This document provides information on reticluocyte count, which assesses bone marrow erythropoietic activity. It describes how a reticluocyte is an immature red blood cell that contains RNA aggregates. The routine reticluocyte count involves staining a blood sample and counting the number of reticluocytes per 1000 red blood cells under a microscope. Quality control measures and different counting methods using a calibrated disk are discussed. The reticluocyte percentage, absolute count, corrected count, and production index are defined and their clinical significance and reference ranges are explained.

1. ReticulocyteReticulocyte
countcount

2. • Reticulocyte is an immature rc that has
lost its nucleus and retains aggregates of
RNA within its ribosomes
• RNA decreases as rc matures
• Reticulocytes remains in BM for 2 days
and 1 day in peripheral blood
> Reticulocyte count – can be used to
assess BM erythropoietic activity

3. Routine Reticulocyte CountRoutine Reticulocyte Count
• Principle and specimen requirements
> ribosomal RNA must be stained
supravitally, that is , with rc in living states
> Reticulocyte is defined as any non-
nucleated rc that contains 2 or more
particles of blue stained ,
granulofilamentous material after the
new methylene blue (supravital) staining
> WB with EDTA , capillary blood is also
acceptable

4. Reagents and EquipmentsReagents and Equipments
No special equipments required
 enumeration can be facilitated by
calibrated disk placed in the ocular of
microscope
Flow cytometry – more rapid , accurate
and precise
Supravital stains – new methylene blue
and brilliant cresyl blue
New methylene blue is recommended

5. Quality controlQuality control
> 4 important reasons for discrepancies
1. interobserver variations in the definition of
reticulocyte (at least 2 ribosomal remnants
should be seen in an erythrocytes )
2. size of sample evaluated ( at least 1000
erythrocytes are examined)
3. type of film examined ( wedge vs spun)
4. lack of standardized area if a calibrated
disk is not used

6. > ways uniformity or increased
accuracy can be achieved
> one technologist count 500 cells on
each two slides
> 2 techs count 500 or 1000 cells
independently
> 2 techs should agree within 20%
> high retic count should correlate
roughly with the number of
polychromatophilic erythrocytes in PS.
The higher the retic count the more
precise the measurement

7. Specimen PreparationSpecimen Preparation
• 1. mix equal amount of WB and stain into a
small tube. Amounts don’t have to exactly
equal . Low HCT – higher proportion of blood,
High HCT – lower proportion of blood
• 2. incubate at room temperature . Incubate for
no less than 10 min or as much as 15min. Rapid
test – not more than 2 min.. Flow cytometric
method – is instantaneous
• 3. after incubation – thoroughly mix and prepare
2-3 films

8. • Several methods of counting reticulocytes
routine light microscope method and
calibrated Miller disk method

9. Routine Light MicroscopeRoutine Light Microscope
MethodMethod
• 1. switch to 100x oil immersion objective
(oculars should be standard 10x)
• 2. select an area where erythrocytes are close
but not overlapping and reticulocytes appear to
be well stained
• 3. count the retic and erythrocytes in each field
using the same pattern normally used for
performing a leukocyte differential count. Retic
should also be counted as erythrocytes
• 4. continue counting until 1000 erythrocytes
• 5. calculate reticulocytes using this formula

10. Reticulocyte
No.OfReticulocyte
1000RBCobserved
X100%

11. Calibrated Miller Disk MethodCalibrated Miller Disk Method
• 1. calibrated miller disk is placed in one microscope
ocular to aid counting process. The miller disk appears in
the field view with 2 squares, one inside the other , the
smaller square (B) 1/9 the size of the larger square (A).
Locate accepatable area to begin count, as in standard
reticulocyte count
• 2. count the erythrocytes in the small square B and the
reticulocytes in large square A in 20 fields where cells
are close but not overlapping. A reticulocyte in square B
is counted as an erythrocytes and is included in the
reticulocyte count , since square B is part of the larger
square A. Cells that touch the top or left lines in both
squares in both squares A and B are counted. Cells that
touch right lines and bottom are not counted.
• 3. compute retic count as….

12. Reticulocyte(%) =
Totalreticulocytes insquare Ax 100
TotalRBC insquareB x 9

13. A
B

14. • Example – if the total of 150 reticulocytes
were seen in square A (inc in square B)
after counting 500 rbcs in 20 fields of
square B the retic count would be
calculated as

15. Reticulocyte(%)=
150
500x9
X100 = 3.3%

16. • Reticulocyte reference ranges:
• Express in percentages or in absolute
number per liter
> adults – 0.5%-1.5% ( 0.5%-2%)
range may be slightly higher in women or
people living in higher altitude ( higher
than 6000feet above sea level)
> newborns (2-6%) but drops within 1-
2weeks
> absolute number – 10-110x109/L

17. Comments and sources
technical error
• 1. When using the Miller disk, failure to follow the “edge”
rule may yield to erroneous results; that is , counting
reticulocytes touching all 4 lines of either squares . Only
reticulocytes touching the top and the left lines should be
counted.
• 2. The use of Romanowsky counterstain is no longer
advised because it may obscure the supravitally stained
granulofilamentous material of the retic.
• 3. Refractive artifacts in erythrocytes caused by moisture
in the air and poor drying of the blood film must not be
confused with RNA filaments, which do not appear
refractory when adjusting the fine focus on the
microscope. The RNA filaments simply disappear when
out of focus.

18. • 4. Blood and stain must be well mixed before making
films, because reticulocytes have a lower specific gravity
than mature erythrocytes and thus goes on the top of the
mixture during incubation.
• 5. Increased levels of glucose in the blood may inhibit
staining of reticulocytes
• 6. Pappenheimer, Howell Jolly and Heinz bodies will also
stain supravitally. Howell jolly bodies stain supravitally as
a deep purple and appear singly or in pairs. If
Pappenheimer bodies and HJ bodies are suspected,
examination of Romanowsky stained peripheral blood
film will confirm their presence, because these inclusions
will stain . Whereas reticulocye granulofilamentous
material will not. Pappenheimer bodies must also be
confirmed by iron staining; they are the most difficult to
distinguish from reticulocytes. Heinz bodies does not
stain with Romanowsky. However , on supravitally
stained films, they stain light blue-green and they may
be differentiated from retic because they are always
found on the periphery of the eryhrocyte and are larger
than ribosomal RNA. Heinz body often make the cell
look like a pitted golf ball

19. Absolute Reticulocyte CountAbsolute Reticulocyte Count
• Principle – The absolute Reticulocyte
Count (ARC) reflects the actual number of
reticulocytes in one liter of whole blood.
This measurement in beneficial in
monitoring BM transplant patients onBM transplant patients on
chemotherapy.chemotherapy. It is not yet routinely
reported; however, some flow cytometric
methods automatically measure the red
cell count and report reticulocytes in
absolute numbers

20. Calculation and Reference Range
ARC= XRBC
Reticulocyte%
100
( )10
12
/L

21. Example : Px retic count = 4%,
RBC ct = 3.3x10 12/L. What is the
ARC?
ARC =
(4) x (3.30x10 /L)
12
100
=132x10 /L9

22. ARC ref range
Ref range = 10 -110x10 /L
9

23. • Reticulocyte percentage may be misleading if
one does not consider the degree of anemia or
of intense erythropoietic stimulation. The
retic count may be truly elevated, indicating
increased effective erythropoiesis, or it may
appear elevated because the total number of
erythrocytes is decreased. Therefore reticulocyte
counts should be corrected for anemia. Several
corrections maybe made to this percentage
taking into consideration total erythrocyte count,
Hct, and early release of erythrocytes from the
bone marrow

24. Corrected Reticulocyte countCorrected Reticulocyte count
• Principle : (CRC)(CRC) is sometimes referred as
reticulocyte index (RI)(RI) or hematocrit
correction. The percentage of reticulocyte
release into the circulation or because of a
decrease in number of mature red cells in
circulation. The CRC corrects the
observed retic count to a “normal” Hct of
0.45L/L to correct the degree of anemia

25. CRC = Reticulocytes (%) x
Hct (L/L)
0.45L/L

26. For example , if the patient retic count is 4.5% and
Hct is 0.30 L/L .What would be the CRC?
CRC= 4.5 x
0.30
0.45
=3.0%

27. • Ref range of CRC – The expected value of CRC
depends on the degree of anemia. Normally
CRC should be approximately 1%. Patient with a
Hct 0.35L/L are expected to have CRC of 2-3%
and those below 0.25L/L should have CRC
greater than 3%
• Comments : CRC is most often used as part of
the reticulocyte production index (RPI)reticulocyte production index (RPI) which
more clinically useful than CRC

28. Reticulocyte Production IndexReticulocyte Production Index
• Principle: (a.k.a shift correction) provides a
further refinement of the CRC. It is a General
indicator of the rate of effective erythrocyte
production in anemias. During intense
erythropoietic stress the maturation time in the
bone marrow may be shortened from usual 3.5
days to as little as 1 day, allowing the
reticulocytes to circulate longer than usual in the
peripheral blood. Cells released early to the
peripheral blood are referred to as shift cells
and have polychromatophilic appearance

29. • Under such intense erythropoietic stress,
the number of reticulocytes in the
peripheral blood may be markedly
increased without the corresponding
increase in the bone marrow. Because the
life span of the reticulocyte in the PB
corresponds to the degree of anemia, the
RPI is corrected for both hematocrit and
maturation time in the PB.

30. Calculation
RPI=
Maturation time in peripheral blood
CRC
RPI=
Reticulocytes(%)x
Hct (L/L)
0.45L/L
Maturation time in peripheral blood
Or

31. • The approximate maturation time varies with
hematocrit as follows and these figures are
used in RPI calculation
Hct (L/L)
0.40-0.45
0.35-0.39
0.25-0.34
0.15-0.24
<0.15
Maturation Time (days)
1.0
1.5
2.0
2.5
3.0

32. For example, If the patient has retic count of
5% and HCT 0f 0.28L/L, the maturation
time correction factor is 2.0. the RPI
calculation is:
RPI =
2.0
=1.6%
5.0x
0.28
0.45

33. • This RPI that erythrocytes production is
increased to 1.6times the normal level,
which is not an effective erythropoietic
response for this degree of anemia
• Reference range
> RPI greater than 3 indicates adequate
marrow response to anemia
> RPI less than 2 represents an
inadequate response

34. • RPI is elevated with chronic hemolysis,
recent hemorrhage and response to
therapy to anemia
• Decreased RPI – bone marrow failure,
ineffective erythropoiesis (vit B12 and
Folate deficiency)

35. • Comments – there are several other methods of
reticulocyte counting, some of which involve
automated instruments that identify reticulocytes
utilizing pattern recognition. Flow cytometry,. Flow cytometry,
combined with flourescent stainingcombined with flourescent staining
techniquestechniques, increases the possibility of a more
accurate and precise assessment of effective
erythrocyte production. These methods count
large number of cells, reducing statistical error,
and are capable of assessing the degree of
maturation of the reticulocyte population. Both
pattern recognition and flow cytometry have
been found to acceptable alternatives to manual
retic counts.