LETTER TO THE EDITOR
T H E PROPOSED HEMOGLOBIN STANDARD OF THE NATIONAL RESEARCH COUNCIL
I am taking the liberty of sending to you my thoughts concerning the proposed
hemoglobin standard of the National Research Council (see Am. J. Clin. Path.,
25: 376-380, 1955), in the hope that they may be of interest to readers of the
JOURNAL.
I feel that all the technical units expressed in the article should be denned
and related to everyday terms so that the "average pathologist" and internist
can be expected to comprehend what is meant without further reference. We
should learn to use the terms "milliatom" and "extinction coefficient" and
pathologists undoubtedly will take to these terms more readily if they are
clearly explained. A few simple definitions may be in order. The following method
is offered to express the concentration of the Standard Stock Hemoglobin
Solution:
(a) With respect to iron Hb:
1 gram of hemoglobin (Hb) contains 3.35 mg. hemoglobin iron (0.0035
Gm. Hb Fe)
1 milliatom of hemoglobin iron equals 55.85 mg. hemoglobin Fe (that
is, the atomic weight of iron in milligrams)
Standard of Reference to contain 1 milliatom of hemoglobin iron per
liter.
1 milliatom Fe (Hb)
55.85 mg. Fe (Hb)
1 Gm. Hb
1 liter Std. of Reference X 1 milliatom Fe (Hb) X 3.35 mg. Fe (Hb)
16.67 Gm. Hb
1 liter Std. of Ref.
(b) With respect to Millimolar Extinction Coefficient:
e (when d = 1 cm. and c = 1 mM/1.)
This constant is derived by measuring the per cent T of a 1:25 dilution of
the Standard of Reference in the Beckman D. U. Spectrophotometer at
a wavelength of 540 millimicrons. The per cent T is 34.5, or converting
to optical density units (2-log percent T), this solution has an optical
density of 0.460. Since the Standard of Reference is 25 times more concentrated than the dilution (1:25) and since optical density increases in
direct proportion with the concentration, therefore:
0.460 optical density units X 25 = 11.5 optical density units for the
undiluted Standard of Reference.
The light path of the Beckman D. U. is 1 cm.; the hemoglobin concentration is 16.67 Gm. per liter or 1 millimole hemoglobin per liter.
Thus the necessary data for calculation of the millimolar extinction
coefficient are available:
e (when d = 1 cm. and c = 1 mM Hb/1.) = '•—;—' '
„—
1 mM Hb/1.
* It may be noted here that any photometer may roughly approximate this figure for
the extinction coefficient.
560
May
1955
LETTER TO THE EDITOR
561
Criteria for Standard of Reference
(a) 1 milliatom hemoglobin iron per liter (55.85 mg. Fe [Hb])
(b) 1 millimole hemoglobin per liter (16.67 grams Hb)
(c) Fractional Molecular Extinction Coefficient
. . .
,
,
, A,I-/I\
11.5 O.D. units
e (when d = 1 cm. and c = 1 mM/I.) = ——_, TT1 ,,—
1 mM Hb/1.
I believe the selection of a definite relationship for the iron content of hemoglobin warrants more explicit units than the term "per cent" which is often
misinterpreted. I would suggest that they be expressed as follows:
Iron content of hemoglobin:
0.335 grams of hemoglobin Fe per 100 grams hemoglobin;
0.00335 grams hemoglobin Fe per 1 gram hemoglobin;
3.35 milligrams hemoglobin Fe per 1 gram hemoglobin.
The selection of 16,700 as the equivalent weight of hemoglobin should be
qualified to explain that a molecule of hemoglobin contains four atoms of iron
and that the true molecular weight of hemoglobin is 66,680. By convention and
for convenience in calculation, however, the weight of hemoglobin containing
one atom of iron is usually employed in expressing hemoglobin relationships.
I am very much interested in having the standard deviation of the mean value
for replicate analyses for each of these properties published, for the laboratory
director or clinician should not expect greater accuracy from a technician in
hemoglobin .determinations than is provided by these reference laboratories
with reference methods performed under research standards of precision.
New England Deaconess Hospital
BRADLEY E. COPELAND, M.D.
195 Pilgrim Road
Clinical Pathologist
Boston 15, Massachusetts