Differential Blood Count

Transcription

1 Differential Blood Count TEAS Related topics Erythrocytes, leucocytes, Pappenheim s stain. Principle In manual blood counts, a droplet of blood is spread on a slide in such a manner that the cells are located separately, side by side in one layer. After application of the Pappenheim's stain, the cells can be classified individually by size, nucleus-plasma relation, shape and structure of nucleus, colour and granulation of cytoplasm. The erythrocytes will be assessed as well. Equipment Student s set Graduated pipette 10 ml, scale 1 division 0.1 ml Pipette filler Test tube rack, PP, 12 places Volumetric flask, 10 ml Volumetric cylinder, 250 ml Staining bench with trough Wash bottle, 5 ml Disposable gloves, M, latex, 1 pcs Object slides Immersion oil, 50 ml Microscope Chemicals Azur eosin methylene blue solution, ml 1 Methanol (fixative solution) May-Grunwald solution Buffer tablets ph 7.2 for microscopy Fig. 1: P P59215 PHYWE Systeme GmbH & Co. KG All rights reserved 1

2 TEAS Differential Blood Count Task 1. Examine a stained blood sample by microscope and differentiate the leucocytes. Procedure Material for the analysis Venous or capillary blood, anticoagulated with EDTA refer to Pre-Analytics. Preparation of a blood smear A droplet of blood is applied to the surface of a slide with the tip of the pipette (Fig. 2) and immediately spread out with a second slide. To do so, the second slide is brought up to the blood droplet from the front, causing it to spread along the rear edge (Fig. 3). Fig. 2 Fig. 3 Smear too thin and too short Correct size and thickness Fig. 4 Fig. 5 Swiftly spread it between specimen slide and smearing slide, holding the latter at an angle of 45. Avoid deformation of blood cells by squeezing. Approx. 3/4 of the slide should be covered with the smear, leaving a small rim along the edges. The cells in the smear should be located separately, side by side, in one mono-cellular layer. They should not touch each other. Leave the smear to air-dry thoroughly. 2 PHYWE Systeme GmbH & Co. KG All rights reserved P59215

3 Differential Blood Count TEAS Note As markings made with ballpoint pen or felt pen will be removed by the fixative and staining solutions, labelling/marking of the smears has to be performed by other means (pencil). Otherwise, mix-ups may easily occur in routine operations. It is recommendable to use object slides with frosted edges (not included in the package) in place of standard object slides. Staining of the blood smear Preparation of the Giemsa-working solution: - Dissolve 1 buffer tablet ph 7.2 in 1l of freshly distilled water, stir if required. The solution is stable for at least 4 weeks in a carefully sealed glass bottle. - Mix 10 ml of Giemsa-stock solution ml buffer solution ph 7.2, leave for 10 min. and filtrate if required. Put specimens into the glass trough on the staining stand and add layers in the sequence shown below for the corresponding times: When the periods shown for each added layer have expired, decant the respective solutions. Methanol 10 min May-Grunwald solution 5 min Giemsa-working solution 8 min Rinse with buffer solution ph 7 1 min Rinse with aqua dest. 1 min Upon conclusion of staining, clean slides from the backside and leave the specimens to air-dry. Note The following errors may occur during preparation of the stained blood smear: - When air-drying is too slow or insufficient, crenation of cells will occur. - When solutions are supersaturated and precipitated dye is rinsed off insufficiently, precipitates will form. - In case of acid vapours or acid distilled water, the smear will be too red (use buffer). - When traces of alkalis are present, staining lasted too long or the smear is too thick, the smear will be too blue. Fig. 6: Microscope: 1. Oculars 2. Lens 3. Focussing gear macro screw 4. Focussing gear fine drive 5. Specimen manipulator (moves the specimen table in 2 planes) Microscopic examination Place smear on the cross table of the microscope. Initially slide in 10x lens. At this magnification, it is usually easy to adjust the smear level with the macro screw and assess the nature of the specimen. P59215 PHYWE Systeme GmbH & Co. KG All rights reserved 3

4 TEAS Differential Blood Count Once the specimen level has been found, apply a droplet of immersion oil to the thin part of the smear (run-out), slide the 1x lens into the beam path, immerse it into the droplet of oil and cautiously turn the fine drive to focus. Caution: Do not contaminate dry 40x and 10x lenses with oil! Name. d.o.b. Fig. 7: Examine the specimen following meander lines When the blood is spread on the slide, the leucocytes will not distribute evenly; at the border of the smear, a higher rate of large leucocytes will be found (granulocytes, monocytes), in the centre, smaller cells will accumulate (lymphocytes). For this reason, it is necessary to examine the specimen in meandering lines (refer to Fig. 7). Carefully clean the oil immersion lens after completion of the differentiation; the same applies to all surfaces which had contact with the oil. Basics Whilst erythrocytes, thrombocytes and leucocytes are mostly counted by automated procedures, microscopic assessments of stained blood smears are indispensable as an additional means of obtaining further important information in haematological diagnostics. Although automated differentiation methods are available, the respective blood smears always have to be stained by hand and follow-up differentiation by microscope is required as well. Leucocyte differentiation is part of full blood counts. The various types of leucocytes differ by size, nucleus-plasma relation, shape and structure of nucleus, colour and granulation of cytoplasm. After application of the Pappenheim's stain, leucocytes can be divided into subpopulations on the basis of morphology and stainability: neutrophil, eosinophil and basophil granulocytes, lymphocytes, monocytes and all precursors. Haematological blood smears are routinely stained by panoptical staining according to Pappenheim (combined May-Grunwald-Giemsa stain). The smears are treated with dye solutions containing acid dyes (eosin) and basic dyes (azure II, methylene blue). Acid cell components like the DNA of the nucleus and the RNA of the nucleoli and the cytoplasm are basophil and take on a blue stain with basic dyes. Basic cell components like proteins and haemoglobin are acidophil and take on a red stain with acid dyes. Evaluation Task 1: Differentiation of leucocytes Differentiation is performed on the basis of: - Cell size Comparison of erythrocytes and granulocytes - Nucleus Shape? Micronuclei? Nucleus-plasma relation? - Cytoplasm Colour blue, blue-grey, pink? Intensity of colour? - Granules Neutro-, eosino-, baso- and azurophil? - Inclusions JOLLY-, HEINZ-granules in erythrocytes / AUER-rods in leucocytes? Leucocytes physiologically occurring in peripheral blood are classified into monocytes, lymphocytes and granulocytes, with the latter being divided further into neutrophil, eosinophil and basophile granulocytes. Haematology atlases are helpful for differentiation. At this point, only the most prominent characteristics will be listed. 4 PHYWE Systeme GmbH & Co. KG All rights reserved P59215

5 Differential Blood Count TEAS Lymphocytes, at a cell diameter of 12 µm, are the smallest cells of the white blood count. The have a round, rarely dented, dark, dense nucleus of an intensive blue colour with a narrow, often hardly visible, light blue rim of cytoplasm ( bare nuclei ). Plasma cells are normally not present in peripheral blood, or only in isolated cases. Their size is µm and their cytoplasm has a deep blue colour, brightening towards the perinucleus. The nucleus is round and usually located eccentrically. The chromatin structure of the nucleus is coarse ( spokes ). Monocytes are the largest cells in peripheral blood (12 20 µm) and are extraordinarily varied in shape and form. The cytoplasm has irregular borders and a grey-blue colour, the nucleus is segmented, dented or rod-shaped. Neutrophil granulocytes with rod-shaped nuclei are of medium size and their cytoplasm is pink. Their nucleus is rod-shaped and red-violet in colour. They are distinguished from the neutrophil granulocytes with segmented nuclei by the fact that the bridge between two segments is narrower than two thirds of the thickness of the neighbouring segments Neutrophil granulocytes with segmented nuclei are very similar in shape to the rod-shaped ones (see above). The, as a rule, 3-5 nucleus segments are characterised by a coarse frame of clotted chromatin and are connected by short bridges or chromatin fibres. Eosinophil granulocytes are of medium size as well and contain oxyphil, pink cytoplasm. They have numerous, large, coarse eosinophil (yellow-orange) granules. These do not cover the rod-shaped or segmented nucleus. Basophil granulocytes are, as a rule, smaller than eosinophil ones. Their pink cytoplasm is in most cases largely covered by the coarse, basophile (blue-violet) granules. Due to this, the shape of the nucleus (segmented, frequently in the shape of a clover-leaf) is often not discernible. Rod-shaped granulocyte Neutrophil granulocyte with segmented nucleus Monocyte Lymphocyte Fig. 8: Examples for cells which may be found in the peripheral blood of a healthy subject. P59215 PHYWE Systeme GmbH & Co. KG All rights reserved 5

6 TEAS Differential Blood Count Eosinophil granulocytes with segmented nuclei Neutrophil granulocyte with segmented nucleus Basophil granulocyte with segmented nucleus The table below additionally includes immature precursors of granulopoesis, which occur in pathologic blood smears only. Among these, promyelocytes, myelocytes, metamyelocytes (juvenile forms) and blast cells are counted. Apart from differentiating leucocytes, the red blood cell count as well as thrombocytes have to be assessed for every smear. When precursors of erythrocytes with nuclei occur, their count per 1 white blood cells has to be indicated. Precarious findings in erythrocyte morphology point to underlying disorders in erythropoesis. However, it is only in extremely rare cases that they prove certain diseases. In case of precarious findings in erythrocyte morphology, it is thus always recommendable to perform further diagnostics. At this point, only the most prominent precarious findings in erythrocyte morphology will be listed. Normal red blood cells (normocytes) have a size of approx µm and a normal haemoglobin content. - In case of hypoferric anaemia, smaller or fewer microcytes or anulocytes stained by haemoglobin to a smaller extent, or not at all, may occur. - Enlarged erythrocytes (>9 µm), when combined with a partially reduced mean corpuscular haemoglobin concentration (MCHC) suggest alcohol abuse or liver damage. - Target cells are erythrocytes which are of a normal size but abnormally thin, with a light sector between the border, which is rich in haemoglobin, and the centre. These cells occur e.g. in thalassaemias, hypochromic anaemias and after splenectomies. In isolated cases, they are also 6 PHYWE Systeme GmbH & Co. KG All rights reserved P59215

7 Differential Blood Count TEAS found in healthy subjects. - In pronounced anaemias, a different ratio of sizes is observed for the red blood cells (anicocytosis). Sometimes, pear- or club-shaped erythrocytes are found as well (Poikilocytosis). - Polychromasy, i.e. plasma stained with basic dyes is an indicator for lead poisoning or accelerated wash-out of erythrocytes from the bone marrow. Sometimes, only small granules in the plasma show basophil staining (punctate basophilia). - In severe anaemias or disorders of the blood-bone marrow barrier, erythrocyte precursors may be detected. Description Type of deviation Occurrence Anisocytosis Different ratio of sizes of red blood cells Practically all anaemias Poikilocytosis Pear- or club-shaped erythrocytes Pronounced anaemias Microcytes Small erythrocytes, stained by haemoglobin to a lesser degree or not at all Hypoferric anaemias Anulocytes Ring forms, abnormally thin cells Severe hypoferric anaemias Target cells Mexican hat shapes, centre rich in Hb Inter alia Thalassaemias, severe iron deficiency, obstructive icterus Sickle-cells Formation of sickles, primarily with O 2 - deficiency Sickle cell anaemia Spherocytes Spherical shapes, central brightening missing Spherocytic anaemia Polychromasy Plasma stained with basic dyes Suggests lead poisoning or accelerated wash-out of erythrocytes from the bone marrow A differentiation scheme (refer to Fig. 9 and Lab- Report) serves two purposes: it facilitates counting of 1 leucocytes and additionally, it provides an overview over the distribution of cells in the smear. When the distribution is not more or less even, another 1 leucocytes (if possible from a second specimen) have to be evaluated. In each vertical column, 10 cells are noted. When 1 leucocytes have been differentiated, cells of the same type are summed up. If the absolute number of cells (total leucocytes) is not known, only the relative distribution of cells in % can be deducted from the differentiation scheme. When the absolute number of leucocytes is known however, the absolute number of cells of the sub groups per µl can be calculated. Fig. 9: Differentiation scheme P59215 PHYWE Systeme GmbH & Co. KG All rights reserved 7

8 TEAS Differential Blood Count Reference values: % Neutrophils Eosinophils 1-6 Basophils 0-1 Monocytes 2-8 Lymphocytes Please note: The indicated normal values refer to the population in Western Europe! Note Due to the fact that the cells have to be located side by side, without touching each other, for an accurate assessment of the erythrocyte morphology, microscopic differentiation is only possible in the thin part of the smear (plume or run-out). The typical, sickle-shaped blood cells occurring in sickle-cell anaemias are not discernible from normal blood cells in differential blood counts. Only when the oxygen content in the sample decreases, deformation of these cells will commence. Regarding the issue of malaria diagnosis from the differential blood count, please refer to instruction P Questions - Which stain is commonly used for differential blood counts? Pappenheim's stain - Which magnification is used for examining differential blood counts under the microscope? 1 x oil immersion - Which purpose serves the May-Grunwald solution in the Pappenheim s stain? It is used for fixing and pre-staining. The actual staining is achieved with the Giemsa-solution. - Which influence has the angle between specimen slide and smearing slide on the smear? The size of the angle between specimen slide and smearing slide determines the thickness of the smear. The smaller the angle the thinner the smear. 8 PHYWE Systeme GmbH & Co. KG All rights reserved P59215

9 Differential Blood Count TEAS Lab Report Enter the result of your differentiation into the following scheme: Cell type Σ % Norma l Blast cells Promyelocytes Myelocytes Juvenile cells Rod nucleus Neutrophil with segmented nuclei Eosinophils Basophils Monocytes Lymphocytes Lymphatic irritation cells Remarks Special observations White blood cell count Assessment Red blood cell count (Toxic granulations, Gumprecht s shadows etc. which may occur) (Erythrocyte morphology, erythroblasts, etc.) up to up to 4 up to 1 up to In each vertical column, 10 cells are noted. When 1 leucocytes have been differentiated, cells of the same type are summed up. When the absolute number of cells (total leucocytes) is not known, only the relative distribution of cells in % can be deducted from the differentiation scheme. Questions - Which stain is commonly used for differential blood counts? - Which magnification is used for examining differential blood counts under the microscope? P59215 PHYWE Systeme GmbH & Co. KG All rights reserved 9

10 TEAS Differential Blood Count - Which purpose serves the May-Grunwald solution in the Pappenheim s stain? - Which influence has the angle between specimen slide and smearing slide on the smear? 10 PHYWE Systeme GmbH & Co. KG All rights reserved P59215