This ppt is about erythrocytes variation in size and shapes, inclusion bodies.

1. Erythrocytes variation in size
and shapes, inclusion bodies
Rabia Mushtaq
Ph.D. Zoology
661- Ph.D- Zoo-2022
G.C. University Lahore

2. 1.1 INTRODUCTION
 The morphology of blood cells in stained films is the
basis of laboratory diagnosis of hematologic disorders
including:
Anemias
Leukemias
Infections
 A careful examination of a well-spread and well-stained
film can be more informative than a series of
investigations
 The film should be covered with a cover glass using a
neutral mounting medium
this step is not, however, mandatory
2

3. 1.1 Introduction cont’d
 The blood film should be inspected under low power
magnification in order to:
get an idea of the quality of the preparation, i.e.,
whether red cell agglutination or excessive rouleaux
is present
get an idea of the number, distribution and staining of
the leucocytes
find an area where the red cells are evenly distributed
and are not distorted
 Having selected a suitable area, the 40x dry or 100x oil
immersion objective is used to appreciate:
variation in red cell size, shape and staining, and
fine details such as cytoplasmic granules and other
red cell inclusions
3

4. Five RBC morphological features are
evaluated on the blood smear:
• Variations in RBC Size
• Variations in RBC Color
• Variations in RBC Shape
• RBC Inclusions
• Variations in RBC Distribution/Pattern

5. 1.2 Morphology of Normal Mature Red cells
(Discocytes)
 In health, red cells are said to be normocytic and
normochromic
 In well spread and stained films the great majority of the
cells have:
Round smooth contours
Have diameters within the comparatively narrow
range of 6.0-8.0m
A thickness of 2.5m at the periphery and 1.0m in
the center
 As a rough guide, normal red cell size appears to be
about the same as that of the nucleus of a small
lymphocyte
5

7. 1.2 Morphology of Normal Mature Red
cells (Discocytes) cont’d
 The hemoglobin stains with the eosin component of
Romanowsky dyes and owing to the biconcavity of the
cell, stains:
More pale at the center, and
Quite deeply at the periphery
This depth and distribution of staining in normal
red cells is described as normochromic
7

8. 1.2 Morphology of Normal Mature Red
cells
8
Peripheral blood film of a healthy subject showing
normal red cells and platelets. The red cells show
little variation in size and shape

9. 1.3 Size Variation (Anisocytosis)
Macrocytes
 Have diameter greater than 8.0m and the mean cell
volume is also increased
 Because of their increased hemoglobin content they
stain darker than discocytes
 Macrocytosis is seen in stress erythropoiesis as seen in
conditions such as hemolytic anemia and also during
recovery from acute blood loss, and Megaloblastic
anemia
9

10. 1.3 Size Variation cont’d
10
The film also shows anisocytosis with
both microcytes and macrocytes

11. 1.3 Size Variation cont’d
Megalocytes
 Large (greater diameter may measure 12m), often oval
shaped cells with increased hemoglobin content
11

12. 1.3 Size Variation cont’d
 True megalocytes are identified only if megaloblasts have been
identified in bone marrow aspirates
 Megalocytes are seen in vitamin B12 and/or folic acid deficiency, in
association with some leukemias and in refractory anemias
Microcytes
 Have diameter less than 6.0m but may appear to have normal size
caused by flattening of the cells during smear preparation
 The mean cell volume is decreased to less than 80.0fl
 Area of central pallor usually increases because of the coexistent
hypochromia
 Are seen in iron deficiency anemia and a slight degree of
microcytosis is seen in inflammation
12

13. 1.3 Size Variation cont’d
13
Microcytosis in a patient with β thalassaemia
trait

14. 1.4 Variation in Shape (Poikilocytosis)
Acanthocytes ('spiny cells')
 Spheroidal cells with 3-12 spicules of uneven length
irregularly distributed over the cell surface.
 Seen in:
disorders of lipid metabolism
alcoholic liver cirrhosis and
Hepatitis
14

15. 1.4 Variation in Shape cont’d
15
Acanthocytes in a patient with anorexia
nervosa

16. 1.4 Variation in Shape cont’d
Dacrocytes ('Tear drop cells')
 These are tear drop or pear shaped red cells
 Could be considered to be discocytes with a single drawn out
spicule.
 It is thought that stretching of the cell membrane beyond a
certain limit results in loss of deformability and ability to revert
to normal discoid shape.
 Seen in:
Myelofibrosis,
Myeloid metaplasia
 Tumour metastases to the bone marrow
 Tuberculosis
Drug-induced Heinz body formation
16

17. 1.4 Variation in Shape cont’d
17
Tear drop cells

18. 1.4 Variation in Shape cont’d
Drepanocytes ('sickle cells')
 These are crescent shaped red cells because of the
formation of rod-like polymers of Hb S or some other
rare hemoglobins
 Have an increased surface area and increased
mechanical fragility which leads to hemolysis and hence
severe anemia
 They are primarily seen in sickle cell anemia where there
is substitution of valine for glutamic acid at position 6 of
the beta chain in the hemoglobin molecule
18

19. 1.4 Variation in Shape cont’d
19
Sickle cells in a patient with sickle cell anemia

20. 1.4 Variation in Shape cont’d
Echinocytes ('crenated cells')
 Red cells showing numerous, short, evenly distributed
spicules of equal length
 These are probably the most common artifacts in a blood
film:
Consistently found in blood samples that have been
stored for some time at room temperature and
 Because of diffusion of alkaline substances from the
slide into the cells resulting in an increase in pH and
thus crenation of the cells
 In vivo they are seen in uremia, pyruvate kinase
deficiency and neonatal liver diseases
20

21. 1.4 Variation in Shape cont’d
21
Echinocytes
Peripheral blood film showing
storage
Artefact-crenation
(echinocytosis), a disintegrated
cell and a neutrophil with a
rounded pyknotic nucleus

22. 1.4 Variation in Shape cont’d
Elliptocytes/ovalocytes
 Elliptical or oval shaped red cells. Normally less than 1%
of the red cells are elliptical/oval shaped.
 Found in almost all anemias where approximately 10%
of the red cells may assume elliptical/oval shape and in
hereditary elliptocytosis where almost all the red cells
are elliptical.
22

23. 1.4 Variation in Shape cont’d
23
Peripheral blood film of a patient with hereditary
elliptocytosis showing elliptocytes and ovalocytes

24. 1.4 Variation in Shape cont’d
Schistocytes ('fragmented cells')
 Two types can be distinguished:
Small fragments of cells of varying shape, sometimes
with sharp angles or spines ('spur cells'), sometimes
round in contour, usually staining deeply but
occasionally palely as a result of loss of hemoglobin
at the time of fragmentation
24

25. 1.4 Variation in Shape cont’d
25
Schistocytes
Peripheral blood
film of a patient
with compound
heterozygosity for
hemoglobin S
and hemoglobin S-
Oman showing
the ‘Napoleon
hat’ red cells that
are characteristic
of hemoglobin S-
Oman

26. 1.4 Variation in Shape cont’d
26
Schistocytes
Peripheral blood
film of a patient
with compound
heterozygosity for
haemoglobin S
and haemoglobin
S-Oman showing
the ‘Napoleon
hat’ red cells that
are characteristic
of haemoglobin
S-Oman

27. 1.4 Variation in Shape cont’d
Larger cells mainly with round contour from which
fragments have been split off, e.g., 'helmet cells'
 They are findings in:
Certain genetically determined disorders, e.g.,
The thalassemias
Hereditary elliptocytosis
Acquired disorders of red cell formation,
megaloblastic and iron deficiency anemias
Direct thermal injury as in severe burns
Burr cells
 Small cells or cell fragments bearing one or a few spines
 Found particularly in uremia
27

28. 1.4 Variation in Shape cont’d
Leptocytes ('target cells'/'Mexican hat cells')
 These are cells showing an area of central staining
 They are abnormally thin cells
 They are common findings in obstructive liver diseases
where there is accumulation of cholesterol and lecithin
due to inhibition of plasma LCAT activity by bile salts
 Variable numbers are seen in iron deficiency anemia and
thalassemia
 There is gross target cell formation after splenectomy
28

29. 1.4 Variation in Shape cont’d
29
Peripheral blood film of a patient with hemoglobin C disease
showing irregularly contracted cells and several target cells

30. 1.4 Variation in Shape cont’d
Stomatocytes
 These are cells with a narrow slit like area of central
pallor
 They are common findings in liver diseases associated
with chronic alcohol abuse
30

31. 1.4 Variation in Shape cont’d
Spherocytes/Microspherocytes
 Dense staining spherical cells with smaller diameter and
greater thickness than normal
 They are formed as a result of loss of membrane due to:
Genetic lack of structural proteins in the red cell
membrane
Chemicals
Bacterial toxins (Clostridium welchii)
Antibody-mediated hemolytic anemias
Burn injury
31

32. 1.4 Variation in Shape cont’d
32
Peripheral blood film of a patient with hereditary spherocytosis as a
result of a band 3 mutation showing pincer or mushroom cells

33. 1.4 Variation in Shape cont’d
33
Peripheral blood film of a patient with clostridial septicaemia
showing many spherocytes

34. 1.4 Variation in Shape cont’d
34
Peripheral blood film of a patient with severe burns showing
spherocytes, microspherocytes and red cells that appear to be
budding off very small spherocytic fragments

35. 1.4 Variation in Shape cont’d
 They are commonly seen in hereditary spherocytosis
that is associated with:
abnormalities in membrane protein
lipid loss and
excessive flux of Na+ across the membrane
35

36. 1.5. Rouleaux formation
(Abnormalities in Distribution)
 Red cells are aligned in formations resembling stacks of
coins
 May be seen as artifacts in the thick areas of the blood
film
 They are often associated with:
Hyperproteinemia
chronic inflammatory disorders
multiple myeloma
macroglobulinemia
36

37. Rouleaux formation
37
Peripheral blood film in Multiple Myeloma

38. Peripheral blood film showing storage
artefact a crenation (echinocytosis), a
disintegrated cell and a neutrophil with a
rounded pyknotic nucleus.
Peripheral blood film showing storage
artefact, a mild crenation and
lobulation of a lymphocyte
nucleus.

39. Peripheral blood film from
a blood specimen that has been
transported in a hot motor vehicle,
showing red cell budding and
fragmentation.
Peripheral blood film from
a patient with hyperlipidaemia
showing misshapen red cells with
fuzzy outlines and blurring of the
outline of the lobes of a neutrophil
consequent on the high concentration
of lipids.

40. 1.5 Abnormalities in Red cell Hemoglobinization
Hypochromia/Hypochromasia
 Hypochromic red cells:
Contain less than the normal amount of hemoglobin
The central pale area is increased to more than one-
third of the cell diameter
In severe hypochromia the hemoglobin appears as a
thin rim at the periphery of the cell
The cells are usually microcytic and may assume
target shape
40

41. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
41
Hypochromic red cells in a patient with iron-deficiency anemia

42. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
 It is a consistent finding in iron deficiency anemia,
thalassemia and sideroblastic anemia.
 In doubtful cases it is wise to compare the staining of the
suspect film with that of a normal film stained at the
same time
 Poor drying of the film may cause a 'false
hypochromia‘
This can be distinguished from a true one in that the
change in the central pale area is sudden while in true
hypochromia it is gradual
42

43. Artefactual changes
produced by 5% water in the
methanol used for fixation.

44. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
Hyperchromia/Hyperchromasia
 Because over-saturation of a red cell can not take place,
true hyperchromia does not exist
 Usually, deep staining of red cells is seen in:
Macrocytosis when the red cell thickness is increased
and the mean cell volume also increased
Spherocytes in which the red cell thickness is greater
than normal and the mean cell hemoglobin
concentration is slightly increased
44

45. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
Polychromasia/Polychromatophilia
 As reticulocytes contain residual RNA:
They will have the affinity for the basic component of
the Romanowsky stain, and
Assume a degree of blue staining proportional to the
amount of RNA
An increase in reticulocytes in the peripheral blood
will be seen as a polychromatic red cell population
which is also macrocytic
45

46. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
46
Fragments including microspherocytes in
the peripheral blood film of a patient with
the hemolytic uraemic syndrome. The film
also shows polychromasia and a nucleated
red blood cell (NRBC)
A polychromatic cell which is
also larger then a normal cell;
it may be designated a
polychromatic macrocyte

47. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
Dimorphism/Anisochromasia
 This is the presence of two populations of red cells,
namely hypochromic and normochromic, in the same
film in approximately equal proportions
 It is a finding in:
Treated iron deficiency anemia where there is the
new normochromic red cell population and the
original hypochromic population, and
Patients with hypochromic anemia who have been
transfused
47

48. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
48
A dimorphic peripheral blood film from a patient with
sideroblastic anaemia as a consequence of a myelodysplastic
syndrome. One population of cells is normocytic and
normochromic while the other is microcytic and hypochromic

49. 1.6 Red cell inclusions
Basophilic stippling/Punctate basophilia
 The red cells contain small irregularly shaped granules
which stain blue in Wright stain and which are found
distributed throughout the cell surface.
 It is a common finding in:
lead poisoning
anemias associated with disorders of hemoglobin
synthesis
49

50. 1.6 Red cell inclusions cont’d
50
Prominent basophilic stippling in the peripheral blood film
of a patient who has inherited both β thalassaemia trait
and hereditary elliptocytosis

51. Case Study

52. 1.6 Red cell inclusions cont’d
Howell-Jolly bodies
 Small, round inclusions that contain DNA and are usually
eccentrically located in the cell
 They stain deep purple
 Found:
In megaloblastic anemia
In some hemolytic anemias, and
After splenectomy
52

53. 1.6 Red cell inclusions cont’d
53
The blood film of a splenectomized post-renal transplant patient
with megaloblastic anaemia caused by azathioprine therapy
showing macrocytosis, acanthocytes and prominent Howell–Jolly
bodies

54. 1.6 Red cell inclusions cont’d
Cabot's rings
 These are incomplete or complete rings, even figures of
'8’
 They appear as reddish - violet fine filamentous
configuration sin Wright- stained films
 They are remnants of the microtubules of the mitotic
spindle
Blood Parasites
 Malaria
 Babesia
54

55. 1.6 Red cell inclusions cont’d
55
Ring forms and an
early gametocyte of
P. falciparum
Blood film from a hyposplenic
patient with babesiosis caused by
Babesia divergens showing
numerous parasites including a
Maltese cross formation and
paired pyriform parasites