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RETICULOCYTE COUNT

RETICULOCYTE
Immature rbc that has lost its nucleus but retains aggregates of RNA.
It contains organelles mitochondria & ribosomes.
Formation of ribosomes ceases with the loss of nucleus
The loss of ribosomes and mitochondria, along with full hemoglobinization of the
cell marks the transition from reticulocyte to full maturation of the erythrocyte.
Found in bone marrow & !eri!heral blood.
Wrights stain: Polychromatophilic macrocyte
o "rythrocytes with bluish tinge #!ink to slightly !inkish gray cyto!lasm$
Supravital stain: Reticulocyte
o Non%nucleated !ale blue erythrocyte with & or more !articles of dark%blue
stained granulofilamentous material.
ROUTINE RETICULOCYTE COUNT
Reflection of the amount of the effecti'e red cell !roduction taking !lace in the bone
marrow #measure effecti'e erythro!oiesis of the bone marrow$.
The bone marrow re!laces a!!ro(imately 1% of the adult red cells e'eryday
Stress reticulc!tes
o )hift or )timulated reticulocytes
o Reticulocytes released !rematurely from the *+ to the !eri!heral blood
o ,ontains more reticulum than normal reticulocytes
o Recognizable on Wright"staine# $l# s%ears by their larger size and
increased blue tint, denser mesh%like network
o +ay be accom!lished by younger erythrocytes like metarubricytes
Ribosomal RNA must be stained su!ra'itally
The re-uired s!ecimen is whole blood anticoagulated with E&T' #.ersene$ but any
common anticoagulant may be used. ,a!illary blood may be used.
SU(R')IT'L ST'IN:
/sed to stain li'ing cells immediately after remo'al from the li'ing body.
Residual RNA is !reci!itated within the reticulocytes.
RNA is !reci!itated as a dye%ribonucleo!rotein com!le(
)u!ra'ital stains
o *rilliant cresyl blue
o New methylene blue recommended by N,,0)
(RINCI(LE:
To detect the !resence of RNA, the red cells must be stained while they are syill
li'ing. 1hole blood is incubated with N+* or *,*.
Any non%nucleated erythrocyte that contains two or more !articles of blue%stained,
reticulum after staining is defined as reticulocyte.
S(ECI*EN (RE('R'TION:
1+ +i( e-ual amount of whole blood and stain in a small tube #, #rps each$.
-+ Allow to stand at r% te%perature, or incubate at ,.

C /r 10"11 %ins+
,+ 2re!are - r , wedge or s!un films & air dry.
ROUTINE LI23T *ICROSCO(E *ET3O&:
3. )witch to oil%immersion ob4ecti'e.
&. )elect an area where red cells are close but not o'erla!!ing and reticulocytes a!!ear
to be well stained.
5. ,ount the reticulocytes and red cells in each field using the same !attern normally
used for !erforming a leukocyte differential.
6. Reticulocyte are also counted as erythrocytes. Red cells stain pale $lue r $lue
green.
7. ,ontinue counting until 1000 r$cs ha'e been obser'ed
8. ,alculate reticulocyte count.
Reticulocyte ,ount #9$: ; of reticulocyte counted ( 3<<
3<<< R*,s
C'LI4R'TE& *ILLER &IS5 *ET3O&:
+iller disk a!!ears in the field of 'iew with two s-uares, one inside the other, the
smaller s-uare being 3=> the size of the larger s-uare.
3. 2lace the calibrated +iller disk in one ocular.
&. ,ount the erythrocytes in * #)mall )-uare$ and the reticulocytes in A #larger s-uare$
in -0 successive /iel#s on the film until 100 re# cells ha'e been counted.

RE6ERENCE R'N2ES:
"(!ressed as !ercentages or in absolute number !er liter.
'#ults: <.79% 3.79
Ne7$rns: &.<9% 8.<9 #higher, but dro!s to adult le'els in 1 t - 7ee8s$.
Range may be slightly higher in menstruating women and !ersons li'ing an altitude
higher than 9:000 /eet abo'e sea le'el.
?ecreased count a!lastic anemia & in conditions in which the bone marrow is not
!roducing cells.
Increased count hemolytic anemias, I?A, recei'ing iron thera!y, thallasemia,
sideroblastic anemia, and in blood loss.
The reticulocyte count may be truly elevate#, indicating increased effecti'e
erythro!oiesis, or it may only a!!ear ele'ated because the total number of
erythrocytes is decreased. Therefore, reticulocyte counts should be corrected for
anemia.
;U'LITY CONTROL:
No ade-uate method of -uality control
Reasons for discre!ancies
o Inter%obser'er 'ariations
o )ize of the sam!le e'aluated
o Ty!e of field e(amined
o 0ack of standardized area
It has been suggested that results obtained by two technologists should agree within
<-0% of each other. If they do not, re!eat the reticulocyte count on the third smear.
@n high reticulcoyte counts, the !ercentage of reticulocytes should correlate roughly
with the number of !olychromato!hilic erythrocytes seen on the !eri!heral blood
film.
Therefore, an increase# number of reticulocytes are indicati'e of a bone marrow
res!onse to anemia.
The higher the reticulocyte count, the lesser error is in'ol'ed in the !rocedure.
CO**ENTS = SOURCES O6 ERROR:
The !ungest er!thrc!te shows a larger amount of RNA whereas the %st
%ature reticulc!te shows only a small amount of RNA.
E&T'"anticagulate# $l# may be stored for -> hurs !rior to staining while
still obtaining acce!table results.
/se of heparin & high $l# sugar may cause reticulocytes to show pale staining.
/se of R%an7s8! counterstain is no longer ad'ised.
+oisture in air & !oor drying of the film must be a'oided.
The RN' sim!ly disa!!ears when out of focus.
*lood and stain must be mi(ed well.
4rilliant cres!l $lue also stains reticulocytes but shows too much inconsistency in
staining for routine use.
(ure a?ure 4 may be used in !lace of new methylene blue with good results #using
the same stain concentration and !rocedure$.
1hen using the +iller disk, failure to follow the @e#geA rule may yield erroneous
resultsA that is, counting the reticulocyte touching all four lines of either of the two
s-uares. @nly reticulocytes touching the to! or left lines should be counted.
There are se'eral re# $l# cell inclusins that are stained with su!ra'ital stain
o Howell-Jolly bodies
&eep purpleA a!!ear singly or in !airs, !resumed to contain
?NA.
o Pappenheimer bodies
)iderotic granules
(urple staining de!osits a!!ear as se'eral granules in a small
cluster & will usually be a darker shade of blue than the
reticulocyte
The darker%staining granules a!!ear near the !eri!hery of a
young erythrocyte
o Heinz bodies
,onsist of denatured globin, stains light $lue"green & usually
cling on R*, membranes & usually larger than ribosomal RNA.
o Hemoglobin H bodies
Round, greenish $lue inclusions
SOURCES O6 )'RI'TION:
The sam!ling error in the manual reticulocyte count is relati'ely large. This means
that if only 1000 er!thrc!tes are e'aluated, the B1% cn/i#ence li%its for a
o 39 count are <.6%3.89
o 79 count are 5.6%8.69
o 3<9 count are B.3%33.>9

CORRECTE& RETICULOCYTE COUNT CCRCD
Creticulocyte inde(D or Chematocrit correctionD
@ften used as a !art of the Reticulocyte 2roduction Inde( #R2I$
9 reticulocyte may a!!ear increase because of early reticulocyte release or a
decrease in the number of mature red cells in the circulation.
)hould reflect the total !roduction of red cell regardless of the concentration of red
cells in the blood #R*, count$.
,orrects the obser'ed reticulocyte count to a normal hematocrit of <.67 0=0
Reference range #de!ends on the degree of anemia$
o Nr%al: 39
o 3ct / 0+,1 LEL: &%59
o 3ct $el7 0+-1 LEL: 5%79
,alculation
,R,: Reticulocyte #9$ ( 2( Ect
<.67 0= 0
'4SOLUTE RETICULOCYTE COUNT C'RCD
It reflects the actual number of reticuolocytes in 3 0 of whole blood
Informati'e inde( of erythro!oietic acti'ity
It is recommended that the absolute count be used in lace of the corrected
reticulocyte count
1hen re!orted in !ercentage, the reticulocyte does not indicate the relationshi!
between the !eri!heral erythrocyte mass & the total number of reticulocytes
!roduced.
This measurement is beneficial in monitoring bone marrow trans!lant !atients and
!atients on chemothera!y.
Reference range Normally &7%F7 #7<$ ( 3<
>
=0 or 39 of circulating erythrocytes
,alculation
AR,:Reticulocyte #9$ ( R*, ,ount
3<<
RETICULOCYTE (RO&UCTION IN&EF CR(ID
Cshift correctionD
2ro'ides further refinement of the ,R,
General indicator of the rate of erythrocyte !roduction increase abo'e normal in
anemias.
PRINCIPLE:
o A sim!le !ercentage calculation of reticulocytes does not account for the
fact that !rematurely released reticulocytes re-uire H to 3 H days longer in
circulating blood to mature & lose their net%like reticulum.
A sim!le !ercentage calculation of reticulocytes does not account for the fact that
!rematurely released reticulocytes re-uire H to 3 H days longer in circulating blood
to mature & lose their net%like reticulum. Therefore, the reticulocyte count, e'en if
CcorrectedD will be ele'ated out of !ro!ortion to the actual increase in erythrocyte
!roduction because of the accumulation of these younger reticulocytes in the
circulating blood.
,ells released early to the !eri!heral blood are referred to as Cshift cellsD or Cstress
reticulocytesD and ha'e a !olychromato!hilic a!!earance.
The !atientIs hematocrit is used to determine the a!!ro!riate correction factor.
+aturation time 'aries in'ersely with hematocrit.
3e%atcrit CLE
LD
Reticulc!te *aturatin Ti%eE Crrectin 6actr
C#a!sD
<.6<% <.67 3.<
<.57% <.5> 3.7
<.&7% <.56 &.<
<.37% <.&6 &.7
J <.37 5.<
,alculation
R2I: ,orrected Reticulocyte ,ount
+aturation Time in 2eri!heral *lood
Reference range
o R2I greater than 5 ade-uate *+ res!onse to anemia
,haracteristic of hemolytic anemias & anemias secondary to
blood loss
o R2I less than & inade-uate *+ res!onse to anemia
Associated with hy!o!roliferati'e anemias & anemias resulting
from maturation disorders.
Increase# R(I:
o ,hronic hemolysis
o Recent hemorrhage
o Res!onse to thera!y for anemia
&ecrease# R(I:
o A!lastic anemia #*one +arrow failure$
o Ineffecti'e erythro!oiesis #e.g. +egaloblastic anemia$
'UTO*'TE& RETICULOCYTE COUNT
?e!ending on the s!ecific model of the analyzer, this !rocess may be semi or
automated.
All methods rely on the addition of a stain or fluorescent dye to detect the RNA
content of R*,.
The amount of dye bound is !ro!ortional to the amount of RNA !resent.
The labeled s!ecimen is !assed in front of laser light source, and cells emitting more
than a threshold amount of fluorescent light are counted as reticulocytes. This
automated methods is more !recise than the manual because many more cells are
analyzed #usually 5<,<<< cells$.
+ethods of detection
o Im!edance
o 0ight scatter
o Absor!tion
o Fluorescence intensity
Automated instruments !ro'ide additional reticulocyte !arameters such as
o Reticulocyte- Hemoglobin concentration (CHr)
?etermined from the direct measurement of reticulocyte 'olume
and Eb concentration by light scattering and absorbance
characteristics.
o Reticulocyte Mean Cell Volume
?eri'ed from reticulocyte cytogram and reflects the a'erage of
the reticulocyte !o!ulation.
o Immature Reticulocyte Fraction or High Intenity Ratio
Reflects those reticulocytes with an increased amount of RNA or
the early immature reticulocytes.
I**'TURE RETICULOCYTE 6R'CTION CIR6D
Eel!ful in e'aluating erythro!oietic bone marrow res!onse to anemia, monitoring
anemia, & e'aluating res!onse & thera!y.
Automated instruments are also ca!able of determining the maturity of reticulocytes
by -uantitating the amount of RNA !resent in reticulocytes.
Reticulocyte fractions are se!arated based on the RNA content with the more
immature cells containing the highest amount of reticulum. The immature
reticulocyte fraction -uantitati'ely describes the youngest reticulocytes with the
greatest staining intensity #highest content of RNA$.
Increased IRF is the first indicator of bone marrow reco'ery following
trans!lantation, ade-uate erythro!oietic res!onse or successful re!lacement thera!y
with erythro!oietin, iron, .itamin *3&, or folate. 1hen the bone marrow increases
!roduction of erythrocytes, there is an obser'able increase in the IRF before there is
an increase in the reticulocyte count or an increase in Eb, Ect, or R*, count.
It is recommended by the N,,0) that the IRF inde( re!lace the R2I.