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Peripheral blood smear examination
(slide preparation and reporting)
Dr. Bahoran Singh
Moderator- Dr. Namrata Shetty
Role of peripheral blood examination
• Evaluation of anemia
• Evaluation of thrombocytopenia/ thrombocytosis
• Identificat...
• Objective:
1. Peripheral Smear Preparation
2. Staining of Peripheral Blood Smear
3. Peripheral Smear Examination
Periphe...
Peripheral Smear Preparation
• Wedge technique
• Coverslip technique
• Automated Slide Making and Staining
WEDGE BLOOD SMEAR
• Specimen:
 Peripheral blood smear made from EDTA-
anticoagulated blood.
 Smears should be made withi...
• Equipment
•Spreaders
• Clean slides
• Blood capillary tube or micropipette 10
µL
SMEAR PREPARATION
1. Place a drop of blood, about 2-3 mm
in diameter approximately 1 cm from
one end of slide.
2. Place th...
The shape of blood film
Characteristics of A Good Smear
1. Good smear is tongue shaped with a smooth
tail.
2. Does not cover the entire area of th...
The thickness of the smear
Is determined by:
1. The angle of the spreader slide. (the
greater the angle, the thicker and s...
The thickness of the spread
1. If the hematocrit is increased, the angle of
the spreader slide should be decreased.
2. If ...
Common causes of a poor blood
smear
1. Drop of blood too large or too small.
2. Spreader slide pushed across the slide in ...
5. Failure to push the spreader slide completely
across the slide.
6. Irregular spread with ridges and long tail: Edge
of ...
Blood films
Slide fixation and staining
Romanowsky staining
It includes:
• May-Grunwald –Geimsa stain,
• Jenner’s stain,
• Wright’s stain,
• Leishman’s stain and,...
Components of Giemsa stain
It mainly composed of
• Eosin Y and,
• Azure B- Methylene Blue
• Eosin-It is acidic component of stain and stain
basic component of cells like hemoglobin.
• Azure B – it is basic compon...
Materials
• Slide holder or rack
• Stain reagent
• Methanol : fix the cells on the slide
• Buffer:
• Used to maintain an a...
Methodology
• Put the smear into methanol jar and fix it for 1 -2 minute.
• Remove excess methanol from the smear
• Insert...
Colour responses of blood cells to
Romanowsky staining
• Cellular component Colour
• Nuclei Chromatin Purple
• Nucleoli Li...
Cytoplasm colour
• Lymphocyte Blue
• Metamyelocyte Pink
• Monocyte Grey–blue
• Myelocyte Pink
• Neutrophil Pink/orange
• P...
Granules
• Promyelocyte(primary granules) Red or purple
• Basophil Purple black
• Eosinophil Red–orange
• Neutrophil Purpl...
Other inclusions
• Auer body Purple
• Cabot ring Purple
• Howell-Jolly body Purple
• Döhle body Light blue
Factors influence smear staining
method
• Blood smear may be under or over stained based
on the following
• Concentration ...
Factors giving rise to faulty staining
• Appearances Causes
Too blue Eosin concentration too low
Incorrect preparation of...
– Too pink Incorrect proportion of azure
B-eosin Y
• Impure dyes
• Buffer pH too low
• Excessive washing in buffer
solution
• Pale staining Old staining solution
• Overused staining solution
• Incorrect preparation of
stock
• Impure dyes, especia...
• Neutrophil granules Insufficient azure B
not stained
• Neutrophil granules Dark Excess azure B
Blue/black (pseudo-toxic)...
• Stain deposit Stain solution left in
on film uncovered jar
Stain solution not filtered
• Blue background Inadequate fixa...
Peripheral blood smear examination
• Macroscopic view : quality of the smear
• Any abnormal particles present
• The Micros...
• find an area where the red cells are evenly
distributed and are not distorted.
• On high power(40x)-
• to obtain a WBC e...
Normal peripheral smear
Area too thin to examine the slide
Too thick area
Evaluation of PBS
• 1. RBC
• Size
• Shape
• Color
• Arrangement
• Inclusions
• 2. WBC
• Total counts
• Differential counts...
RBC
• In the blood from healthy person RBCs are
– Circular , Homogenous disc nearly of uniform
size(6–8 µm)
– deep pink cy...
Various changes in RBCs
1. COLOUR:
It is determined by hemoglobin content of RBC.
1. Normochromic- Normal intensity of sta...
Hypochromic
• Dcrease in Hemoglobin
content of RBC
• increase in central
pallor(>1/3rd)
• Decrease in MCH and
MCHC
• Seen ...
Hyperchromia
• Red cells stain
deeply
• Have less central
pallor,
• Increase in MCH
• Seen in
Megaloblastic
anemia
• Hered...
• Anisochromia – presence of hypochromic cells and
normochromic cells in the same film. Also called
dimorphic anemia.
• Se...
Dimorphic blood picture
Polychromatophillia
Blue grey tint of red cells
Due to Hb and
RNA(Residual) in young
cells.
• Larger than normal and
may l...
Variation In Size
• Anisocytosis- Variation in size of the red blood
cells
• Normal MCV is -80-100 fl
• Microcytes ( MCV <...
Microcytes
• Size of RBC is
reduced(<80fl)
• Seen when hemoglobin
synthesis is defective
1. Iron deficiency anemia
2. Thal...
Macrocytes
• When MCV of RBC is
Increased(>100fl)
• Macro- ovalocytes are
seen in Megaloblastic
anemia
• Myledysplastic
sy...
Shape
• Variation in shape is called Poikilocytosis.
• It is of following types-
• Elliptocytes
• Spherocytes
• Target cel...
Elliptocytes
• Elipitical in shapes
• Most abundant in
hereditary
elliptocytosis
• Seen in –
1. Iron deficiency anemia
2. ...
Spherocytes
• Nearly spherical
• Diameter is smaller than
normal
• Lack central pale area or
have a smaller , eccentric,
p...
Target cells
• Cells in which central
round stained area and
peripheral rim of
cytoplasm
• Seen in Thalassaemia
• Chronic ...
Schistocytes
• These are fragmaented
erythrocytes.
• Smaller than normal red
cells and of varying shape
• Seen in
• Geneti...
Acanthocytes
Red cells with small no of
spicules of inconstant length,
thickness and shape ,
irregularly disposed over the...
Echinocytes
• Also called crenated cells
• Numerous, short, regular
projection
• Commonly occur as an artifact
during prep...
Keratocytes
• Have pairs of spicules
either one or two pairs.
• Sometimes termed as
Bite cell or helmet cell
• Seen in
– M...
Leptocytes
• Thin red cells with large
unstained central area.
• Seen in
– Severe iron deficiency
anemia
– Thaleasaemia
Stomatocytes
• Red cells with central
biconcave area appears slit
like in dried film.
• Wet film it appears as cup-
shaped...
Sickle cell
• Cells are sickle (boat
shape) or crescent
shape
• Present in film of
patient with
homozygosity for Hb S.
• U...
Tear drop cells
• One side of cells is
tapered and other is
blunt.
• Seen in
– Myelofibrosis
– thalassemia
– Hemoglobin E
...
Structure
• Basophilic stippling (Punctate basophilia)
• Howell – jolly Bodies
• Cabot Rings
• Malarial Stippling
• Roulea...
Red blood cell inclusions
• Name of Inclusion Content
• Howell-Jolly body DNA
• Basophilic stippling RNA
• Pappenheimer bo...
Basophilic Stippling
• Presence of irregular basophilic
granules with in Rbc which are
variable in size .
• Stain deep blu...
Howell-Jolly Bodies
• Smooth single large round
inclusions which are remnant of
nuclear chromatin.
• Seen in
• Single –
– ...
Pappenheimer Bodies
• These are small single or
multiple peripherally sited
angular basophilic (almost
black) erythrocyte
...
Heinz bodies
• Seen on supravital stains
• Not seen on Romanowsky stain.
• Purple, blue, large, single or
multiple inclusi...
Cabot Rings
• These are Ring shaped
,figure of eight or loop
shaped
• Red or Reddish purple
with Wright’s stain and
have n...
Malrial stippling
• Fine granules of
plasmodium vivax
• On wright stain these
are fine , purplish red
• Red cells are larg...
Rouleaux Formation
• Alignment of red cells one
upon another so that they
resemble
stacks of coins.
• Occurs in
• Paraprot...
Agglutination
• It is more irregular and
round clumping than
linear rouleaux
• Seen with cold
agglutinin
• Anti RBC antibo...
WBC MORPHOLOGY
 Before evaluating leucocyte following must be
seen-
Film is well made
Distribution of cells is uniform
Staining is satis...
• Ten microscopic fields are examined in a vertical
direction from bottom to top or top to bottom
• Slide is horizontally ...
Manual differential counts
• These counts are done in the same area as
WBC and platelet estimates with the red cells
barel...
•If 10 or more nucleated RBC's (NRBC) are
seen, correct the
White Count using this formula:
Corrected WBC Count =
WBC x 10...
Leukocytes Normally Present in Blood
• GRANULOCYES
Neutrophils ( polymorphonuclear leucocytes)
Eosinophils
Basophils
• Agr...
POLYMORPHONUCLEAR
NEUTROPHILS
• 40 to 80 percent of total
WBC count(2.0–7.0 ×109/l )
• Diameter - 13 µm
• segmented nucleu...
Band forms
• neutrophils has either a
strand of nuclear material
thicker than a filament
connecting the lobes, or a U-
sha...
• Left-shift: non-
segmented neutrophil >
5%
– Increased
bands Means acute
infection, usually
bacterial
Granules
• Toxic granulation-
increase in staining
density and number of
granules
• Seen with Bacterial
infections and oth...
• Hypogranular and
agranular neutophils
poorly stained
• seen in Myelodysplastic
syndrome
Alder–Reilly anomaly
• Granules are large,
– discrete,
– stain deep red
– may obscure the
nucleus
– Neutrophil function
is...
Chédiak-Higashi Syndrome
• Granules are
– also seen in other
leukocytes like
lymphocytes
– Giant
– Scanty azurophilic
– fu...
Dohle Bodies
• Small, round or oval, pale
blue-grey structure
• Found at periphery of
neutrophil.
• Contains Ribosomes and...
May–Hegglin anomaly
• inclusions occur in all
types of leucocytes
except lymphocytes.
• contain small basophilic
cytoplasm...
Vacuoles in neutrophils
• In Fresh blood smear
• vacuoles seen in
– severe sepsis
– as an artifact with
prolonged standing
Nuclei
• Hypersegmentated
neutrophil
• def.-presence of
neutrophils with six or
more lobes or the
presence of more
than 3%...
Pelger–Huët Cells
• Pelger–Huët anomaly
• Benign inherited
condition.
• Neutrophil nuclei fail to
segment properly.
• Majo...
Pseudo-Pelger cells
• Pseudo-Pelger cells or
the acquired Pelger–
Huët anomaly
• Acquired condition
• Morphologically simi...
Pyknotic Neutrophils (Apoptosis)
• Small numbers of dead
or dying cells may
normally be found in
the blood
• seen in infec...
EOSINOPHILS
• Normally 1-6%( 0.02–0.5
× 109
/l)
• Size- 12–17 µm
• Nucleus- Bilobed
(spectacle shaped)
• Cytoplasm- Pale b...
Eosinopenia- seen with prolonged steroid
administration.
• Eosinophilia- allergic conditions hay fever,
asthama
• severe e...
BASOPHILS
• Rarest <1%
• Nucleus segments fold up
on each other resulting
compact irregular dense
nucleus(closed lotus flo...
MONOCYTES
• 2-10% of total wbc count
• Size- largest circulating leucocyte,
15–18µm in diameter
• Cytoplasm- grey blue
• N...
LYMPHOCYTES
• 20-40% of total wbc count
• two types
1. Small lymphocyte(6-10µm)
2. Large lymphocyte(12-15µm)
• Nucleus-sin...
Turk cells
• Türk’ cell (immunoblasts)-
Transformed lymphocyte seen
in bacterial and viral infection
• Size 10-15 µm
• Nuc...
Reactive lymphocytes
• Have slightly larger
nuclei with more
open chromatin
• Abundant cytoplasm
that may be
irregular.
• ...
Malignant lymphoid cells
• Commonest malignancy
is Chronic lymphocytic
leukemia- composed
almost exclusively of
small lymp...
• Lymphocytes predominate in the blood films
of infants and young children.
Platelates
• Size -1-3µm
• Normal count - 280 ±130×109/µl
• Non nucleated cells derived from cytoplasmic
fragments of Mega...
Thrombocytopenia
• Decreased production
– Aplastic anemia
– Acute leukemia
– Viral infections *Parvovirus *CMV
−Amegakaryo...
Thrombocytosis
• Reactive thrombocytosis
Post infection
Inflammation
Juvenile rheumatoid arthritis
Collagen vascular d...
Platelet morphology: Giant platelets
• Platelates seems to be
size of rbcs.
• Seen in
– May –Hegglin anomly
– Bernard Soul...
Peripheral Blood Cells
changes on prolonged storage of
sample
• EFFECT ON COUNT-
• Less marked in blood in ACD, CPD or Alsever’s solution
than in...
Changes in Morphology
• At 3 h changes start occure
• By 12–18 h these become striking
• RBC- progressive crenation and sp...
Changes in netrophils
Changes in monocytes
changes in lymphocytes
Apoptotic changes
• Cell shrinkage
• Cytoplasmic condensation
around the nuclear
membrane
• Indentations in the nucleus
• ...
Disadvantages of the Peripheral Blood
Smear
• Experience is required to make technically
adequate smears.
• Non-uniform di...
EXAMINATION OF BLOOD FILMS
FOR PARASITES
• thick film- when parasites are scanty
• thin film – identification of species
•...
Plasmodium falciparum
Erythrocytes throughout
this series are not
enlarged or distorted.
• Early trophozoites
• Accole for...
double chromatin dot
Schizonts are commonly seen in P. vivax infection and appear
as large bodies containing 12 to 24 nuclei and a loose
pigmen...
Ealy trophozoit in the form of thick ring with large chromatin
dot
Leishmaniasis (Leishman–Donovan
bodies)
African trypanosomiasis
(Trypanosoma brucei gambiense)
American trypanosomiasis ( T.
cruzi);
microfilaria
Thank you
Peripheral blood smear examination
Peripheral blood smear examination
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Peripheral blood smear examination

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Peripheral blood smear examination

  1. 1. Peripheral blood smear examination (slide preparation and reporting) Dr. Bahoran Singh Moderator- Dr. Namrata Shetty
  2. 2. Role of peripheral blood examination • Evaluation of anemia • Evaluation of thrombocytopenia/ thrombocytosis • Identification of abnormal cells (blasts/abnormal promyelocytes/atypical lymphoid) • Infections like malaria, microfilaria • Inclusions like basophilic stippling, Howell-Jolly bodies, Cabot ring
  3. 3. • Objective: 1. Peripheral Smear Preparation 2. Staining of Peripheral Blood Smear 3. Peripheral Smear Examination Peripheral Blood Smear
  4. 4. Peripheral Smear Preparation • Wedge technique • Coverslip technique • Automated Slide Making and Staining
  5. 5. WEDGE BLOOD SMEAR • Specimen:  Peripheral blood smear made from EDTA- anticoagulated blood.  Smears should be made within 1 hour of blood collection from EDTA specimens stored at room temperature to avoid distortion of cell morphology  Blood smears can also be made from finger prick blood directly onto slide.
  6. 6. • Equipment •Spreaders • Clean slides • Blood capillary tube or micropipette 10 µL
  7. 7. SMEAR PREPARATION 1. Place a drop of blood, about 2-3 mm in diameter approximately 1 cm from one end of slide. 2. Place the slide on a flat surface, and hold the other end between your left thumb and forefinger. 3. With your right hand, place the smooth clean edge of a second (spreader) slide on the specimen slide, just in front of the blood drop. 4. Hold the spreader slide at a 30°- 45 angle, and draw it back against the drop of blood 6. Allow the blood to spread almost to the edges of the slide. 7. Push the spread forward with one light, smooth moderate speed. A thin film of blood in the shape of tongue. 8. Label one edge with patient name, lab id and date. 9. The slides should be rapidly air dried by waving the slides or using an electrical fan.
  8. 8. The shape of blood film
  9. 9. Characteristics of A Good Smear 1. Good smear is tongue shaped with a smooth tail. 2. Does not cover the entire area of the slide. 3. Has both thick and thin areas with gradual transition. 4. Does not contain any lines or holes.
  10. 10. The thickness of the smear Is determined by: 1. The angle of the spreader slide. (the greater the angle, the thicker and shorter the smear). 2. Size of the blood drop. 3. Speed of spreading
  11. 11. The thickness of the spread 1. If the hematocrit is increased, the angle of the spreader slide should be decreased. 2. If the hematocrit is decreased, the angle of the spreader slide should be increased
  12. 12. Common causes of a poor blood smear 1. Drop of blood too large or too small. 2. Spreader slide pushed across the slide in a jerky manner. 3. Failure to keep the entire edge of the spreader slide against the slide while making the smear. 4. Failure to keep the spreader slide at a 30° angle with the slide
  13. 13. 5. Failure to push the spreader slide completely across the slide. 6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide 7. Holes in film: Slide contaminated with fat or grease and air bubbles. 8. Cellular degenerative changes: Delay in fixing, inadequate fixing time or methanol contaminated with water.
  14. 14. Blood films
  15. 15. Slide fixation and staining
  16. 16. Romanowsky staining It includes: • May-Grunwald –Geimsa stain, • Jenner’s stain, • Wright’s stain, • Leishman’s stain and, • Field’s stain.
  17. 17. Components of Giemsa stain It mainly composed of • Eosin Y and, • Azure B- Methylene Blue
  18. 18. • Eosin-It is acidic component of stain and stain basic component of cells like hemoglobin. • Azure B – it is basic component and stains the acidic component of cells like DNA and RNA (nucleus of WBC)
  19. 19. Materials • Slide holder or rack • Stain reagent • Methanol : fix the cells on the slide • Buffer: • Used to maintain an adequate pH. • 0.05M Na2PO4 (pH 6.4) • Distill water kept in glass bottle for at least 24hours (pH 6.4-6.8)
  20. 20. Methodology • Put the smear into methanol jar and fix it for 1 -2 minute. • Remove excess methanol from the smear • Insert the smear into Wright’s stain jar and leave the stain for 2 minutes. • Insert smear into a buffer jar and allow to stand for 4-8 minutes • Rinse thoroughly with a steam of distilled water • Allow to air dry • Note: time varies from manufacturers, thus ensure to follow the exact time in the kit manual of each procedure
  21. 21. Colour responses of blood cells to Romanowsky staining • Cellular component Colour • Nuclei Chromatin Purple • Nucleoli Light blue • Cytoplasm • Erythroblast Dark blue • Erythrocyte Dark pink • Reticulocyte Grey–blue
  22. 22. Cytoplasm colour • Lymphocyte Blue • Metamyelocyte Pink • Monocyte Grey–blue • Myelocyte Pink • Neutrophil Pink/orange • Promyelocyte Blue • Basophil Blue
  23. 23. Granules • Promyelocyte(primary granules) Red or purple • Basophil Purple black • Eosinophil Red–orange • Neutrophil Purple • Toxic granules Dark blue • Platelet Purple
  24. 24. Other inclusions • Auer body Purple • Cabot ring Purple • Howell-Jolly body Purple • Döhle body Light blue
  25. 25. Factors influence smear staining method • Blood smear may be under or over stained based on the following • Concentration of the stain used – Low concentration: pale coloured cells (under staining) – High concentration: dark stained smear (over stained) • Time of exposure the stain and the buffer – Too long: overstaining, – Too short: understaining
  26. 26. Factors giving rise to faulty staining • Appearances Causes Too blue Eosin concentration too low Incorrect preparation of stock stock stain exposed to bright daylight Batch of stain solution overused • Impure dyes • Staining time too short • Staining solution too acid • Smear too thick • Inadequate time in buffer solution
  27. 27. – Too pink Incorrect proportion of azure B-eosin Y • Impure dyes • Buffer pH too low • Excessive washing in buffer solution
  28. 28. • Pale staining Old staining solution • Overused staining solution • Incorrect preparation of stock • Impure dyes, especially azure A and/or C • High ambient temperature
  29. 29. • Neutrophil granules Insufficient azure B not stained • Neutrophil granules Dark Excess azure B Blue/black (pseudo-toxic) • Other stain anomalies Various contaminating dyes and metal salts
  30. 30. • Stain deposit Stain solution left in on film uncovered jar Stain solution not filtered • Blue background Inadequate fixation or prolonged storage before fixation • Blood collected into heparin as anticoagulant
  31. 31. Peripheral blood smear examination • Macroscopic view : quality of the smear • Any abnormal particles present • The Microscopic analysis • begins on lower power (10x), • to assess quality of the preparation • assess whether red cell agglutination, excessive rouleaux formation or platelet aggregation is present; • Assess the number, distribution and staining of the leucocytes; and
  32. 32. • find an area where the red cells are evenly distributed and are not distorted. • On high power(40x)- • to obtain a WBC estimate • All of the detailed analysis of the cellular elements using high power or oil immersion(100x).
  33. 33. Normal peripheral smear
  34. 34. Area too thin to examine the slide
  35. 35. Too thick area
  36. 36. Evaluation of PBS • 1. RBC • Size • Shape • Color • Arrangement • Inclusions • 2. WBC • Total counts • Differential counts • Abnormal WBC • 3. Platelets • Counts • Abnormality •4. Parasites
  37. 37. RBC • In the blood from healthy person RBCs are – Circular , Homogenous disc nearly of uniform size(6–8 µm) – deep pink cytoplasm with Central pallor <1/3rd
  38. 38. Various changes in RBCs 1. COLOUR: It is determined by hemoglobin content of RBC. 1. Normochromic- Normal intensity of staining. 2. Hypochromic- 3. Hyperchromic- • Normal Hb conc. Male-150±20 g/l • Female- 135±15 g/l
  39. 39. Hypochromic • Dcrease in Hemoglobin content of RBC • increase in central pallor(>1/3rd) • Decrease in MCH and MCHC • Seen in Iron Deficiency anemia • thalassaemia • hypochromia
  40. 40. Hyperchromia • Red cells stain deeply • Have less central pallor, • Increase in MCH • Seen in Megaloblastic anemia • Hereditary spherocytosis(MC H is normal but MCHC is increased)
  41. 41. • Anisochromia – presence of hypochromic cells and normochromic cells in the same film. Also called dimorphic anemia. • Seen in – Sideroblastic anemia – Some weeks after iron therapy for iron deficiency anemia – hypochromic anemia after transfusion with normal cells.
  42. 42. Dimorphic blood picture
  43. 43. Polychromatophillia Blue grey tint of red cells Due to Hb and RNA(Residual) in young cells. • Larger than normal and may lack central pallor. • Implies Reticulocytosis • Seen in • Hemolysis • Acute blood loss
  44. 44. Variation In Size • Anisocytosis- Variation in size of the red blood cells • Normal MCV is -80-100 fl • Microcytes ( MCV <80 fl) • Macrocytes (MCV >100fl) • Anisocytosis is a feature of most anemias.
  45. 45. Microcytes • Size of RBC is reduced(<80fl) • Seen when hemoglobin synthesis is defective 1. Iron deficiency anemia 2. Thalassemia 3. Anemia of chronic disease 4. Sideroblastic anemia
  46. 46. Macrocytes • When MCV of RBC is Increased(>100fl) • Macro- ovalocytes are seen in Megaloblastic anemia • Myledysplastic syndrome. • Round Macrocytes seen in Alcoholism, Liver disease.
  47. 47. Shape • Variation in shape is called Poikilocytosis. • It is of following types- • Elliptocytes • Spherocytes • Target cells • Schistocytes • Acanthocytes • Keratocytes • Echinocytes
  48. 48. Elliptocytes • Elipitical in shapes • Most abundant in hereditary elliptocytosis • Seen in – 1. Iron deficiency anemia 2. Myelofirosis with myeloid metaplasia 3. Megaloblastic anemia 4. Sickle cell anemia
  49. 49. Spherocytes • Nearly spherical • Diameter is smaller than normal • Lack central pale area or have a smaller , eccentric, pale area • Seen in – hereditary spherocytosis – Some cases of autoimmune hemolytic anemia – direct physical or chemical injury
  50. 50. Target cells • Cells in which central round stained area and peripheral rim of cytoplasm • Seen in Thalassaemia • Chronic liver disease • Hereditary hypo- betalipoproteinemia • Iron deficiency anemia • Hemoglobinopathies (Hb C, Hb H, Sickel cell anemia • Postsplenectomy
  51. 51. Schistocytes • These are fragmaented erythrocytes. • Smaller than normal red cells and of varying shape • Seen in • Genetic disorder – Thalassaemia – congential dyserythropoietic anemia. • Acquired disorder of RBC formation – Megaloblastic – Dyserythropoietic • Mechanical stress MAHA • Direct thermal injury
  52. 52. Acanthocytes Red cells with small no of spicules of inconstant length, thickness and shape , irregularly disposed over the surface. • Seen in Abnormal phospholipid metabolism • Abetalipoproteinemia • Inherited abnormalities of red cell membrane protein • Splenectomy
  53. 53. Echinocytes • Also called crenated cells • Numerous, short, regular projection • Commonly occur as an artifact during preparation of film • Hyperosmolarity • discocyte–echinocyte transformation • Overnight stored blood at 20 C before films are made. • Premature infant after exchange transfusion • water contaminating the Wright’s stain (or absolute methanol)
  54. 54. Keratocytes • Have pairs of spicules either one or two pairs. • Sometimes termed as Bite cell or helmet cell • Seen in – Mechanical damage – Removal of Heinz body by pitting action of spleen.
  55. 55. Leptocytes • Thin red cells with large unstained central area. • Seen in – Severe iron deficiency anemia – Thaleasaemia
  56. 56. Stomatocytes • Red cells with central biconcave area appears slit like in dried film. • Wet film it appears as cup- shaped. • Seen in – Artifact – south-east Asian ovalocytosis – liver disease, – alcoholism, – myelodysplastic syndromes.
  57. 57. Sickle cell • Cells are sickle (boat shape) or crescent shape • Present in film of patient with homozygosity for Hb S. • Usually absent in neonates and rare in patients with high Hb F percentage
  58. 58. Tear drop cells • One side of cells is tapered and other is blunt. • Seen in – Myelofibrosis – thalassemia – Hemoglobin E heterozygous + homozygous ●HbH disease ●HbC trait ●Hb Lepore heterozygous + homozygous ●HbO Arab disease ●HbD disease ●Iron defciency ●Hb Lepore trait
  59. 59. Structure • Basophilic stippling (Punctate basophilia) • Howell – jolly Bodies • Cabot Rings • Malarial Stippling • Rouleaux formation
  60. 60. Red blood cell inclusions • Name of Inclusion Content • Howell-Jolly body DNA • Basophilic stippling RNA • Pappenheimer body Iron • Heinz body(supravital only) Denatured hemoglobin • Crystals Hemoglobin-C • Cabot rings Mitotic spindle remnants • Nucleus DNA
  61. 61. Basophilic Stippling • Presence of irregular basophilic granules with in Rbc which are variable in size . • Stain deep blue with Wright’s stain • Fine stippling seen with – Increased polychromatophilia – Increased production of red cells. • Coarse stippling – Lead and heavy metal poisoning – Disturbed erythropoiesis • Megaloblastic anemia • Thalassaemia • infection • liver disease – Unstable Hb – Pyrimidine-5’-nucleotidase def.
  62. 62. Howell-Jolly Bodies • Smooth single large round inclusions which are remnant of nuclear chromatin. • Seen in • Single – – Megaloblastic anemia – Hemolytic anemia – Postsplenectomy • MULTIPLE – – Megaloblastic anemia – Abnormal erythropoiesis • Howell-Jolly Bodies
  63. 63. Pappenheimer Bodies • These are small single or multiple peripherally sited angular basophilic (almost black) erythrocyte inclusions. • Smaller than Howell–Jolly bodies. • composed of haemosiderin. • Their nature can be confirmed by Perls’ stain. • Seen in – Sideroblastic erythropoiesis – Hypospenism – Myelodysplastic syndrome – Hemolytic anemia
  64. 64. Heinz bodies • Seen on supravital stains • Not seen on Romanowsky stain. • Purple, blue, large, single or multiple inclusions attached to the inner surface of the red blood cell. • Represent precipitated normal or unstable hemoglobins. • seen – Postsplenectomy • Oxidative stress – Glucose-6-phosphate dehydrogenase deficiency, – Glutathione synthetase deficiency – Drugs – Toxins – Unstable hemoglobins
  65. 65. Cabot Rings • These are Ring shaped ,figure of eight or loop shaped • Red or Reddish purple with Wright’s stain and have no internal structure • Observed rarely in – Pernicious anemia, – Lead poisoning,
  66. 66. Malrial stippling • Fine granules of plasmodium vivax • On wright stain these are fine , purplish red • Red cells are larger than normal
  67. 67. Rouleaux Formation • Alignment of red cells one upon another so that they resemble stacks of coins. • Occurs in • Paraproteinemia ( monoclonal gammopathy) • Elevated plasma fibrinogen or globulin level
  68. 68. Agglutination • It is more irregular and round clumping than linear rouleaux • Seen with cold agglutinin • Anti RBC antibody • Autoimmune hemolytic anemia • Macroglobulinemia
  69. 69. WBC MORPHOLOGY
  70. 70.  Before evaluating leucocyte following must be seen- Film is well made Distribution of cells is uniform Staining is satisfactory  While scanning estimate the total leucocyte count  Differential count is done at oil immersion
  71. 71. • Ten microscopic fields are examined in a vertical direction from bottom to top or top to bottom • Slide is horizontally moved to the next field • Ten microscopic fields are counted vertically. • Procedure is repeated until 100 WBCS have been counted (zig zag motion) Scanning technique for WBC differential count and morphologic evaluation
  72. 72. Manual differential counts • These counts are done in the same area as WBC and platelet estimates with the red cells barely touching. • This takes place under × 100 (oil) using the zigzag method. • Count 100 WBCs including all cell lines from immature to mature. Reporting results • Absolute number of cells/µl = % of cell type in differential x white cell count
  73. 73. •If 10 or more nucleated RBC's (NRBC) are seen, correct the White Count using this formula: Corrected WBC Count = WBC x 100/( NRBC + 100) Example : If WBC = 5000 and 10 NRBCs have been counted Then 5,000× 100/110 = 4545.50 The corrected white count is 4545.50
  74. 74. Leukocytes Normally Present in Blood • GRANULOCYES Neutrophils ( polymorphonuclear leucocytes) Eosinophils Basophils • Agranulocytes Lymphocytes Monocytes
  75. 75. POLYMORPHONUCLEAR NEUTROPHILS • 40 to 80 percent of total WBC count(2.0–7.0 ×109/l ) • Diameter - 13 µm • segmented nucleus and pink/orange cytoplasm with fine granulation(0.2-0.3µm) stain tan to pink with Wright’s • Lobes -2-5 • Neutrophils usually have trilobed nucleus. • small percent has four lobes and occasionally five lobes.
  76. 76. Band forms • neutrophils has either a strand of nuclear material thicker than a filament connecting the lobes, or a U- shaped nucleus of uniform thickness. • Up to 8% of circulating neutrophils are unsegmented or partly segmented (‘band’ forms)
  77. 77. • Left-shift: non- segmented neutrophil > 5% – Increased bands Means acute infection, usually bacterial
  78. 78. Granules • Toxic granulation- increase in staining density and number of granules • Seen with Bacterial infections and other inflammation • Administration of G-CSF • Anaplastic anemia
  79. 79. • Hypogranular and agranular neutophils poorly stained • seen in Myelodysplastic syndrome
  80. 80. Alder–Reilly anomaly • Granules are large, – discrete, – stain deep red – may obscure the nucleus – Neutrophil function is Normal
  81. 81. Chédiak-Higashi Syndrome • Granules are – also seen in other leukocytes like lymphocytes – Giant – Scanty azurophilic – functional defect occur
  82. 82. Dohle Bodies • Small, round or oval, pale blue-grey structure • Found at periphery of neutrophil. • Contains Ribosomes and Endoplasmic reticulum • Seen in – Bacterial infection – inflammation – administration of G-CSF – during pregnancy
  83. 83. May–Hegglin anomaly • inclusions occur in all types of leucocytes except lymphocytes. • contain small basophilic cytoplasmic granules
  84. 84. Vacuoles in neutrophils • In Fresh blood smear • vacuoles seen in – severe sepsis – as an artifact with prolonged standing
  85. 85. Nuclei • Hypersegmentated neutrophil • def.-presence of neutrophils with six or more lobes or the presence of more than 3% of neutrophils with at least five lobes. • seen in Megaloblastic anemia – uraemia – iron deficiency. – Drugs-cytotoxic treatment with Methotrexate – hydroxycarbamide
  86. 86. Pelger–Huët Cells • Pelger–Huët anomaly • Benign inherited condition. • Neutrophil nuclei fail to segment properly. • Majority of circulating neutrophils have only two discrete equal-sized lobes connected by a thin chromatin bridge.
  87. 87. Pseudo-Pelger cells • Pseudo-Pelger cells or the acquired Pelger– Huët anomaly • Acquired condition • Morphologically similar to Pelger–Huët anomaly • seen in Myelodysplastic syndromes, • Acute myeloid leukaemia with dysplastic maturation, • Occasionally in chronic myelogenous leukaemia
  88. 88. Pyknotic Neutrophils (Apoptosis) • Small numbers of dead or dying cells may normally be found in the blood • seen in infections – invitro after standing for 12-18 hrs – Nuclei-round dense, featureless – Cytoplasm-dark pink
  89. 89. EOSINOPHILS • Normally 1-6%( 0.02–0.5 × 109 /l) • Size- 12–17 µm • Nucleus- Bilobed (spectacle shaped) • Cytoplasm- Pale blue • Granules - Coarse spherical gold/orange
  90. 90. Eosinopenia- seen with prolonged steroid administration. • Eosinophilia- allergic conditions hay fever, asthama • severe eosinophilia- parasitic infection – reactive eosinophilia – Eosinophilic leukaemia – Idiopathic hypereosinophilic syndrome – T-cell lymphoma, B-cell lymphoma and acute lymphoblastic leukaemia.
  91. 91. BASOPHILS • Rarest <1% • Nucleus segments fold up on each other resulting compact irregular dense nucleus(closed lotus flower like) • Granules-large, variable size dark blue or purple often obscure the nucleus • Granules are rich in histamine, serotonin and heparin • Increase in myeloproliferative disorder- CML
  92. 92. MONOCYTES • 2-10% of total wbc count • Size- largest circulating leucocyte, 15–18µm in diameter • Cytoplasm- grey blue • Nucleus- large , curved , horse shoe shape • No segmentation occur • Chromatin- fine evenly distributed • Increase in chronic infections and inflammatory conditions such as – Tuberculosis and Crohn’s disease, – Chronic myeloid leukaemias – Acute leukaemias with a monocytic component – Infectious mononucleosis
  93. 93. LYMPHOCYTES • 20-40% of total wbc count • two types 1. Small lymphocyte(6-10µm) 2. Large lymphocyte(12-15µm) • Nucleus-single, sharply defined, stain dark blue on Wright’s stain • Cytoplasm- Pale blue • Large lymphocytes less densely stain nuclei & abundant cytoplasm • Few round purple(azure) granules are present
  94. 94. Turk cells • Türk’ cell (immunoblasts)- Transformed lymphocyte seen in bacterial and viral infection • Size 10-15 µm • Nucleus- Round, • Large nucleolus, and abundant, deeply basophilic cytoplasm
  95. 95. Reactive lymphocytes • Have slightly larger nuclei with more open chromatin • Abundant cytoplasm that may be irregular. • Seen in -infectious mononucleosis – viral infections
  96. 96. Malignant lymphoid cells • Commonest malignancy is Chronic lymphocytic leukemia- composed almost exclusively of small lymphocytes. • Some times few larger nucleolated cells
  97. 97. • Lymphocytes predominate in the blood films of infants and young children.
  98. 98. Platelates • Size -1-3µm • Normal count - 280 ±130×109/µl • Non nucleated cells derived from cytoplasmic fragments of Megakaryocytes • Has purple red granules. • Liliac color
  99. 99. Thrombocytopenia • Decreased production – Aplastic anemia – Acute leukemia – Viral infections *Parvovirus *CMV −Amegakaryocytic thrombocytopenia (AMT) • Increased destruction – Immune thrombocytopenia – Idiopathic thrombocytopenic purpura (ITP) – Neonatal alloimmune thrombocytopenia (NAITP) – Disseminated intravascular coagulation (DIC) – Hypersplenism • Pseudothrombocytopenia- due to clumpping of pltelates in EDTA bulb
  100. 100. Thrombocytosis • Reactive thrombocytosis Post infection Inflammation Juvenile rheumatoid arthritis Collagen vascular disease • Essential thrombocythemia
  101. 101. Platelet morphology: Giant platelets • Platelates seems to be size of rbcs. • Seen in – May –Hegglin anomly – Bernard Soulier syndrome – Alport syndrome – Storage pool syndrome
  102. 102. Peripheral Blood Cells
  103. 103. changes on prolonged storage of sample • EFFECT ON COUNT- • Less marked in blood in ACD, CPD or Alsever’s solution than in EDTA. • At room temperature blood is stable up to 8 h. • RBC – Swell up the PCV and MCV increases – Osmotic fragility increases – Erythrocyte sedimentation rate decreases – At 4ͦ C up to 24 h – Reticulocyte count- Unchanged upto 24 h at 4 C – Hemoglobin Unchanged upto 2-3 days
  104. 104. Changes in Morphology • At 3 h changes start occure • By 12–18 h these become striking • RBC- progressive crenation and sphering • Netrophils- nuclei stain more homogeneously – Nuclear lobes may become separated – Cytoplasmic margin may appear ragged – Vacuoles appear in the cytoplasm – Lymphocytes and monocytes undergo similar changes
  105. 105. Changes in netrophils
  106. 106. Changes in monocytes
  107. 107. changes in lymphocytes
  108. 108. Apoptotic changes • Cell shrinkage • Cytoplasmic condensation around the nuclear membrane • Indentations in the nucleus • Followed by nuclear fragmentation. • Cell remnants form dense basophilic masses (the apoptotic bodies)
  109. 109. Disadvantages of the Peripheral Blood Smear • Experience is required to make technically adequate smears. • Non-uniform distribution of white blood cells • Larger leukocytes concentrated near edges and lymphocytes scattered throughout. • Non-uniform distribution of RBCs – Small crowded red blood cells at the thick edge – Large flat red blood cells without central pallor at the feathered edge
  110. 110. EXAMINATION OF BLOOD FILMS FOR PARASITES • thick film- when parasites are scanty • thin film – identification of species • STAINING OF FILM – by Leishman’s stain at pH 7.2
  111. 111. Plasmodium falciparum Erythrocytes throughout this series are not enlarged or distorted. • Early trophozoites • Accole form • Crescent (‘banana- shaped’) gametocyte
  112. 112. double chromatin dot
  113. 113. Schizonts are commonly seen in P. vivax infection and appear as large bodies containing 12 to 24 nuclei and a loose pigmented body. This photograph shows an early schizont of P. vivax on the left and mature schizonts
  114. 114. Ealy trophozoit in the form of thick ring with large chromatin dot
  115. 115. Leishmaniasis (Leishman–Donovan bodies)
  116. 116. African trypanosomiasis (Trypanosoma brucei gambiense)
  117. 117. American trypanosomiasis ( T. cruzi);
  118. 118. microfilaria
  119. 119. Thank you

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