Acute inflammation:

Answer 16

• acute inflammation: neutropenia, neutrophilia, left shift, regenerative left shift, degenerative left shift, leukemoid reaction, toxic neutrohils, dohle-bodies.
• the very first lab. sign of inflammation is the increase of positive acute phase proteins (APP) in the blood and the decrease of negative acute phase proteins

Question 17

Neutropenia:

Answer 17

• during the first period of the inflammatory process, or widespread severe inflammation neutropenia occurs.
• it is caused by the migrating factors (i.e leukotriens, interleukins etc) produced by the 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 peaople are convinced that the inflammation always cause high WBC count.

Question 18

Neutrophilia:

Answer 18

• during later phases of 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.

Question 19

left shift:

Answer 19

• more young WBc´s 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 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.
• there are two different types of left shift: Regenerative left shift and Degenerative left shift.

Question 20

Regenerative left shift:

Answer 20

• increased WBC count, neutrophilia, and left shift (younger neutrophils).
• after the first neutropenic phase, bone marrow regenerates the utilised 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 the young neutrophils.
• regenerative left shift is a sign of favourable prognosis.

Question 21

Degenerative left shift:

Answer 21

• 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 abscesses, periotonitis, pleuritis, phlegmone, pyometra) and 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.

Question 22

Leukemoid reaction:

Answer 22

• when there is a great stimulus for producing neutrophil granulocytes 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, pyometra and in case of some neoplastic (but not leukaemia) diseases.
• during blood smear analysis, leukemoid reaction can be easily confused with chronic myelogenous leukemia.

Question 23

Toxic neutrophils:

Answer 23

• in very severe inflammatory processes the granulocytopoesis can be distributed.
• when the granule production 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.

Question 24

Döhle-bodies:

Answer 24

• due to toxic effects som angular, basophilic inclusion bodies can be seen in som neutrophil graulocytes.
• these are the remnants of the endoplasmatic reticulum.
• appearance is mostly common in cats.

Question 25

Chronic inflammation:

Answer 25

Right shift:

• means that many segmented and hypersegmented, old neutrophils (3-4 segments are on one nucleus) are seen in the smear.
• the appearance of these older forms while there is an increased WBC count is typical for chronic inflammatory process.
• this phenomenon is also typical for the effects of glucocorticoids.
• glucocorticoids inhibit cellular proliferation (disappearance of young cells) and have membrane stabilising effect (which let the neutrophils to grow old and become hypersegmented).
• glucocorticoids are lympholytic and inhibit the outflow of eosinophils from the bone marrow (can cause lymphopenia and eosinopenia).
• common fincing in macrocytosis of poodles.
• Right shift, leukocytosis, neutrophilia, lymphopenia and eosinopenia together are clled Stress Leukogram.
• typical signs of chronic inflammation in the CBS is similar to glucocorticoid effect (leukocytosis, neutrophilia, right shift), the difference is that in this case leukocytosis is sometimes associated with lymphocytosis, monocytosis and/or eosinophilia.

Question 26

Haematology of Addison´s disease (hypoadrenocorticism):

Answer 26

• 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 polyuria caused increased PCV), increase in young neuotrophils, left shift (no inhibition cell proliferation), lymphocytosis and eosinophilia.

Question 27

Pelger-Huet anomaly:

Answer 27

Normocytaemia, and left shift (metamyelocytes, band forms of neutrophils)

• inheritable problem causes diagnostic problem

Question 28

cyclic neutropenia

Answer 28

• inheritable disease of Grey Collies.
• due to cyclic bone marrow activity, neutropenia occurs in weekly, monthly inervals.
• during these periods the animals are sensitive to infections

Question 29

Bone marrow damage:

Answer 29

• leukopenia, and neutropenia occurs in case of bone marrow damage or decreased bone marrow function.
• thormbocytopenia and aplastic anemia is often accompanied.
• when other cells suppress the hemopoetic cells in bone marrow, it is called myelophtysis.

Question 30

Glutaric aldehyde test:

Answer 30

This method is used to examine the increase of fibrinogen and globulin concentration in plasma.

• as fibrinogen is an acute phase protein it can show a marked increase during acute inflammatory processes.
• this method is used in clinical practice to indicate inflammatory processes in adult cattle (more than one year old).

Question 31

What does Glutaric aldehyde solution cause?

Answer 31

It causes a rapid coagulation of fibrinogen and labile globulins, so blood mixed with this solution can show coagulation within seconds in case of acute inflammatory processes (high fibrinogen and globulin level in the blood), even if the blood sample contains heparin or EDTA.

Question 32

Glutaric aldehyde test method:

Answer 32

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 sek by turning them upside down.

Question 33

Aid for the evaluation of glutaric aldehyde test in cattle:

• coagulation time
• presence of the inflammatory proteins in the sample
• character of the pathological process

Answer 33

Question 34

What do we diagnose with the Glutaric Aldehyde test?

Answer 34

Reticuoperitonitis, severe mastitis or endometritis of cattle.

These animals can be extremely indolent, sometimes they show just vagye clinical signs, or no signs at all, even if they are suffering from very severe diseases.

If the symptoms and the restults are not correlated to each other, it is necessary to repeat the test 24-48 hr later.

Question 35

What is Erythrocyte sedimentation rate (ESR)?

Answer 35

ESR is the increased sedimentation of RBC´s due to inflammatory proceses, as the acute phase proteins and other globulins tend to attach onto the surface of RBC´s.

• Normally RBC´s have negatively charged albumin molecules on their surface, this prevents RBC aggregation.
• In case of high globulin level in the blood (severe inflammation or some type of neoplastic disease), the surface of RBCs is covered by globulins, instead of albumins. Globulins have no negative charge, therefore RBCs are able to attatch to each other, form big aggregates and sediment quickly.

Question 36

How is the Erythorcyte sedimentation rate (ESR) test method performed?

Answer 36

• Specific tubes (made of glass called Westergreen) contains Na-citrate and there is a mm scale on it.
• The tube has a hole on the bottom and on the top, so when it is driven thorugh the rubber plug of another tube containg the blood sample, blood will flow up in the tube with the scale until zero point.
• sedimentation rate of RBC´s should be checked in the tube after 1hr.

Question 37

Physiological ESR is?

Answer 37

0,5-3 cm/hour.

• ESR is inversely proportional with the Ht (PCV).

Question 38

What happens in case of immunhemolytic anemia?

Answer 38

• the seidmentation can be biphasic, when the young RBCs sediment later than the older forms.
• This is common im case of immunhemolytic anemia (regneratie anemia, many young RBCs are produced). The older and even aggreagated RBcs sediment much earlier then the younger forms.

Question 39

What i special about ESR in horses?

Answer 39

• Sedimentation is very fast in horses, so ESR must be evaluated after 20 min.
• it is found that the speed of the sedimentation decreases (!) due to inflammatory processes contrary to other species.
• ESR is inversely proportional with the Ht (PCV) and proportional with the serum viscosity, total protein and fibrinogen concentration.

Question 40

ESR in dogs and horses in case of different Ht (PCV):

Answer 40

Question 41

What is the origin of the name of C-reactive protein (CRP)?

Answer 41

origin of the name of this acute phase protein: it can bind non-specifically to a protein of Pneumococcus bacteria, called protein-C and cause precipitation.

Question 42

What happens in case of acute inflammatory process?

Answer 42

• the concentration of acute phase proteins becomes increased in the blood plasma. Such acute phase proteins is CRP, haptoglobin, serum amyloid A (SAA) etc.
• some negative acute phase proteins (i.e transferrin, lactoferrin) show a decreased concentration due to acute inflammatory processes.

Question 43

Where is CRP produced?

Answer 43

• In the liver and in the beginning of the inflammatory process cytokins (interleukins) stimulate its production

Question 44

What are used for measurment of CPR?

Answer 44

• we use serum samples
• the determination is based upon immunological (ELISA or immunturbidimetric) method. Today species (dog, horse) specific ELISA methods are available.

Question 45

General physiological value of CRP?

Answer 45

8 mg/l

Question 46

typical for Acute leukemic diseases:

Answer 46

• blast cells that have coarse chromatin pattern, and have nucleoli.
• the origin of the cell can be evaluated by immune(cytochemical), and bone marrow analysis.
• in some cases neoplastic cells do not appear in peripheric blood, but anemia, a leukopenia and thrombocytopenia.

Question 47

Types of Acute leukemic diseases:

Answer 47

• acute lymphoblastic leukemia
• acute myeloblastic, promyelocytic leukemia
• acute erythroblastic leukemia
• lymphoma of Stage V (metastatisis to bone marrow)

Question 48

Typical for Chronic leukemic diseases:

Answer 48

• mature differentiated or well differentiated cells, that appear in enormous number in the peripheric blood.
• this presentation does not cause diagnostif difficlties in case of lymphoid, thrombocytes, or erythroid forms, but it can be mixed with leukemoid reaction.
• bone marro evaluation is essentially required for diagnosis.

Question 49

Types of Chronic leukemic diseases:

Answer 49

• chronic small lymphocytic leukemia
• chronic myeloid (neutrophil granulocytic, eosinophil granulocytic, basophil granulocytic, monocytic leukemia)
• polycythaemia absoluta vera (overproduction of mature erythorcytes)
• essential thrombocytosis (overproduction of mature thrombocytes)

Question 50

Types of hemopoetic tumours?

Answer 50

leukemic and lymphomas

Question 51

typical for lymphomas? (a hemopoetic tumour)

Answer 51

• 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.
• in these special viral infection is causing the disease: Bovine Leukosis Virus (BVL) and Feline Leukemia Virus (FeLV)

Question 52

Absolute WBC numbers calculation

Answer 52

Question 53

General normal values for WBC numbers in dog, cat, horse, humans

Answer 53