This document provides an overview of red blood cell morphology assessment. It discusses the morphology of normal red blood cells and various abnormal red blood cell forms including variations in size, shape, hemoglobinization, and distribution. Examples of different abnormalities such as macrocytosis, microcytosis, elliptocytes, schistocytes, and rouleaux formation are described. The purpose is to enable students to identify red blood cell abnormalities and correlate them with underlying clinical conditions.

1. CHAPTER TWO
RED CELL MORPHOLOGY STUDY
1

2. Acknowledgements
• Addisa Ababa University
• Jimma University
• Haramaya University
• Hawassa University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-Ethiopia
2

3. Objectives
At the end of this chapter, the students shall be able to:
 Explain the purpose of red cell morphology assessment
 Describe the morphology of normal red cells
 List and describe the different abnormal erythrocyte forms
 Correlate the different abnormal RBC morphology with
clinical entities
 Perform assessment of red cell morphology on a stained
blood film
 Understand and apply the reporting system of the red cell
morphology assessment findings
3

4. 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
4

5. 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
5

6. 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

7. 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
7

8. 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
8

9. 1.2 Morphology of Normal Mature Red
cells
9
Peripheral blood film of a healthy subject showing
normal red cells and platelets. The red cells show
little variation in size and shape

10. 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
10

11. 1.3 Size Variation cont’d
11
The film also shows anisocytosis with
both microcytes and macrocytes

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

13. 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
13

14. 1.3 Size Variation cont’d
14
Microcytosis in a patient with β thalassaemia
trait

15. 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
15

16. 1.4 Variation in Shape cont’d
16
Acanthocytes in a patient with anorexia
nervosa

17. 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
17

18. 1.4 Variation in Shape cont’d
18
Tear drop cells

19. 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
19

20. 1.4 Variation in Shape cont’d
20
Sickle cells in a patient with sickle cell anemia

21. 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
21

22. 1.4 Variation in Shape cont’d
22
Echinocytes
Peripheral blood film showing
storage
Artefact-crenation
(echinocytosis), a disintegrated
cell and a neutrophil with a
rounded pyknotic nucleus

23. 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.
23

24. 1.4 Variation in Shape cont’d
24
Peripheral blood film of a patient with hereditary
elliptocytosis showing elliptocytes and ovalocytes

25. 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
25

26. 1.4 Variation in Shape cont’d
26
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

27. 1.4 Variation in Shape cont’d
27
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

28. 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
28

29. 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
29

30. 1.4 Variation in Shape cont’d
30
Peripheral blood film of a patient with hemoglobin C disease
showing irregularly contracted cells and several target cells

31. 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
31

32. 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
32

33. 1.4 Variation in Shape cont’d
33
Peripheral blood film of a patient with hereditary spherocytosis as a
result of a band 3 mutation showing pincer or mushroom cells

34. 1.4 Variation in Shape cont’d
34
Peripheral blood film of a patient with clostridial septicaemia
showing many spherocytes

35. 1.4 Variation in Shape cont’d
35
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

36. 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
36

37. 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
37

38. Rouleaux formation
38
Peripheral blood film in Multiple Myeloma

39. 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.

40. 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.

41. 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
41

42. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
42
Hypochromic red cells in a patient with iron-deficiency anemia

43. 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
43

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

45. 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
45

46. 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
46

47. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
47
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

48. 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
48

49. 1.5 Abnormalities in Red cell
Hemoglobinization cont’d
49
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

50. 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
50

51. 1.6 Red cell inclusions cont’d
51
Prominent basophilic stippling in the peripheral blood film
of a patient who has inherited both β thalassaemia trait
and hereditary elliptocytosis

52. Case Study

53. 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
53

54. 1.6 Red cell inclusions cont’d
54
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

55. 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
55

56. 1.6 Red cell inclusions cont’d
56
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

57. Review of Abnormal RBC Morphology

58. Objectives
 Distinguish normal and abnormal RBC morphology with
respect to the following:
 Size, including alterations caused by a heterogeneous or
dimorphic RBC population
 Haemoglobin content
 RNA concentration (polychromasia/reticulocytes)
 Shape alterations
 RBC inclusion bodies
 Variations in red cell pattern
 Artifact
 List synonyms for abnormal RBC morphology

59. Objectives
 Identify the composition of RBC inclusion bodies, including
the following stains used to identify:
 Wright’s Romanowsky stain
 Perl’s Prussian blue iron stain
 Supravital stain
 Correlate abnormal RBC morphology with associated
conditions

60. 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

61. 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

62. Normocytic RBCs, MCV 80-
100 fL
Homogeneous RBC population
RDW < 14.0% (low), uniform size

63. RBC Size Variations -
Anisocytosis
Heterogeneous RBC population,
RDW > 14.0% (high)

64. Variations in RBC Size
Microcytosis (MCV < 82.0 fL)
Macrocytosis (MCV > 98.0 fL)

65. The red cells shown in
A, B and C appear:
A - Macrocytic
B - Microcytic (& hypo)
C - Normocytic
A
B
C

66. The red cell picture shown
demonstrates:
Anisocytosis with high RDW – RBC population is
heterogeneous with microcytic and macrocytic red cells,
aka dimorphic with two RBC populations

67. 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

68. Variations in RBC Size and/or
Hgb Content (Color)

69. The red cells shown in A and B
exhibit:
A – Hypochromia, B - Polychromasia
B
A

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

71. End-stage Liver disease
Spherocytes
Schistocytes
Poikilocytosis - Abnormal RBC
Shape, Rigid RBCs
Normal Discoid Shape –
Deformable Red Cells
Damaged red cells
Variations in RBC Shape

72. Spherocytes – No pallor
Acanthocytes – No pallor
Variations in RBC Shape
Echinocytes – Have a pallor
Includes crenated & burr cells

73. The red cells at the arrows are:
Acanthocytes – no pallor area (a
‘spherocyte with projections’)

74. Variations in RBC Shape
Target Cells/Codocytes
C Crystals
Sickle Cells/Drepanocytes

75. Variations in RBC Shape
C crystal Hgb C
SC crystals 
Hgb S & Hgb C
Sickle Cells  Hgb S
Note the target cells

76. The red cells at the arrows are:
a.Sickle cells that contain Hgb S
b.C crystals that contain Hgb C
c. SC crystals that contain Hgb S AND Hgb C
c. SC crystals (bizarre shape)

77. Variations in RBC Shape
Pencil forms – microcytic & hypochromic
Ovalocytes/Elliptocytes
Normocytic Ovalocytes
Macrocytic Ovalocytes

78. Variations in RBC Shape
Stomatocytes
Teardrops/Dacrocytes

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

80. RBC Inclusions
Basophilic stippling,
a stippled RBC with
punctate dark ‘dots’
Howell-Jolly bodies
Pappenheimer
bodies
NucRBC
Nucleated RBC
HJB
Pap
body

81. RBC Inclusions
Reticulocytes
Supravital stain
Polychromasia,
Wright’s stain
NRBC
Lymph
Pappenheimer bodies
Prussian blue iron stain
Pappenheimer
bodies Wright’s stain
Jun
k
Jun
k

82. RBC Inclusions
B
A
A - Diffuse Polychromasia, faint ‘dots’ B -
Stippled RBCs, punctate ‘dots’
Cabot Ring (figure 8)
Supravital stain
Heinz Bodies,
Wright’s stain
(not visible)
Heinz Bodies
Supravital stain

83. RBC Inclusions
Malarial gamete
form, P. vivax
Malarial gamete form,
P. falciparum
Malarial ring forms, P. falciparum
Platelet on RBC
Cabot rings
(right arrow)

84. Identify the RBC inclusions at
A, B and C
A – Basophilic stippling
B – Howell-Jolly body
C – Pappenheimer bodies
B
C
A

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

86. Variations in RBC Distribution
Rouleaux
RBC Agglutination
Normal RBC Distribution

87. Erroneous RBC Morphology
due to Artifact
Oil Artifact
Precipitated Stain
Crenated cells & spherocytes due to poor smear/wrong area

88. Summary for RBC Morphology
RBC Morphology:
 Size, including alterations caused by a
heterogeneous or dimorphic RBC population
 Haemoglobin content
 RNA concentration (polychromasia/reticulocytes)
 Shape alterations
 RBC inclusion bodies
 Variations in red cell pattern
 Artifact
 synonyms for abnormal RBC morphology
 composition of RBC inclusion bodies, including stains
used to identify
 abnormal RBC morphology with associated
conditions

89. Review Questions
1. What parameters of the red cell morphology are
appraised in red cell morphology study on a stained
blood film? Supplement your answers with examples.
89

90. Bibliography
• MA Lichtman, E Beutler, U Seligsohn, K Kaushansky, TO
Kipps (Editors). William’s Hematology. 7th Ed. McGraw-
Hill Co. Inc. 2008.
• Dacie, John V and Lewis, S.M. Practical Hematology
10th Edition Churchill-Livingstone 2006.
• Wintrobe, Maxwell M. Clinical Hematology 11th Edition
Lea and Febiger, Philadelphia 2003.
90