The document discusses blood film examination and abnormalities seen in red blood cells. Key points include: Blood films are prepared manually or automatically and stained using Romanowsky stains like Giemsa or Leishman's. Films are examined under low then high power microscopy to assess cell morphology and identify abnormalities. Abnormal red blood cell morphology seen in various anemias includes microcytosis in iron deficiency, macrocytosis in megaloblastic anemia, poikilocytosis in thalassemia, and hypochromia in iron deficiency. Specific abnormalities include target cells, sickle cells, spherocytes, and nucleated red blood cells. The differential diagnosis is considered based on the

1. BLOOD FILM EXAMINATION

2. BLOOD FILM PREPARATION
made on clean glass slides
Films may be spread by hand or by
means of an automated slide spreader
Manual Method

3. • Hemogram:
measured and
calculated
parameters
• Histograms:
size distribution of
WBC, RBC and Plt
• Cytogram: WBC
differential
CBC on automated analyzers
Flagging for abnormalities
necessitates a manual
PBS review

4. STAINING BLOOD AND BONE MARROW
FILMS
Romanowsky stains are used universally for
routine stainingof blood films
 Giemsa’s Stain
 Leishman’s Stain
Automatic staining machines are available that
enable large batches of slides to be handled

5. EXAMINATION OF BLOOD FILMS
 examined systematically
 starting with macroscopic observation of the
stained film assess whether the spreading
technique was satisfactoryand to judge its
staining characteristics and either any
abnormal particles present

6. EXAMINATION OF BLOOD FILMS
Microscopic examination- progressing from low-
power to high-power
Under low magnification to:
(a) get an idea of the quality of the preparation
(b) assess whether red cell agglutination, excessive
rouleaux formation or platelet aggregation is present
(c) assess the number, distribution and staining
of the leucocytes
(d) find an area where the red cells are evenly distributed
and are not distorted

7. EXAMINATION OF BLOOD FILMS
Having selected a suitable area,
Inspection under high-power magnification
for the final examination of unusual cells and for
looking at fine details

8. 1. RBC
 Size, Shape
 Stain
 Arrangement
 Inclusions
 nucleated RBCs
2. WBC
 Total counts
 Differential counts
 Abnormal /immature WBC
3. Platelets
 Counts
 Abnormality
4. Parasites
EXAMINATION OF BLOOD FILMS

9. RED CELL MORPHOLOGY
NORMAL
SHAPE - round, smooth contours
SIZE- range of 7–8 μm (about the same as that of the nucleus
of a small lymphocyte on the dried film)
STAIN - Central pallor 1/3 of total area
stain quite deeply with the eosin component of
Romanowsky dyes, particularly at the periphery of
the cell as a result of the cell’s normal biconcavity

10. Biconcave disc
Diameter : 7 - 8 μm
Central pallor occupy 1/3
rd of total
Size : approx. same as
nucleus of mature
lymphocyte
NORMAL RED CELL MORPHOLOGY

11. blood film from a healthy adult

12. ABNORMAL ERYTHROPOIESIS
Anisocytosis- variation in size of RBC
result of the presence of cells larger than normal
(macrocytosis), cells smaller than normal
(microcytosis) or both; frequently both
macrocytes and microcytes are present

13. Microcytes
 results from a defect in haemoglobin
formation
 characteristic of iron deficiency anaemia
various types of thalassaemia and severe
cases of anaemia of chronic disease.
Anisocytosis

14. Anisocytosis
Macrocytes
 classically found in megaloblastic anaemias
 also present in some cases of aplastic anaemia,
myelodysplastic syndromes ,other
dyserythropoietic states, in patients being
treated with hydroxyurea, chronic alcoholic &
chronic liver disease

15. Poikilocytosis –variation in shape of RBC
produced in many types of abnormal
erythropoiesis
for example
- megaloblastic anaemia
- iron deficiency anaemia
- Thalassaemia
- Myelofibrosis
ABNORMAL ERYTHROPOIESIS

16. Poikilocytosis
Elliptocytes and ovalocytes
 present when there is dyserythropoiesis
 in megaloblastic anaemia (macro-ovalocytes)
 in iron deficiency anaemia (‘pencil cells’)

17. Target Cells
refers to a cell in which there is a central round
stained area and a peripheral rim of
haemoglobinized cytoplasm separated by non-
staining or more lightly staining cytoplasm
result from cells having a surface that is
disproportionately large compared with their
volume
are seen in films in chronic liver diseases, iron
deficiency anaemia and thalassaemia
Poikilocytosis

18. Sickle Cells - vary in shape between boat-shaped forms
and sickles
SPICULATED CELLS AND RED CELL FRAGMENTATION
- Schistocytosis – Fragmented red cells
- Acanthocytosis- abnormality of the red cell in which
there are a small number of spicules of inconstant length,
thickness
-Stomatocytosis ,
Red cell inclusion bodies- Howell–Jolly Bodies
Pappenheimer Bodies
Basophilic stippling or punctate basophilia
Poikilocytosis

19. Basophillic stippling Hereditary spherocytosis
target cells

20. seen when there is extramedullary erythropoiesis
Tear drop cells / dacrocytes
• Osteopetrosis
• Myelofibrosis
• Bone marrow infiltrated with
hematological or
non-hematological
malignancies
• Iron deficiency anemia
• Pernicious anemia

21. INADEQUATE HAEMOGLOBIN
FORMATION
Hypochromia
 refers to the presence of red cells that stain
unusually palely
 two possible causes: a lowered haemoglobin
concentration and abnormal thinness of the
red cells

22. Hyperchromasia (Hyperchromia)
 deep staining of the red cells with a lack of
central pallor
 seen in two circumstances
- in the presence of macrocytes
- cells are abnormally rounded
Abnormally rounded cells may be either
spherocytes or irregularly contracted cells

23. Spherocytosis
 cells that are more spheroidal (i.e. less disc like)
than normal red cells but maintain a regular
outline
 diameter is less and thickness is greater than
normal
 result from genetic defects of the red cell
membrane as in hereditary spherocytosis,
haemolytic anaemia

24. Anisochromasia-
Dimorphic Red Cell Population
 abnormal variability in staining of red with two
distinct populations
 is characteristic of a changing situation
It can occur during the development or resolution
of iron deficiency anaemia

25. Polychromasia
Blue-gray coloration of RBCS.
Due RNA remnants
Increased - Increased erythropoietic
activity. Decreased - Hypoproliferative
states.
Hemolytic anemias
•Blood loss anemias
•Recovering anemia

26. Hypochromic Microcytic
Anaemia
 Iron deficiency Anaemia
 Thalasemia
 Anemia of chronic disease

27. Iron deficiency anemia

28. Iron deficiency anemia

29. Hypochromic microcytic anemia of iron
deficiency (peripheral blood smear)

30. Examination of peripheral blood film
RBC shows Hypochromic Microcytic anaemia with
moderate anisocytosis & poikilocytosis with the
presence of elliptical forms, elongated pencil
shaped cells
WBC - white cell count and differential are normal
Platelet - normal
Comment: Hypochromic Microcytic anaemia
Differential diagnosis: Iron deficiency anaemia
Thalasaemia

31. Investigation of a microcytic hypochromic
anaemia

32. Microcytosis Macrocytosis Target cells Spherocytes Red cell
fragments
Nucleated
red blood Howell-Jolly body
Basophilic stippling
Polychromasia
abnormal ribosom
al RNA appears as
blue dots

33. Shows marked anisocytosis, marked poikilocytosis,
one unusually large macrocyte and one severely
hypochromic cell

34. Blood film showing macrocytes, oval macrocytes & a
hypersegmented neutrophil

35. Blood film showing
hypochromia, microcytosis and poikilocytosis

36. Investigation of a macrocytic anaemia