Examination%20of%20Peripheral%20Blood%20Smear

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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
• The cover glass smear.
• The wedge smear .
• The spun smear.
• The are two additional types of blood smear used
  for specific purposes
• Buffy coat smear for WBCs lt 1.0109/L
• 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
• Many smears can be done in just a single draw
• Immediate preparation of the smear is not
  necessary
• 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

• Wedge technique
• Coverslip technique
• Automated Slide Making
• and Staining

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Peripheral Smear Preparation

• Wedge technique
• Push Type wedge preparation
• Pull Type wedge prepartion
• Easiest to master
• Most convenient and most commonly used technique

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• Material needed
• Glass slide 3 in X 1in
• Beveled/chamfered edges

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Peripheral Smear Preparation
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Peripheral Smear Preparation

• Procedures
• 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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• 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
• 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

• Cold agglutinin
• RBCs will clump together.
• Warm the blood at 37 C for 5 minutes, and then
  remake the smear.
• Lipemia
• holes will appear in the smear.
• There is nothing you can do to correct this.
• Rouleaux
• RBCs 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

• 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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• 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
• Overall bluer color increased blood proteins
  (multiple myeloma, rouleaux formation)
• Grainy appearance RBC agglutination (cold
  hemagglutinin diseases)
• Holes increased lipid
• Blue specks at the feathery edge Increased WBC
  and Platelet counts

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• Microscopic
• 10x Objective
• 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
• Snow-plow effect more than 4x/cells per field on
  the feathery edge Reject
• Fibrin strands Reject
• Rouleaux formation, large blast cell assessment

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Too acidic Suitable
Too basic
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• Too Acid StainRBC too pale, WBC barely visible
• insufficient staining time
• prolonged buffering or washing
• old stain
• Correction
• lengthen staining time
• check stain and buffer pH
• shorten buffering or wash time

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• Too Alkaline StainRBC gray, WBC too dark,
  Eosinophil granules are gray
• thick blood smear
• prolonged staining
• insufficient washing
• alkaline pH of stain components
• heparinized sample
• Correction
• check pH
• shorten stain time
• 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
• humidity in the air as you air dry the slides.
• Water absorbed from the humid air into the
  alcohol based stain
• Solution
• Drying the slide as quickly as possible.
• Fix with pure anhydrous methanol before staining.
• Use of 20 v/v methanol

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• AUTOMATED SLIDE STAINERS
• It takes about 5-10 minutes to stain a batch of
  smears
• Slides are just automatically dipped in the stain
  in the buffer and a series of rinses
• Disadvantages
• Staining process has begun, no STAT slides can be
  added in the batch
• 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 physicians clinic
  only

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Manual differential
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Principle

• White Blood Cells
• Check for even distribution and estimate the
  number present (also, look for any gross
  abnormalities present on the smear).
• Perform the differential count.
• Examine for morphologic abnormalities.

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• Red Blood Cells, Examine for
• Size and shape ( Anisocytosis,Poikilocytosis
• Relative hemoglobin content.
• Polychromatophilia.
• Inclusions.
•  Rouleaux formation or agglutination
• Platelets.
• Estimate number present.
•  Examine for morphologic abnormalities.

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• Observations Under 10X
• Check to see if there are good counting areas
  available free of ragged edges and cell clumps.
• Check the WBC distribution over the smear.
• Check that the slide is properly stained.
• 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

• Use the oil immersion lens estimate the number of
  platelets per field.
• Look at 5-6 fields and take an average.
• Multiply the average by 20,000.
• Note any macroplatelets.

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• Platelets per oil immersion field (OIF)
• lt8 platelets/OIF decreased
• 8 to 20 platelets/OIF adequate
• gt20 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 gt 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 leukocytosis- neutrophilia
• Lymphocytic leukocytosis - lymphocytosis
• Eosinophilic leukocytosis - eosinophilia
• Monocytic leukocytosis - monocytosis
• Basophilic leukocytosis- basophilia

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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

• 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

• 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
• 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)
• 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. non-immune
  
• 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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