Peripheral blood smear examination

(Slide preparation and reporting)

Caesar Abu Arra
MSc Life sciences
BSc Medical lab. Sciences
Medicare Labs
 Aim of blood smear

 Evaluation of anemia
 Infections
• bacteria, malaria, microfilaria..etc.
 Abnormal cells
• blasts, inclusions..etc.
 Cells morphology and count

Abu Jad Caesar

 Making blood films

 Three basic steps to make blood film:

 Preparation of blood smear.

 Fixation of blood smear.

 Staining of blood smear.

Abu Jad Caesar

 Preparation of blood smear

 Different types of blood smears:

 The wedge smear slide to slide method.
 The spun smear.
 Two additional types of blood smears (used for specific
purposes):
o Buffy coat smear for WBCs < 1.0×109/L.
o Thick blood smears for blood parasites .

Abu Jad Caesar

 Wedge blood smear

 Smears should be made within 1 hour of blood

collection from EDTA specimens stored at
room temperature to avoid distortion of cell
morphology.
 Small drop (10 µl)
 Hold the spreader slide at a 30°- 45° angle,
and draw it back against the drop of blood.
 Air dry at room temperature.

Abu Jad Caesar

Wedge blood smear

Abu Jad Caesar

 Procedure notes

1. The smear should be made without delay.

Any delay results in an abnormal distribution
of the white blood cells, with many of the
large white cells accumulating at the thin
edge of the smear.
2. HCT↑ ↓ The angle of the spreader slide
HCT↓ ↑ The angle of the spreader slide

Abu Jad Caesar

 Procedure notes

high HCT

small angle

low HCT

large angle
Abu Jad Caesar
 Spin method

 Automated method
 Place a drop of blood in the center of a glass slide.
 Spin at a high speed in a special centrifuge cytospin.
 Blood spreads uniformly.

Abu Jad Caesar

Characteristics of a Good Smear

 Thick at one end, thinning out to a smooth

rounded feather edge.
 Should occupy 2/3 of the total slide area.
 Should not touch any edge of the slide.

Abu Jad Caesar

Characteristics
tail of a Good
body head Smear

Abu Jad Caesar

Common causes of a poor blood smear

 Drop of blood too large or too small.

 Spreader slide pushed across the slide in a
jerky manner.
 Failure to keep the spreader slide at a 30°
angle with the slide.
 Failure to push the spreader slide completely
across the slide.

Abu Jad Caesar

Common causes of a poor blood smear

 Holes in film: Slide contaminated with fat or

grease
 Cellular degenerative changes:
delay in fixing
 Inadequate fixing time
Methanol contaminated with water.

Abu Jad Caesar

 Biologic causes of a poor smear

 Cold agglutinin - RBCs will clump together.

Warm the blood at 37° C for 5 minutes.
 Lipemia - holes will appear in the smear.
Nothing you can do to correct this.
 Rouleaux - RBC’s will form into stacks as coins.
Nothing you can do to correct this.

Abu Jad Caesar

 Staining of blood smear
 Romanowsky stain:
Any Combination of:

Eosin Y (Acidic or anionic dye):

o Binds to cationic sites (cytoplasm & Hb)

Methylene blue (Basic, cationic or Azure B dye):

o Binds to anionic sites (nucleus and RNA)

Abu Jad Caesar

 Staining of blood smear
 Buffer:
 Used to maintain an adequate pH.
 0.05M Na2PO4 (pH 6.4) or dH2O (pH 6.4-6.8)

Abu Jad Caesar

 pH of the phosphate buffer
 If the pH is too acidic:
RBC :Pinker
WBC nuclei : very pale staining (very pale purple)

 If the pH is too basic:

RBC :Grayish blue
WBC nuclei :very deeply purple

Abu Jad Caesar

pH of the phosphate buffer

Abu Jad Caesar

 Staining Troubleshooting
 Too Acid Stain:
Insufficient staining time
Prolonged buffering or washing
Old stain

 Correction:
 Lengthen staining time
 Check stain and buffer pH
 Shorten buffering or wash time
Abu Jad Caesar
 Staining Troubleshooting
 Too Alkaline Stain:
Thick blood smear
Prolonged staining
Insufficient washing
Alkaline pH of stain components

 Correction :
 Check pH
 Shorten stain time
 Prolong buffering time
Abu Jad Caesar
Abu Jad Caesar
 Blood film examination - preliminary

 Macroscopic view:
 Quality of the smear

 The microscopic view:

 Progressing from low power to high power:

Abu Jad Caesar

Blood film examination - preliminary

Abu Jad Caesar

 Blood film examination - preliminary

 Low-Power (10x) Scan

 Determine the overall staining quality of the blood
smear.

 Determine if there is a good distribution of the cells.

o Discussed later

 Find an optimal area for the detailed examination and

enumeration of cells

Abu Jad Caesar

 Blood film examination - preliminary

 High-Power (40x) Scan

 Determine the WBC estimate.
o WBCs are counted in 10 fields and averaged X 2000
o The estimate could be reported according to the table below:

Abu Jad Caesar

 Blood film examination - preliminary

• High-Power (40x) Scan

 Grading scale for WBC count (X103 cell/µl)

Low WBC High WBC

Mild 3-4.5 Mild 10-15

Moderate 2-3 Moderate 15-30

Severe <2 Marked 30-100

Extreme >100

Abu Jad Caesar

 Blood film examination - preliminary

 Oil Immersion (100x) Examination

 Perform a 100 WBC differential count.

 Correct WBC count that has greater than 10 NRBCs per

100 WBCs.
o Report as: WBC count was corrected due to presence of
NRBCs.

Abu Jad Caesar

 Blood film examination - preliminary

 Oil Immersion (100x) Examination

 to correct a WBC count (/µl):

Uncorrected WBC (/ ) X l

Abu Jad Caesar

 Blood film examination - preliminary

 Oil Immersion (100x) Examination

 Evaluate the RBCs for:
o Anisocytosis
o Poikilocytosis
o Inclusions
o Hypochromasia
o Polychromasia.

Abu Jad Caesar

 Blood film examination - preliminary

 Oil Immersion (100x) Examination

 Perform a platelet estimate and evaluate platelet
morphology:
o Platelets are counted in 10 fields and averaged:
• X 15  Automated smear.
• X 20  Wedge smear is used.

Abu Jad Caesar

 Blood film examination - preliminary

 Oil Immersion (100x) Examination

 Grading scale for platelets count (X103 cell/µl)
Low Platelets High Platelets
Mild 100-150 Mild 500-700

Moderate 50-100 Moderate 700-900

Severe <50 Severe 900-1000

Extreme >1000

Abu Jad Caesar

 Blood film examination - preliminary

 Oil Immersion (100x) Examination

 The absolute WBC count (/µl) may be determined:

Relative count (%) X Total WBC count (/µl)

Abu Jad Caesar

 Blood film examination - preliminary

 Relative vs. Absolute value:

 Relative value:
o Measures the percentage of corresponding WBC type in
peripheral blood.

Abu Jad Caesar

 Blood film examination - preliminary

 Relative vs. Absolute value:

 Absolute value:
o Measures the total count of corresponding WBC type /µl
in peripheral blood.
o Gives more meaningful information than the percentage.
o It is the preferred reporting method for the WBC
differential.
• Reported as absolute cytosis or absolute cytopenia.

Abu Jad Caesar

 Blood film examination - preliminary

 Relative vs. Absolute value:

Example: leukopenic patient and increased susceptibility
to infection and sepsis:

 WBC=5000 Relative Neutrophils=50%

o Absolute= 2500 cell/µl Normal

 WBC=2000 Relative Neutrophils=85%

o Absolute= 1700 cell/µl Low

Abu Jad Caesar

Abu Jad Caesar
 Examination of blood films
 Examination of blood films should include:

 RBC
 Size, Shape, color
 Hemoglobin distribution
 Arrangement and distribution
 Inclusions
 nucleated RBCs

Abu Jad Caesar

 Examination of blood films
 Examination of blood films should include:

 WBC
 Total counts
 Differential counts
 Abnormal and immature WBC

Abu Jad Caesar

 Examination of blood films
 Examination of blood films should include:

Platelets
 Counts should be verified by estimation
 Size
 Clumping

Abu Jad Caesar

 Examination of blood films
 Examination of blood films should include:

Parasites

Abu Jad Caesar

 Terms

 Pancytopenia
Deficiency in all the three cell lines RBC, WBC, and platelets.
 Aplastic anemia
 When report, recommend bone marrow assessment.

 Bicytopenia
Deficiency in two of the three cell lines
 Viruses and drugs

Abu Jad Caesar

 PARASITES

 Thick film When parasites are scanty

 Thin film  Identification of species

Abu Jad Caesar

 PARASITES

Plasmodium falciparum Microfilaria

Trypanosoma spp.
Abu Jad Caesar
 Platelets
Megakaryopoiesis

Abu Jad Caesar

 Platelets

 1 to 4 μm in diameter and vary in shape.

 Reddish-purple granules
 Life span 9-12 days
 One megakaryocyte can release several thousand platelets
(4000).
 Report as platelets are adequate with normal-sized ones
OR platelets are normal

Abu Jad Caesar

 Platelets

 Large and Giant platelets

Large platelets (4-7 µm)
oITP

Abu Jad Caesar

 Platelets

 Large and Giant platelets

Giant platelet (7-20 µm)
o Platelets seems to be size of RBC.
o Bernard Soulier syndrome

Abu Jad Caesar

 Platelets

 Large and Giant platelets

NOTE:
o When estimate, Large platelets and platelet aggregates
NOT counted.
Report as Occasional, Few, Many

Abu Jad Caesar

 Platelets

 Platelet anisocytosis:
Small large giant platelets will be seen
 Report as: Platelets anisocytosis ranging in size from
tiny to large giant platelets were seen.

Abu Jad Caesar

 Platelets

 Thrombocytosis
 Post infection and Inflammation
 Report as:
o Thrombocytosis (with grading).
o Platelets were estimated and approved to be about ( /µl)
 In case of microcytosis (RBC and PLT diameters are
similar) report as :
o Thrombocytosis due severe microcytosis.

Abu Jad Caesar

 Platelets

 Thrombocytopenia:
Could be due to:
o Decreased production Aplastic anemia
o Increased destruction ITP

Abu Jad Caesar

 Platelets

 Thrombocytopenia:
 In case of no aggregates report as:
o Thrombocytopenia (with grading).
o No platelet clumps are noted OR Negative for
microaggregates.
o Platelets were estimated and approved to be about ( /µl)

Abu Jad Caesar

 Platelets

 Pseudothrombocytopenia:
In vitro EDTA dependent phenomena
o Platelet clumping

Abu Jad Caesar

 Platelets

 Pseudothrombocytopenia:
In vitro EDTA dependent phenomena
o Platelet satellitism
• Platelets adhere to neutrophil surface

Abu Jad Caesar

 Platelets

 Pseudothrombocytopenia:
Repeat on new samples (EDTA and sodium citrated)
Scan for platelet clumping in thin and thick portion of
smear

Abu Jad Caesar

 Platelets

 Pseudothrombocytopenia:
Reporting criteria:
 Two samples were drawn to rule out EDTA- induced
pseudothrombocytopenia:
o EDTA smear revealed platelet clumps / Platelet
satellitism
o Na+ citrated smear revealed normal platelets count and
distribution.
 Conclusion: Platelets are adequate

Abu Jad Caesar

 RBC

Morphology of Normal RBC

 Biconcave disc
 Diameter 7 ~ 8 μm
 Central pallor occupy 3rd of total size
 Approx same as nucleus of mature lymphocyte
 Stained with eosin component of Romanowsky dyes

Abu Jad Caesar

Erythropoiesis

Abu Jad Caesar

 RBC Abnormality

 Examination of blood films for RBC’s should

include:
 Anisocytosis
 Poikilocytosis
 Hemoglobin distribution
 Arrangement and Distribution
 Inclusions
 NRBCs

Abu Jad Caesar

RBC Abnormality

Abu Jad Caesar

 Arrangement and Distribution
 Normal arrangement and distribution:
 The thin portion of the smear where RBC’s are slightly
separated from one another or at most, barely touching,
with no overlap.
 The thin area should represent at least 3rd of the entire
film.

Abu Jad Caesar

 Arrangement and Distribution
 Normal arrangement and distribution:
 The reviewer should avoid the thicker portion of the
slide where cells are overlapping and the edges of smear
where cells may be distorted in size, shape, and color.
o An exception is to be made when scanning for platelet
clumping

Abu Jad Caesar

 Arrangement and Distribution
 Rouleaux
 RBC’s appear as stacks of coins (linear)
 Due to ↑ Globulins or fibrinogens
 seen in:
o Multiple myeloma
o Macroglobulinemia

Abu Jad Caesar

 Arrangement and Distribution
 Agglutination
 More irregular and round clumping than linear rouleaux
 Seen in:
o Cold agglutinin
o Anti RBC antibodies
o Autoimmune HA
o Macroglobulinemia

Abu Jad Caesar

 Inclusions
 Basophilic stippling (Punctate basophilia)
 Aggregates of Ribrosomes.
 Multiple blue black inclusions (coarse or punctate )
 Seen in:
o Lead and arsenic poisoning
o Thalassemias
o Alcoholism
o Megaloblastic anemias

Abu Jad Caesar

 Inclusions
 Howell Jolly bodies
 Large round inclusions which are remnant of nuclear
chromatin
 Appear singly or doubly in an eccentric position on the
cell periphery
 Deep purple

Abu Jad Caesar

 Inclusions
 Howell Jolly bodies
 Seen in:
o Postsplenectomy
o Megalobalstic anaemia
o Haemolytic anaemia

Abu Jad Caesar

 Inclusions
 Pappenheimer Bodies

 Ferric compound complexed with protein

 Small dark blue bodies of uniform size.

Abu Jad Caesar

 Inclusions
 Pappenheimer Bodies
 Unlike basophilic stippling:
o Basophilic stippling appears homogeneously over the cell
whereas Pappenheimers tend to appear as single or clusters
in the cells periphery.
 Unlike Howell–Jolly bodies:
o Howell–Jolly bodies appear to be
larger

Abu Jad Caesar

 Inclusions
 Pappenheimer Bodies
 Seen in:
o Sideroblastic erythropoiesis
o Postsplenectomy
o Hemolytic anemia
o Thalassemia

Abu Jad Caesar

 Inclusions
 Heinz bodies
 Result of denatured or precipitated hemoglobin
 Purple, blue, large, single or multiple inclusions
attached to the inner surface of the red blood cell.

Abu Jad Caesar

 Inclusions
 Heinz bodies
 Stained by supravital stains
 Not Stained by Romanowsky stain.
 Seen in:
o Postsplenectomy
o oxidant stress of drugs and
chemicals

Abu Jad Caesar

 Inclusions
 Cabot rings
 Represent a part of the mitotic spindle, remnants of
microtubules, or a fragment of the nuclear membrane.
 Have no internal structure

Abu Jad Caesar

 Inclusions
 Cabot rings
 Red or reddish purple
 Could be appear in a figure-of-eight conformation

Abu Jad Caesar

 Inclusions
 Cabot rings
 Seen rarely in
o Megaloblastic anemias
o Postsplenectomy

Abu Jad Caesar

 Poikilocytosis
 Shape variation
 Mention the abnormal shapes rather than the term
poikilocytosis.
 Grading scale:

Abu Jad Caesar

 Poikilocytosis
 Spherocytes
 Loss of biconcavity
o ↓ Surface-to-volume ratio
o ↑ Osmotic fragility
 Smaller diameter
 Dense-staining
o No central pallor area

Abu Jad Caesar

 Poikilocytosis
 Spherocytes
 Seen in:
o Hereditary spherocytosis
o Immune hemolytic anemia

Abu Jad Caesar

 Poikilocytosis
 Target cell
 Codocytes
 ↑ membrane cholesterol and phospholipid and ↓ cellular
hemoglobin.
 ↑ surface-to-volume ratio

Abu Jad Caesar

 Poikilocytosis
 Target cell
 Seen in:
o Obstructive liver disease
o Iron deficiency anemia
o Thalassemia
o Post-transfusion

Abu Jad Caesar

 Poikilocytosis
 Leptocyte
 Synonymous with target cell, BUT:
o Red cells with large unstained central area.

Abu Jad Caesar

 Poikilocytosis
 Leptocyte
 Seen in:
o IDA
o Thalassemia

Abu Jad Caesar

 Poikilocytosis
 Elliptocyte and ovalocyte
 Elliptocytes
o Pencil, rod, or cigar shaped
o Hb appears to be concentrated on both ends of the cell
o Seen in IDA

Abu Jad Caesar

 Poikilocytosis
 Elliptocyte and ovalocyte
 Ovalocyte
o More egg-shaped
o Have a greater tendency to vary in their Hb content
o Seen in megaloblastic anemia

Abu Jad Caesar

 Poikilocytosis
 Elliptocyte and ovalocyte

Abu Jad Caesar

 Poikilocytosis
 Teardrop cell
 Dacrocytes
 Physiologic mechanism is unknown
 Seen in:
o IDA
o Pernicious anemia

Abu Jad Caesar

 Poikilocytosis
 Stomatocyte
 RBC with narrow slit-like area of central pallor
 Normal size, but are not biconcave
 Seen in:
o Acute alcoholism
o Hemolytic anemia
o Malignancies

Abu Jad Caesar

 Poikilocytosis
 Acanthocyte
Thorn Cells, Spur Cells
3 to 12 irregular, uneven length spicules distributed
along the periphery of the cell membrane

Abu Jad Caesar

 Poikilocytosis
 Acanthocyte
 Seen in:
o Severe hepatic disease
o Autoimmune HA
o Postsplenectomy
o Vitamin E deficiency

Abu Jad Caesar

 Poikilocytosis
 Echinocyte
 Burr Cells, crenated cells
 10 to 30 regular spicules, evenly placed over the surface
of RBC
 Considered pathologic and should be reported.

Abu Jad Caesar

 Poikilocytosis
 Echinocyte
 Seen in:
o Liver disease
o Renal disease
o Severe burns

Abu Jad Caesar

 Poikilocytosis
 Fragmented cells
 Several types
1- Schistocytes
2- Helmet Cells (bite cell) usually 2 projections
3- Keratocytes

Abu Jad Caesar

 Poikilocytosis
 Fragmented cells
 Sometimes counted as platelets
o Estimate platelets and report thrombocytosis due to presence
of RBC fragments
 Seen in : DIC, TTP, HUS

Abu Jad Caesar

 Poikilocytosis
 Fragmented cells
 Several types
4- Prekeratocyte (Blister cell) resemble a women’s handbag
o RBC’s containing a peripherally located vacuole.
o Keratocytes are RBC’s that have been caught on fibrin strands in
circulation, and rather than splitting, the cell hangs over the fibrin
fusing two sides of the cell together, creating a vacuole.
o Seen in:
 Oxidative stress G6PD deficiency

Abu Jad Caesar

 Anisocytosis
 Size variation
 Grading scale:

Abu Jad Caesar

 Anisocytosis
 Size variation
 Normocytic
 Microcytic
 Macrocytic

Abu Jad Caesar

 Anisocytosis
 Normocyte
 MCV 80-100 fl
 Diameter 7 to 8 μm

Abu Jad Caesar

 Anisocytosis
 Microcytes
 MCV <80 fl
 Diameter <7 μm
 Results from a defect in hemoglobin formation
 Seen in:
o IDA
o Thalassemia

Abu Jad Caesar

 Anisocytosis
 Macrocytes
 MCV >100 fl
 Diameter >9 μm
 Result from impaired DNA synthesis.
 Seen in:
 Megaloblastic anemia (↓ B12 and folic acid).
 Aplastic anaemia

Abu Jad Caesar

 Anisocytosis
 Anisocytosis is a feature of most anemias.
o Report as anisocytosis (with Qual. or Quan. Grading):

RDW: 16-18  Slight

RDW: 18-22  Moderate
RDW: >22 Marked or severe

Abu Jad Caesar

 Anisocytosis
 Mixture of large and small with normal MCV and high
RDW usually due:
Recent blood transfusion
Anemia or recovery stages of anemia.
o Report as Anisocytosis with dimorphic RBC population
(with grading):

RDW: 16-18  Slight

RDW: 18-22  Moderate
RDW: >22 Marked or severe

Abu Jad Caesar

 Hemoglobin Distribution
 MCHC used in conjunction with MCV used
do describe anemias
Normochromia
Hypochromia
Hyperchromia
Polychromasia
Anisochromia

Abu Jad Caesar

 Hemoglobin Distribution
 Normochromia: MCHC 32-36%
 Normal intensity of staining.
 The central pallor < 3µm (< 3rd of total size)

Abu Jad Caesar

 Hemoglobin Distribution
 Hypochromia : MCHC < 32%
 Unusually palely RBC (↓Hb content)
 The central pallor > 3µm (>1/3 rd of total size)
 Seen in:
o IDA
o Thalassemia

Abu Jad Caesar

 Hemoglobin Distribution
 Hypochromia : MCHC < 32%
 Grading:

Abu Jad Caesar

 Hemoglobin Distribution
 Hyperchromia : MCHC > 36%
 Decreased or absent central pallor
 Seen in:
o Sperocytosis (↓surface-to-volume ratio)
o hemolytic anemias (hemolysis caused by burns)

Abu Jad Caesar

 Hemoglobin Distribution
 Hyperchromia : MCHC > 36%
 Even though true hyperchromia does exist it is not
reported as such:
o It is reported in terms of the cell abnormalities resulting
from the increased volume of hemoglobin and the
decreased surface area (e.g spherocytes).

Abu Jad Caesar

 Hemoglobin Distribution
 Polychromasia
 Premature RBC in circulation called Polychromatophilic
cells actually reticulocytes.
 Gray-blue in color and usually larger than normal RBC
o Result of the residual RNA involved in Hb synthesis.

Abu Jad Caesar

 Hemoglobin Distribution
 Polychromasia
 Seen in:
o Acute and chronic blood loss.
o Recovering from anemias

Abu Jad Caesar

 Hemoglobin Distribution
 Polychromasia
 Grading excellent indicator of therapeutic effectiveness
when patient is given iron or vitamin therapy as treatment
of anemia

Abu Jad Caesar

 Hemoglobin Distribution
 Anisochromia
 Hypochromic cells and normochromic cells in the
same film
 Seen in:
o Development or recovering from anemias
o Post-transfusion

Abu Jad Caesar

 Hemoglobin Distribution
 MCHC used in conjunction with MCV used do
describe anemias

Abu Jad Caesar

 White blood cells
 WBC categories:
 Granulocytes (PMN):
o Neutrophils, Eosinophils and basophiles
o Granules in their cell cytoplasm.
o Multilobed nucleus
 Agranulocytes (MNC):
o Lymphocytes and monocytes.
o Do not have granules (contain non specific azurophilic
granules)
o Nonlobular nuclei.

Abu Jad Caesar

Neutrophil

Abu Jad Caesar

 Neutrophil
 Terms
 Shift to the left
o Release of younger granulocytes (specifically bands and
metamyelocytes from the bone marrow).

Abu Jad Caesar

 Neutrophil
 Terms
 Shift to the right
o An abnormal cell maturation situation that occurs when
hypersegmented cells are seen.
• It is indicative of vitamin B12 and/or folate deficiency.

Abu Jad Caesar

 Neutrophil
 Cytoplasm
o Pink
 Nucleus
o Dark purple blue
o Dense chromatin
o 3-5 lobes

Abu Jad Caesar

 Neutrophil
 Report as:
o Mature/normal appearing neutrophils.
o In case of leukocytosis neutrophilic leukocytosis
o Absolute / relative neutrophilia or neutropenia

Abu Jad Caesar

 Neutrophil
 Neutrophilia
o Acute bacterial infection.
o Many inflammatory processes.

 Neutropenia
o Typhoid fever
o Brucellosis
o Viral diseases.

Abu Jad Caesar

 Abnormal neutrophil
 Abnormal neutrophil morphology:
Band or stab forms
Hypersegmentation (≥ 6 lobes)
Vacuolization
Toxic granulation
Dohle bodies
Nuclear pyknosis and karyorrhexis

Abu Jad Caesar

 Band or Stab
 Cytoplasm
o Pink
 Nucleus
o U shaped nucleus of uniform thickness.
o Purplish red
o Dense chromatin

Abu Jad Caesar

 Band or Stab
 Normally form 5-10% of WBC’s
 Increased bands:
o Acute infection (usually bacterial)
o Non infectious inflammatory disease

Abu Jad Caesar

 Band or Stab
 Include it when calculate absolute neutrophil
 Report as:
o Many/Few neutrophilc bands OR with shift to the
left

Abu Jad Caesar

 Hypersegmentation
 Neutropils with ≥ 6 lobes
 Normally < 3% of WBC’s
 Seen in megaloblastic anemia

Abu Jad Caesar

 Hypersegmentation
 Report as:
o Few/many hypersegmented neutrophils OR with shift to
the right.
o Further hematological examinations are recommended
(B12 & folic acid).

Abu Jad Caesar

 Vacuolization
 Vacuoles in neutrophil contain ingested microorganisms.
 Seen in:
o Severe infection (leukemoid reaction)
o Non infectious inflammation

Abu Jad Caesar

 Toxic granulation
 ↑ staining density and number of granules
 Seen in:
o Bacterial infection and leukemoid reaction
o Other inflammation

Abu Jad Caesar

 Döhle bodies
 Pale blue inclusions at the periphery of the cytoplasm
 Strands of RER that have aggregated
o Bacterial infection and leukemoid reaction
o Inflammation

Abu Jad Caesar

 Nuclear pyknosis and karyorrhexis
 Degenerative changes in nucleus of neutrophil :
 Pyknosis: Condensation and shrinkage of cells through degeneration.
 Karyorrhexis: A necrotic stage with fragmentation of the nucleus,
whereby chromatin is distributed irregularly throughout the cytoplasm.

Abu Jad Caesar

 Nuclear pyknosis and karyorrhexis
 Due to:
 Prolonged time in circulation.
 Increased numbers in inflammatory and neoplastic disorders
 Old blood samples.
 New fresh sample is preferred.

Abu Jad Caesar

Nuclear pyknosis and karyorrhexis
 Could be mistaken with NRBC’s.

Abu Jad Caesar

Eosinophil

Abu Jad Caesar

 Eosinophil
 Cytoplasm
o Full of orange-red granules
 Nucleus
o Blue purple
o Coarsely granular chromatin
o 2 Lobes (like a pair of glass)

Abu Jad Caesar

 Eosinophil
 Eosinophilia
o Parasitic infection
o Allergic Disorders
o Eosinophilic leukemia

Abu Jad Caesar

Basophil

Abu Jad Caesar

 Basophil
 Cytoplasm
o Pale blue with coarse violet-blue granules
 Nucleus
o Blue purple
o Coarsely granular chromatin
o 2 Lobes

Abu Jad Caesar

 Basophil
 Basophilia
o Allergic Reactions
o Infections
Small pox
Chicken pox
Influenza

Abu Jad Caesar

Monocyte

Abu Jad Caesar

 Monocyte
 Cytoplasm
o Pale gray-blue
o Vacuoles and granules may be observed
 Nucleus
o Blue-purple
o large irregularly, folded, kidney shaped, ….etc

Abu Jad Caesar

 Monocyte

Often mistaken for a large lymphocyte

Abu Jad Caesar

 Monocyte
 Monocytosis
o Tuberculosis
o Brucellosis
o Malaria

Abu Jad Caesar

Lymphocyte

Abu Jad Caesar

 Lymphocyte
 Cytoplasm
o Light sky blue
 Nucleus
o Dark blue
o Dense chromatin
o Round

Abu Jad Caesar

 Lymphocyte
 Report as:
o Mature-appearing lymphocytes with homogenous chromatin
pattern (Resting lymphocytes).
o In case of leukocytosis Lymphocytic leukocytosis

Abu Jad Caesar

 Lymphocyte
 Lymphocytosis
o Lymphocytic leukemia
o Hepatitis
o Mumps
o Syphilis
 Lymphocytopenia
o Severe bacterial infection

Abu Jad Caesar

Lymphocyte

Abu Jad Caesar

 Abnormal lymphocyte
 Abnormal lymphocyte morphology:
Reactive lymphocytes
Smudge cells (Basket cell)
Turk cell (Transformed lymphocyte or immunoblast)
Atypical Lymphocytes (Malignant-appearing cell)

All should be reported

Abu Jad Caesar

 Reactive lymphocyte
 Normally < 10% of the total lymphocytes
 Nucleus:
o Slightly large with more open chromatin
 Cytoplasm:
o Abundant and irregular (↓N:C)

Abu Jad Caesar

 Reactive lymphocyte
 Seen in:
o Infectious mononucleosis
o Viral infections
 The term atypical should not be used interchangeably with
reactive lymphocyte.

Abu Jad Caesar

 Smudge cell
 Normally < 1%
 Remnants of cells that lack:
o Identifiable cytoplasmic membrane and Nuclear structure.
 Associated with abnormally fragile lymphocytes (mostly
CLL)
 Should be reported.

Abu Jad Caesar

 Turk cell
 Reactive lymphocyte with large nucleolus, abundant and
deeply basophilic cytoplasm
 Seen in bacterial and viral infection
 Round nucleus

Abu Jad Caesar

 Atypical Lymphocyte
 Malignant-appearing cells
 Discussed later

Abu Jad Caesar

 Leukemia
 Two major types according to cell maturity:
 Acute
o The malignant cells are immature (stem cells, blasts, or other
immature precursors.
 Chronic
o The cells are predominantly mature.

Abu Jad Caesar

 Leukemia
 Two major types according to cell lineage:
Myeloid (Myelogenous)
o Granulocytic leukemia
o Monocytic leukemia
o Megakaryocytic leukemia
o Erythrocytic leukemia
Lymphoid (Lymphocytic)

Abu Jad Caesar

 Leukemia
 So, four major types of leukemias:
 Acute myeloid leukemia (AML)
 Chronic myeloid leukemia (CML)
 Acute lymphoblastic leukemia (ALL)
 Chronic lymphocytic leukemia (CLL)

Abu Jad Caesar

Acute vs. Chronic

Abu Jad Caesar

 WBC Reporting
 WBC Reporting should includes:
Leukocytosis/Leukocytopenia.
Maturation stage
Absolute value of major cell.
Chromatin (fine, coarse, clumped)
Nucleolus ??
Amount of cytoplasm ??.

Abu Jad Caesar

 WBC Reporting
 WBC Reporting should includes:
For Leukemia, recommend flow cytometry for
immunological markers
For CML Recommend Philadelphia chromosome
(BCR–ABL fusion genepositive >95%)

Abu Jad Caesar

Granulocytopoiesis

Abu Jad Caesar

Granulocytopoiesis

Abu Jad Caesar

Granulocytopoiesis

Abu Jad Caesar

Granulocytopoiesis

Abu Jad Caesar

Erythropoiesis

Abu Jad Caesar

Lymphopoiesis

Abu Jad Caesar

Monopoiesis

Abu Jad Caesar

Thrombopoiesis

Abu Jad Caesar

 CML
 CML is characterized by three phases
Chronic phase
Accelerated phase
Blast phase

Abu Jad Caesar

 CML
 Chronic phase
 Neutrophilic leukocytosis
o From segmented neutrophils to occasional blasts (≤10%)
 But mainly mature

 Basophilia/eosinophilia

Abu Jad Caesar

 CML
 Chronic phase
 Thrombocytosis (~50% of cases).
 Normocytic anemia
 ↑ uric acid , LDH, Vitamin B12

Abu Jad Caesar

 CML
 Accelerated phase
 Worsening of anemia
 Basophils (>20%).
 Shift to the more immature myeloid forms (blasts 10-19%).

Abu Jad Caesar

 CML
 Accelerated phase
 Persistent thrombocytopenia ( 100 X 109/L)
 Persistent thrombocytosis ( 1000 X 109/L)

Abu Jad Caesar

 CML
 Blast phase
 Blast crisis (≥20%)
 Conversion of CML to AML

Abu Jad Caesar

CML

Abu Jad Caesar

 Leukemoid reaction
 Moderate or marked leukocytosis
Usually
o >30000 cell/µl
Absolute neutrophilia
Shift to the left
o Mostly neutrophilic metamyelocytes and band forms.
o Immaturity is similarly observed in the early stages of
CML

Abu Jad Caesar

 Leukemoid reaction
 Not a result of a leukemic disease
Infectious disease
o Bacterial (most common)
o Toxoplasma and viral (less common)
Other inflammatory process
o Uremia, acute gout and burns
Drug and chemical intoxication

Abu Jad Caesar

 Leukemoid reaction
 Reactive changes of neutrophils
 Usually appear as follow arrangement
1- Toxic granulation
o Nonspecific reactive changes
2- Döhle bodies
o Nonspecific reactive changes
3- Cytoplasmic vacuolization
o Strongly indicates a serious bacterial infection

Abu Jad Caesar

 Leukemoid reaction
 Reactive changes of neutrophils
 Usually appear as follow arrangement
1- Toxic granulation
o Nonspecific reactive changes
2- Döhle bodies
o Nonspecific reactive changes
3- Cytoplasmic vacuolization
o Strongly indicates a serious bacterial infection

Abu Jad Caesar

Leukemoid reaction

Abu Jad Caesar

 Leukemoid reaction
 Report as:
WBC immaturity.
Neutrophils reactive changes
Features in keeping with marked response to infectious
or inflammatory processes (Leukemoid reaction).

Abu Jad Caesar

 Mild infection
 Slight leukocytosis with neutrophilia
With or without the shift to the left.
Toxic granulation
 Report as:
Features in keeping with mild response to infection
or inflammatory process.

Abu Jad Caesar

 AML & ALL
 Mild to severe normocytic normochromic anemia
 ↓ platelet counts
 WBC count is variable (↓ to ↑↑↑)
 Auer rods in blasts cytoplasm (for AML)
 The blood smear usually reveals blasts or other immature
cells
 Circulating NRBC are occasionally seen

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

 AML & ALL
For AML

Abu Jad Caesar

 AML & ALL
For ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

AML & ALL

Abu Jad Caesar

 CLL
 Normochromic normocytic anemia
 ↑ Indirect serum bilirubin level
 Thrombocytopenia is not common.

Abu Jad Caesar

 CLL
 Lymphocytic leukocytosis From mature appearing
lymphocytes to prolymphocytes (10%)
o Small lymphocytes or slightly larger than a normal
lymphocyte and have a hypercondensed nuclear
chromatin pattern
 Smudge cells are common

Abu Jad Caesar

 CLL
Smudge cells

Abu Jad Caesar

 WBC Reporting for CLL
 WBC Reporting
o Leukocytosis/Leukocytopenia.
o Maturation stage
o Smudge cell
o Chromatin (fine, coarse, clumped)
o Nucleolus ?? (for prolymphocyte)
o Amount of cytoplasm ??.
o For definitive diagnosis, recommend flow cytometry for
immunological markers.

Abu Jad Caesar

 WBC Reporting for CLL
 WBC Reporting
o Mostly reported as: Mature-appearing lymphocyte with
hypercondensed nuclear chromatin pattern and few/many
prolymphocytes

Abu Jad Caesar

 CLL
 A lymphoblasts
 B lymphocytes
 C smudge cells

Abu Jad Caesar

THANK YOU

ABU JAD CAESAR