HEMATOLOGY WBC Anaemia classification

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1 HEMATOLOGY WBC Anaemia classification Tropical Laboratory Medecine Unit February 2009 P. Gillet

2 WBC Leukocyte number concentration (WBC count) Man and women (Asia) : leukocytes/µl Man and women (Africa) : leucocytes/µl RBC count : /µl Platelet count : /µl Destruction of the RBC Dilution and counting WBC in a known volume

3 WBC Leukocyte number concentration (WBC count) Destruction of the RBC (hemolysis) TÜRCK SOLUTION dilution 1/20 (v/v) (20 µl blood µl Türck) Dilution and counting WBC in a known volume COUNTING CHAMBER

4 WBC Neubauer counting chamber

5 WBC Neubauer (double improved) counting chamber: Area : 3 mm x 3 mm = 9 mm². Depth : 0,1 mm. 1 4 Field of vue 10x 2 3

6 WBC Neubauer (double improved) counting chamber: Area : 3 mm x 3 mm = 9 mm². Depth : 0,1 mm. Field of vue 40x

7 WBC Leukocyte number concentration (WBC count) Dilution factor : 20 µl of blood µl Turck solution, gives a 20 x blood dilution Volume factor : 4 large squares counted, or 4 x 0.1 mm³. [10/4 = 2,5 x]. Total factor : 50 x Number of leukocytes par mm3 (or µl) of blood = Number of leukocytes counted x 50

8 WBC Count Leukocyte type number fraction (% of the different types of WBC) preparation of thin blood film staining of thin blood films

9 WBC Count Preparation of thin blood film Let dry (fix with methanol) Stain with (May-Grünwald-Giemsa) or with Giemsa

11 WBC Count Film too thick Film too thin

13 Giemsa Dry thin blood film. Fixation with methanol. Cover the complete preparation with a 3.5 % diluted Giemsa buffered solution [5 drops of concentrated Giemsa + 4 ml of buffered water per slide]. Let stain for 20 to 30 minutes. (coloration) Rinse by gently dripping tap water on the preparation. Let dry and examine under the microscope with an oilimmersion objective 100x

14 May-Grünwald-Giemsa Dry thin blood film. Cover completely with May-Grünwald solution. Let the solution act for 3 minutes. (fixation) Without removing the May-Grünwald solution, add (drop by drop) 4 ml of buffered water. Let the preparation stand for 1 minute (coloration 1). Remove the staining solution. Do not rinse the slide Without rinsing the slide, cover the complete preparation with a 3.5 % diluted Giemsa buffered solution [5 drops of concentrated Giemsa + 4 ml of buffered water per slide]. Let stain for 20 to 30 minutes (coloration 2). Rinse by gently dripping tap water on the preparation. Let dry and examine under the microscope with an oil-immersion objective 100x

15 Differential WBC Count CELL TYPE SIZE NUCLEUS CYTOPLASM GRANULOCYTES µm FORM COLOUR CHROMATIN STRUCTURE QUANTITY COLOUR GRANULES LEUKOCYTES : POLYMORPHONUCLEAR (GRANULOCYTES) IMMATURE NON SEGMENTED NEUTROPHILS Band forms or S shaped Horseshoe, Central curvature is maximum a third part of the width of the lobes Clear blue purple Strands of fine chromatin abundant +++ Dusty rose (=very small granules) Small granules, light purple or violet Not always present SEGMENTED NEUTROPHILS to 5 lobes Deep blue purple Rather thick and coarse +++ Rose Small granules, Pink or pink mauve EOSINOPHILS Usually a bi-lobed nucleus Blue purple Rather thick and coarse +++ Rose Many large, uniform granules, red orange BASOPHILS Hardly visible lobes, not well separated (polymorph) Blue purple Rather thick and coarse, covered by granules +++ Light rose Very large, well separated, variable granules Deep purple Small in number Non segmented Neutrophil Segmented Neutrophil Eosinophil Basophil

16 Differential WBC Count CELL TYPE SIZE NUCLEUS CYTOPLASM AGRANULOCYTES µm FORM COLOUR CHROMATIN STRUCTURE QUANTITY COLOUR GRANULES LEUKOCYTES : MONOMORPHONUCLEAR (AGRANULOCYTES) SMALL LYMPHOCYTES 7-10 Round or oval Or slightly indented Deep purple Big clumps of intensely stained chromatin (-) or + Sky blue (Often absent) LARGE LYMPHOCYTES Round or oval Or slightly indented Red, purple Clumps of deep stained chromatin and other clumps which are less intensely stained ++ Sky blue Absent or a few granules azurophils (rose violet) MONOCYTES Round, oval, indented or bean form Blue to slightly violet Fine, spongy like +++ Vacuoles often demonstrable. Grey or blue grey Very fine granules (dusty like), azurophils (rose violet) ERYTHROCYTES 6,7 7,7 Biconcave discus shape Rose None TROMBOCYTES 1,5 2 (5) Slightly blue Reddish Lymphocyte Monocyte

18 Hemato 2

19 Hemato 3

20 Hemato 4

21 Hemato 5

22 2. Anaemia classification Haemoglobin concentration Erythrocyte volume fraction (PCV) RBC count indices

23 Anaemia classification (1) RED CELL INDICES 1 : Haemoglobin and PCV : MCHC Mean Corpuscular Haemoglobin Concentration MCHC (g/100 ml) = Haemoglobin concentration (g/100 ml) / PCV (%) x 100 MCHC < 30 g% MCHC between 30 and 36 g% Hypochromic anaemia. Normochromic anaemia.

24 Anaemia classification (2) 2 : PCV and RBC count : MCV Mean Corpuscular Volume PCV (%) MCV (fl) = x 10 x l Red blood cell count per µl (in millions) FOR ADULTS microcytic anaemia < 82 normocytic anaemia > 92 macrocytic anaemia Reference range : 82 to 92 fl (for adults; age related variation for children) [a femtolitre (fl) is of a litre]

25 Anaemia classification (3) 3 : Haemoglobin and RBC count : MCH Mean Corpuscular Haemoglobin Haemoglobin (in g/l) MCH (pg) = Red blood cells count per µl (in millions) Reference range : 28 to 32 pg (for adults; age related variation for children) FOR ADULTS > 32 hyperchromic anaemia

26 Other useful tests Thin blood films examination (Giemsa) Test for sickle-cell anaemia Determination of the reticulocyte number fraction/number Parasites detection (faeces, blood, )

27 RED CELL MORPHOLOGY Shape, size or color Inclusions (DNA) (Ribosomes) Anisocytosis : Different sizes (Haemoglobin) Poikilocytosis : Different shapes Anisochromasia : Different colors

29 a a : Normal. b : Anisocytosis, poikilocytosis, anisochromasia, microcytes, elliptocytes, c : Anisocytosis, poikilocytosis, microcytes, d: Hypochromia, anisocytosis, microcytes, elliptocytes, fragment, c b d

30 RED CELL MORPHOLOGY Shape, size or color Inclusions (DNA) (Ribosomes) Anisocytosis : Different sizes (Haemoglobin) Poikilocytosis : Different shapes Anisochromasia : Different colors

31 a b c a: b: c: Anisocytosis Anisocytosis, Anisocytosis Spherocytes Target cells, Basophilic stippling Polychromatic macrocytes Poikilocytes, Ovallocytes Howell-Jolly body Acanthocytes Sprerocytes

32 a : Anisocytosis Fragments Acanthocytes a b : Anisocytosis Macrocytes Microcytes b C: Anisocytosis Anisochromasia Sperocytes Macrocytes Microcytes Fragments c

33 Corpuscules de Heinz (haemoglobin) Siderocytes (Pappenheimer) Howell-Jolly body (DNA) Pappenheimer (iron) b c a

34 Acanthocytes Echinocytes

36 Hypochromic anaemia : Iron deficiency a and b : moderately severe Hypochromasia Microcytosis Poikilocytosis Anisocytosis Elliptocytes c: very severe Hypochromasia, Microcytosis Poikilocytosis Anisocytosis Elliptocytes

37 Hypochromic anaemia : Iron deficiency Before the treatment During the treatment

38 Hypochromic anaemia : β thalassaemia minor (a, b) β thalassaemia major (c) a : microcytosis, anisocytosis, poikilocytosis b : microcytosis, anisocytosis, Poikilocytosis Basophilic stippling (Hypochromic cells) a : normoblasts, double population (trf) Howell Jolly Basophilic stippling

39 Hypochromic anaemia : β thalassaemia minor a b

41 Normochromic anaemia : Sickel cells

42 Other useful tests Thin blood films examination (Giemsa) Test for sickle-cell anaemia Determination of the reticulocyte number fraction/number Parasites detection (faeces, blood, )

43 SS AS

45 hyperchromic anaemia: MACROCYTIC ANAEMIA i

46 Hyperchromic anaemia : Megaloblastic anaemia

48 Other useful tests Thin blood films examination (Giemsa) Test for sickle-cell anaemia Determination of the reticulocyte number fraction/number Parasites detection (faeces, blood, )

49 Determination of the reticulocyte number fraction/number Reticulocytes can be distinguished from mature RBCs because they still contain remnant genetic material (RNA) inside them, a characteristic not found in mature RBCs Blood briefly incubated with supravital stain, the RNA is precipitated as a dye-rna-protein complex that can be seen microscopically as a dark blue network (reticulum) or as dark blue granules on a thin blood film.

50 Determination of the reticulocyte number fraction/number count the number of reticulocytes in 5 fields of 200 RBCs. Must be a thin area where cells are evenly distributed and not touching each other.

51 Reticulocytes count Percentage : Adults and children : Infants at birth : 2-15 / RBC / RBC Absolute number : Adults and children : / µl Infants at birth : up to /µl

Hematology Morphology Critique

Hematology Morphology Critique Survey Slide: History: 60-year-old female presenting with pneumonia Further Laboratory Data: Hgb : 90 g/l RBC : 2.92 10 12 /L Hct : 0.25 L/L MCV : 87 fl MCH : 30.8 pg MCHC : 355 g/l RDW : 17.7 % WBC :