LESSON 8: Reticulocyte Count

OUTLINE • New Methylene Blue Table 1. Normal Reticulocyte Count

I Introduction • Microscope slides Normal Reticulocyte 0.5-2.5%
II Principle
• Miller Disc Count
III Materials
IV Procedure • Aspirator
V Methods for Reticulocyte Count • Microscopes METHODS FOR RETICULOCYTE COUNT
A. Miller Disc • Test tubes MILLER DISC
B. Absolute Reticulocyte Count • Because large numbers of red blood cells should
C. Corrected Reticulocyte Count PROCEDURE be counted to obtain a more precise reticulocyte
D. Reticulocyte Production Index
1. Mix equal amounts of blood and new methylene count, the MILLER DISC WAS DESIGNED TO
blue stain (2 to 3 drops, or approximately 50 mL REDUCE THIS LABOR-INTENSIVE PROCESS.
INTRODUCTION each), and allow to incubate at room temperature • The disc is composed of two squares, with the
Reticulocytes are the LAST IMMATURE RED for 3 to 10 minutes. area of the smaller square measuring 1/9 the area
BLOOD CELL STAGE. Normally, it only spends 2 days in the 2. Remix the preparation. of the larger square.
bone marrow and 1 day in the peripheral blood before 3. Prepare two wedge films (PBS).
developing into a mature red blood cell. The reticulocyte 4. In an area in which cells are close together but not
contains remnant cytoplasmic ribonucleic acid (RNA) and touching, count 1000 RBCs under the oil
organelles such as the mitochondria and ribosomes. The immersion objective lens (1000x total
reticulocyte count is USED TO ASSESS THE magnification). Reticulocytes are included in the
ERYTHROPOIETIC ACTIVITY OF THE BONE MARROW. total RBC count.
5. To improve accuracy, have another laboratorian
RECALL! Stage of Differentiation count the other film; counts should agree within
RUBRIBLAST → PRORUBRICYTE → RUBRICYTE → 20%. • RBCs are COUNTED IN THE SMALLER SQUARE,
METARUBRICYTE → RETICULOCYTE → ERYTHROCYTE 6. Calculate the % reticulocyte count: and reticulocytes are COUNTED IN THE LARGER
• Differentiation from Rubriblast to Reticulocyte – 3 Reticulocytes (%) =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑥 100 SQUARE. A minimum of 112 cells should be
1000 (𝑅𝐵𝐶𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑) counted in the small square, because this is
to 5 DAYS
For example, if 15 reticulocytes are counted,
• Reticulocytes remains in the BM – 1 to 2 DAYS 15 𝑥 100
equivalent to 1008 red cells in the large square
• Reticulocytes goes to the peripheral blood and it Reticulocytes (%) = = 1.5% and satisfies the College of American Pathologists
1000
takes 1 DAY for them to mature OR (CAP) hematology standard for a manual
Number of reticulocytes counted x 0.1 reticulocyte count based on at least 1000 red
PRINCIPLE cells.
Whole blood, anticoagulated with EDTA, is • This is to ensure that the smear is evenly
stained with a SUPRAVITAL STAIN, SUCH AS NEW distributed.
METHYLENE BLUE. Any non-nucleated red blood cell that Reticulocytes (%) =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐴 (𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑎𝑟𝑒) 𝑥 100
contains two or more particles of blue stained 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑅𝐵𝐶𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐵 (𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒) 𝑥 9
granulofilamentous material after new methylene blue For example, if 15 reticulocytes are counted in the
staining is defined as a RETICULOCYTE.
large square and 112 red blood cells are counted
in the small square,
MATERIALS 15 𝑥 100
Figure 1. A-Reticulocyte and B-Heinz body stained with Reticulocytes (%) = = 1.5%
• Anticoagulated Blood (EDTA) Supravital stain (blue) 112 𝑥 9

DIVINA GRACIA A. MARGES | BSMT 3-3 1

 LESSON 8: Reticulocyte Count, Platelet Count and Peripheral Blood Smear

ABSOLUTE RETICULOCYTE COUNT NOTE! Polychromasia is the presentation of

• It is the actual number of retics in 1L or microliter multicolored RBCs in a blood smear test. This
of blood. is an indication of red blood cells being
released prematurely from bone marrow
during formation.
• The patient’s hematocrit is used to determine the
For example, if a patient’s reticulocyte count is 2% appropriate correction factor (reticulocyte
and the RBC count is 2.20 x 1012/L, the ARC is maturation time in days)
calculated as follows:
Table No. 3 Reticulocyte maturation time in days
PATIENT’S HEMATOCRIT CORRECTION FACTOR
VALUE (%) (maturation time, days)
NOTE! The calculated result has to be converted 40-45 1
from 1012/L to 109/L) 35-39 1.5
25-34 2
Table 2. Normal Absolute Reticulocyte Count 15-24 2.5
Normal Absolute 50-100x109/L <15 2
Reticulocyte Count
𝐻𝐶𝑇(%)
𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 (%) 𝑥 [ ]
45
RPI =
CORRECTED RETICULOCYTE COUNT 𝑚𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
OR
• In specimens with a low hematocrit, the 𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡
percentage of reticulocytes may be falsely RPI =
𝑚𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
elevated because the WHOLE BLOOD CONTAINS For example,
FEWER RED BLOOD CELLS (anemic). A correction 30
7.8 𝑥 [ ]
45
factor is used, with the average normal RPI = = 2.6
2
hematocrit considered to be 45%.
• Used in the diagnosis of Anemia

Corrected Reticulocyte Count (%) =

𝑝𝑎𝑡𝑖𝑒𝑛𝑡 𝐻𝐶𝑇 (%)
Reticulocyte (%) =
45

RETICULOCYTE PRODUCTION INDEX

• Reticulocytes that are released from the marrow
prematurely are called SHIFT RETICULOCYTES.
• These reticulocytes are “shifted” from the bone
marrow to the peripheral blood earlier than
usual to compensate for anemia. Instead of
losing their reticulum in 1 day, as do most normal
circulating reticulocytes, these cells take 2 to 3
DAYS TO LOSE THEIR RETICULA.
• For this reason, the reticulocyte count is falsely
increased when POLYCHROMASIA is present,
because the count no longer represents the cells
maturing in just 1 day.

DIVINA GRACIA A. MARGES | BSMT 3-3 2

 LESSON 8: Platelet Count

OUTLINE 4. Platelets are counted using the HPO. The shape

VI Introduction and color help distinguish the platelets from
VII Materials highly refractile dirt and debris.
VIII Procedure
5. Count the number of platelets in the 25 small
squares in the center square of the grid. Platelets
INTRODUCTION should be counted on each side of the
Platelets adhere to foreign objects and to each hemacytometer, and the difference between the
other which makes them difficult to count. totals should be less than 10%.
A platelet count is the number of platelets in 1 6. Calculate the platelet count.
liter (L) or 1 microliter (mL) of whole blood. In this
procedure, whole blood, with EDTA as the anticoagulant, is
diluted 1:100 with 1% ammonium oxalate to lyse the non-
nucleated red blood cells (to prevent hindrances in counting
the platelets). The platelets are counted in the 25 small
squares in the large center square (1mm2) of the
hemacytometer using a phase-contrast microscope in the
reference method described by Brecher and Cronkite. A
light microscope can also be used, but visualizing the
platelets may be more difficult.

MATERIALS
• Anticoagulated Blood
• 1% Ammonium Oxalate
• Hematocytometer
• Test tube
• Aspirator
• Microscope

PROCEDURE
1. Make a 1:100 dilution by placing 20 uL of well-
mixed blood into 1980 uL of 1% ammonium
oxalate in a small test tube.
2. Mix the dilution thoroughly and charge the
chamber.
3. Place the charged hemacytometer in a moist
chamber for 15 minutes to allow the platelets to
settle.

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 LESSON 8: Peripheral Blood Smear

OUTLINE THICK BLOOD SMEAR

IX Introduction • This is prepared for detecting blood parasites such
X Types of Peripheral Blood Smear (PBS)
as MALARIA and MICROFILARIA.
A Thick Blood Smear
B Thin Blood Smear • Procedure
i. Cover Glass method 1. Place a large drop of blood in the center
ii. Spin Method of a clean glass slide.
iii. Slide Method 2. Spread it in a circular area of 1.5cm with
XI Parts of a Good Blood Smear
the help of a stick or end of another
XII Characteristics of a Good Blood Smear
XIII Materials glass slide. Figure 3. Cover Glass Method
XIV Procedure 3. Dry the slide then proceed to staining
A Preparation of Blood Smear 2. SPIN METHOD
B Fixation of Blood Smear THIN BLOOD SMEAR 1. Place a drop of blood in the center of a glass slide.
C Staining of Blood Smear • This can be prepared from anticoagulated blood 2. Spin at a high speed in a special centrifuge
i. Romanowsky Stain (EDTA) or from free-flowing finger prick blood by (cytospin).
any of the following techniques: 3. Blood spreads uniformly in the slide.
INTRODUCTION o Cover glass method 4. Dry the slide and then stain it.
Blood films are usually examined to investigate o Spin method
hematological problems. It is important in the investigation o Slide method 3. SLIDE/MANUAL WEDGE/PUSH WEDGE METHOD
and management of amenia, infections, and other o Automated method
• Blood specimen may come from the EDTA tube or
conditions which produce changes in the appearance of
directly from finger pricked blood.
blood cells and differential white blood cell count. 1. COVER GLASS METHOD
• Produces a good distribution of leukocytes in all areas 1. Place a small drop (0.05 mL) of well-mixed blood
NOTE! Blood films are also evaluated to identify viral of preparation either directly from the freshly wiped fingertip
infections or distinguish bacterial from viral infection. • Due to a small specimen amount, 15 leukocytes are puncture or with an applicator stick
This is also used to look for inclusion bodies like counted per cover slip. Owing to the small size of the approximately 0.5 inch from one end of the slide.
basophilic stippling, Howell-Jolly body, cabot rings and cover slip, it is usually mounted by conventional glass a. If frosted slides are used, place the
diagnose blood-borne parasites. slide for staining. blood near the frosted end of the slide.
(letter A in Figure 4)
TYPES OF PERIPHERAL BLOOD SMEAR 1. Hold two clean cover slips by their edges with the 2. Place another slide (spreader) with smooth and
thumb and forefinger of each hand. Touch the clean edge at an angle of 30-45° near the drop of
center of one coverslip to a small drop of blood. blood. (letter B in Figure 4)
2. Immediately place the second coverslip on top of 3. Move the spreader backwards so that it makes
a very small drop of blood in a diagonal position. contact with the drop of blood.
3. Allow the blood to spread by capillary action. Just 4. Then rapidly move the spreader forward over the
before the spreading action has almost stopped, slide.
evenly and smoothly pull the coverslips apart in 5. Wait for the slide to dry and then stain it.
the horizontal plane
Figure 2. Thin and Thick Blood Smear 4. Place the smears in an upright position and allow
to air dry before staining.

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Figure 4. Push Wedge Method
PARTS OF A GOOD BLOOD SMEAR
• Good smear is tongue-shaped, with a smooth tail
Figure 5. Parts of a good blood smear
CHARACTERISTICS OF A GOOD BLOOD SMEAR
1. It progresses from being thick at the point of
origin to thin with a uniform edge at the
termination point / feathery edge.
2. It does not touch the outer borders of the slide or
run off the sides or ends of the slide.
3. It appears smooth, without waves or gaps and
holes.
4. It does not have any streaks, ridges, or troughs,
which indicate an increased number of leukocytes
carried to that area.
5. It is prepared with a proper amount of blood and
spread to occupy approximately two thirds of the
length of the glass slide.
LESSON 8: Reticulocyte Count, Platelet Count and Peripheral Blood Smear
Figure 6. Acceptable and unacceptable blood smears
Unacceptable blood for smear:
• Cold agglutinin – RBCs clump
o Remedy: warm the blood for 5 mins.
at 37oC
• Lipemia/Lipemic – it has fats that may produce
holes in the smear
o No remedy; impossible to smear
properly
• Rouleaux formation – RBCs clump
o No remedy; impossible to smear
properly
MATERIALS
• Anticoagulated Blood (EDTA)
• Glass slides
• Wright stain
PROCEDURE
A. PREPARATION OF BLOOD SMEAR
1. Place a small drop (0.05 mL) of well-mixed blood
either directly from the freshly wiped fingertip
puncture or with an applicator stick
approximately 0.5 inch from one end of the slide.
DIVINA GRACIA A. MARGES | BSMT 3-3
If frosted slides are used, place the blood near the
frosted end of the slide.
2. Place another slide (spreader) with smooth and
clean edge at an angle of 30-45° near the drop of
blood.
3. Move the spreader backwards so that it makes
contact with the drop of blood.
4. Then rapidly move the spreader forward over the
slide.
B. FIXATION OF BLOOD SMEAR
1. Blood and other types of specimens can be
stained using Romanowsky-based stains. These
stains can be prepared in the laboratory or
purchased in a ready-to-use form (already has a
fixative and stain; slide can be readily soaked).
2. In some laboratories, blood smears are fixed
separately in alcohol before the staining
procedure is performed. This step enhances the
retention of granules in blood cells. The usual
fixative is methyl alcohol. Slides can be placed in
anhydrous and acetone-free methanol for 1
minute or longer.
3. Wright and other Romanowsky-based stains are
dissolved in methyl alcohol; therefore, fixation
normally takes place when the stain is applied to
the blood smear, manual or automated
techniques can be used.
NOTE! Fixation is done for the smear to not wash out.
C. STAINING OF BLOOD SMEAR
1. After fixation, place a thoroughly dried and
labeled slide on a level staining rack with the
smear side facing up.
2. Place freshly filtered stain slowly on the slide until
the smear is completely covered. Do not add
excess stain (because cells color may thicken and
appear dark). Commonly, 3 to 10 minutes may be
needed for an acceptably stained blood smear.
3. At the end of the staining time, gently add buffer
(pH 6.4) to the slide without removing the stain.
4. Mix the stain and buffer by gently blowing on the
slide. A well-mixed slide will have a metallic green
sheen rise to the surface of the slide. The timing
for this stage ranges from 2 to 5 minutes.
5. Wash the stain and buffer off the slide with a
gentle flow of tap water. Allow the slide to air dry.
5
 LESSON 8: Reticulocyte Count, Platelet Count and Peripheral Blood Smear

ROMANOWSKY STAIN
• This stains are universally employed for staining
blood films.
• Main components of a Romanowsky Stain:
o Cationic or Basic Dye
▪ Affinity for acidic component
(i.e. nucleus)
▪ Methylene blue
▪ Gives a blue-grey color
o Anionic or Acidic Dye
▪ Affinity for basic component
(i.e. cytoplasm)
▪ Eosin Y or B
▪ Gives an orange-red color
• Various Romanowsky Stain
o Leishman stain – for the identification
of blood-borne parasites
o Giemsa stain
o Wright stain
o Field stain
o Jenner stain
o JSB stain

Figure 7. Battlement method / Battlement scan pattern

NOTE! Battlement method – is done to avoid

overlapping count of cells

REFERENCES
Notes from the discussion of Mr. Jo Hanes Contemprato
RMT, MSMT
Cavite State University powerpoint presentation: Lesson 8
– Reticulocyte Count, Platelet Count and Peripheral
Blood Smear

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