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Routine microestimation of iron in hemoglobin
CLINICA CHIWCA
G86
ROUTINE
MICROESTIMATION
ACT.4
voL.
OF IRON
(1953)
4
IN HEMOGLOBIN
J. FISCHL Chemical Laboratory,
“.4ssaf
Harofe”
Government
Hospital,
Zrifcn
(Israel)
INTRODUCTION Accurate A
and easily performed
“plus-minus
one gram”
therapeutically
has made
in the hemoglobin standards’, stability, within
biochemical ment
for iron determination
in a large
principle
furic
acid
is added,
iron
and persulfate, and after
methods3s4.
then
is not only
can be used as a routine number
that
gy9
to find suitable
standard
SUSDER-
because
of its
iron estimation
We have suitable
that it can compete
is
attempted
to
for hemoglobin
with any of the usual
test for the exact
measure-
of specimens. AND >IETHODS
is similar
to that
and thiocyanate.
mixing
suitable
use of iron
as 0.3-0.3
in order
laboratories5.
which
and reliable
of the method
between
of as little
done
required.
extensive
He has also shown
of most clinical
nlATERIALS The
been
and to develop
availability.
tests and therefore
the reaction
is increasingly the
of the use of iron as a reference
but is so simple
of hemoglobin
and
up changes
has already
analysis
of possibility
out a method
standardization,
measurement
inaccurate,
to follow
work
and ready
the bounds
work
Much
the desirability
reliability
is too
it essential
level.
2 for hemoglobin
iXAN 4 stresses
hemoglobin
report
Blood
the reagent,
and clarifying
of THOIQPSON~ and W’ONG’; is treated
with
concentrated
a thiocyanate-isobutyl the colour
intensity
alcohol
and sul-
solution,
is measured.
REAGEKTS I.
Sulfuric
acid,
2. Potassium
cont.
C.P.
persulfate
IOO ml of distilled 3. Colour
reagent
(saturated
: dissolve
standard
amount dilute
solution:
of distilled iron;
der the conditions. from
dissolve add
distilled
shaken
with
containing
in IOO ml of distilled
2 ml of concentrated water.
sulfuric
in the minimum acid,
One ml of this standard used, corresponds
a certified
water,
alcohol.
0.8635 g of FeNH4(S04)z.~zH,0
0.1 ml, the amount
If obtainable,
pure iron wire,
thiocyanate
and 500 ml of isobutyl
water,
to IOOO ml with
IOO pug of ferric
: 8 g of a good grade reagent
30 g of potassium
and add 4 ml of acetone 4. Iron
solution)
water.
iron standard
the same amount
let
solution
cool
and
contains
to 50 mg O/9of iron unsolution
of iron as given
or one prepared above should be
used. PROCEDURE In placed.
a centrifuge Exactly
potassium added References
tube
persulfate
solution
and the mixture p. 690
of about
0.02 ml of blood, were
Is-ml followed added.
was immediately
capacity, by After shaken
5 drops
of distilled
0.3 ml of sulfuric cooling,
acid
3 ml of colour
vigorously
water and
reagent
and centrifuged
were
0.5 ml of were
at high
MICROESTIMATION
VOL. 4 (1959)
OF
Fe IN HEMOGLOBIN
637
speed for IO min. Enough of the clear, coloured supernatant solution was removed for calorimetry with a dry pipette *. Readings were taken on the Klett** calorimeter. (Any similar instrument is suitable.) For every batch of tests one blank containing the reagents only, and one standard using 0.1 ml of iron standard solution (equivalent to IO ,ug of ferric iron) were carried out simultaneously. The zero setting of the colorimeter was made against isobutyl alcohol. CALCULATION ~(S?B)
. (U -
B) = mg 74 of iron in the sample.
The hemoglobin can be directly calculated by the following formula: -l=.(U-B)=gO/bofHb. (S-B)
since mg of iron per 100 ml --___ = g of Hb per IOO m, ___ 3.40 where S = reading of standard, B = reading of blank, U = reading of unknown. DISCUSSION
We have repeated the check on hemoglobinometrywhich was done in the U.S.A.” by requesting various laboratories to measure the hemoglobin contents of two blood samples. The purpose of comparison was to produce a value for precision and to reduce technical errors. However, the range of variation was very great (Table I). Most of the TABLE HEMOGLOBIN
VALUES
OBTAINED LABORATORIES
Laboratory A B
G H I
Acid hematine
USING
11.4 IO.1 13.1 II.9 ‘5.7
-
Carboxy -
ANALYSIS
OF
THEIR
I THE
SAME
ROUTINE
Hb value (g “/b) specim. I specim.
Method
C D E F
ON
Hb
TWO
SPECIMENS
BY
DIFFERENT
METHODS
Deviation 2
from
real value (g Oi)
Specim. 1
s.1 7.8
-0.2
-1.2
-1.4
-1.5
9.7
$1.5
j-o.4
8.6
-0.7
10.8
9.6
+0.3 +4.’ -0.8
12.4 14.1 13.1
10.3 1I.Z 9.9
+0.8 +2.5 +I.5
+ 1.0 +I.9 +0.3
-0.1 +0.1
+0.5 +0.6
+0.6
iI.
10.6
J, L
Cyanomet-Hb -
11.5 11.7 12.2
9.8 9.9 10.4
M N 0
-
13.6 ICI.1 15.0
12.3 IO.0 13.0
P S
-
13.1 11.4
9.3 9.8
+2.0
-1.5 +3.4
S4.1 +0.3
i-3.0 +I.7 +3.7
+1.5 -0.2
* To improve speed of work a rubber bulb for pipetting is recommended. ** A Klett-Summerson photoelectric calorimeter was used in the present work. References p. 690
Specim. 2
+:.5
J. FISCHL
688
VOL. 4 (1959)
laboratories used the cyanomethemoglobin3 method but only two had the standards prepared by DRABKIS. Some others had used them previously but had abandoned them, and one worker found them unsatisfactory owing to too high values. We concluded from the survey that there is still need for a new method of high reliability in hemoglobinometry. TABLE I1
Helnoglobin No. of sample
method
WONG'S~
(g 9;)
DRABKIN’S~ method
method
Presmt
I
IL.4
II.4
12.0
2
‘5.3
16.1
‘5.’
3 4
‘4.4 16.3
‘4.i rg.0
13.7 16.3
5 b
17.8
18.j
18.0
‘4.7 IO.‘? 16.1
‘4.3 9.4 1j.j
‘4.7 IO.0 Ij.8
;; 9
16.1
16.6
I j.0
TO II
16.5 IL.0
16.3 13.6
16.3 I2.4
12
14.3
I4.r
‘4.3
I3 ‘4
16.4 X.1
16.8 7.8
I5 16
‘5.3 18.2
‘7.4 9.2 16.4 LO. I
‘7 18 19 20
5.4 IL.2 II.3 16.4
6.3 II.4 IO.7 16.3
LI 22 23
14.6 18.1 IO.3
74.4 16.5 11.6
‘4.4 17.8 10.3
24
5.5
6.0
5.7
25 26 2, 28
Ij.0
14.1 8. I
‘4.7 13.0 9.8
‘4.4 8.4
I7.7 9.6 Ij.I
18.4 10.4 I7.L
3 h 36 min
44 min
29 30 Time of performance
‘5.7 18.1
6.0
1I.8 II., 16.1
1j.l
‘7.5 9.8 ___5!L 81 min
The method proposed is simple enough for routine work and its results compare favourably with WOSG’S’ (Table II). The linearity of light absorption is very good, and Beer’s law is obeyed over a wide range of concentration (Fig. I). WONG’S7 method is most often used for hemoglobin standardization. It is not suitable for routine hemoglobinometry because it lasts too long, (see Table II), requires too much blood, and the colour produced in the reaction fades; therefore, the greater Referencesp.
690
VOL. 4 (1959)
Fe INHEMOGLOBIS
MICROESTIMATIONOF
689
the number of tests, the less reliable the results. The test proposed takes little time if performed by experienced technicians (Table II). It requires the same amount of blood as the other routine hemoglobin methods (e.g. 0.02 ml) and the colour is stable for a long time in all the necessary concentrations (Table III). 300r
D250.k & L200Y
5 m 1 150 ;; P 100
I
0
I
1 2
t
3 4
1
I
5
6
I
t
7 8
13
9 10
1.
11
12
1
I
13
14
Few Fig.
I.
Curve
of iron standards.
As far as possible, good grades of analytical reagents should be used, but glassdistilled water is unnecessary, since all iron contaminations are deducted with the blank readings. Deproteinization is omitted and with it the main source of error of WONG’S method7;
sodium tungstate
is not used. TABLE111
STABILITY
OF COLOUR
Hb concentration (g 74)
-..__
_
: 9 I2 15 I8 Blank
2:
5
min I.21 87
OF THE
IRON-THIOCYANATE
COMPLEX
Calorimeter reading 10 min 30 min 60 min 90 124
120 min
90 ‘24
91 124
93 125
‘73 213
216
253 290 330 52
253 291 329 52
170
17=
‘73
20s
212
212
244 282
25’ 285
25’ 286
325 50
328 51
328 5’
173
However, a few precautions must be taken in order to ensure precise results. a) The blood and the colour reagent must be exactly measured. b) Clean glassware must be used, and any contact with iron avoided to prevent further reaction of the coloured solution. The tube must be shaken well immediately after addition of the colour reagent, otherwise the colour fades. c) The calorimeter tube and the pipette used must be free from water to avoid emulsification with the isobutyl alcohol and consequently faulty light absorption. References p.
690
J. FISCHL
690 However, drop
if emulsification
of propyleneglycol With
occurs, which
the observance
of these
could be very
useful in routine
working
for greater
time
the sample
clarifies
voL.
might
be saved
4
by the addition
(1959) of one
the solution.
precautions
and with
hemoglobinometry,
average
whenever
skill,
this
it is possible
method
to exchange
precision. ACKNOWLEDGEMENTS
Our thanks Prof.
are due to Dr. H. MENACHEM for calling
F. RAPPAPORT
help in preparing
for his help
and many
useful
our attention
suggestions,
to the problem,
and Dr. MUNDEL
for
the manuscript. SUMMARY
A new method It is reliable,
easily
the standardization
for hemoglobinometry performed, of other
based on micro iron estimation
and suitable
for routine
examinations,
is presented. as well
as for
methods. REFERENCES
1 R. K.
CANNAN, Am. J. Clin. Pathol., 25 (1955) 376. 2 R. K. CANNAN, Am. J. Clin. Pathol., 30 (1958) 211. 3 D. L. DRABKIN AND J. H. AUSTIN, /. Biol. Chem., 98 (1932) 719. 4 F. W. SUNDERMAN, B. E. COPELAND, R. P. MACFATE, V. E. MARTENS, G. F. STEVENSON, Am. J. C&z. Pathol., 25 (1955) 489. 5 F. W. SUNDERMAN, B. E. COPELAND, R. P. MACFATE, V. E. MARTENS, G. F. STEVENSON, Am. J. Clin. Pathol., 25 (1955) 695. B J. B. THOMPSON, Ind. Eng. Chem.. Anal. Ed., 16 (1944) 646. 7 S. Y. WONG, J. Biol. Chem., 77 (1928) 409.
Received
H. N. SAUMANN
AND
H. N. NAUMANN
AND
February
Iqth,
1959
G86
ROUTINE
MICROESTIMATION
ACT.4
voL.
OF IRON
(1953)
4
IN HEMOGLOBIN
J. FISCHL Chemical Laboratory,
“.4ssaf
Harofe”
Government
Hospital,
Zrifcn
(Israel)
INTRODUCTION Accurate A
and easily performed
“plus-minus
one gram”
therapeutically
has made
in the hemoglobin standards’, stability, within
biochemical ment
for iron determination
in a large
principle
furic
acid
is added,
iron
and persulfate, and after
methods3s4.
then
is not only
can be used as a routine number
that
gy9
to find suitable
standard
SUSDER-
because
of its
iron estimation
We have suitable
that it can compete
is
attempted
to
for hemoglobin
with any of the usual
test for the exact
measure-
of specimens. AND >IETHODS
is similar
to that
and thiocyanate.
mixing
suitable
use of iron
as 0.3-0.3
in order
laboratories5.
which
and reliable
of the method
between
of as little
done
required.
extensive
He has also shown
of most clinical
nlATERIALS The
been
and to develop
availability.
tests and therefore
the reaction
is increasingly the
of the use of iron as a reference
but is so simple
of hemoglobin
and
up changes
has already
analysis
of possibility
out a method
standardization,
measurement
inaccurate,
to follow
work
and ready
the bounds
work
Much
the desirability
reliability
is too
it essential
level.
2 for hemoglobin
iXAN 4 stresses
hemoglobin
report
Blood
the reagent,
and clarifying
of THOIQPSON~ and W’ONG’; is treated
with
concentrated
a thiocyanate-isobutyl the colour
intensity
alcohol
and sul-
solution,
is measured.
REAGEKTS I.
Sulfuric
acid,
2. Potassium
cont.
C.P.
persulfate
IOO ml of distilled 3. Colour
reagent
(saturated
: dissolve
standard
amount dilute
solution:
of distilled iron;
der the conditions. from
dissolve add
distilled
shaken
with
containing
in IOO ml of distilled
2 ml of concentrated water.
sulfuric
in the minimum acid,
One ml of this standard used, corresponds
a certified
water,
alcohol.
0.8635 g of FeNH4(S04)z.~zH,0
0.1 ml, the amount
If obtainable,
pure iron wire,
thiocyanate
and 500 ml of isobutyl
water,
to IOOO ml with
IOO pug of ferric
: 8 g of a good grade reagent
30 g of potassium
and add 4 ml of acetone 4. Iron
solution)
water.
iron standard
the same amount
let
solution
cool
and
contains
to 50 mg O/9of iron unsolution
of iron as given
or one prepared above should be
used. PROCEDURE In placed.
a centrifuge Exactly
potassium added References
tube
persulfate
solution
and the mixture p. 690
of about
0.02 ml of blood, were
Is-ml followed added.
was immediately
capacity, by After shaken
5 drops
of distilled
0.3 ml of sulfuric cooling,
acid
3 ml of colour
vigorously
water and
reagent
and centrifuged
were
0.5 ml of were
at high
MICROESTIMATION
VOL. 4 (1959)
OF
Fe IN HEMOGLOBIN
637
speed for IO min. Enough of the clear, coloured supernatant solution was removed for calorimetry with a dry pipette *. Readings were taken on the Klett** calorimeter. (Any similar instrument is suitable.) For every batch of tests one blank containing the reagents only, and one standard using 0.1 ml of iron standard solution (equivalent to IO ,ug of ferric iron) were carried out simultaneously. The zero setting of the colorimeter was made against isobutyl alcohol. CALCULATION ~(S?B)
. (U -
B) = mg 74 of iron in the sample.
The hemoglobin can be directly calculated by the following formula: -l=.(U-B)=gO/bofHb. (S-B)
since mg of iron per 100 ml --___ = g of Hb per IOO m, ___ 3.40 where S = reading of standard, B = reading of blank, U = reading of unknown. DISCUSSION
We have repeated the check on hemoglobinometrywhich was done in the U.S.A.” by requesting various laboratories to measure the hemoglobin contents of two blood samples. The purpose of comparison was to produce a value for precision and to reduce technical errors. However, the range of variation was very great (Table I). Most of the TABLE HEMOGLOBIN
VALUES
OBTAINED LABORATORIES
Laboratory A B
G H I
Acid hematine
USING
11.4 IO.1 13.1 II.9 ‘5.7
-
Carboxy -
ANALYSIS
OF
THEIR
I THE
SAME
ROUTINE
Hb value (g “/b) specim. I specim.
Method
C D E F
ON
Hb
TWO
SPECIMENS
BY
DIFFERENT
METHODS
Deviation 2
from
real value (g Oi)
Specim. 1
s.1 7.8
-0.2
-1.2
-1.4
-1.5
9.7
$1.5
j-o.4
8.6
-0.7
10.8
9.6
+0.3 +4.’ -0.8
12.4 14.1 13.1
10.3 1I.Z 9.9
+0.8 +2.5 +I.5
+ 1.0 +I.9 +0.3
-0.1 +0.1
+0.5 +0.6
+0.6
iI.
10.6
J, L
Cyanomet-Hb -
11.5 11.7 12.2
9.8 9.9 10.4
M N 0
-
13.6 ICI.1 15.0
12.3 IO.0 13.0
P S
-
13.1 11.4
9.3 9.8
+2.0
-1.5 +3.4
S4.1 +0.3
i-3.0 +I.7 +3.7
+1.5 -0.2
* To improve speed of work a rubber bulb for pipetting is recommended. ** A Klett-Summerson photoelectric calorimeter was used in the present work. References p. 690
Specim. 2
+:.5
J. FISCHL
688
VOL. 4 (1959)
laboratories used the cyanomethemoglobin3 method but only two had the standards prepared by DRABKIS. Some others had used them previously but had abandoned them, and one worker found them unsatisfactory owing to too high values. We concluded from the survey that there is still need for a new method of high reliability in hemoglobinometry. TABLE I1
Helnoglobin No. of sample
method
WONG'S~
(g 9;)
DRABKIN’S~ method
method
Presmt
I
IL.4
II.4
12.0
2
‘5.3
16.1
‘5.’
3 4
‘4.4 16.3
‘4.i rg.0
13.7 16.3
5 b
17.8
18.j
18.0
‘4.7 IO.‘? 16.1
‘4.3 9.4 1j.j
‘4.7 IO.0 Ij.8
;; 9
16.1
16.6
I j.0
TO II
16.5 IL.0
16.3 13.6
16.3 I2.4
12
14.3
I4.r
‘4.3
I3 ‘4
16.4 X.1
16.8 7.8
I5 16
‘5.3 18.2
‘7.4 9.2 16.4 LO. I
‘7 18 19 20
5.4 IL.2 II.3 16.4
6.3 II.4 IO.7 16.3
LI 22 23
14.6 18.1 IO.3
74.4 16.5 11.6
‘4.4 17.8 10.3
24
5.5
6.0
5.7
25 26 2, 28
Ij.0
14.1 8. I
‘4.7 13.0 9.8
‘4.4 8.4
I7.7 9.6 Ij.I
18.4 10.4 I7.L
3 h 36 min
44 min
29 30 Time of performance
‘5.7 18.1
6.0
1I.8 II., 16.1
1j.l
‘7.5 9.8 ___5!L 81 min
The method proposed is simple enough for routine work and its results compare favourably with WOSG’S’ (Table II). The linearity of light absorption is very good, and Beer’s law is obeyed over a wide range of concentration (Fig. I). WONG’S7 method is most often used for hemoglobin standardization. It is not suitable for routine hemoglobinometry because it lasts too long, (see Table II), requires too much blood, and the colour produced in the reaction fades; therefore, the greater Referencesp.
690
VOL. 4 (1959)
Fe INHEMOGLOBIS
MICROESTIMATIONOF
689
the number of tests, the less reliable the results. The test proposed takes little time if performed by experienced technicians (Table II). It requires the same amount of blood as the other routine hemoglobin methods (e.g. 0.02 ml) and the colour is stable for a long time in all the necessary concentrations (Table III). 300r
D250.k & L200Y
5 m 1 150 ;; P 100
I
0
I
1 2
t
3 4
1
I
5
6
I
t
7 8
13
9 10
1.
11
12
1
I
13
14
Few Fig.
I.
Curve
of iron standards.
As far as possible, good grades of analytical reagents should be used, but glassdistilled water is unnecessary, since all iron contaminations are deducted with the blank readings. Deproteinization is omitted and with it the main source of error of WONG’S method7;
sodium tungstate
is not used. TABLE111
STABILITY
OF COLOUR
Hb concentration (g 74)
-..__
_
: 9 I2 15 I8 Blank
2:
5
min I.21 87
OF THE
IRON-THIOCYANATE
COMPLEX
Calorimeter reading 10 min 30 min 60 min 90 124
120 min
90 ‘24
91 124
93 125
‘73 213
216
253 290 330 52
253 291 329 52
170
17=
‘73
20s
212
212
244 282
25’ 285
25’ 286
325 50
328 51
328 5’
173
However, a few precautions must be taken in order to ensure precise results. a) The blood and the colour reagent must be exactly measured. b) Clean glassware must be used, and any contact with iron avoided to prevent further reaction of the coloured solution. The tube must be shaken well immediately after addition of the colour reagent, otherwise the colour fades. c) The calorimeter tube and the pipette used must be free from water to avoid emulsification with the isobutyl alcohol and consequently faulty light absorption. References p.
690
J. FISCHL
690 However, drop
if emulsification
of propyleneglycol With
occurs, which
the observance
of these
could be very
useful in routine
working
for greater
time
the sample
clarifies
voL.
might
be saved
4
by the addition
(1959) of one
the solution.
precautions
and with
hemoglobinometry,
average
whenever
skill,
this
it is possible
method
to exchange
precision. ACKNOWLEDGEMENTS
Our thanks Prof.
are due to Dr. H. MENACHEM for calling
F. RAPPAPORT
help in preparing
for his help
and many
useful
our attention
suggestions,
to the problem,
and Dr. MUNDEL
for
the manuscript. SUMMARY
A new method It is reliable,
easily
the standardization
for hemoglobinometry performed, of other
based on micro iron estimation
and suitable
for routine
examinations,
is presented. as well
as for
methods. REFERENCES
1 R. K.
CANNAN, Am. J. Clin. Pathol., 25 (1955) 376. 2 R. K. CANNAN, Am. J. Clin. Pathol., 30 (1958) 211. 3 D. L. DRABKIN AND J. H. AUSTIN, /. Biol. Chem., 98 (1932) 719. 4 F. W. SUNDERMAN, B. E. COPELAND, R. P. MACFATE, V. E. MARTENS, G. F. STEVENSON, Am. J. C&z. Pathol., 25 (1955) 489. 5 F. W. SUNDERMAN, B. E. COPELAND, R. P. MACFATE, V. E. MARTENS, G. F. STEVENSON, Am. J. Clin. Pathol., 25 (1955) 695. B J. B. THOMPSON, Ind. Eng. Chem.. Anal. Ed., 16 (1944) 646. 7 S. Y. WONG, J. Biol. Chem., 77 (1928) 409.
Received
H. N. SAUMANN
AND
H. N. NAUMANN
AND
February
Iqth,
1959