Peripheral blood smear

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Peripheral blood smear

• Dr Ajit Nambiar

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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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Selection of Site

• Very important to get adequate volume
• Will be decided by
• Age of the child Infants and Neonates HEEL
  stick puncture(Lateral plantar bottom surface)
• Older child gt one yr age finger prick
  adults-finger prick or venepuncture
• Equipment available eg routine needle syringe
  vacutainers, etc

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Specimen Collection

• Venipuncture
• should be collected on an EDTA Tube
• EDTA liquid form preferred over the powdered
  form
• Chelates calcium
• Disodium or Tripotassium ethylenediamine
  tetra-acetic acid

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In What to collect blood ?
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HEMOLYSIS OF SAMPLES

• Contact with water
• Excessive heat or cold
• Rigorous mechanical injury to RBS ( thin gauge )
• Prolonged storage
• Hemolyzed samples will give erroneous results

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PREVENTION OF HEMOLYSIS

• Equipment used to collect absolutely dry
• Minimum constriction of limb
• Use correct gauge needle
• Collect slowly and steadily
• Remove needle and put immediately into the bulb
• Easier if collection in Vacutainer

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Collection of blood

• Specimen EDTA blood within 2 to 3 hours
  collected to the mark on tube.
• Not's May change RBCs morphology such as
  Spiculated (crenated) cells if
• Excessive amount of anticoagulant to specimen
• Old blood - long standing.
• Warm environment (room temperature) may hasten
  changes.

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Procedure of making smear

• placing a drop of blood from mixed sample on a
  clean glass slide.
• Spreader slide using another clean glass slide at
  30-45 degree angle.
• 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

• Procedures
• Drop 2-3 mm blood at one end of the slide
• Diff safe Blood Dispenser can be used
• a. Easy dropping
• b. Uniform drop

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Peripheral Smear Preparation

• Precaution Too large drop too thick
  smear
• Too small drop too thin smear

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Peripheral Smear Preparation

• 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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Peripheral Smear Preparation
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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.
• Should be margin free, except for point of
  application.

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tail body head
TAIL end of smear
Jujunction of body and tail
Head end of 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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MORPHOLOGIC CHANGES DUE TO AREA OF SMEAR

• Thin area- Spherocytes which are really
  "spheroidocytes" or flattened red cells. True
  spherocytes will be found in other (Good) areas
  of smear.
• 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 spreader slide at the right
   degree of angle with the slide.
4. Failure to push the spreader slide completely
   across the slide.
5. Irregular spread with ridges and long tail Edge
   of spreader dirty or chipped dusty slide
6. Holes in film Slide contaminated with fat or
   grease
7. Cellular degenerative changes delay in fixing,
   inadequate fixing time or methanol contaminated
   with water.

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Slide Fixation Staining

• LEISHMAN'S STAIN

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Principle like Romanowsky Principle

• 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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Staining 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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too acidic suitable too basic
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Causes correction

• 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

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• Too Alkaline Stain
• thick blood smear
• prolonged staining
• insufficient washing
• alkaline pH of stain components
•  Correction
• check pH
• shorten stain time
• prolong buffering time

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Staining of Peripheral Blood Smear
HEMA-TEK STAINER
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Performing A Manual differential And assessing
blood cell Morphology
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Principle

• White Blood Cells.
• estimate the number present .
• Perform the differential count.
• Examine for morphologic abnormalities.

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Principle

• Red Blood Cells, Examine for
• Size and shape.
• Relative hemoglobin content.
• Polychromatophilia.
• Inclusions.
•  Rouleaux formation or agglutination
• parasites

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Principle

• Platelets.
• Estimate number present.
•  Examine for morphologic abnormalities.

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Observing direction
Observe one field and record the number of WBC
according to the different type then turn to
another field in the snake-liked direction avoid
repeat or miss some cells
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RBC abnormalities
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Normal RBCs

• Round, elastic, non-nucleated, bi-concave discs
• Many RBCs have an area of central pallor which
  covers about one-third of the cell.
• The pallor occurs as a result of the disc-shaped
  cells being spread on the slide.

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Normal RBCs

• Average diameter of 7.2 microns with a range of
  6-9 microns, almost the same size as the nucleus
  of a small lymphocyte,

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Size Variation
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Size variation

• Red blood cells can vary in size from smaller
  than normal, microcytes, to larger than normal,
  macrocytes.
• When red cells of normal size, microcytes and
  macrocytes are present in the same field, the
  term anisocytosis is used.

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Normal size

• Size of normal RBC is almost the size of the
  nucleus of the lymphocyte.

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Microcyte

• Smaller than a nucleus of the lymphocyte, central
  pallor is greater than 1/3 of the cell

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Microcyte, increased central pallor
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Microcyte, normal Hb content
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Microcytes
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summary

• microcytes have a diameter of less than 7 microns
  and an MCV of less than 80 cubic microns.
• Two types of microcytes can be seen, those with
  increased central pallor and those with normal
  central pallor.

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Macrocyte (megalocyte)

• diameter of 9-14 microns (1.5 - 2 times larger
  than normal red cells)
• MCV is 100 cubic microns or more.

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Macrocytes (Megalocytes)

• Megalocytes are the result of decreased DNA
  synthesis, frequently due to vitamin B12 and/or
  folic acid deficiencies.
• Decreased DNA synthesis causes the nucleus in the
  developing red cells to mature at a slower than
  normal rate.
• Since hemoglobin production is not affected, the
  mature red cell is larger than normal

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Macrocytes
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Pseudomacrocytes

• appears larger than the lymphocyte but in
  contrast to megalocytes has an area of central
  pallor.
• size is the result of exaggerated flattening and
  thus the presence of the central pallor.
• in patients with cirrhosis of the liver,
  obstructive jaundice, post splenectomy.

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Psudomacrocytes
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Summary

• two types of macrocytes-
• True macrocytes (megalocytes). Increased MCV, MCH
• Pseudomacrocytes. Normal MCV, MCH

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Anisocytosis

• Increased variation in size of the red cell
  population present on a blood smear.
• Normal, small and large cells can be seen in one
  field.
• Normal MCV, high RDW
• As the severity of the anemia increases, the
  amount of significant anisocytosis present also
  increases.

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Anisocytosis
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Anisocytosis
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RBCs
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RDW

• RDW is an expression of the homogeneity of the
  RBC population size.
• A large RDW says there's a wide variation in the
  RBC diameters within the test pool. 
• It doesn't say the cells are large or small,
  rather that the population is not homogenous. 
• Younger cells are larger (reticulocytes).
• Older RBCs are smaller.

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RDW
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Microcytic Anemia
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Macrocytic Anemia
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RBC Color
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RBC Color

• Erythrocytes, when spread on a glass slide, show
  varying degrees of central pallor
• This central pallor is related to the hemoglobin
  concentration present in the red cells.

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RBC Color

• the central area (1/3 of the cell) is white,
  while buff-colored hemoglobin is visible in the
  outer 2/3 of the cell.
• The MCHC (32-36 gm/dl) is the index value which
  is used to verify the presence of adequate
  hemoglobin concentration in the cells visible on
  the peripheral smear.

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RBC Color

• A decreased amount of hemoglobin is referred to
  as hypochromasia or hypochromia.
• MCHC values of 30 or less reflect this
  condition.
• Hyperchromasia and hyperchromia, refer to a
  hypothetical situation rather than an actual
  occurrence.

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RBC Color

• Cells located in the "too thin" portion of the
  smear often appear to be "hyperchromic".
• Megalocytes (macrocytes) are normochromic.

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Normochromic cells
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Hypochromic cells
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Hyperchromia
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Hypochromia
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Polychromasia
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Poikilocytosis
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Poikilocytosis

• Variations in shape.

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Acanthocytes

• 3-12 thorn-like projections irregularly spaced
  around the cell.
• Smaller than normal and have little or no central
  pallor.
• Acanthocytes have an excess of cholesterol
• Large numbers of these cells on a smear can be of
  diagnostic significance.

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Acanthocytes

• Abetalipoproteinemia
• Hereditary acanthocytosis,50 100 of blood
  cells.
• Alcoholic cirrhosis
• lipid disorders
• splenectomy

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Acanthocyte
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TARGET CELLS (Codocyte)

• Target cells are thin-walled cells showing a
  darkly-stained centre area of hemoglobin which
  has been separated from the peripheral ring of
  hemoglobin.

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Codocyte

• Codocytes appear in conditions which cause the
  surface of the red cell to increase
  disproportionately to its volume.
• This may result from a decrease in hemoglobin, as
  in iron deficiency anemia, or an increase in cell
  membrane.
• Thalassemias, Hb C disease, post splenectomy,
  obstructive jaundice.

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Dacrocyte

• Dacryocytes are pear-shaped or teardrop shaped
  cells.
• myelofibrosis/
• thalassemia

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sickle cells (Drepanocytes

• Drepanocytes or sickle cells are formed as a
  result of the presence of hemoglobin S in the red
  cell.
• As the red cell ages, it becomes less flexible or
  deformable and becomes rigid as it passes through
  the low oxygen tension atmosphere of the small
  capillaries in the body.
• In the absence of oxygen, hemoglobin S
  polymerizes into rods, causing the sickle cell
  shape.
• Sickle cells can be somewhat pointed at the ends

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• Most sickled cells can revert back to the discoid
  shape when oxygenated.
• About 10 of sickled cells are unable to revert
  back to their original shape after repeated
  sickling episodes.

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Echinocyte (Urchin)

• Echinocytes are reversible,
• The projections are rounded and evenly spaced
  around the cell.
• Acanthocytes have irregularly spaced thorn-like
  projections.

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Echinocyte

• Uremia,
• Following heparin injection,
• Pyruvate kinase deficiency.
• Artificial

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Elliptocytes

• Elliptocytes can vary in appearance from slightly
  oval to thin pencil-shaped forms. Less than 1 of
  red cells in normal blood are oval.

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Elliptocytes

• Hereditary Elliptocytosis
• Thalassemia, megaloblastic anemia, iron
  deficiency.

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Elliptocytes
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Keratocytes

• Keratocytes are cells which have been damaged due
  to contact with fibrin strands.

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• intravascular coagulation
• microangiopathic hemolytic anemia
• glomerulonephritis
• rejection of renal transplants.

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Shistocytes

• Schistocytes are red cell fragments which are
  formed when fibrin strands come in contact with
  circulating red cells. The strands cut a small
  piece from the original cell.

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Spherocytes

• cells which have a decreased surface-to-volume
  ratio.
• cell is thicker in diameter than normal red cells
  
• they appear to be round, darkly-stained cells
  without central pallor.

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Spherocytes
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causes

• Hereditary spherocytosis
• Immune hemolytic anemia
• Severe burns
• In-vitro prolonged storage of blood

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Stomatocytes
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Stomatocytes

• cup-shaped erythrocytes which have an elongated
  or slit-like central pallor.
• hereditary stomatocytosis, neoplastic disorders,
  liver disease and Rh null disease, in-vitro
  change in pH

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Rouleaux
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Rouleaux

• most of the red cells, in the proper viewing
  area, are stacked together like coins.
• Four or more cells make up each formation,
  leaving much of the field empty of cells
  (increased white space).
• Rouleaux is clinically significant when increased
  globulins are present, as in multiple myeloma.

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Autoagglutination

• Cells clumping together rather than stacked like
  coins.
• Autoagglutination is caused by the presence of
  antibody in the plasma.

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IV -Erythrocyte inclusion bodies

• 1- Howell-Jolly Bodies
• MorphologySmall round cytoplasmic red cell
  inclusion with same staining characteristics as
  nuclei
• Found in- Post splenectomy- Megaloblastic
  anemia

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IV -Erythrocyte inclusion bodies

• 2- Siderotic Granules (Pappenheimer Bodies)
• RBCs which contain no hemoglobin iron granules.
  They appear as dense blue, irregular granules
  which are unevenly distributed in Wright stained
  RBCs. Pappenheimer bodies can be increased in
  hemolytic anemia, infections and post-splenectomy.

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IV -Erythrocyte inclusion bodies

• 3- Basophilic stippling
• MorphologyConsiderable numbers of small
  basophilic inclusions in red cells.
• Found in- Thalassaemia- Megaloblastic anemia-
  Hemolytic anemia - Liver disease- Heavy metal
  poisoning.

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IV -Erythrocyte inclusion bodies

• 4- Heinz Bodies
• Represent denatured hemoglobin (methemoglobin -
  Fe) within a cell. With a supravital stain
  like crystal violet, Heinz bodies appear as round
  blue precipitates. Presence of Heinz bodies
  indicates red cell injury and is usually
  associated with G6PD-deficiency.

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IV -Erythrocyte inclusion bodies

• 5- Cabot Rings
• Reddish-blue threadlike rings in RBCs of severe
  anemia's. These are remnants of the nuclear
  membrane and appear as a ring or figure 8
  pattern. Very rare finding in patients with
  Megaloblastic anemia, severe anemia's, lead
  poisoning, and dyserythropoiesis.

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blood-borne parasites

• Plasmodium, which causes malaria (P. vivax, P.
  falciparum)
• Trypanosoma cruzi is a parasite that causes
  Chagas disease.
• Babesia microti parasites cause the condition
  babesiosis.
• Leishmania donovanii parasites cause the disease
  leishmaniasis
• Wucheria bancrofti cause filiariasis

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Trophozoites,schizonts and gametocytes in malaria
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Wuchereria bancrofti
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Bone marrow smear showing Leishmania donovani
parasites in a bone marrow histiocyte
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B. microti ring forms with a typical Maltese
Cross (four rings in cross formation).
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blood smear from a patient with trypanosomiasis
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