Transcript Examination of Peripheral Blood Smear

Examination of Peripheral
Blood Smear
Dr S Homathy
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Complete blood count
• The most common test used in clinical
medicine
• Determine type and severity of blood cell
abnormalities
• Nowadays, CBC is fully automated and highly
reproducible.
• Correct interpretation of automated CBC can
reduce rate of unnecessary blood smear
examination
• Provide useful information for provisional
diagnosis of RBC and WBC diseases
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A well Made and well Stained Smear can
provide:
• Estimates of cell count
• Proportions of the different types of WBC
• Morphology
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Preparation of blood smear
There are three types of blood smears:
1.The cover glass smear.
2.The wedge smear .
3.The spun smear.
The are two additional types of blood smear
used for specific purposes
1.Buffy coat smear for WBCs < 1.0×109/L
2.Thick blood smears for blood parasites .
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Peripheral Blood Smear
• Objective
1. Specimen Collection
2. Peripheral Smear Preparation
3. Staining of Peripheral Blood Smear
4. Peripheral Smear Examination
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Specimen Collection
• Venipuncture
– should be collected on an EDTA (Disodium or
Tripotassium ethylene diamine tetra-acetic acid)
Tube
– EDTA liquid form preferred over the powdered
form
– Chelates calcium
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Specimen Collection
Advantages
1. Many smears can be done in just a single draw
2. Immediate preparation of the smear is not
necessary
3. Prevents platelet clumping on the glass slide
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Specimen Collection
Disadvantages:
PLATELET SATELLITOSIS
• causes pseudothrombocytopenia and
pseudoleukocytosis
• Cause: Platelet specific auto antibodies that
reacts best at room temperature
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Specimen Collection
• Platelet satellitosis
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Peripheral Smear Preparation
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Wedge technique
Coverslip technique
Automated Slide Making
and Staining
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Peripheral Smear Preparation
Wedge technique
1. Push Type wedge preparation
2. Pull Type wedge prepartion
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Easiest to master
Most convenient and most commonly used
technique
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• Material needed
1. Glass slide 3 in X 1in
2. Beveled/chamfered edges
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Peripheral Smear Preparation
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Peripheral Smear Preparation
• Procedures:
1. Drop 2-3 mm blood at one end of the slide
Diff safe can be used
a. Easy dropping
b. Uniform drop
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Precaution:
Too large drop = too thick smear
Too small drop = too thin smear
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2. The pusher slide be
held securely with the
dominant hand in a
30-45 deg angle.
- quick, swift and
smooth gliding motion
to the other side of the
slide creating a wedge
smear
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• Control thickness of the smear by changing the
angle of spreader slide
• Allow the blood film to air-dry completely
before staining.
• Do not blow to dry.
• The moisture from your breath will cause RBC
artifact
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Peripheral Smear Preparation
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Peripheral Smear Preparation
Precautions:
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Ensure that the whole drop of blood is
picked up and spread
•
Too slow a slide push will accentuate
poor leukocyte distribution, larger cells are
pushed at the end of the slide
• Maintain an even gentle pressure on the
slide
• Keep the same angle all the way to the end
of the smear.
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Peripheral Smear Preparation
Precautions:
Angle correction:
1. In case of Polycythemia:
–
–
high Hct
angle should be lowered
- ensure that the smear made is not
to thick
2. Too low Hct:
–
Angle should be raised
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Feature of a Well Made Wedge Smear
• Smear is 2/3 or 3/4 the entire slide
• Smear is finger shaped, very slightly rounded at
the feathery edge:
– widest area of examination
• Lateral edges of the smear visible
• Should not touch any edge of the slide.
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• Should be margin free, except for point of application
• Smear is smooth without irregularities, holes or
streaks
• When held up in light:
– feathery edge should show rainbow appearance
• Entire whole drop of blood is picked up and spread
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Cover Slip Technique
• rarely used
• used for Bone marrow aspirate smears
• Advantage:
– excellent leukocyte distribution
• Disadvantage:
– labeling, transport, staining and storage is a
problem
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• 22 x 27mm clean coverslip
• More routinely used for bone marrow aspirate
• Technique:
1. A drop of marrow aspirate is placed on top of 1
coverslip
2. Another coverslip is placed over the other
allowing the aspirate to spread.
3. One is pulled over the other to create 1 thin
smears
4. Mounted on a 3x1 inch glass slide
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Precautions:
• Very light pressure should be applied
between the index finger and the thumb
• Crush preparation technique
• Too much pressure causes rupture of the cells
making morphologic examination impossible
• Too little pressure prevents the bone spicules
from spreading satisfactorily on the slide
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tail
body
head
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Thin area:
• Spherocytes which are really "spheroidocytes"
or flattened red cells.
• True spherocytes will be found in other (Good)
areas of smear.
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Thick area:
• Rouleaux, which is normal in such areas.
• Confirm by examining thin areas.
• If true rouleaux, two-three RBC's will stick
together in a "stack of coins" fashion.
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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.
5.Failure to push the spreader slide completely
across the slide.
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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
8.Cellular degenerative changes:
delay in fixing, inadequate fixing time or methanol
contaminated with water
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Biologic causes of a poor smear
1.Cold agglutinin:
– RBCs will clump together.
– Warm the blood at 37° C for 5 minutes, and then
remake the smear.
2.Lipemia:
– holes will appear in the smear.
– There is nothing you can do to correct this.
3.Rouleaux:
– RBC’s will form into stacks resembling coins.
There is nothing you can do to correct this
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• Automatic Slide Making and Staining
• SYSMEX 1000i
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Peripheral Smear Preparation
• Drying of Smears
– Fan
– Heating pans
– No breath blowing of smears – may produce
crenated RBCs or develop water artifact (drying
artifact)
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Slide fixation and
staining
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Romanowsky staining
Leishman's stain : a polychromatic stain
• Methanol : fixes cells to slide
• methylene blue stains RNA,DNA
blue-grey color
• Eosin stains hemoglobin, eosin granules
orange-red color
• pH value of phosphate buffer is very important
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• Pure Wright stain or Wright Giemsa stain
– Blood smears and bone marrow aspirate
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Procedure
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Thin smear are air dried.
Flood the smear with stain.
Stain for 1-5 min.
Experience will indicate the optimum time.
Add an equal amount of buffer solution and mix the
stain by blowing an eddy in the fluid.
• Leave the mixture on the slide for 10-15 min.
• Wash off by running water directly to the centre of
the slide to prevent a residue of precipitated stain.
• Stand slide on end, and let dry in air.
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Features of a well-stained PBS
• Macroscopically: color should be pink to
purple
• Microscopically:
RCS: orange to salmon pink
WBC: nuclei is purple to blue
cytoplasm is pink to tan
granules is lilac to violet
Eosinophil: granules orange
Basophil: granules dark blue to black
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•
1.
2.
3.
4.
Optimal Assessment Area:
RBCs are uniformly and singly distributed
Few RBC are touching or overlapping
Normal biconcave appearance
200 to 250 RBC per 100x OIO
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Trouble shooting
• Macroscopic
1. Overall bluer color: increased blood proteins
(multiple myeloma, rouleaux formation)
2. Grainy appearance: RBC agglutination (cold
hemagglutinin diseases)
3. Holes: increased lipid
4. Blue specks at the feathery edge: Increased
WBC and Platelet counts
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Microscopic:
10x Objective
1. Assess overall quality of the smear i.e feathery
edge, quality of the color, distributin of the cells
and the lateral edges can be checked for WBC
distribution
2. Snow-plow effect: more than 4x/cells per field
on the feathery edge: Reject
3. Fibrin strands: Reject
4. Rouleaux formation, large blast cell assessment
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Too acidic
Suitable
Too basic
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Too Acid Stain:RBC too pale, WBC barely
visible
1.insufficient staining time
2.prolonged buffering or washing
3.old stain
Correction:
1)lengthen staining time
2)check stain and buffer pH
3)shorten buffering or wash time
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Too Alkaline Stain:RBC gray, WBC too dark,
Eosinophil granules are gray
1.thick blood smear
2.prolonged staining
3.insufficient washing
4.alkaline pH of stain components
5.heparinized sample
Correction :
1.check pH
2.shorten stain time
3.prolong buffering time
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Problem encountered during
staining
Water artifact:
• moth eaten RBC,
• heavily demarcated central pallor on the
RBC surface,
• crenation,
• refractory shiny blotches on the RBC
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• What contributes to the problem:
1. humidity in the air as you air dry the slides.
2. Water absorbed from the humid air into the
alcohol based stain
• Solution:
1. Drying the slide as quickly as possible.
2. Fix with pure anhydrous methanol before
staining.
3. Use of 20% v/v methanol
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• AUTOMATED SLIDE STAINERS
1. It takes about 5-10 minutes to stain a batch of
smears
2. Slides are just automatically dipped in the
stain in the buffer and a series of rinses
• Disadvantages:
1. Staining process has begun, no STAT slides
can be added in the batch
2. Aqueous solutions of stains are stable only
after 3-6 hours
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QUICK STAINS
• Fast, convenient and takes about 1 minute to
be accomplished
• Modified Wrights-Giemsa Stain, buffer is
aged distilled water
• Cost effective
Disadvantage:
Quality of stains especially on color acceptance
For small laboratories and for physician’s clinic
only
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Manual differential
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Principle
White Blood Cells
1.Check for even distribution and estimate the
number present (also, look for any gross
abnormalities present on the smear).
2.Perform the differential count.
3.Examine for morphologic abnormalities.
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Red Blood Cells, Examine for :
1.Size and shape ( Anisocytosis,Poikilocytosis
2.Relative hemoglobin content.
3.Polychromatophilia.
4.Inclusions.
5. Rouleaux formation or agglutination
Platelets.
1.Estimate number present.
2. Examine for morphologic abnormalities.
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Observations Under 10X
1.Check to see if there are good counting areas
available free of ragged edges and cell clumps.
2.Check the WBC distribution over the smear.
3.Check that the slide is properly stained.
4.Check for the presence of large platelets,
platelet clumps, and fibrin strands.
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WBC estimation Under 40X
• Using the × 40 high dry with no oil.
• Choose a portion of the peripheral smear
where there is only slight overlapping of the
RBCs.
• Count 10 fields, take the total number of white
cells and divide by 10.
• To do a WBC estimate by taking the average
number of white cells and multiplying by
2000.
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Platelet estimation Under 100X
1.Use the oil immersion lens estimate the
number of platelets per field.
2.Look at 5-6 fields and take an average.
3.Multiply the average by 20,000.
4.Note any macroplatelets.
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Platelets per oil immersion field (OIF)
1)<8 platelets/OIF = decreased
2)8 to 20 platelets/OIF = adequate
3)>20 platelets/OIF = increased
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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
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• 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
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Determine a quantitative scale
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• Left-shift: non-segmented neutrophil > 5%
– Increased bands Means acute infection, usually
bacterial
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• Right-shift: hypersegmented
neutrophil
• Increased hypersegmented neutrophile
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• Leukocytosis, a WBC above 10,000, is usually
due to an increase in one of the five types of
white blood cells
Neutrophilic leukocytosisneutrophilia
Lymphocytic leukocytosis - lymphocytosis
Eosinophilic leukocytosis - eosinophilia
Monocytic leukocytosis monocytosis
Basophilic leukocytosisbasophilia
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Morphology of WBC
• Normal blood smear
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Segmented neurophile
• Diameter: 12-16
• Cytoplasm : pink
• Granules: primary
secondary
• Nucleus: dark purple blue
• dense chromatin
• 2-5 lobes
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Eosinophil
• One eosinophil - mature. Normal blood 100X.
• Orange colour granules.
• Bi-lobed nucleus
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Basophil
•One mature basophil.
•Blackish granules overlying the nucleus
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Normal lymphocytes
• Lymphocytes are the smallest WBC.
• They have large condensed nucleus, with a
scanty bluish cytoplasm.
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Normal monocyte
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Monocytes are the largest WBC.
The nucleus is slightly indented .
The cytoplasm is abundant, sky blue in colour.
Some have vacuoles in the cytoplasm.
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Red cells
• Normal red cells or erythrocytes show only
slight variation in size and shape.
• The blood film should be examined in the area
where the red cells are touching but not often
overlapping.
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•In this area many red cells
have an area of central
pallor which may be up to a
third of the diameter of the
cell.
•This is consequent on the
shape of a normal red cell,
which resembles a disc that
is thinner in the centre.
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Summarizing RBC Parameters
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RBC Count )RBC x 1012/L)
Hb (g/dl)
Hct (5 or L/L)
Mean Cell Volume (MCV. Fl)
Mean Cell Hb (MCH, pg)
Mean Cell Hb Concentration (MCHC. %, g/dl)
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• RBC distribution
• Morphology
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Size
Shape
Inclusions
Young rbcs
Color
Arrangement
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Platelets
• Normal platelets are also apparent.
• They are small anuclear fragments between the
red cells containing small purple-staining
granules.
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Platelet aggregates
• Platelet aggregates may be
composed of
– apparently intact platelets,
– degranulated pale grey platelets
– or a mixture of both, as in this
example.
– If the platelet count is low it is
essential to examine the blood
film carefully for platelet
aggregates
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Platelet satellitism
• Platelet satellitism describes
the phenomenon of adherence
of platelets to white cells.
• It is an in vitro phenomenon of
no clinical significance.
• However it is important that it
is detected since the platelet
count will be factitiously low.
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Staining of Peripheral Blood Smear
HEMA-TEK STAINER
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Component of automated CBC
Blood count basic parameters:
Hb, Hct,RBC, WBC, platlet.
• Red cell indices: MCV, MCH, MCHC, RDW
• WBC differentials
• Cytogram or Scattergram
• Reticulocyte count
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• Red cell parameters
Direct Measurement
• Erythrocyte Concentration (RBC) x 106/ml
• Mean Corpuscular Volume (MCV) Femtolitre (fl)
• Hemoglobin (Hb) Gram/decilitre (g/dl)
Indirect Measurement
• Hematocrit (Hct) = RBC x MCV/10 %
• Mean Corpuscular Hemoglobin (MCH) = HB x 10 / RBC
(pg)
• Mean Corpuscular Hemoglobin Concentration (MCHC) =
Hb/Hct x 100 (g/dl)
• Red Cell Distribution Width (RDW) %
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Reticulocyte count
• Reticulocyte =
– non-nucleated RBC with polyribosomal RNA
– as stained by supravital stain (new methylene blue or
brilliant cresyl blue)
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Polychromasia underestimates Reticulocytes
Three methods of reticulocyte Enumeration
Manual count on slide per 1,000RBC
Automated CBC with reticulocyte counter
(Coulter VCS, Cell-Dyne 4000, Technicon-H3)
• Flow cytometry with fluorescent dyes
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• RBC disorders
Hypochromic microcytic anemia
• Iron deficiency anemia
• Thalassemia and hemoglobinopathy
Macrocytic anemia
• Megaloblastic anemia
• Non-megaloblastic macrocytic anemia
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Hemolytic anemia
• Immune hemolytic anemia: AIHA, DHTR
• Microangiopathic hemolytic anemia (MAHA)
• Red cell enzymopathies: G-6-PD deficiency
• RBC membrane defects: spherocytosis,
• ovalocytosis, elliptocytosis, stomatocytosis
RBC inclusion bodies and parasites
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WBC disorders
Leukopenia
• with absolute neutropenia: bone marrow
failure, agranulocytosis
• with atypical lymphocytes: viral infection,
chronic lymphoproliferative disorders
• with immature myeloid cells: acute leukemia,
MDS or myelopthisis
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Leukocytosis
• Reactive leukocytosis: leukemoid reaction
• Acute leukemia: AML vs. ALL
• Chronic myeloproliferative disorders
• Chronic lymphoproliferative disorders
Leukoerythroblastosis
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Platelet disorders
Quantitative disorders
• Isolated thrombocytopenia: Immune vs. nonimmune
• Thrombocytopenia associated with other
hematologic abnormalities
• Thrombocytosis
Qualitative disorders
• Giant platelets (megathrombocytes)
• Platelet inclusion or granule abnormality
• Bizarre in shape and size
• Megakaryocytes or megakaryoblasts
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