RETICULOCYTE COUNT
Manual reticulocyte estimation
Objective
At the end of this section, the student should be able to
• Define reticulocytes and explain it use as an
erythropoietic index.
• Prepare supravital stain for retic counts.
• Perform reticulocytes test - identify retics, count and
express the results in the appropriate units.
• Interpret the result of reticulocytes count.
• Explain the limitations associated with manual
retic count.
Introduction
• Reticulocytes are immature RBCs that contain
remnant cytoplasmic ribonucleic acid (RNA)
and organelles such as mitochondria and
ribosomes.
• They are formed when the nucleus of a maturing
red cell is either dissolved or extruded at the late
normoblast stage.
• Reticulocytes are seen in peripheral blood
because after their release from BM, it takes 1-2
days for the developing RBC to fully mature.
RBCs go through six stages to mature:
1) Pronormoblast
2) Basophilic normoblast
Bone Marrow
3) Polychromatophilic normoblast 2-3 days B.M
4) Orthochromic normoblast
5) Reticulocyte 1 day P.B
Peripheral blood
6) Mature red blood cell.
The average life-span of an RBC is ~ 120days.
The B.M. replaces approx. 1 % of the adult RBCs (normal
range; 4.5-5.5 x 1012/L) every day and reticulocytes take ~ 1
day to mature, thus:
The normal value of retic in the PB is :
0.5 - 1.5% in adults (absolute count : 0.025 – 0.075 X 1012/L)
2.0 – 6.0% in neonates.
• Reticulocytes count, thus is a reflection of the
amount of effective red blood cell production
taking place in the bone marrow.
Causes of low retic count (reticulocytopenia)
• A low reticulocyte count indicates that the bone marrow
is not producing adequate number of red blood cells.
This could be due to:
• Aplastic anaemia.
• Exposure to radiation or radiation therapy.
• Chronic infection.
• Untreated pernicious anaemia, megaloblastic anaemia
and iron deficiency anaemia
• Chemotherapy medications
• Viral infections eg: parvo-virus
• Marrow infiltration eg: leukaemias
Causes of increased Retic count (reticulocytosis)
• Thalassaemia, sideroblastic anaemia.
• Acute and chronic blood loss (haemorrhage).
• Haemolytic anaemias (~25% are associated with normal retic count
due to immune destruction of RBC precursors).
• Pregnancy.
• Medications.- haematinic to treat IDA or Pernicious
Anaemia
• High altitude
• Smoking
• Newborns
Importance of reticulocytes count
• Reticulocytes count is used as an index to
determine erythropoietic activity (red cell
production) by the bone marrow.
• Retic count can also help to determine the cause
of anaemia and aid in classifying the different
types of anaemia.
• Retic count can be used as a tool to monitor the
effectiveness of [Link]: IDA treatment.
Demonstration of Retics in the lab
• Retics are demonstrated by staining them with
supravital dyes such as new methylene blue,
brilliant cresyl blue or pure Azure B which
precipitate the remnant of RNA and other
organelles to form a filamentous network of
reticulum observable with a microscope.
• On thin blood films stained with Leishman`s stain
or any of the Romanowsky`s stain, retics appear as
polychromatophilic macrocytes which has a
diffuse cytoplasmic basophilia due to inability of
the stain to ppt. reticular material.
Reticulocytes
Blood films showing reticulocytes demonstration
DEMONSTRATION OF RETICS AS POLYCHROMATIC CELL IN
THIN BLOOD FILM STAINED WITH LEISMAN`S STAIN
POLYCHROMATIC
MACROCYTES
MICROSPHEROCYTES
NORMAL RBC
Characteristics of supra-vital stains for retics
Reticulocytes are supra-vitally stained (ie; they are
stained while still alive), hence the staining
solution must not kill the cells. Supra-vital stain
for retic must therefore meet the ff requirement.
• It must be non-toxic to prevent killing the live
red blood cells.
• It must be readily assimilated by the cells to
facilitate staining.
• Must stain specifically the cellular structures
being demonstrated.
Components of reticulocyte fluid
A standard reticulocytes fluid consists of
• An anticoagulant to prevent blood from clotting
– tri-sodium citrate.
• A dye that will stain the cells - Brilliant Cresyl
Blue, New Methylene Blue or Pure Azure B.
• Preservative to provide similar physiological
conditions to the cells as prevails in the body –
physiological saline.
Preparation of reticulocytes fluid
To prepare 100ml of reticulocytes fluid
• Brilliant cresyl blue or new methylene blue – 1g
• Tri-sodium citrate (3.0g/l) – 20ml
• Physiological saline(9.0g/l) – 80ml
Mix to dissolve and filter before use.
Procedure for setting reticulocytes test
• Deliver 3-4 drops of well mixed blood into
75×12mm test tube.
• Add 2-3 drops of reticulocytes fluid and mix
• Incubate at room temperature for 10 -15mins
(incubation at 37oC had no superior advantage)
• Re-suspend the cells and prepare standard thin
blood films for examination.
Observation
• Mature red cells stain greenish-blue without any
demonstrable inclusions whereas reticulocytes
contain deep-blue filamentous network, strands or
fragments of reticular material in their cytoplasm.
• Other cellular inclusion bodies such as Heinz
bodies, Pappenheimer bodies, Howell-Jolly
bodies are demonstrable only after counter-
staining the reticulocytes film.
Reticulocytes counting
• Oil immersion objective (×100) is preferred.
• An area of the field where the cells are not over-lapping
is chosen.
• Ehlich`s diaphragm (an eye-piece with a special in-built
diaphragm) is used to reduce the field size so that few
numbers of RBCs are in focus to facilitate counting.
• Total red cells (both matured and retics) in each field are
counted whiles noting number of RBCs that are retics.
• At least 500 total RBC should be counted preferably
1000 RBC. The greater the number of RBC counted, the
lesser the margin of error in the reticulocytes estimation.
How to report retic. result
Percentage reticulocytes count
% Retic count = Number of retic counted x 100
Total RBC counted
EXAMPLE: 25 reticulocytes in 1,000 total RBC’s
• Reticulocyte count = 25 x 100 = 2.5%
1000
% retics may not accurately reflect a patient`s
erythropoietic status because an apparent increase in %
retic count may just reflect normal RBC production with
reduced RBC count rather than increased erythropoiesis.
• This is particularly true in presence of anaemia,
where number of RBC is reduced and
percentage reticulocytes count does not reflect
reticulocytes production because each
reticulocyte released is diluted into fewer
numbers of matured RBCs.
Absolute reticulocytes count
This is the actual number of reticulocytes in 1L of whole
blood. It is given by;
Absolute retic count = % Retic count x RBC count
The result must always be interpreted in relation to total
RBC count. However, an accurate erythropoietic activity in
BM is obtained when patient`s haematocrit is compares
with a normal haematocrit value. This will enable us to
know if when the patient has a normal PCV, the retic count
obtained is either low, normal or increased.
Thus, a Corrected Reticulocyte Count (retic index) is
calculated based on a normal haematocrit of 45%.
Reticulocytes production index (RPI)
• This is a better measure of erythropoietic activity of the
BM. It adjust the retic count to reflect patient PCV and
adjust for the maturation time of retics since retics are
released earlier in patients with low PCV.
• It is given by
Corrected retic count (%)/ # Days (Maturation time)
• This is calculated by first correcting the percentage
reticulocytes count into a normal haematocrit (PCV)
of 45% for males and 42% for females.
• The RPI and maturation time vary with the haematocrit.
Using this formula
CRC = PCV of patient x % Retic count of patient
45%
• Next, correct for the duration reticulocytes take to
mature which is 2days (48hrs) in peripheral circulation
by dividing the equation by 2.
• In normal conditions where there is no reticulocytosis, a
24hr maturation period for reticulocytes (ie. One day) is
used in the formula.
RPI = PCV of patient x % Retic of patient /45
2
Maturation Time Haematocrit (%)
• 1 day 45
• 1.5 days 35
• 2 days 25
• 3 days 15
Interpretation of Retic. production index result
• A normal marrow has RPI =1.
• In haemolytic anaemia with functionally normal
marrow, RPI could range from 3 – 7.
• RPI ≥ 2 indicate adequate response to anaemia.
• In situation of bone marrow damage, erythropoietin
suppression, iron deficiency, vitamin B12 or folate
deficiency, the RPI is less than expected for the anaemic
condition. ie (≤ 2).
• Ineffective erythropoiesis is present if numerous NRBC
are seen in peripheral blood film yet the RPI is low.
