Lab 4 Flashcards
What type of sample are used for WBC count?
Anticoagulated blood, Na2EDTA, K2EDTA or Na-citrate. In case of bird, reptile fish: Li-, or Ca heparin is used.
WBC counting by using Bürker-chamber:
- 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
WBC counting by hematology analyser:
- 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.
Impedance method -> haematology analyser:
- 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.
Laser cell counters technique:
- 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.
Laser cell counters graph:
- 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.
Qualitative blood count:
- 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.
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
White blood cell (WBC) pools in the body:
- 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
physiological leukocytosis:
- 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.
In cats, stress can cause?
an extreme neutrophilia, as the marginal pool is bigger (two third of the total neutrophilic count) in cats.
Acute inflammation:
- 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
Neutropenia:
- 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.
Neutrophilia:
- 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.
left shift:
- 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.
Regenerative left shift:
- 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.
Degenerative left shift:
- 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.