AN IMPROVED METHOD OF AND APPARATUS FOR THE QUANTITATIVE ESTIMATION OF HEMOGLOBIN IN THE BLOOD
Year: 1885

page_text: COMPLETE SPECIFICATION,
I, ALEXANDER MELVILLE CLARK, of the firm of A. M. and Wm. Clark, of
53, Chancery Lane, in the County of Middlesex, Fellow of the Institute of Patent
Agents, do hereby declare the nature of the said Invention for AN IMPROVED
METHOD OF AND APPARATUS FOR THE QUANTITATIVE ESTIMATION OF HEMOGLOBIN
IN THE BLOOD (a communication to me from abroad by Dr Ernst Fleischl von
Marxow of 11 Währingerstrasse, Vienna in the Empire of Austria, Professor of
Physiology at the Vienna University) and in what manner the same is to be
performed, to be particularly described and ascertained in and by the following
statement:-
The method of estimating haemoglobin, which forms the subject of this invention,
is a colorimetric one, ie: the quantity of haemoglobin contained in the blood is
estimated by the thickness of a layer of uniformly stained standard glass,
presenting under certain conditions, the same tone of colour as an aqueous solution
of blood.
As conveniently stained glass seems to afford and really affords an invariable
and-in every other respect-very suitable standard for a colorimetric method of
estimating haemoglobin, attempts have been made long since to devise such a
method, but these attempts have failed for the following reason.
In order that colorimetric comparison between two coloured substances shall be
possible, it is necessary that said substances not only present identical colour when
examined in layers of one certain thickness, for each substance, or with one certain
intensity of the light transmitted by each substance, but the colours must remain
equal, when the thickness of the layers of both substances is doubled or tripled.
That is to say, that the absorbing power of both substances for all rays of which
light is composed, ought to increase in the same proportion, when the thickness of
the layers is multiplied.
Now, that is not the case for blood and the so called ruby glass, the colour of
which most resembles that of blood. It is true that the stated condition is

page_text: sufficiently accomplished with respect to those absorption phenomena, which cause
the so called Stokes' bands of the blood spectrum to appear, but this condition_is
not complied with in respect of the absorption, which the violet rays (between the
lines G and H of the solar spectrum) undergo in blood and in ruby-glass. Indeed
while the ratio of the absorption of the violet rays to the absorption of the green
rays in a layer of blood solution of a given thickness, changes at a certain rate
when the thickness of the layer is doubled, the change of this ratio between the
absorption of violet and of green rays will not be the same when the thickness
of the glass plate, whose tone of colour corresponded to that of the single layer of
blood solution, is likewise doubled.
To obviate this inconvenience, by which the practical use of red glass is hindered,
it suffices to eliminate from the light which is caused to pass through both
substances, the violet rays, as the absorbing-power of red glass for violet rays does
not change in the same ratio, according to the thickness of the layer, as the
absorbing power of a solution of haemoglobin. This purpose is most simply
accomplished by the use of "yellow" light (of lamps, candles or gas) for the
colorimetric observations. This yellow light, when passing through blood
solution layers of different thicknesses, and thus being transmitted with different
degrees of intensity, produces upon the eye of the observer exactly the same series
of impressions, as under like circumstances these luminous rays would produce,
when passing through plates of ruby glass of different thicknesses. The human
eye has in a considerably high degree, the faculty of distinguishing different tones
of the same colour, and hence of recognizing the identity of two tones, when the
same are in juxtaposition; consequently, and as by the elimination of the violet
rays, the solution of the colouring matter of the blood, and the ruby glass are
allowed to appear in nearly identical colours, all necessary means for combining a
method of and constructing an apparatus for the estimation of the haemoglobin
contained in the blood are provided.
After having found, by means of the described comparison, the thickness of the
ruby-glass plate, which corresponds in tone to the normal quantity of haemoglobin
contained in the blood, it will be easy to find out, whether a blood to be examined
contains more or less than the normal quantity of haemoglobin. This can be done.
either by ascertaining to what degree a given quantity of the blood to be examined.
must be diluted in order that it shall present the same tone of color as a ruby-glass
plate corresponding in tone to the same quantity of normal blood diluted to a
known degree, or by diluting the blood to be examined to the same degree as the
normal one and by ascertaining the ratio of the thicknesses of the ruby glass plates
corresponding respectively in tone to the blood to be examined and to the normal
blood, the thickness of the latter plate being taken as a unit.
As the impression produced by a luminous ray upon the human eye only depends
on the nature and number of the coloured molecules, which said ray traverses
before reaching the eye, while the number and distribution of the colourless mole-
cules exert no influence, a known volume of blood, contained in a vessel with a,
transparent bottom and vertical sides, will obviously always present the same
tone of colour, whatsoever the degree of dilution may be, when the observer looks
through the liquid upon a source of light placed in the axis of the vessel, the
visual ray being perpendicular to the surface of the blood solution and to the
bottom of the vessel.
To carry this invention into practice, I make use of an apparatus which consists
essentially of a cylindric or prismatic vessel divided into two chambers by a
vertical partition and having its bottom formed by a perfectly colourless glassplate,
under one chamber of which a very acute wedge of ruby glass is so placed and
made adjustable, in a direction perpendicular to its acute edge, that portions of the
wedge of different thicknesses may be successively presented beneath the said
chamber, said wedge being slightly and uniformly stained, and one longitudinal
border of the same being placed exactly below the partition of the aforesaid
cylindric or prismatic vessel,

page_text: !
In the annexed sheet of drawings, figure 1 is a perspective view of the apparatus,
having certain parts broken away, and figure 2 is a sectional end elevation of the
same.
The plate a rigidly connected to the stand is provided with a hole, above which
rests the colourless glass plate c, carrying the cylindric or prismatic vessel b. This
vessel, destined to receive the blood to be examined, is advantageously made of a
piece of a glass-tube, having its ends ground and the bottom end cemented to the
colourless glass-plate c, by means of glass-cement or Canadian turpentine (Canada
balsam). By cementing into the cylindric or prismatic vessel b a partition b¹,
made of glass or metal, said vessel is divided into two compartments.
Below the plate a is arranged a metal frame d, having a downwardly projecting
flange, which is formed into a 'rack meshing with a pinion e, whose spindle turns
in a bearing in the standard and is provided with a milled head f. To the frame d
is fastened the wedge made of slightly and uniformly stained ruby glass, so that
when the head fis turned, the frame d with the wedge h is moved longitudinally
in either direction in a straight line, guides g g preventing any lateral deviation of
the wedge.
As repeatedly stated, the glass wedge is made of slightly and uniformly stained
red glass, by preference of the so called true ruby glass. To such a wedge, one
surface of a large piece of red glass is ground perfectly plane, and it is then
cemented to a perfectly colourless glass plate i, of the same size and rectangular
section, the cement employed being a colourless one or Canada Balsam. Then, the
second surface of the red glass is ground at such an angle to the first as to form a
wedge, whose thickness diminishes to zero at one end. The supporting colourless
glass plate i is allowed to remain cemented to the wedge h, as said plate protects
the wedge and affords facility for mounting it on the metal frame d. As it is
necessary that the outer border of the glass wedge h be always exactly in the plane
of the partition b¹ of the cylindric or prismatic vessel b, said free border of the
wedge ought to be perfectly rectilinear and parallel to the direction in which the
wedge is to be guided.
To prepare the apparatus for the estimation of haemoglobin contained in blood,
that portion of the wedge h, whose tone of colour corresponds to a given quantity
of normal blood, must be ascertained.
For determining said portion of the wedge and gauging the same, the blood of
a certain number of perfectly healthy, strong, middle-aged men is placed, in a given
quantity, into the front compartment of vessel b, both compartments of which have
previously been filled with water (preferably to the same level). As soon as the
blood has become fully dissolved in the water, the glass plate c with the vessel b is
su placed above the opening of the table a, that the partition b' stands exactly
vertical and in the plane of the outer longitudinal border of the red glass wedge h.
The glass wedge h is then moved beneath the water-filled compartment of vessel b,.
by means of the pinion e, until both compartments of said vessel present a perfectly
identical aspect. This will occur, when that part of the wedge which is beneath
the compartment containing the pure water,' is of such thickness as to have the
same absorbing power for the transmitted luminous rays, as the solution of blood
contained in the other compartment of the vessel b.
When this process is repeated several times with the blood of divers healthy
individuals, the operator will observe that in every case one and the same portion
of the wedge shows the quality in question; that is to say that the notion of
"normal amount of haemoglobin in the blood" is a palpable one, based on facts.
The portion of the wedge thus ascertained as corresponding to the normal amount
of haemoglobin in blood, is marked upon the wedge or upon the metal frame
carrying the same. In this manner, the value of the glass of which the wedge is
made, with respect to its colorimetric equivalency with haemoglobin is determined,
and the amount of haemoglobin corresponding to any other portion of the wedge
can easily be calculated from the distances of said portion from the edge of the
wedge and of the known thickness of the wedge opposite said mark.

page_text: Consequently, if the space between the mark and the edge of the wedge is
divided, for instance, into 10 equal parts, the ninth divisional line, reckoned from
the edge, will answer to 90 per cent of the normal amount of haemoglobin.
As all wedges made of the contents of one melting-pot will really be of uniform
quality, only one of these wedges needs to be gauged in the described manner.
Moreover, it is not necessary to make use of blood, when gauging a new set of
wedges; it suffices to determine that thickness of the new glass, which corresponds
to the tone of colour of normal blood (100 per cent) in the standard wedge. In
the wedges of this apparatus, each millimeter corresponds to one per cent. When
making such wedges, the grinder is directed to grind the edge at the distance of
10 centimetres from that thickness of the wedge, which has been recognized to
correspond to the tone of colour of normal blood, from 3.5 millimetres for instance,
As stated above, the observations should be made under conditions which exclude
violet rays, and this is most easily complied with by using yellow light, such as the
artificial light of oil lamps, candles or gas. In order to cause the luminous rays to
pass through the vessel b in a suitable direction, a conveniently inclined reflector /
is mounted on the stand of the apparatus.
From what has been stated, it is obvious in what manner blood is examined by
means of an apparatus provided with a gauged wedge h, the graduation being
read off through a slot in the table a. In the described way, the amount of
haemoglobin contained in a blood to be examined may be exactly ascertained.
Having now particularly described and ascertained the nature of the said
invention and in what manner the same is to be performed, I declare that what
I claim is
1st For the purpose of estimating, in a colorimetric way, the amount of
haemoglobin contained in blood, the use of wedge-shaped plates of glass, stained
red, and the diminution of the relative intensity of the violet rays in the light
caused to be transmitted by the glass and the blood solution.
2nd An apparatus for estimating, in a colorimetric way, the amount of haemoglobin
contained in blood, said apparatus essentially consisting of a cylindrical vessel
having a transparent bottom and being divided, by means of a vertical partition
into two chambers, one containing water and the other an aqueous solution of the
blood to be examined,-and of a graduated wedge of red glass, movable beneath
the compartment filled with water, said wedge being adjusted until the portion
thereof which has by reason of its thickness the same absorbing power as the
blood solution, is presented below the water filled compartment.
3rd Making the standard-wedge by grinding a plate of so called true ruby glass
in the shape of a wedge, marking the portion, which transmits light, poor in violet
rays, with the same intensity as a solution of a given quantity of normal blood,
and finally graduating the portion between said mark and the edge.
Dated this 15th day of May 1885,
A. M. CLARK.
By A. M. & W Clark,
53, Chancery Lane, London, W.C.