Practical Clinical Hematology

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Practical Clinical Hematology
Reticulocyte Count
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Reticulocytes

• Reticulocytes are young , premature, non
  nucleated red blood cells, contain reticular
  material (RNA) that stain gray blue.
• Reticulum is present in newly released blood
  cells for 1-2 days before the cell reach its full
  mature state.

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Reticulocytes Stains

• Reticulocytes are visualized by supra-vital
  staining (such as new methylene blue, Brilliant
  Cresyl Blue, Pure azure blue) that precipitate
  the RNA and organelles, forming a filamentous
  network of reticulum
• On Wright stain. the Reticulocyte appears
  polychromatophilic or as a Macrocytic blue red
  cell.

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PRINCIPLE

• Whole blood is incubated with supra-vital
  staining (new methylene blue). The vital stain
  causes the ribosomal and residual RNA to
  coprecipitate with the few remaining mitochondria
  and ferritin masses in living young erythrocytes
  to form dark-blue clusters and filaments
  (reticulum).
• Smears of this mixture are then prepared and
  examined. The number of reticulocytes in 1000 red
  blood cells is determined. This number is divided
  by 10 to obtain the reticulocyte count in percent.

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SPECIMEN

• Whole blood that is anticoagulated with either
  EDTA or heparin is suitable.
• Capillary blood drawn into heparinized tubes or
  immediately mixed with stain may also be used.
• Red blood cells must still be living when the
  test is performed therefore it is best to perform
  it promptly after blood collection.
• Blood may be used up to 8 hours after collection.
  
• Stained smears retain their color for a prolonged
  period of time.

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REAGENTS, SUPPLIES AND EQUIPMENT

1. Commercially prepared liquid new methylene blue
   solution. It should be stored in a brown bottle.
   If precipitate is a problem on the smear, the
   stain should be filtered prior to use.
2. Microscope slides
3. Microscope
4. 10 x 75 mm test tubes
5. Pasteur pipets (with bulb if pipets are glass)
6. Capillary tubes
7. Miller ocular (if available)

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PROCEDURE

• Preparation of smears
• Add 3-4 drops of new methylene blue solution to
  3-4 drops of thoroughly mixed EDTA anticoagulated
  blood to a glass 10 x 75 mm test tube.
• Mix the contents by gently shaking and allow to
  incubate at room temperature for a minimum of 10
  minutes.
• At the end of 10 minutes, gently mix the
  blood/stain solution.
• Using a capillary tube, place a drop of the
  mixture on each of three slides near the frosted
  edge as you would when making a peripheral smear.
• Using the wedge smear technique, make acceptable
  smears not too thick or thin.
• Label the slides with patient name, ID and date.
• Allow to air dry. (Do not blow to hasten to
  drying.)

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counting The Reticulocytes Cells

1. Place the first slide on the microscope stage
   and, using the low power objective (10x), find an
   area in the thin portion of the smear in which
   the red cells are evenly distributed and are not
   touching each other. Carefully change to the oil
   immersion objective (100x) and further located an
   area in which there are approximately 100 red
   cells per oil immersion field. Do this by finding
   a field where the cells are evenly distributed
   and mentally divide the field into 4 quadrants.
   Count the cells in 1 quadrant. If there are about
   25, you are in a good area. There will be a lot
   of open space between the red cells.

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• Be sure to count all cells that contain a
  blue-staining filament or at least 2 or more
  discrete blue aggregates of reticulum in the
  erythrocyte.
• Count 1000 red cells in consecutive oil immersion
  fields. Record the number reticulocytes seen.
• You may count 500 cells on two slides each. They
  should agree within 15 of each other. If they
  do not, repeat the reticulocyte count on the
  third smear.
• Calculate the result as follow

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Method using the Miller disc

• Use a 100x objective and a 10x ocular secured
  with a Miller disc.
• The Miller disc imposes two squares (one 9 times
  the area of the other) onto the field of view.
• Find a suitable area of the smear. A good area
  will show 3-10 RBCs in the smaller square of the
  Miller disc.
• Count the reticulocytes within the entire large
  square including those that are touching the
  lines on the left and bottom of the ruled area.
  Count RBCs in the smaller square whether they
  contain stained RNA or not. A retic in the
  smaller square should be counted as an RBC and a
  retic. Record RBC counted and retic counted
  separately.
• Continue counting until a minimum of 111 RBCs
  have been observed (usually 15-20 fields). This
  would correspond to 999 RBCs counted with the
  standard procedure.

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Miller Disc Method of Counting

• The Miller disc may be placed in one of the
  ocular lenses to aid in the counting of the
  reticulocytes.

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Normal Ranges

• RBCs life span 100 days, 20 days
• Reticulocyte 1 day in peripheral blood, Then
  the B.M. replaces approximately 1 of the adult
  red blood cells every day.
• Normal value
• 0.5 to 1.5/100 red blood cells (or, 0.5 to 1.5)
• Absolute count 25 to 75 X 109/L
• Newborn (0-2 weeks) 2.5-6.0
• Normal Reticulocyte Index 1-3

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Reporting Results

• Absolute Reticulocyte Count (ARC) is the actual
  number of reticulocytes in 1L of whole blood.
  This is calculated by multiplying the
  reticulocytes by the RBCs count and dividing by
  100.
• Corrected Reticulocyte Count is calculated based
  on a normal hematocrit of 45.
• Reticulocyte Production Index (RPI) Corrected
  retic count () / Days (Maturation time)

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Calculation of corrected reticulocyte

• In states of anemia, the reticulocyte percentage
  is not a true reflection of reticulocyte
  production. A correction factor must be used so
  as not to overestimate marrow production, because
  each reticulocyte is released into whole blood
  containing few RBCs - a low hematocrit (Hct) -
  thus relatively increasing the percentage.
• The corrected reticulocyte count my be calculated
  by the following formula

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Calculation of the reticulocyte production index
(RPI)

• The RI is a measurement for reticulocytes when
  anemia is present
• Estimating RBC production by using the corrected
  reticulocyte count may yield erroneously high
  values in patients when there is a premature
  release of younger reticulocytes from the marrow
  (owing to increased erythropoietin stimulation).
• The premature reticulocytes are called stress or
  shift reticulocytes. These result when the
  reticulocytes of the bone marrow pool are shifted
  to the circulation pool to compensate for anemia.
  The younger stress reticulocytes present with
  more filamentous reticulum. The mature
  reticulocyte may present with granular dots
  representing reticulum. Normally, reticulocytes
  lose their reticulum within 24 to 27 hours after
  entering the peripheral circulation.

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• The premature stress retics have increased
  reticulum and require 2 to 2.5 days to lose their
  reticulum, resulting in a longer peripheral blood
  maturation time.
• The peripheral blood smear should be reviewed
  carefully for the presence of many
  polychromatophilic macrocytes, thus indicating
  stress reticulocytes and the need for correction
  for both the RBC count and the presence of stress
  reticulocytes. The value obtained is called the
  reticulocyte production index (RPI).

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Maturation Time Hematocrit
1 day 45
1.5 day 35
2 day 24
3 day 15
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Interpretation

• The Reticulocyte count is an important diagnostic
  tool The number of Reticulocytes is a good
  indicator of bone marrow activity, because it
  represents recent production. It is used to
  differentiate anemia's caused by bone marrow
  failure from those caused by hemorrhage or
  hemolysis.
• It used also to check the effectiveness of
  treatment in prenicious anemia and folate and
  iron deficiency.
• To assess the recovery of bone marrow function in
  aplastic anemia and to determine the effects of
  radioactive substance on exposed workers.
• A low reticulocyte count may mean a need for a
  bone marrow biopsy. This can tell if is a problem
  with how new reticulocytes are made by the bone
  marrow.

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• Reticulocytosis (Increased RBC Production)
• Reticulocyte Index gt3, Reticulocyte Count gt1.5
• Acute blood loss or hemorrhage
• Post-Splenectomy
• Acute Hemolytic Anemia (Microangiopathic Anemia)
• Hemoglobinopathy
• Sickle Cell Anemia
• Thalassemia major
• Post-Anemia Treatment
• Folate Supplementation
• Iron Supplementation
• Vitamin B12 Supplementation

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• Reticulocytopenia (Decreased RBC Production)
• Reticulocyte Index lt1, Reticulocyte Count lt0.5
• Aplastic Anemia
• Bone Marrow infiltrate
• Bone Marrow suppression or failure
• Sepsis
• Chemotherapy or radiotherapy
• Disordered RBC maturation
• Iron Deficiency Anemia
• Vitamin B12 Deficiency
• Folate Deficiency
• Sideroblastic Anemia
• Anemia of Chronic Disease
• Hypothyroidism
• Blood transfusion
• Liver disease

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What can affect the Test

• Reasons you may not be able to have the test or
  why the results may not be helpful include
• Taking medicines, such as levodopa,
  corticotropin, azathioprine (Imuran),
  chloramphenicol (Chloromycetin), dactinomycin
  (Cosmegen), medicines to reduce a fever,
  medicines to treat malaria, and methotrexate and
  other cancer chemotherapy medicines.
• Getting radiation therapy
• Taking sulfonamide antibiotics (such as Bactrim
  or Septra)
• Being pregnant
• Having a recent blood transfusion

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Sources of error

• A refractile appearance of erythrocytes should
  not be confused with reticulocytes.
• Filtration of the stain is necessary when
  precipitated material is present which can
  resemble a reticulocyte.
• Erythrocyte inclusions should not be mistaken for
  Reticulocytes.
• Howell-Jolly bodies appear as one or sometime
  two, deep-purple dense structures.
• Heinz bodies stain a light blue-green and are
  usually present at the edge of the erythrocyte.
• Pappenheimer bodies are more often confused with
  reticulocytes and are the most difficult to
  distinguish. These purple-staining iron deposits
  generally appear as several granules in a small
  cluster. If Pappenheimer bodies are suspected,
  stain with Wright-Giemsa to verify their
  presence. Hemoglobin H inclusions will appear as
  multiple small dots in every cell.

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• Falsely decreased reticulocyte counts can result
  from under staining the blood with new methylene
  blue. Be sure the stain/blood mixture incubates
  the full 10 minutes.
• High glucose levels can cause reticulocytes to
  stain poorly.
• There is high degree of inaccuracy in the manual
  reticulocyte count owing to error ( 2) in low
  counts and 7 in high counts) and a lack of
  reproducibility because of the inaccuracy of the
  blood film. This inaccuracy has been overcome by
  the use of automated instruments using flow
  cytometry.
• If no reticulocytes are observed after scanning
  at least two slides, report none seen.

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Thank you