BLOOD FILM EXAMINATION

BLOOD FILM PREPARATIONmade on clean glass slidesFilms may be spread by hand or bymeans of an automated slide spreaderManual Method

• Hemogram:

measured and

calculated

parameters

• Histograms:

size distribution of

WBC, RBC and Plt

• Cytogram: WBC

differential

CBC on automated analyzers

Flagging for abnormalitiesnecessitates a manualPBS review

STAINING BLOOD AND BONE MARROWFILMSRomanowsky stains are used universally forroutine stainingof blood films

Giemsa’s Stain

Leishman’s Stain

Automatic staining machines are available thatenable large batches of slides to be handled

EXAMINATION OF BLOOD FILMS examined systematically

starting with macroscopic observation of thestained film assess whether the spreadingtechnique was satisfactoryand to judge itsstaining characteristics and either anyabnormal particles present

EXAMINATION OF BLOOD FILMSMicroscopic 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 stainingof the leucocytes(d) find an area where the red cells are evenly distributedand are not distorted

EXAMINATION OF BLOOD FILMSHaving selected a suitable area,

Inspection under high-power magnification

for the final examination of unusual cells and forlooking at fine details

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

RED CELL MORPHOLOGY

NORMAL

SHAPE - round, smooth contours

SIZE- range of 7–8 μm (about the same as that of the nucleusof a small lymphocyte on the dried film)

STAIN - Central pallor 1/3 of total area

stain quite deeply with the eosin component ofRomanowsky dyes, particularly at the periphery ofthe cell as a result of the cell’s normal biconcavity

Biconcave discDiameter : 7 - 8 μmCentral pallor occupy 1/3rd of totalSize : approx. same asnucleus of maturelymphocyte

NORMAL RED CELL MORPHOLOGY

blood film from a healthy adult

ABNORMAL ERYTHROPOIESIS

Anisocytosis- variation in size of RBCresult of the presence of cells larger than normal(macrocytosis), cells smaller than normal(microcytosis) or both; frequently bothmacrocytes and microcytes are present

Microcytes results from a defect in haemoglobin

formation

characteristic of iron deficiency anaemiavarious types of thalassaemia and severecases of anaemia of chronic disease.

Anisocytosis

AnisocytosisMacrocytes classically found in megaloblastic anaemias

also present in some cases of aplastic anaemia,myelodysplastic syndromes ,otherdyserythropoietic states, in patients beingtreated with hydroxyurea, chronic alcoholic &chronic liver disease

Poikilocytosis –variation in shape of RBC

produced in many types of abnormalerythropoiesis

for example

- megaloblastic anaemia

- iron deficiency anaemia

- Thalassaemia

- Myelofibrosis

ABNORMAL ERYTHROPOIESIS

PoikilocytosisElliptocytes and ovalocytes present when there is dyserythropoiesis

in megaloblastic anaemia (macro-ovalocytes)

in iron deficiency anaemia (‘pencil cells’)

Target Cellsrefers to a cell in which there is a central roundstained area and a peripheral rim ofhaemoglobinized cytoplasm separated by non-staining or more lightly staining cytoplasm

result from cells having a surface that isdisproportionately large compared with theirvolume

are seen in films in chronic liver diseases, irondeficiency anaemia and thalassaemia

Poikilocytosis

Sickle Cells - vary in shape between boat-shaped formsand sicklesSPICULATED CELLS AND RED CELL FRAGMENTATION

- Schistocytosis – Fragmented red cells

- Acanthocytosis- abnormality of the red cell in whichthere are a small number of spicules of inconstant length,thickness

-Stomatocytosis ,Red cell inclusion bodies- Howell–Jolly BodiesPappenheimer BodiesBasophilic stippling or punctate basophilia

Poikilocytosis

Basophillic stippling Hereditary spherocytosis

target cells

seen when there is extramedullary erythropoiesis

Tear drop cells / dacrocytes• Osteopetrosis• Myelofibrosis• Bone marrow infiltrated with

hematological ornon-hematological

malignancies• Iron deficiency anemia• Pernicious anemia

INADEQUATE HAEMOGLOBINFORMATION

Hypochromia refers to the presence of red cells that stain

unusually palely

two possible causes: a lowered haemoglobinconcentration and abnormal thinness of thered cells

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 eitherspherocytes or irregularly contracted cells

Spherocytosis cells that are more spheroidal (i.e. less disc like)

than normal red cells but maintain a regularoutline

diameter is less and thickness is greater thannormal

result from genetic defects of the red cellmembrane as in hereditary spherocytosis,haemolytic anaemia

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 resolutionof iron deficiency anaemia

Polychromasia

Blue-gray coloration of RBCS.Due RNA remnants

Increased - Increased erythropoieticactivity. Decreased - Hypoproliferativestates.

Hemolytic anemias•Blood loss anemias•Recovering anemia

Hypochromic MicrocyticAnaemia

Iron deficiency Anaemia

Thalasemia

Anemia of chronic disease

Iron deficiency anemia

Iron deficiency anemia

Hypochromic microcytic anemia of irondeficiency (peripheral blood smear)

Examination of peripheral blood film

RBC shows Hypochromic Microcytic anaemia withmoderate anisocytosis & poikilocytosis with thepresence of elliptical forms, elongated pencilshaped cells

WBC - white cell count and differential are normal

Platelet - normal

Comment: Hypochromic Microcytic anaemia

Differential diagnosis: Iron deficiency anaemiaThalasaemia

Investigation of a microcytic hypochromicanaemia

Microcytosis Macrocytosis Target cells Spherocytes Red cellfragments

Nucleatedred blood Howell-Jolly body

Basophilic stipplingPolychromasiaabnormal ribosomal RNA appears asblue dots

Shows marked anisocytosis, marked poikilocytosis,one unusually large macrocyte and one severelyhypochromic cell

Blood film showing macrocytes, oval macrocytes & ahypersegmented neutrophil

Blood film showinghypochromia, microcytosis and poikilocytosis

Investigation of a macrocytic anaemia