A SIMPLE METHOD FOR MEASUREMENT OF HEMOGLOBIN
IN SERUM AND URINE
HAL E. FIELDING, B.S., M.T. (ASCP), AND PAUL E. LANG LEV, M.T. (ASCP)
Department of Pathology, St. Vincent Hospital, Santa Fe, New Mexico
Various methods have been proposed for
determining hemoglobin in body fluids other
than whole blood. Flink and Watson 5 used
pyridine, but this was inaccurate at lower
concentrations. The use of benzidine has
been the most widely used, and was first
proposed by Wu.9 Several modified benzidine methods have followed.1"4,7 None of
these procedures has proved entirely
satisfactory owing to interfering substances
in plasma and urine, and owing to the
complexity and instability of the reagents.
The following method eliminates the
problems of the benzidine methods, is
simple, reliable, and utilizes an easily
available stable, reagent tablet. The procedure is based on the use of a single reagent
composed of orthotolidine, strontium peroxide, tartaric acid, and calcium acetate
supplied in combined form in Hematest
tablets.*
The mechanism of the reaction is believed
to be one in which calcium acetate and
tartaric acid with strontium peroxide react
to liberate hydrogen peroxide. Hemoglobin
and some of its degradation products possess
properties comparable to those of peroxidase
enzymes and, accordingly, liberate active
oxygen from hydrogen peroxide. The colorless orthotolidine acts as an oxygen acceptor
and is oxidized to a blue reaction product. 6
MATEItlALS AND
METHODS
Reagents
1. Hemoglobin reagent. Thoroughly crush
1 Hematest reagent tablet (Ames Company, Inc., Elkhart, Indiana), and add 25
ml. of distilled water. Mix thoroughly by
shaking 3 or 4 min. Allow to stand 30 min.
Received, April 16, 195S; revision received,
June 9; nccepted for publication Jul.y 14.
Messrs. Fielding and Langley are Medical
Technologists.
* Ames Company, Inc., Elkhart, Indiana.
52S
Filter through a fine filter paper. This reagent should be crystal clear and keeps at
least 3 months. Store in the refrigerator.
2. Standard hemoglobin solution. Determine the hemoglobin concentration of
oxalated blood by a reliable method such as
the iron method of Wong.8 Determination of
the concentration of hemoglobin by ordinary
hemoglobinometry will suffice for routine
purposes. Dilute this blood to appropriate
concentrations with distilled water. The
resulting turbid solutions will clear on adding to the hemoglobin reagent.
Equipment
1. Photometer.
2. Test tubes of 15-ml. capacity.
3. Sahli pipet, 0.02-ml., accurately calibrated, and 10-ml. volumetric pipets. Glassware must be chemically clean.
Procedure
1. Measure 10 ml. of hemoglobin reagent
into a test tube or cuvet for each unknown.
2. Add 0.02 ml. of serum or filtered urine
to the hemoglobin reagent, and wash out
the pipet. Mix thoroughly by inversion.
3. In exactly 8 min., measure the optical
density of the test against a distilled water
blank at wavelength 630 mju using a 19-mm.
cuvet. The maximal development of color is
attained in 7 min. at room temperature, and
is stable for 2 to 3 min.
4. Read the concentration of hemoglobin
from a prepared standard curve.
Calibration
1. Prepare several concentrations of
hemoglobin solutions with distilled water
ranging from 5 mg. per 100 ml. to 100 mg.
per 100 ml.
2. Perform a hemoglobin determination
on each standard as described in the procedure for unknowns.
3. Plot a curve on ordinary graph paper
Dec. 1958
HEMOGLOBIN
I N SERUM
529
AND U R I N E
with accuracy (Table 1), and as readily as
ordinary clinical hemoglobinometry. The
materials used are available in most laboratories.
SUMMARY
A simple, reliable method for measuring
hemoglobin in serum and urine is described.
The method is based on the use of a single,
easily available reagent tablet in which
orthotolidine, strontium peroxide, tartaric
acid, and calcium acetate are combined.
SUMMARIO
50
100
F I G . 1. A typical standardization curve for
hemoglobin, demonstrating conformity t o Beer's
law.
TABLE 1
Sample
Calculated
Recovery
Actual
Recovery
mg./lOO ml.mg./lOO ml.mg.1100 ml.
Serum 1 (3.0 mg./lOO
nil.)
Serum 2 (8.1 mg./lOO
ml.)
15.0
30.0
60.0
15.0
30.0
60.0
18.0
33.0
63.0
23.1
3S.1
6S.1
17.2
32.3
62.0
23.2
37.3
67.1
INTERLINGUA
Es describite un simple methodo pro le
mesuration de hemoglobina in sero e urina.
Le methodo usa un sol reagente que es
facilemente disponibile in le forma de un
tabletta continente orthotolidina, peroxydo
de Strontium, acido tartaric, e acetato de
calcium.
RECOVERY OF HEMOGLOBIN FROM SERUM
Hemoglobin
Added
IN
REFERENCES
1. B I N G , F . C , AND B A K E R , R . W.: T h e deter-
mination of hemoglobin in minute amounts
of blood by Wu's method. J . Biol. Chem.,
92: 5S9-600', 1931.
2. CREDITOR, M. C : T h e q u a n t i t a t i v e determination of plasma hemoglobin by t h e benzidine
reaction. J . L a b . & Clin. Med., 41: 307311, 1953.
3. CROSBY,
W.
H.,
AND DAMESHEK,
W.:
The
significance of hemoglobinemia and associated hemosiderinuria, with
particular
reference to various tvpes of hemolvtic
anemia. J . L a b . & Clin" Med., 38: S29-S41,
1951.
4. CROSBY, W. H . , AND F U R T H , F . W.: A modifi-
with optical density
concentration.
versus hemoglobin
cation of the benzidine method lor measurement of hemoglobin in plasma and urine.
Blood, 11: 3S0-3S3, 1956.
5. F U N K , E . B . , AND W A T S O N , C. J . : A method
COMMENTS
Unknowns with values more than 100 mg.
per 100 ml. should be repeated on diluted
amounts. Unknowns with values less than
15 mg. per 100 ml. should be repeated using
larger amounts of specimen and sufficient
hemoglobin reagent to make 10 ml. Increased
amounts of specimen will not cause turbiditv. The development of color follows
Beer's law (Fig. 1).
When the proposed method is used, serum
and urine hemoglobin may be performed
for the q u a n t i t a t i v e determination of hemoglobin and related heme pigments in feces,
urine a n d blood plasma. J . Biol. Chem.,
146: 171-17S, 1942.
6. HEMINWAY, N . L . : (Ames Company, Inc.,
E l k h a r t , Indiana) Personal communication.
7. M C F A R L A N E , W. D., AND HAMILTON, R. C. M . :
On the factors influencing the blood-benzidine reaction as applied to the micro
determination of haemoglobin. Biochem.
J., 26: 1050-1060, 1932.
S. WONG, S. Y . : Colorimetric determination of
iron and hemoglobin in blood. J . Biol.
Chem., 55: 421-425, 1923.
9. Wu, H . : Studies on hemoglobin: Ultra-micromethod for determination of hemoglobin as
peroxidase. J . Biochem., 2 : 1S9-194, 1923.