L a u ra S te ria n W a rd , R e n a ta B ritto N o v is , V e rid ia n a T o le d o N a s c im e n to , M iria m S ie s le r N o b re g a , M a rio J o s e A b d a lla S a a d
D eterm in atio n o f g lu co se lev els u sin g d ried filter p ap er
b lo o d sp o ts: n ew p ersp ectiv e in h o m e m o n ito rin g
Department
of Internal Medicine, Faculdade de Medicina,
University of Campinas - Campinas, Brazil
W e present a m ethod for the determ ination of blood glucose using dried filter paper blood spots. T o validate this m ethod, w e com pared our results .using filter paper and sim ultaneously collected venous blood. W e dem onstrated that there is a linear relationship betw een the filter paper glucose levels and those determ ined in w hole blood (r=O .98). T here w as no significant difference betw een the results of the tw o m ethods (p>O .05). T his m ethod is a cheap alternative w hich m ay im prove the control of diabetes m ellitus, and m ay also be very useful in the diagnosis of postprandial hypoglycem ia and other special situations.
U N IT E R M S : D ia b e te s m e llitu s . G lycem ic control. B lood glucose self-m onitoring.
IN T R O D U C T IO N
T
h e re s u ltsC o m p lic a tio n so f th e T ria l),D C C T (D ia b e te sa la rg e C o n tro lm u ltic e n te ra n dp ro s p e c tiv e tria l d e s ig n e d to e x a m in e th e e ffe c t o f
m e ta b o lic c o n tro l o n th e d e v e lo p m e n t o f d ia b e tic
c o m p lic a tio n s , re c e n tly p ro v id e d c o n v in c in g e v id e n c e th a t
g ly c e m ic c o n tro l a m e lio ra te s s u c h c o m p lic a tio n s 4 .
A s s e s s m e n t o f th e b lo o d g lu c o s e le v e ls d u rin g th e
p a tie n t's d a ily a c tiv itie s o u ts id e th e h o s p ita l m a y b e a n
im p o rta n t a id in d ia b e te s m a n a g e m e n t5 .1 4 .1 5 .S e v e ra l m e th o d s
h a v e b e e n in tro d u c e d fo r th is p u rp o s e , in c lu d in g u rin e
g lu c o s e m e a s u re m e n ts , g lu c o s e o x id a s e re a g e n t s trip s a n d
re fle c ta n c e m e te rs . It is w e ll k n o w n th a t u rin e g lu c o s e
m e a s u re m e n ts a re n o lo n g e r s a tis fa c to ry a s th e o n ly m e a n s
o f c o n tro l o u ts id e th e h o s p ita l'l. 1 5 .T h e re fo re , th e ra p e u tic
d e c is io n s d e p e n d o n , a m o n g o th e r th in g s , th e re lia b ility o f
th e b lo o d g lu c o s e d e te rm in a tio n s 5 . W h e n g lu c o s e
A d d re s s fo r c o rre s p o n d e n c e : M a rio J o s e A b d a lla S a a d
D e p a rtm e n t o f In te rn a l M e d ic in e F C M - U N IC A M P P . 0 .B o x 1 1 7 0
C a m p in a s /S P - B ra s il- C E P 1 3 0 8 1 -9 7 0
c o n c e n tra tio n s a re d e te rm in e d o u ts id e o f th e la b o ra to ry u s in g
te s ts s trip s a n d re fle c to m e rs , o n e m u s t e n s u re p e rfe c t q u a lity
o f th e s trip s , re lia b le o p e ra tio n o f th e re fle c to m e r a n d
re p ro d u c ib le a n a lity c a l h a n d lin g o f th e s y s te m 1 6 . In th is
re g a rd , th e s o c io -c u ltu ra l a n d e c o n o m ic s ta tu s o f o u r p a tie n ts
a re s e v e re h a n d ic a p s to h o m e m o n ito rin g .
G lu c o s e m e a s u re m e n t u s in g d rie d c a p illa ry b lo o d s p o ts
o n filte r p a p e r is a n a lte rn a tiv e m e a n s o f h o m e m o n ito rin g .
T h is p rin c ip le h a s p re v io u s ly b e e n d e s c rib e d6
.1 7 . T he m ethod
is s im p le a n d c h e a p fo r th e p a tie n t to u s e , re q u ire s little
e q u ip m e n t, a n d is s u ita b le fo r in te rm itte n t a s s e s s m e n t o f
d iu rn a l b lo o d g lu c o s e c u rv e s o r th e te m p o ra ry a s s e s s m e n t
o f p a rtic u la r p ro b le m s . T h e s ta n d a rd iz a tio n o f s u c h a m e th o d
fo r b lo o d g lu c o s e d e te rm in a tio n c o u ld , th e re fo re , p ro v id e a
n e w p e rs p e c tiv e in d ia b e te s m a n a g e m e n t.
T h e p u rp o s e o f th is s tu d y w a s to d e s c rib e a n d e v a lu a te
a re lia b le a n d re p ro d u c ib le te c h n iq u e fo r b lo o d g lu c o s e
q u a n tific a tio n w h ic h c o u ld b e e m p lo y e d in v a rio u s s itu a tio n s o u ts id e th e h o s p ita l.
M A T E R IA L A N D M E T H O D S
P a tie n ts : W e s tu d ie d a to ta l o f 5 4 b lo o d s a m p le s . F O lty
-tw o o f th e s e w e re fro m n o rm a l s u b je c ts , e ig h t w e re fro m
1101
1 1 :1 5 p .m .
"2 h rs . a fte r lu n c h " 4 /1 7
"B e fo re lu n c h "
F ig u re 1 - F ilte r p a p e r w ith p a tie n t's p e rs o n a l n o te s .
S in c e w e c o lle c te d b lo o d fro m p a tie n ts u n d e r v e ry
d iffe re n t c o n d itio n s , w e o b ta in e d a w id e ra n g e o f b lo o d
g lu c o s e v a lu e s (2 0 - 4 5 0 m g /d l). T h is w a s im p o rta n t to v a lid a te
th e m e th o d in c o n d itio n s o f h y p o - a n d h y p e rg ly c e m ia .
T h e filte r p a p e r b lo o d g lu c o s e le v e ls w e re
c o m p a re d w ith th o s e o b ta in e d u s in g th e ro u tin e
m e th o d fo r v e n o u s b lo o d g lu c o s e d e te rm in a tio n s .
T h e re s u lts a re s h o w n in F ig u re 2 . T h e re w a s a
lin e a r re la tio n s h ip b e tw e e n th e filte r p a p e r g lu c o s e
v a lu e s a n d th o s e d e te rm in e d in w h o le b lo o d
(r= 0 .9 8 ). T h e re w a s n o s ig n ific a n t d iffe re n c e
b e tw e e n th e re s u lts o f th e tw o m e th o d s (p > 0 .0 5 ).
T h e s ta n d a rd c u rv e w a s lin e a r. T h e in tra
-a s s -a y a n d in te ra s s a y c o e ffic ie n t v a ria tio n s w e re
b o th 6 p e rc e n t. F ig u re 3 s h o w s th a t th e g lu c o s e
c o n c e n tra tio n in th e filte r p a p e r b lo o d s a m p le s in
th e a b s e n c e o f g ly c o ly s is in h ib ito rs w a s s ta b le
u n d e r la b o ra to ry c o n d itio n s fo r 7 d a y s . A fte r 2 0
d a y s , th e re w a s a 3 7 p e rc e n t fa ll in th e g lu c o s e
le v e ls o f th e s a m p le s . U s in g s o d iu m -flu o rid e
tre a te d filte r p a p e rs , g lu c o s e v a lu e s w e re s ta b le
fo r m o re th a n 3 0 d a y s (F ig . 3 ).
C O S T C O M P A R IS O N : A c o s t c o m p a ris o n
c a n o n ly b e a p p ro x im a te b u t m a y s till b e o f in te re s t.
R E S U L T S
o f b lo o d . T h e d is c s w e re tra n s fe rre d to la b e le d ' te s t tu b e s
c o n ta in in g 3 0 0 u l o f a 5 p e rc e n t tric h lo ro a c e tic a c id s o lu tio n
a n d w e re e lu te d b y s h a k in g a t 1 0 -m in u te in te rv a ls fo r o n e
h o u r a t ro o m te m p e ra tu re . T h e s a m p le s w e re th e n c e n trifu g e d
a t 3 0 0 0 rp m fo r 1 0 m in u te s .
A s p o s itiv e c o n tro ls , 1 0 0 u l s a m p le s o f w h o le b lo o d
w e re p re p a re d in a n id e n tic a l m a n n e r. T h e s u p e rn a ta n ts o f
th e s a m p le s fro m b lo o d s p o ts , fro m w h o le b lo o d a n d fro m
th e s ta n d a rd s , w e re tra n s fe rre d to s a m p lin g tu b e s a n d
im m e d ia te ly a n a ly s e d u n d e r id e n tic a l c o n d itio n s , b y th e s a m e
m e th o d d e s c rib e d h e re in 1 0 .
S ta n d a rd c u rv e p re p a ra tio n :
6 m l o f E D T A -tre a te dv e n o u s b lo o d w e re s to re d a t ro o m te m p e ra tu re fo r 2 4 h o u rs
to re a c h n e a r z e ro g lu c o s e c o n c e n tra tio n (le s s th a n 2 m g /
d L ). O n e m illilite r o f th e b lo o d w a s th e n tra n s fe re d to s o d iu m
flu o rid e -c o n ta in in g tu b e s a n d g lu c o s e w a s a d d e d to o b ta in
fin a l c o n c e n tra tio n s o f 5 0 , 1 0 0 ,2 0 0 a n d 4 0 0 m g /d l. S a m p le s
fro m e a c h o f th e s e tu b e s w e re th e n s p o tte d o n to filte r p a p e r
to th e n b e e lu te d a n d p ro c e s s e d a s p re v io u s ly d e s c rib e d .
S ta tis tic a l
a n a ly s is :
D iffe re n c e s b e tw e e n g ro u p sw e re c o m p a re d b y o n e -w a y a n a ly s is o f v a ria n c e . P e a rs o n 's
re g re s s io n a n a ly s is w a s u s e d w h e n a p p ro p ria te . P v a lu e s
< 0 .0 0 5 w e re c o n s id e re d to in d ic a te s ta tis tic a l s ig n ific a n c e .
1 0 :5 0 a .m .
9 /1 4 6 :0 0 a .m .
p e rs o n s w ith u n c o n tro lle d d ia b e te s , a n d fo u r w e re c o lle c te d
d u rin g a n in s u lin to le ra n c e te s t (lIT ) a fte r 1 5 m in u te s o f
in s u lin in fu s io n .
P a p e r:
W e u tiliz e d a s p e c ia lly -p rin te d filte r p a p e r fro mF IT E C In d . &C o m . d e F iltro L td a . T h is p a p e r is 1 0 0 p e rc e n t
c o tto n . E a c h filte r c o n ta in e d fiv e n u m b e re d z o n e s , e a c h 2
c m in d ia m e te r (F ig . 1 ). A ll z o n e s w e re id e n tifie d a n d d a te d
b y w ritin g d ire c tly o n th e filte r p a p e r.
B lo o d s a m p lin g :
A ll s u b je c ts w e re a s k e d to c o lle c t ad ro p o f b lo o d a fte r p lic k in g th e la te ra l s id e o f a fin g e r tip w ith
a c o m m o n d is p o s a b le n e e d le a n d w e re in s tru c te d to p u t e n o u g h
b lo o d o n th e filte r p a p e r to fill a t le a s t o n e o f th e fiv e c irc le s
c o m p le te ly . F o llo w in g a p p lic a tio n o f th e s p e c im e n , th e s p o t
w a s e x a m in e d to m a k e s u re th a t its s iz e w a s a p p ro x im a te ly
th e s a m e o n b o th s id e s o f th e p a p e r. T h e s p o tte d p a p e rs w e re
s to re d in a n e n v e lo p e a t ro o m te m p e ra tu re , w h e re th e y w e re
a llo w e d to d ry . W e s im u lta n e o u s ly c o lle c te d 3 m l o f v e n o u s
b lo o d fro m a ll p a tie n ts in to tu b e s c o n ta in in g 2 p e rc e n t s o d iu m
flu o rid e a n d c a lc iu m o x a la te .
In o rd e r to im p ro v e th e re p ro d u c ib ility , w e s o a k e d s o m e
o f th e filte r p a p e rs in a 2 p e rc e n t s o d iu m flu o rid e s o lu tio n
p rio r to u s e ; th e s e p a p e rs w e re th e n a ir-d rie d a t ro o m
te m p e ra tu re . T o c h e c k th e re p ro d u c tiv e a b ility o f th e m e th o d ,
w e c a lc u la te d th e c o e ffic ie n t v a ria tio n o f th e a b s o rb a n c e
v a lu e s o f a ll th e s ta n d a rd p o in ts a n d s a m p le s fro m b o th
s o d iu m flu o rid e -tre a te d a n d n o n -tre a te d p a p e rs , u s in g s in g le
a n d d u p lic a te s p o ts . T h e a s s a y s w e re re p e a te d d a ily fo r e ig h t
d a y s a n d th e re a fte r s o m e s a m p le s w e re a n a ly z e d a t s e t
in te rv a ls fo r u p to fo u r w e e k s .
P re p a ra tio n
fo r a n a ly s is :
T h e c e n te rs o f th e b lo o ds p o ts w e re s a m p le d w ith a h a n d h e ld p u n c h 5 m m in d ia m e te r.
T h e b lo o d c o n te n t o f s u c h d is c s c o rre s p o n d e d to a b o u t 1 0 u l
1102
F ig u r e 2 - R e la tio n s h ip b e tw e e n th e filte r p a p e r g lu c o s e v a lu e s a n d th o s e d e te r m in e d in w h o le b lo o d .
T h e m eth o d d escrib ed is sim p le to u se. T h e o n ly asp ect
p atien ts h av e to b e co n cem ed
ab o u t is th e ap p ro p riate
size
T h e filter p ap er m eth o d is ch eap an d sim p le, req u irin g o n ly
th e p ap er an d fin g er-p rick in g
eq u ip m en t.
In co n trast, h o m e
m o n ito rin g
w ith g lu co se reag en t strip s in v o lv es acq u isitio n
an d m ain ten an ce
o f a reflecto m er
an d strip s. In B razil, m o st
o f th e reag en t strip s are im p o rted .
T h e fin al co st o f b lo o d
g lu co se
d eterm in atio n s
u sin g
filter
p ap er
is, th erefo re,
ap p ro x im ately
fiv e tim es less th an th e d eterm in atio n
w ith
th e reflecto m eters.
o
440
o f th e sp o ts
an d th at th ey
b eco m e
co m p letely
so ak ed w ith b lo o d . S in ce d iab etic p atien ts are u sed
to n eed les, th e n eed fo r rep eated
fin g erp rick s
is
n o t a serio u s p ro b lem
I .L ik e W A K E L IN
et aL ,
w e h av e o b tain ed
accep tab le
resu lts
fo llo w in g
sto rag e o f th e sam p les
at ro o m tem p eratu re
lO•In
ad d itio n , th e stab ility o f g lu co se w as fo u n d to b e
en h an ced b y p retreatin g
th e filter p ap ers w ith a 2
p ercen t so lu tio n o f so d iu m
flu o rid e. T h e sam p le
stab ility an d th e sim p licity
o f h an d lin g
th e filter
p ap er are o f p ractical
im p 0 l1 an ce
as th ey p erm it
sam p les to b e co llected
o v er sev eral d ay s an d to
b e sen t to th e lab o rato ry
b y m ail!!.
In
th e
ab o v e
m eth o d ,
filter
p ap er
sp ecially
d esig n ed
fo r
sam p lin g
is u sed .
In
ad d itio n ,
an
en zy m atic
p ro ced u re
th at
is
p ractically
free fro m in terferen ce
is em p lo y ed .
T h e m eth o d
is d efin itely
ch eap er
th an th e u se
o f g lu co se
stri p s an d reflectan ce
m eters,
an d
th e
resu lts
are
m o re
reliab le
sin ce
th e
fin al
an aly sis
is p erfo rm ed
in a lab o rato ry
2 .T h e m eth o d is su itab le fo r p erio d ic d eterm in atio n s
o f
b lo o d g lu co se lev els d u rin g ev ery d ay
activ ity . It also u sefu l
fo r th e in term itten t
assessm en t
o f d iu rn al
b lo o d
g lu co se
p ro files, o r th e tem p o rary assessm en t o f p articu lar p ro b lem s.
S in ce little eq u ip m en t
is n eed ed an d b lo o d sam p lin g
tak es
little tim e, th e m eth o d
is also su ited to v ario u s
situ atio n s
o u tsid e th e p atien t's
h o m e. A s p o in ted
o u t b y th e D C C T ,
th is is v ery im p o rtan t
in d iab etes care sin ce th ere is a clear
co rrelatio n
b etw een g o o d g ly cem ic
co n tro l an d a red u ctio n
in ch ro n ic
d iab etes
co m p licatio n s
su ch
as retin o p ath y ,
n eu ro p ath y an d n ep h ro p ath y '.
T h e m eth o d
d escrib ed
m ay
also
b e u sefu l
in th e
d iag n o sis
o f p o stp ran d ial
h y p o g ly cem ia.
P o stp ran d ial
o r
reactiv e h y p o g ly cem ia
p o ses a d ifficu lt d iag n o stic ch allen g e
400 320 360
2 8 0
240 200
1 6 0
G L U C O S E IN F IL T E R P A P E R
( m g /d l)
1 2 0
80
1 6 4 0
D IS C U S S IO N
400
360
0 320
0 0
...J 280
a J
W 240
...J
0
: r :
-~ -~ 200
~
0 1
E 160
W
C J ')
0 120
U
::> ...J
80
C >
40
14
'" 0 1 2 0
"
0 \1 0 0
E
w
8 0( f ) 6 0
0
U4 0
:::::>
- - I 2 0
(9
4 8 1 2 1 6 2 0 2 4 2 8
" '0
3 2 0
"
0 \B
E
2 5 0W 2 0 0
( f ) / 5 0
0
U1 0 0
:::::>
- - I
(9
5 06 1 2 1 8 2 4 3 0 3 6 4 2
F ig u r e 3 - G lu c o s e c o n c e n tr a tio n in th e filte r p a p e r b lo o d s a m p le s in th e a b s e n c e o f in h ib ito r s o f ( A ) g ly c o ly s is a n d u s in g s o d iu m flu o r id e ( 8 ) .
1103
because the symptoms are nonspecific, the diagnosis is often
made by the patients themselves,
and there is no reliable
diagnostic test. The evaluation of patients with postprandial
symptoms
suggestive
of hypoglycemia
requires
the
measurement
of blood glucose at the time of spontaneously
occuring
symptoms
and avoidance
of the oral
glucose-tolerance test. The filter-paper method for the measurement
of blood glucose when symptoms are present can therefore
help to obtain a correct diagnosis!)'
12. 13.The disadvantage
for the patients of not getting the
results immediately may be overcome by taking notes. Our
experience is that the filter paper method is an aid to improved
diabetes control.
In conclusion,
we have described
a method for the
determination of blood glucose using dried filter paper blood
spots. This method is a cheap alternative that may improve
the control of diabetes mellitus, and may also be useful in
the diagnosis of postprandial hypoglycemia.
RESUlVlO
Apresentamos um metoda de dosagem de glicemia em papel de filtro. Para comprovar seu valor, comparamos as resultados
obtidos no papel de filtro e no sangue venoso colhidos simultaneamente. Demonstramos que existe uma relay80 linear entre
a
glicemla
dosada no papel de filtro e a do sangue perifenco. Nao houve diferenya estatistica entre as daiS metodoS (p>O,05).o
metoda se constitui em uma alternativa barata no controle do diabetes melittus e pode ser usado tambem no diagn6stico dahlpoglicemla reativa e em outras situa<;oes especiais.
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