MLAB 1415 Hematology Laboratory #3

Reticulocyte
Reticulocytes
Laboratory #3: Reticulocyte Count
Points= 30
Objectives:
1. To perform, it!i" #$% accuracy of i"structor&s 'alues, reticulocyte
cou"ts o" !ole bloo( specime"s.
#. To perform reticulocyte cou"ts it! a"( it!out a Miller ocular.
3. List refere"ce ra"ges for reticulocytes.
4. )orrelate t!e reticulocyte cou"t results it! "ormal a"( (isease states.
5. List sources of error i" t!e reticulocyte cou"t.
*. )alculate t!e reticulocyte cou"t a"( correcte( reticulocyte cou"t.
+. )alculate t!e Reticulocyte ,ro(uctio" -"(e. /R,-0.
1. List # commo" supra'ital stai"s.
Principle:
Reticulocytes are immature RB)s t!at co"tai" rem"a"t cytoplasmic
ribo"ucleic aci( /R2A0 a"( orga"elles suc! as mitoc!o"(ria a"( ribosomes.
Reticulocytes are 'isuali3e( by stai"i"g it! supra'ital stai"s /met!yle"e
blue or brillia"t cresyl blue0 t!at precipitate t!e R2A a"( orga"elles. T!ese
stai"s cause t!e ribosomal a"( resi(ual R2A to coprecipitate it! t!e fe
remai"i"g mitoc!o"(ria a"( ferriti" masses i" li'i"g you"g eryt!rocytes to
form microscopically 'isible (ar45blue clusters a"( filame"ts /reticulum0.
T!e reticulocyte cou"t is a mea"s of assessi"g t!e eryt!ropoietic acti'ity of
t!e bo"e marro. T!e "umber of reticulocytes is e.presse( as a perce"tage
of t!e total "umber of eryt!rocytes cou"te(.
T!e reticulocyte cou"t is ele'ate( i" patie"ts it! !emolytic a"emia,
!emorr!age, a"( folloi"g t!e treatme"t of iro" (eficie"cy a"emia. T!e
reticulocyte cou"t is (ecrease( i" cases of aplastic a"emia a"( (isor(ers
resulti"g i" i"effecti'e eryt!ropoiesis.
Page 1 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
Specimen:
6!ole bloo( t!at is a"ticoagulate( it! eit!er 78TA or !epari" is suitable.
)apillary bloo( (ra" i"to !epari"i3e( tubes or imme(iately mi.e( it! stai"
may also be use(. RB)s must still be li'i"g !e" t!e test is performe(9
t!erefore, it is best to perform it promptly after bloo( collectio". Bloo(
may be use( up to 1 !ours after collectio". :tai"e( smears retai" t!eir
color for a prolo"ge( amou"t of time.
Quality Control:
T!ree sli(es s!oul( be ma(e for eac! retic cou"t performe(. T!e fi"al
calculate( retic% from t!e first to sli(es s!oul( agree it!i" 15% of eac!
ot!er. -f t!is criterio" is "ot met, cou"t t!e t!ir( sli(e.
Reagents Supplies an! "#uipment:
1. 1)ommercially prepare( li;ui( "e met!yle"e blue solutio". -t s!oul( be
store( i" a bro" bottle. -f precipitate is a problem o" t!e smear, t!e
stai" s!oul( be filtere( prior to use.
#. 1Microscope sli(es
3. Microscope
4. 1$ . +5 test tubes
5. ,asteur pipets /it! bulb if pipets are glass0
*. )apillary tubes
+. Miller ocular /if a'ailable0
Proce!ure:
Page 2 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
Preparation o$ Smears
1. Label a 1$ . +5 mm test tube it! patie"t "ame a"( -8.
#. A(( 5 (rops of "e met!yle"e blue solutio" to t!e labele( test tube
3. A(( 5 (rops of ell mi.e( 78TA a"ticoagulate( bloo( to t!e labele( test
tube.
4. Mi. t!e co"te"ts by ge"tly s!a4i"g.
5. -"cubate at room temperature /#$5#4<)0 for %0 mi"utes.
*. After t!e 1$ mi"ute i"cubatio", ge"tly mi. t!e stai"=bloo( solutio".
+. >si"g t!e e(ge smear tec!"i;ue, ma4e t&o a!!itional smears /"ot to
t!ic4 or t!i".
1. Allo to A-R (ry. /8o "ot blo to !aste" (ryi"g0
?. Label t!e sli(es it! t!e (ate a"( t&o patient i!enti$iers /i.e.@ patie"t&s
"ame, patie"t&s -8 # or (ate of birt!0.
'anual 'et(o! o$ Counting )*+,-O., 'iller !isc/
1. ,lace t!e first sli(e o" t!e microscope stage a"(, usi"g t!e 1$. /lo
poer0 obAecti'e, fi"( a" area i" t!e t(in portio" of t!e smear i" !ic!
t!e re( cells are e'e"ly (istribute( a"( 2BT touc!i"g eac! ot!er.
#. 6it!out mo'i"g t!e sli(e, c!a"ge to 1$$. /oil immersio" obAecti'e0, a((
oil, a"( fi"( a" area of t!e smear i" !ic! t!ere are appro.imately 1$$
re( cells 'isible per fiel(. A met!o( for fi"(i"g a fiel( it! arou"( 1$$
RB)s is to me"tally (i'i(e t!e fiel( i"to 4 ;ua(ra"ts a"( cou"t o"e of
t!ose 4 ;ua(ra"ts. -f you cou"t ##5#1 RB)s i" o"e ;ua(ra"t, t!e" t!e
area is acceptable. -" ot!er or(s, t!ere s!oul( be arou"( #5 RB): i" C
of t!e oil immersio" fiel( 'isible.
3. Begi" cou"ti"g bot! t!e RB)s a"( t!e retics. Be sure to cou"t all cells
t!at co"tai" a blue5stai"i"g filame"t or at least # or more (iscrete blue
aggregates of reticulum i" t!e eryt!rocyte as retics an! RB)s. /:ee
proce(ure "otes for i"clusio"s t!at may be co"fuse( it! reticulum.0
Page 3 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
4. )o"ti"ue cou"ti"g co"secuti'e oil immersio" fiel(s u"til you !a'e reac!e(
1$$$ total re( cells /RB)s D Retics0. Recor( t!e "umber of Retics
cou"te( o" sli(e #1 o" t!e (ata s!eet.
5. ,lace t!e seco"( sli(e o" t!e microscope stage a"( procee( it! steps 15
4. Recor( t!e "umber of Retics cou"te( o" sli(e ## o" t!e (ata s!eet.
*. -f t!e to sli(es agree it!i" E#$%, a'erage t!e "umber of Retics from
sli(e #1 a"( sli(e ## t!e" perform t!e perce"t reticulocyte formula,
belo.
+. -f t!e to sli(es (o "ot agree it!i" E#$% /roug!ly to u"its0, perform a
retic cou"t o" t!e t!ir( sli(e.
1. )alculate t!e perce"t of reticulocytes as follos@
0 ReticsF # of total reticulocytes cou"te( i" 1$$$ total re( cells
1$
7.ample@
:li(e 1F 4* i" 1$$$ eryt!rocytes
:li(e #F 41 i" 1$$$ eryt!rocyte
A'erageF 4+
Retics cou"te( F 4+ i" 1$$$ eryt!rocytes
% Retics F 4+=1$ F4.+%
'et(o! using t(e 'iller !isc:
T!e Miller (isc or eyepiece is place( i"si(e t!e microscope eyepiece as a" ai( to cou"ti"g
reticulocytes. T!e Miller (isc pro'i(es a sta"(ar(i3e( area i" !ic! to cou"t bot! RB)s
a"( reticulocytes. T!ere are to s;uares t!at ma4e up t!e (isc. :;uare A, t!e retic
cou"ti"g s;uare is "i"e times t!e area of s;uare B, t!e RB) cou"ti"g s;uare /see (iagram0.
Page 4 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
Giel( of Hie
'iller !isc
Retic cou"ti"g s;. /:;. A0
RB) cou"ti"g s;./:;. B0
/cou"t RB)s a"( Retics as RB)s0
1. 1>se a 1$$. obAecti'e a"( a 1$. ocular secure( it! a Miller (isc.
#. ,erform t!e Miller (isc proce(ure usi"g a"ot!er stu(e"t&s set of retic
sli(es. 1
3. Gi"( a suitable area of t!e smear t!at s!os 351$ RB)s i" t!e Retic
cou"ti"g s;uare.
4. )ou"t t!e retics it!i" t!e large retic cou"ti"g s;uare /:;uare A0.
5. )ou"t all cells i" t!e RB) cou"ti"g s;uare/:;uare B0. )ou"t retics i" t!is
s;uare as a RB). )ells touc!i"g t!e li"es s!oul( o"ly be cou"te( o" t!e
bottom a"( left si(es.
*. )o"ti"ue cou"ti"g i" ot!er acceptable fiel(s.
+. :top cou"ti"g !e" you !a'e reac!e( 111 total RB)s /RB)s DRetics0 from
t!e RB) cou"ti"g s;uare. T!is "umber of cells s!oul( be reac!e( i" 155
#$ fiel(s a"( correspo"(s to ??? RB)s cou"te( it! t!e sta"(ar( ma"ual
proce(ure.
1. )alculate t!e % Reticulocytes usi"g t!e Miller (isc met!o( by t!e
folloi"g formula@
0 Retics = Total Retics cou"te( / :;uare A0 I 1$$
Total "umber of RB)s cou"te( /:;uare b0 I ?
7.ample@
Retics cou"te( i" retic cou"ti"g s;. /large s;.0F#$
Page 5 of 14
T!e factor ? corrects
for t!e (iffere"ce i"
area betee" t!e
smaller a"( t!e larger
s;uare.
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
RB)s cou"te( i" RB) cou"ti"g s;. /small s;0F111
%reticsF #$=/111 J?0 I 1$$F #%
Calculation o$ correcte! reticulocyte $or Lo& -ematocrit
T!e reticulocyte cou"t is most ofte" e.presse( as a perce"tage of total re(
cells. -" a" a"emic state it! a lo !ematocrit, t!e reticulocyte % is not a
true reflectio" of t!e reticulocyte pro(uctio" because t!e !ole bloo( i"
t!ese patie"ts !as feer RB)s resulti"g i" a" ele'ate( perce"tage.
Correcte! retic countFRetic% . patie"t&s Hct%
A'g. "ormal Hct %
Ke"(er /a(ult0 A'erage "ormal Hct %
Male 45
Gemale 4#
7.ample@
>"correcte( retic% F 5.$%
,atie"t HctF #5.$% /male0
)orrecte( retic% F 5.$% . #5%=45% F #.1%
Page 6 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
Calculation o$ t(e reticulocyte pro!uction in!e1 )RP+/
17stimati"g RB) pro(uctio" by usi"g t!e correcte( reticulocyte cou"t may
yiel( erro"eously !ig! 'alues i" patie"ts !e" t!ere is a premature release
of you"ger reticulocytes from t!e marro /oi"g to i"crease(
eryt!ropoieti" stimulatio"0. T!e premature reticulocytes are calle( Lstress
or s!iftM reticulocytes. T!ese result !e" t!e reticulocytes of t!e bo"e
marro pool are s!ifte( to t!e circulatio" pool to compe"sate for a"emia.
T!e you"ger stress reticulocytes prese"t it! more filame"tous reticulum.
T!e mature reticulocyte may prese"t it! gra"ular (ots represe"ti"g
reticulum. 2ormally, reticulocytes lose t!eir reticulum it!i" #4 to #+ !ours
after e"teri"g t!e perip!eral circulatio". T!e premature stress retics !a'e
i"crease( reticulum a"( re;uire # to #.5 (ays to lose t!eir reticulum,
resulti"g i" a lo"ger perip!eral bloo( maturatio" time.
Maturatio" Time Hematocrit %
1 (ay 45
1.5 (ays 35
# (ays #5
3 (ays 15
1T!e 'alue obtai"e( is calle( t!e reticulocyte pro(uctio" i"(e. /R,-0. A"
R,- N# represe"ts a" a(e;uate respo"se to a"emia by t!e bo"e marro,
!ereas a" R,- O# is co"si(ere( a" i"a(e;uate respo"se of eryt!opoiesis by
t!e bo"e marro to a state of a"emia.
Calculation o$ t(e RP+:
RP+F)orrecte( retic cou"t i" %
Maturatio" time i" (ays
7.ample@
,atie"t&s correcte( retic F 1#%
,atie"t&s Hct F #5%
Page 7 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
R,-F 1#%=#F *
Proce!ural 2otes:
-"terpretatio" of results@
1. 1T!e reticulocyte cou"t is ele'ate(@ 10 i" patie"ts it! !emolytic a"emia,
#0 i" t!ose it! !emorr!age /acute a"( c!ro"ic0, 30 folloi"g treatme"t
of iro"5(eficie"cy a"emia a"( t!e megaloblastic a"emias, a"( 40 i"
patie"ts it! uremia.
#. 1T!e reticulocyte cou"t is (ecrease( i" cases of@ 10 aplastic a"emia, #0
aplastic crises of !emolytic a"emias, 30 i"effecti'e eryt!ropoiesis as
see" i" t!alassemia, per"icious a"emia a"( si(eroblastic a"emia.
3. 1Reticulocytope"ia i" t!e prese"ce of a suggeste( !emolytic a"emia may
ofte" ma4e (iag"osis (ifficult. T!e (iag"osis of a !emolytic a"emia ca"
be ma(e because t!e combi"atio" of bot! !emolysis a"(
reticulocytope"ia results i" a rapi(ly falli"g !emoglobi" a"( !ematocrit.
:ources of 7rror
1. 1A refractile appeara"ce of eryt!rocytes s!oul( "ot be co"fuse( it!
reticulocytes. Refractile bo(ies are (ue to poor (ryi"g oi"g to moisture
i" t!e air. To rule out refractile material, fi"e focus up a"( (o". T!e
refractile material ill get s!i"y a"( reticulum ill remai" blue.
#. -"cubatio" P15 mi"utes falsely increases t!e retic cou"t (ue to (ye
a(!eri"g to mature RB)s.
3. 1Giltratio" of t!e stai" is "ecessary !e" precipitate( material is
prese"t !ic! ca" resemble a reticulocyte.
4. 17ryt!rocyte i"clusio"s s!oul( "ot be mista4e" for reticulocytes.
-"clusio" Appeara"ce
Page 8 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
Hoell5Qolly bo(ies o"e or sometime to, (eep5purple
(e"se structures.
Hei"3 bo(ies stai" a lig!t blue5gree" a"( are usually
prese"t at t!e e(ge of t!e
eryt!rocytes.
,appe"!eimer bo(ies Most of co"fuse( (ue to (ifficulty
(isti"guis!i"g. T!ese purple5stai"i"g
iro" (eposits ge"erally appear as
se'eral 1gra"ules i" a small cluster. 1-f
,appe"!eimer bo(ies are suspecte(,
stai" it! 6rig!t5Kiemsa to 'erify
t!eir prese"ce.
Hemoglobi" H i"clusio"s multiple small (ots i" e'ery cell.
5. 1Galsely (ecrease( reticulocyte cou"ts ca" result from u"(erstai"i"g t!e
bloo( it! "e met!yle"e blue. Be sure t!e stai"=bloo( mi.ture
i"cubates t!e full 1$ mi"utes.
*. 1Hig! glucose le'els ca" cause reticulocytes to stai" poorly.
+. 1T!ere is !ig! (egree of i"accuracy i" t!e ma"ual reticulocyte cou"t
oi"g to error /E #%0 i" lo cou"ts a"( E +% i" !ig! cou"ts0 a"( a lac4 of
repro(ucibility because of t!e i"accuracy of t!e bloo( film. T!is
i"accuracy !as bee" o'ercome by t!e use of automate( i"strume"ts usi"g
flo cytometry.
1. 1-f "o reticulocytes are obser'e( after sca""i"g at least to sli(es,
report L"o"e see"M.
Reporting Results:
Refere"ce 'alues@
2ebor" /$5# ee4s0@ #.55*.$%
Page 9 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
A(ult@ $.55#.5%
Re$erences:
1Harme"i"g., 8e"ise, )li"ical Hematology a"( Gu"(ame"tals of Hemostasis,
3
r(
e(itio", pp. 5??*$1.
Turgeo", Mary Louise, )li"ical Hematology 5 T!eories a"( ,roce(ures, 3
r(

e(itio", pp3#453#*.
Page 10 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte Reticulocytes
Laboratory #3: Reticulocyte 3ata S(eet
Points=30
:tu(e"t "ame@ RRRRRRRRRRRRRRRRRRRRRRRRRRR 8ate@RRRRRRRRRRRRRRRRRRRRRRRRR
Retic stai" lot #@ RRRRRRRRRRRRRRRRRRRRRRRRRR 7.piratio" (ate@RRRRRRRRRRRRRRRR
-"structio"s@ ,erform T6B reticulocyte cou"ts o" T6B (iffere"t patie"ts. S(o& your calculations4 Remember to use
correct units &(en reporting t(e !ata4
,atie"t
2ame
,atie"t
-8
,atie"t
Age=
Ke"(er
Retics cou"te( ,atie"t
!ematocrit
)alculatio" Retic
Result
-s t!e
retic
result
"ormalS
)orrecte( Retic Reticulocyte
,ro(uctio"
-"(e.
1. :li(e 1 RRRRR
:li(e # RRRRR
/:li(e 3 RRRR0
A'erage@ RRRRR
Calculation:
Result:55555
Calculation:
Result:5555
5
#. :li(e 1 RRRRR
:li(e # RRRRR
/:li(e 3 RRRR0
A'erage@ RRRRR
Calculation:
Result@RRRRR
Calculation:
Result@RRRRR
Page 11 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
Laboratory #3: Reticulocyte
Stu!y Questions
67 points
:tu(e"t 2ame@RRRRRRRRRRRRRRRRR
8ate@RRRRRRRRRRRRRRRRRRRRRRRR
1 pt. 1. 1What is the reticulocyte used to assess?
2pts. 2. What constitutes reticulum?
1pt. 3. What is the ratio of blood to stain in the manual retic procedure?
1pt. 4. How long must the blood incubate with the new methylene blue before making
the retic smear?
1pts. . What is a !stress reticulocyte"?
3pts. #. $ist 3 conditions in which the reticulocyte count is increased.
a. %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
b. %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
c. %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
3pts &. $ist 3 conditions in which the reticulocyte count is decreased.
a. %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
b. %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
c. %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
#pts. '. $ist 3 ()* inclusions that could be confused with reticulocytes on a retic
smear and state how you could differentiate them from retics.
Page 12 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
a.%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%
b.%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%
c.%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%
1pt. +. How can you distinguish reticulum from artifact?
2pts. 1,. -tate the reference range for the following patient populations.
/dult.%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
1 week old baby.%%%%%%%%%%%%%%%%%%%%
2pts. 11. *alculate the following retic count which was done using the 0/12/$
method. -how calculations.
1
st
slide3 1 retics counted in 1,,, cells
2
nd
slide3 1& retics counted in 1,,, cells
1pt. 12. *orrect the retic count obtained in 4uestion 11. 5he patient is an adult
female with a hematocrit of 26. -how calculation.
Page 13 of 14
MLAB 1415 Hematology Laboratory #3
Reticulocyte
Reticulocytes
2pts. 14. *alculate the following corrected retic count which is done using the 0iller
disc method. 5he patient is an adult male. -how your calculation.
7 retics counted in larger s4uare. 2#
7 ()*s 8all cells9 in smaller s4uare. 111
2pts. 1. Why would a doctor re4uest and (P:?
Page 14 of 14