Reticulocytes are immature red blood cells that are released from the bone marrow into circulation. They contain remnants of RNA and ribosomes. Reticulocyte counts are used to assess bone marrow response to anemia and erythropoietin therapy. Reticulocytes can be manually counted using supravital dyes like new methylene blue that stain the RNA, or automatically using cell counters that detect nucleic acid content. Normal ranges are 0.5-2.5% and increased counts indicate bone marrow response to anemia or therapy, while decreased counts suggest bone marrow suppression.

1. RETICULOCYTE
ASHA KUMARI
HAEMATOPATHOLOGY, TMH

2. What is Reticulocyte ?
Reticulocytes are young or juvenile ,
immature red cells released from
the bone marrow into the
bloodstream and that contain
remnants of ribonucleic acid (RNA),
protoporphyrin, ribosome's.

3. Formation of Reticulocyte
In Bone Marrow In Peripheral
blood

4. Why Reticulocyte count ?
• The number of Reticulocytes in the peripheral
blood is a fairly accurate reflection of
erythropoietic activity
• To diagnose anemia due to ineffective
erythropoiesis
• To assess response to specific therapy in iron
deficiency and megaloblastic anemia's
• Bone marrow recovery (after bone marrow
transplantation or intensive chemotherapy)
• To assess response to erythropoietin therapy
in anemia of chronic renal failure.
• Red cell production and regeneration

5. Reticulocyte count methods
1.Manual :
Supravital dye
Fluorescent dye
2. Automated:
Cell counters
Flowcytometer

6. Specimen collection and Storage
 K2EDTA is the anticoagulant of choice
 Venous or capillary blood
 At Room Temperature- within six hours after
blood collection.
 If sample analysis is delayed, the sample
should be refrigerated. Samples stored at 2 to
8 °C may be stable for up to 72 hours.
NCCLS guidelines H44-A2 Vol. 24 No. 8,2004

7. Manual Reticulocyte count by
Supravital dyes
Supravital staining refers to staining of cells in a living
state before they are killed by fixation or drying or
with passage of time
After staining with a Supravital dye such as New
Methylene blue / Brilliant Cresyl blue or Azure B,
RNA appears as blue precipitating granules or
filaments within the red cells. Following supravital
staining, any non nucleated red cell containing 2 or
more granules of blue stained material is considered
as a reticulocyte.
The College of American Pathology /ICSH

8. Manual Reticulocyte count by
Supravital dyes
Better and more reliable results are
obtained with New methylene blue than with
brilliant cresyl blue
New Methylene blue stains the
reticulofilamentous material in reticulocytes
more deeply and more uniformly than brilliant
cresyl blue
Azure B is a satisfactory substitute for
New Methylene blue, it has the advantage
that the dye does not precipitate and it is
available in pure form

9. PRINCIPLE
A equal volume of blood (collected in EDTA)
are incubated at 370C with Brilliant cresyl blue
solution which stains granules of RNA in red
cells. A thin smear is prepared on a glass slide
from the mixture and reticulocytes are
counted under the microscope. Number of
reticulocytes is expressed as a percentage of
red cells

10. Staining solution
1% Brilliant cresyl blue or 1% New methylene blue
• Dissolve 1gm of BCB /NMB in 100ml of iso-osmotic
phosphate buffer
• Iso osmotic phosphate buffer : pH 6.5
(A) NaH2PO4 2H2O (150mmol/L) 23.4gm/L
+
(B) Na2HPO4 (150mmol/L) 21.3gm/L
Mix 64 ml of A + 36 ml of B
• Check pH
• Filter with Whatmann’s No.1 filter
• Keep in dry bottle
• Stable for 4 – 6 weeks

11. Procedure
• Mix equal quantities of EDTA anticoagulated
blood and dye solution
• The exact volume of blood to be added to the
dye solution for optimal staining depends on
the RBC count.
• A larger proportion of anemic blood, and a
smaller proportion of polycythemic blood,
should be added than of normal blood

12. Procedure
 Incubate the mixture at 370C in water
bath for 15-20 mins
 Resuspend the red cells by gentle mixing
 Make films on glass slides in the usual way
 When dry, Examine the films under the
microscope using oil-immersion objective
 In a successful preparation, the
reticulofilamentous material stained deep
blue .

13. Reticulocyte

14. on Romanowsky stains:
Reticulocytes appear
as Polychromatophilic
(grey/blue) cells

15. Maturation stages of reticulocytes
• Stage I: Dense
cohesive reticulum in
non-nucleated red
cell (0.1% of
Reticulocyte count in
normal individuals)
• Stage II: Extensive
network of loose
reticulum
(0.7% of reticulocyte count
in normal individuals)
Heilmeyer and Westhaeuser, 1932

16. Maturation stages of reticulocytes
• Stage III: Small reticulum
along with scattered
granules (32% of
reticulocyte count in
normal individuals)
• Stage IV: Scattered
granules (61% of
reticulocyte count in
normal individuals).

17. Calculation
• There are two commonly used methods for
counting reticulocytes.
1. Standard counting procedure
2. Miller Reticle procedure
Standard counting procedure
 Dry smear is examined under oil immersion.
 Total of 1,000 RBCs are counted.
 Number of reticulocytes are recorded per
1,000 RBCs.
 Calculate the percentage of Reticulocyte and
absolute Reticulocyte count.

18. Miller Reticle procedure
Reticulocyte Count % =
Total reticulocytes counted in square A × 100
Total red cells counted in square B × 9
Miller ocular disk to facilitate
counting of reticulocytes.
The Miller ocular disk contains
two squares: A and B. Square
A is 9 times larger than square B.
Square B is used for counting
red cells, while reticulocytes are
counted in square A

19. • Reticulocyte count (%)= no. of Retic x100/1000 RBC
• Absolute Reticulocyte count (ARC) is the actual number
of reticulocytes in 1L of whole blood
Absolute Reticulocyte count = Retic % × RBC
• Reticulocyte Production Index (RPI) = Corrected Retic count
(%) ÷ # Days (Maturation time)

20. Reticulocyte Index(corrected
Reticulocyte count)
• This is a corrected reticulocyte count used in
anemias. In anemias, a falsely elevated reticulocyte
count can occur. A decrease in RBCs automatically
increases the reticulocyte count even if
erythropoiesis has not taken place because the
count represents a percentage in comparison to
RBCs. It is possible the appropriate increase of
erythropoiesis has not taken place in response to
the anaemia. This value, therefore, corrects for this
issue.
corrected reticulocyte count > 2% indicates reticulocyte release
appropriate for the degree of anemia.
If < 2%, reticulocyte release is inappropriate

21. Difference between Reticulocytes and other
red cell inclusions
Heinz bodies: These are precipitated globulin
Chains attached to the red cell membrane. They
stain light blue and are seen in glucose-6-phosphate
dehydrogenase deficiency following exposure to
oxidant stress.

22. Pappenheimer bodies:
These are iron-containing
granules which appear as
one or more small dots
in red cells.
Hb H inclusions: These are round,
golf ball like inclusion bodies seen in
α-thalassemias. Stain greenish blue
These can be easily differentiated
from reticulofilamentous material

23. • Howell-Jolly bodies: These are
nuclear remnants in
red cells seen in certain anemias
and following
splenectomy. As supravital dyes
stain both DNA
and RNA, these structures are also
stained.
• Granules of DYE: superimposed
on Red cells, if stain is not
filtered before use.

24. REFERENCE RANGE
Reticulocyte percentage:
Adults and Children : 0.5-2.5%
At birth or cord blood : 2 - 5 %
Dacie and Lewis Practical Hematology Twelfth edition 2017

25. Causes of increased reticulocyte
• High altitudes
• Hemolytic anemias
• Blood loss
• Following specific therapy of nutritional
anemia
(like iron in iron deficiency anemia, folate in
Folate deficiency anemia, Vit B12 in B12
deficiency anemia)
• Hemoglobinopathies, e.g. sickle cell anemia.

26. Causes of decreased reticulocyte
• Aplastic anemia and pure red cell aplasia
• Bone marrow infiltration (leukemia, lymphoma,
myelofibrosis, metastatic malignancy)
• Renal disease
• Anemia of chronic disease
• Alcoholism
• Myxedema
• Ineffective erythropoiesis: Megaloblastic anemia,
sideroblastic anemia, thalassemia, myelodysplasia
• Following blood transfusion

27. Fluorescence method
Reticulocytes can be counted manually by
fluorescence microscopy on appropriately stained
films.
Add 1 volume of acridine orange solution (50
mg/100 ml of 9 g/L NaCl) to 1 volume of blood. Mix
gently for 2 min; make films on glass slides, dry
rapidly and examine with a fluorescence microscope.
RNA gives an orange–red fluorescence, whereas
nuclear material (deoxyribonucleic acid, DNA)
fluoresces yellow
Although the amount of fluorescence
is proportional to the amount of RNA

28. Automated methods (Cell counters)
SIEMENS ADVIA 2120i
The ADVIA auto RETIC reagent
contains a zwitter ionic detergent
(surfactant) that isovolumetrically
spheres the red cells. It also
contains a cationic dye, Oxazine
750, that stains cells according to
their RNA content.

29. Measurement
A constant volume of the cell suspension from the
Retic reaction chamber passes through the flowcell
where the low-angle light scatter (2° to 3°), the
high-angle light scatter (5° to 15°), and the
absorption signatures for each cell are measured.
The low-angle and high-angle light scatter
signatures are proportional to cell size and
hemoglobin concentration. Light absorption is
proportional to RNA content. The stained
reticulocytes will absorb more light than the
mature RBCs

30. Reticulocyte analysis

31. The RETIC Scatter cytogram is the graphical representation of the
absorption and light-scatter measurements:
the high-gain, absorption (cell maturation) is plotted along the x axis
and the high-angle, low-gain light scatter (cell size) is
plotted along the y axis.
1 RTC Platelet threshold
2 RTC Coincidence threshold
3 RTC threshold
4 Low/Medium RTC threshold
5 Medium/High RTC threshold
A Mature RBCs
B Low absorption Retics
C Medium absorption Retics
D High absorption Retics
E Platelets
F Coincidence events

32. Reticulocyte Reported Parameters
• % RETIC
• # RETIC
• CHr
• MCVr
• CHCMr

33. Reticulocyte Reported Parameters
• %Retic : 100 x (RETIC Count ÷ #RTC Gated
Cells) x % RETIC Cal Factor
• #Retic : RBC x (%Retic ÷ 100) x 1000
• CHr : Mean of the Retic CH histogram for
the Reticulocyte which reflect the functional
availability of iron for the cell and the cell
incorporation of iron into hemoglobin over
the last several days
• Detects functional iron deficiency with more
sensitivity than biochemical parameters

34. Immature Reticulocyte Fraction (IRF)
• IRF helpful in evaluating marrow
erythropoietic response to anemia, in patient
receiving human recombinant
erythropoietin(rHuEPO) or iron therapy
• IRF earliest marker of marrow response it
increases before retic count ,hemoglobin and
hematocrit, Rbc count
• After marrow transplantation an increased IRF
has been observed as one of the first sign of
cell recovery

35. Clinical utility of (MCVr)
Reticulocyte mean volume
• Decrease – Iron deficiency, Thalassemia
• Depleted iron stores - ↑ when iron
supplements are given- ↓ during iron-deficient
erythropoiesis
• Increase - Macrocytic anemia
• Rapid normalization with treatment - Inversion
of MCVr/MCV ratio
• Sickle cell anemia- Hydroxyurea therapy- Fetal
Hgb synthesis- Increased hydration of sickle
reticulocytes- MCVr

36. BECKMEN COULTER DxH 800
• Reticulocyte analysis uses new methylene
blue stain to identify reticulated red cells by
precipitating the residual RNA.
• A portion of the blood sample is diluted and
treated with a hypo-osmotic ghosting solution
to clear the red cells of hemoglobin while
preserving the stained RNA contained within
reticulocytes.

37. Reticulocyte immaturity is related to cell volume
and light scatter. Since more immature
reticulocytes are larger, contain more RNA and
cause increased light scatter, the cell volume and
light scatter will increase with immaturity of the
cell
 Volume (V) is
plotted on the y-
axis,
 Linear light scatter
(llsn) is plotted on
the x-axis.
Measurement

38. The Dataplot shows mature red cells and
Reticulocytes
1.RBC
2.RETIC
3.OTHER
4.WBC

39. REFERENCE
• Dacie and Lewis Practical Hematology Twelfth
edition 2017
• S B McKenzie and J L Williams Clinical
Laboratory Hematology 3rd edition
• Siemens ADVIA 2120I Operator’s manual
• Beckman coulter DxH800 Operator’s manual

40. TANK YOU