PERIPHERAL BLOOD SMEAR.pptx preparation staining fixation

28/01/2025
•Presented By- Dr.Amrit Kour
•PG SCHOLAR
•R.G.G.P.G.Ayurvedic College and Hospital,
Paprola
PERIPHERAL BLOOD SMEAR
(Method of preparation,staining procedure and formation of RBC’s)
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Introduction:
 A blood smear or film is a specimen for microscopic
examination
 prepared by spreading a drop of blood across a glass slide
 followed by staining with one of the Romanowsky's stains.
 The peripheral blood slide review is a comprehensive
qualitative examination of the blood film to detect
clinically significant abnormalities in the morphology of
 leukocyte,
 erythrocyte, and
 platelet.
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Alternative Names :
Peripheral blood film (PBF)
Blood smear
Peripheral smear
Generalised blood picture(GBP)
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USES:
 1. Blood smear is helpful in suggesting the cause of anemia or
thrombocytopenia,
 identifying leukemia and types of leukemia, and
 in diagnosing hemoparasitic infections (malaria, filaria, and
trypanosomiasis).
 It is also helpful in the management of these conditions.
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 2. To monitor the effect of chemotherapy and
radiotherapy on bone marrow.
 3. To provide direction for further investigations
that will help in arriving at the correct diagnosis
(e.g. in infections, drug toxicity, etc.).

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INDICATIONS OF PBF-
Blood smear examination is therefore indicated in-
 Clinically suspected cases of anemia.
 Thrombocytopenia.
 Hematological malignancies (leukemia, lymphoma,
multiple myeloma).
 Disseminated intravascular coagulation.
 Parasitic infections (like malaria or filaria).
 Infectious mononucleosis.
 Various inflammatory, or malignant diseases.
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PREPARATION OF BLOOD SMEAR:
WEDGE TECHNIQUE.
COVERSLIP TECHNIQUE.
AUTOMATED SLIDE MAKING AND
STAINING.
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(WEDGE METHOD)
 A small drop of blood (2-3 mm in diameter) is placed in
the center line about 1 cm away from one end of a glass
slide (typical size of slide is 75 × 25 mm; thickness about
1mm) with a wooden stick or glass capillary.
 Blood sample may be venous (anticoagulated with EDTA)
or capillary (finger prick).
 Better blood cell morphology is obtained if smear is made
directly from a skin puncture.
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 If EDTA-anticoagulated venous blood is used, smear
should be prepared and stained within 2 hours of blood
collection.
 If venous blood collected in a syringe is used, the last
drop of blood in the needle after withdrawing (or first
drop while dispensing) should be used.
 A 'spreader' slide is placed at an angle of 30° in front of
the drop and then drawn back to touch the drop of blood.
Blood spreads across the line of contact of two slides.
 Smear is made by smooth, forward movement of the
'spreader' along the slide.
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 Smear is rapidly dried by waving it in the air or keeping
it under an electric fan.
 Slow drying causes shrinkage artifact of red cells.
 Patient's name or laboratory number and date are
written (with a lead pencil, a permanent marker pen, or
a diamond pencil) on the thicker end of the smear.
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A well-stained smear shows following features:
• Red cells: pink-red or deep pink
• Polychromatic cells (Reticulocytes): Gray-blue
• Neutrophils: Pale pink cytoplasm; mauve-
purple granules
• Eosinophils: Pale-pink cytoplasm; orange-red
granules
• Basophils: Blue cytoplasm; dark blue-violet
granules
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• Monocytes: Gray-blue cytoplasm; fine reddish (azurophil)
granules
• Small lymphocytes: Dark blue cytoplasm
• Platelets: Purple
• Nuclei of all cells: Purple-violet
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Definition
 Erythropoiesis is the process of the origin, development and
maturation of erythrocytes.
 It is a part of the process of development of blood cells called
Hemopoiesis or hematopoiesis which includes.
 Erythropoiesis
 Leucopoiesis – formation of W.B.Cs
 Thrombopoiesis – formation of platelets
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Stages of Erythropoiesis
 There are three stages of erythropoiesis –
 1. Mesoblastic Stage
 2. Hepatic Stage
 2. Myeloid Stage
 1. Mesoblastic Stage –
 During the first two months of intrauterine life, erythropoiesis
takes place in the mesoderm of yolk sac.
 During this stage, erythropoiesis is intravascular.
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 2. Hepatic Stage –
 From third month of intrauterine life, the main site for erythropoiesis is
liver.
 Spleen and lymphoid organs are also involved in erythropoiesis.
 3. Myeloid Stage –
 During the last three months of intrauterine life, the RBCs are produced
from red bone marrow and liver.
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After birth, bone marrow becomes the sole site of
erythropoiesis.
In young children, active hematopoietic bone marrow is
found in axial skeleton and bones of extremities.
Active hematopoietic bone marrow is red in colour due to
marked cellularity – Red Bone Marrow.
However, there occurs a progressive fatty replacement
throughout the long bones converting red bone marrow to
yellow bone marrow.
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 After 20 – 30 years, erythropoiesis is mostly limited
 sternum
 Ribs
 Vertebrae
 Skull
 Pelvis &
 Pelvic Girdle
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Steps of Erythropoiesis
 There are four major cell stages –
 Stem cells
 Progenitor cells
 Precursor cells
 Mature cells
During erythropoiesis, following cellular changes occur
–
Cell size progressively decreases
Size of nucleus & number of nucleoli decreases.
Chromatin material condenses & nucleus disappears.
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Stem cells
 Pleuripotent stem cells are the mother stem cells for
different cell lines.
It possess two fundamental properties :
 Self Replication – they are capable of giving rise to
more stem cell.
 Differentiation – they have the ability to differentiate
into specalized cells called progenitor cells.
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COMMITED STEM CELLS
 These cells develop from pleuripotent stem cells.
 They are of two categories :
 Myeloid stem cell and
 Lymphoid stem cell
 Myeloid stem cell form
 erythroid series,
 monocytic granulocytic series &
 Megakaryoid series.
 Erythroid stem cells - Give rise to progenitor cells for erythroid cell lines.
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PRECURSOR CELLS/ BLAST CELLS
 The precursor for erythrocytes –
 erythroblasts or normoblasts
 Normoblast develop from Pronormoblasts.
Normoblasts have three successive forms :
Early Normoblasts
Intermediate Normoblasts
Late Normoblasts
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PRONORMOBLAST
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 First blast cell to appear in the bone marrow and it is first
identifiable cell of the erythroid series.
 MORPHOLOGICAL FEATURES –
 1. Size- 15-20 µ.
 2.Shape – irregular rounded and slightly oval.
 3. Cytoplasm – less, basophilic – due to the presence of
polyribosomes, high content of RNA.
 4. Nucleus – Large, multiple nucleoli – cover almost whole
cytoplasm.
 5. Mitosis – present
 6. Hemoglobin – Not yet formed.

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EARLY/ BASOPHILIC NORMOBLAST
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 Pronormoblast progresses into early normoblast.
 1. Size- 12 - 16 µ.
 2.Shape – irregular rounded and slightly oval.
 3. Cytoplasm – scanty, blue and basophilic.
 4. Nucleus –nucleoli disappear & Condensation of
chromatin network occurs.
 6. Hemoglobin – Not present.

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INTERMEDIATE NORMOBLAST
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 This appears following mitotic division of early
normoblast.
 Also known as polychromatophilic normoblast.
 1. Size- 10 - 14 µ.
 2. Cytoplasm – Polychromatophilic (contains
admixture of basophilic RNA & Acidophilic
Hemoglobin).
 4. Nucleus –coarse, condense, deeply
basophilic with no nucleoli.
 6. Hemoglobin – starts appearing.

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LATE NORMOBLAST
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 Last nucleated cell of the erythroid series.
 Also known as orthochromatophilic normoblast.
 1. Size- 8 - 10 µ.
 2. Cytoplasm –Deeply acidophilic with diffuse
basophilic hue. It gives an appearance of
orthochromatic cell.
 4. Nucleus – Small, pyknotic with dark chromatin.
 5. Mitosis – absent
 6. Hemoglobin –Present.

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RETICULOCYTE
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 Immediate precursors of red cells.
 Also known as juvenile red cells.
 1. Size- 7– 7.5 µ.
 2. Cytoplasm –contain small amount of RNA. With supravital stains
like brilliant cresyl blue, the RNA appears in the form of reticulum
and hence the cell is called reticulocyte.
 4. Nucleus – absent
 5. Mitosis – absent
 6. Hemoglobin – Increases.

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ERYTHROCYTES
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 Final cells in erythropoiesis.
 Reticulocytes spend 1 – 2 days in the marrow and circulate for
1-2 days in the pheripheral blood before maturing to form
erythrocytes.

 1. Size- 7.5 µ.
 2. shape – biconcave disc.
 3. Nucleus – absent
 4. Hemoglobin – present..

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 Almost the size of
nucleus of a mature
lymphocyte.
 Central 1/3rd
portion is
pallor.
 It lacks nucleus,
mitochondria.
 Semi-permeable
membrane is present.
 Contains Antigens –
ABO & Rh – Blood
typing.

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DURATION
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 The total period of erythropoiesis is 7 to
9 days.
 It takes 5 – 7 days for progenitor cells to
become reticulocytes and
 another 2 days for reticulocytes to
become red cells.

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APPLIED CONDITIONS
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 Abnormal count of RBCs –
 1. Increase in erythrocytes – Polycythemia
 2. Decrease in erythrocytes – Anemia
 Abnormal size of RBCs –
 1. Microcytosis – Erythroctes are smaller than 7 µm.
 2. Macrocytosis – Erythroctes are larger than 9 µm.
 3.Anisocytes (cells with different sizes).
 Abnormal shape of RBCs –
 Sickle cell: Crescentic shape as in sickle cell anemia.
 Poikilocytosis: Unusual shapes due to deformed cell membrane.
 The shape will be of flask, hammer or any other unusual shape

PERIPHERAL BLOOD SMEAR.pptx preparation staining fixation

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PERIPHERAL BLOOD SMEAR.pptx preparation staining fixation