Studies in the Osteopathic
Cells of the Blood: Volume
Louisa Burns, M.S., D.O., D.Sc.O.
TECHNIQUE OF BLOOD CELL EXAMINATIONS
ESTIMATION OF HEMOGLOBIN
The amount of hemoglobin in the blood is estimated
by means of some kind of color scale. Different instruments
use different types of scale and different methods of preparing the
blood. Older methods based upon chemical analysis for iron are
much less accurate and are very cumbersome; they are not suitable
for ordinary laboratory work in diagnosis.
The method of securing the blood is the same
for all the newer methods of determining hemoglobin.
It is important to secure the drop of blood
in such a manner as to prevent its changing from its condition within
the vessels as little as possible. The lobe of the ear, the
palmar surfaces of the fingers and the balls of the toes are almost
devoid of sensory nerves. The skin at the sides of the fingers
is thinner than that upon the heavier parts of the balls and is
often less sensitive. In persons of certain occupations, such
as piano or violin playing the finger tips may have thick skin.
It is necessary to avoid any area which is more than usually liable
to injury after the prick is made, the violinist might find some
difficulty in playing with the injured finger for a few hours after
the prick has been made, for example. The lobe of an ear is
the best site in adults, the side of a toe is best in babies.
Having selected the site of the puncture, the
skin should be cleaned in any way that is convenient. Probably
a good washing with clean water is best for ordinary conditions.
Various antiseptics are sometimes employed, but these must be sufficiently
dilute to prevent irritation to the skin; this means little or no
antiseptic value. Any of the ordinary antiseptics strong enough
to injure bacteria injure t he tissues still more seriously.
But a washing with anything which removes the dust and perhaps a
few desquamating epithelial cells takes away the chief source of
infection and does not prevent the most speedy repair. Care
must be taken in the washing that the skin is not irritated enough
to cause overfilling of the vessels since this modifies the hemoglobin
The first drop of blood must be wiped away,
and the second or later drops taken up in the manner suitable for
each instrument. The tissues must not be squeezed or handled
roughly but the blood musts come gently and freely from the puncture.
The puncture is best made with a sharp, angular
lancet. The lancet which is provided with a spring makes a
sudden thrust which is not painful at all. A pen which has
been broken longitudinally in half, so that only one sharp point
remains, is useful. A dozen or more pens can be prepared and
sterilized and a new one used for each patient. Glove needles,
being triangular, make a sharp, clean puncture which heals at once.
Round needles are not quite so suitable because they cause more
pain and leave a rougher puncture; the slight bruising which occurs
may affect the concentration of the blood in the vicinity of the
cut. The error due to this factor is negligible in most cases.
The blood drop as it wells up from the tiny
wound made for the purpose gives a general idea of its hemoglobin
content, by its color. When the blood is more viscid, this
drop stands up round and high, and appears to be richer in hemoglobin
than it really is, when it is of low viscidity it spreads out over
the surrounding skin in a thinner layer, and appears to be lower
in hemoglobin content than it is. The character of the skin
modifies the height of the drop, also, as does the size of the wound
and the rapidity of the flow.
The simplest, cheapest, and easiest method of
estimating the hemoglobin is by means of the Tallquist Hemoglobin
Scale. It consists of a scale of ten colored paper slips for
comparison, each corresponding to a certain percentage of normal
hemoglobin, and strips of filter paper of uniform thickness to take
the blood. In using this scale the drop is secured by the
usual means, and a strip of the filter paper laid gently and quickly
over the drop, until the blood is soaked up into the paper over
an area about one-fourth of an inch in diameter. This is held
for a few seconds or until the first shiny look of the blood stain
has disappeared, then it is compared with the color scale.
Some of the scales have an opening in the center of each of the
ten color-tint blocks, and the blood-stained paper is then placed
beneath these openings, one after another, up and down the scale
in succession until the matching tint of the scale is found.
If the blood is apparently half way between two blocks as 70% and
80%, the hemoglobin may be considered as 75%. In this way,
those who are experienced in the test can make estimations of the
hemoglobin which are fairly useful though they cannot be considered
accurate within more than 20% under the best of circumstances in
the most skillful hands. The method is perhaps the most convenient
of all, since there is no apparatus to wash, no special light requirements
are necessary and the scale is so small that it can easily be carried
in the pocket. Its lack of accuracy is the only factor against
its use. The method is useful for a preliminary test or in
Next in ease of use is Dave’s Hemoglobinometer.
This instrument has a revolving color scale of glass, a telescoping
tube for securing distinct vision, and two slips of glass which
hold a measured film of blood between them. The slips of glass
are placed in the holder and screwed closely but not too tightly
together; the blood flows between them by capillary attraction.
The holder than slips into its place at the back of the instrument.
The telescope is pulled out until the edges of the two circles show
very plainly and in good focus. The revolving color scale
is moved by means of the screw at the upper part of the instrument,
until the tint of the two circles appears exactly the same.
The amount of hemoglobin is read off in percentages of the normal
from a scale which is attached to the side of the glass circle,
and which is seen through a tiny window at the side of the instrument.
In using the Dave’s instrument it is necessary
to have a yellowish light, not too brilliant, and to make the examination
in a rather dark room. It is better to look with the eyes
alternately, thus giving each eye an opportunity to rest, in turn.
The correctness of the reading may be gauged by the fact that one
is able to return to about the same figures several times in succession,
after changing the position of the scale and resting the eyes.
It is probably correct within 5% of the actual amount. This
scale is made so that blood containing 137.7 grams per liter is
considered as 100%, or normal. It is evident that in using
this instrument correction must be made for the age of the patient.
Gower’s hemoglobinometer is not now in general
use. It consists of three tubes, one filled with gelatine
stained with picrocarmine to be used for artificial light, one filled
with gelatine stained slightly differently, to be used with daylight,
and one to be filled with diluted blood, the latter graduated.
The graduated tube is filled to a certain point with blood, then
water is added until the tint exactly matches the tube filled with
gelatine, using the one or the other according as the light is artificial
(yellowish) or is daylight. The dilution is difficult; the
gelatine tubes fade quickly if they are exposed to the light often
(as they must be if they are used at all frequently) and the newer
instruments are much more accurate and convenient.
Sahli’s hemoglobinometer is somewhat like Gower’s
but has certain advantages over the older instrument. It consists
essentially of two tubes, one filled with a stock solution of 1%
acid hematin, and one tube for the blood. Twenty cubic millimeters
of blood are taken into the blood tube, and this is mixed with deci-normal
hydrochloric acid. (15 c.c. HCI: 1 liter, H2O is sufficiently
accurate.) The acid changes the hemoglobin of the blood into
the stable acid hematin. The blood mixture is then to be diluted
with water until it matches accurately the color of the stock solution
in the other tube The advantages of this instrument are that
it can be used in any light; since both the stock solution and the
blood mixture are colored by the same compound—acid hematin,--they
are equally affected by different lightings, so that yellow light,
daylight, dim lights or brilliant lights all act upon both tubes
in the same way. A moderate light, however, gives the most
nearly accurate readings. This instrument is, in the hands
of reasonably skillful observers, accurate within about five per
cent. The readings are made in percentages of the normal,
which is thus subject to the necessity for correction for the age
of the patient. In this instrument 172 grams of hemoglobin
per liter of blood are taken as 100%.
The most accurate, the least convenient and
the most expensive hemoglobinometer is that of Fleischl as modified
by Miescher. This has for its scale a long, slender, delicate
glass bar which is thick at one end and thin at the other, so colored
as to be spectroscopically identical in color with hemoglobin.
This has a scale connect ed with it, and is set beneath a stage
which is perforated in its center. A second perforation near
the edge of the stage permits the scale to be read easily.
The colored prism is moved across the central perforation.
Beneath the prism is a reflecting dull white surface and a candle
or other dim yellowish light is placed in front of this dull reflector.
The blood is taken in a pipette somewhat like
those used for blood dilution for counting, except that the hemoglobinometer
pipette is so graduated that dilutions of 1-200, 1-300, or 1-400
can be made. Dilution is made with 0.1% sodium carbonate;
ordinary tap water is really better, in our experience.
If the blood appears very pale as it emerges
through the puncture use the dilution 1-200. If it appears
almost or quite normal in tint use a dilution of 1-400. For
milder anemias uses 1-300 dilution. In taking the blood into
the pipette keep the lower end of the pipette within the blood drop
but do not allow it to touch the skin. If by accident the
blood should be drawn above the selected mark on the tube, or should
not quite reach that mark, there are accessory graduations by means
of which adequate correction can be made after the work is completed.
Draw the diluting fluid into the pipette to the mark at the top
of the bulb, and then immediately thoroughly shake the pipette from
side to side. The shaking must be gentle, and the pipette
must not be shaken endways lest some of the blood be forced into
the capillary portions of the pipette.
Two chambers, each divided into compartments,
are used for the examination. Each chamber has a glass cover
and a glass bottom, and a perforated dark metal cap to be placed
over the cover glass. One chamber is 12 millimeters in depth,
the other 15 millimeters, and readings are made with both chambers.
Put the glass bottom into the 15 millimeter
chamber and fix it in place by means of the screw-like arrangement
provided for the purpose. Fill one compartment with water
or the solution used for dilution. By means of a rubber bulb
blow out and discard four drops from the pipette used for diluting
the blood. (This removes the fluid in the capillary bore of
the tube, which presumably contains no blood.) Still using
the rubber bulb, fill the other compartment with the diluted blood.
Be very careful that both compartments are completely filled, that
none of the clear fluid reaches the blood compartment, and that
none of the diluted blood reaches the clear fluid. Cover with
the glass; there must not be any air drops in either compartment.
Place the metal cap over the cover in such a manner that two equal
squares are visible, one of the diluted blood, the other of the
clear fluid. Place the chamber in the perforation provided
for the purpose in the stage of the hemoglobinometer in such a manner
that the clear square is exactly over the colored bar. Arrange
the candle and the reflecting surface so that a dim light is thrown
upward through the colored prism and the clear fluid, and through
the diluted blood.
Move the prism back and forth by means of the
screw until the two squares have exactly the same tint. Use
first one eye then the other. Do this quickly; if too much
time is taken vision becomes less acute. Read and note the
figures on the scale. Move the prism away from matching tint.
Close the eyes for a few seconds, then repeat the test. In
this way make ten different readings. If the readings do not
vary more than five points the readings are sufficiently accurate.
Fill the 12 millimeter chamber in exactly the
same manner and make ten readings in the same manner.
Make an average of the ten readings from the
12 millimeter chamber and make an average of the ten readings from
the 15 millimeter chamber.
Divide the first average (from the 12 millimeter
chamber) by 4 and multiply by t, this should equal the average made
from the ten readings from t he 15 millimeter chamber. If
the figures thus secured do not agree within five points the work
is not sufficiently accurate and should be repeated.
Compare the figures secured, corrected for the
15 millimeter chamber, with a scale provided with the instrument.
This gives the grams of hemoglobin per liter of the diluted blood.
If the blood was diluted 1-200, multiply the scale reading by 200,
which gives the grams of hemoglobin per liter of whole blood.
If the dilution was 1-300 or 1-400, multiply by these figures.
This instrument requires an absolutely dark
room; it is too cumbersome to carry about easily; its use requires
much time for the actual work and for the washing of the apparatus.
Parts are easily broken and they cannot be easily replaced.
It is by far the most accurate instrument on the market and for
this reason has an important place in hospitals and research laboratories.
We use it for checking up on other simpler instruments, and in cases
in which unusual accuracy is required.
SOURCES OF ERROR
The errors that may be made in the determination
of the hemoglobin are not many. By the Tallquist scale, two
errors are not infrequent; the drop of blood may be small, thus
the area of stained paper is too small and the reading too low.
Or, in the endeavor to secure a larger area of stained paper, from
an insufficient amount of blood, the paper may be pressed again
and again over the wound, until the same area of paper may be soaked
several times in the blood; naturally the reading is then very much
higher than it should be. In severe anemias and in blood of
very low viscosity, the serum soaks into the paper around the cells,
leaving a rather dark center in a very pale ring. The reading
is thus too high. This source of error is especially apt to
occur in chlorosis, and it frequently occurs in pernicious anemia
and in secondary anemias of similar type. Sometimes the examination
is delayed, and the blood takes on a brown color, due, probably,
to the formation of methemoglobin. When this occurs it is
impossible to secure anything like an exact match of the tints of
the scale; the resulting readings are certainly inaccurate, and
they may be too high or too low.
In using Dare’s hemoglobinometer, the slides
may not be thoroughly dry, especially if it has been necessary to
clean them after errors in technique. The slightest
film of moisture upon the surfaces of the glass slides affects the
reading by diluting the blood. Sometimes the slides are not
screwed closely together; the film of blood is then too thick, and
the reading too high. Sometimes the slides are screwed too
tightly together, whereupon they break.
The finger should be removed from the wheel
which turns the scale, before the telescope-tube is removed form
the eyes. Else, when the telescope is taken from the eyes,
the fingers which have been turning the scale may still turn it
slightly. The scale may thus be turned and the reading correspondingly
The use of Sahli’s or Gower’s instrument depends
for its accuracy upon the care with which the blood is taken, is
blown into the larger diluting pipette, and with which the readings
and dilutings are performed. For if the drop of blood should
not be removed from the end of the tube, after the requisite 20
c.c. have been taken, a part of this is almost sure to be drawn
into the tube with the diluting fluid, and the determination is
increased. If the tubes are moist, the blood is unduly diluted,
and the readings are too low. If the blood is not thoroughly
rinsed into the mixing pipette, the readings are thus too low.
In adding the last of the water to the blood in the mixing pipette,
it is best to read off the hemoglobin percentage, and write it down,
before the later and decisive readings are taken. Then, if
by accident or by choice, the dilution should be sufficient to carry
the color past that of the control, the reading can be corrected
In using the original Fleischl instrument sources
of error are plentiful and for this reason it is not now used in
many laboratories. The Miescher modification is subject to
much less error but still it is necessary to use great care in making
every step of the procedure. In very important cases for research
work it is our practice to take the blood in two different dilutions
and to compare these; discarding the results if the results do not
agree within five points of the final computation, and making an
average of the two final results if they do so agree.
For each method of determining hemoglobin accuracy
is gained only by experience and carefulness; with these the hemoglobin
can be determined reasonably well, within the limits of accuracy
for the instrument employed, in any case.
THE ACTUAL COUNT OF CELLS
The enumeration of the cells by the use of a
counting chamber is called the “actual” count, to distinguish it
from the “differential” count, which is made upon a smear of blood.
The actual count gives the actual number of cells per cubic millimeter
of blood; the differential count gives the percentages of the different
types of white cells, and the relative number of normoblasts, malarial
corpuscles and any other items desired may also be determined.
From the differential count and the actual count, the number of
these various structures in an average cubic millimeter of blood
can be determined. The actual count is not a complicated process
but errors must be very carefully avoided or the findings may be
APPARATUS NEEDED FOR THE ACTUAL COUNT
Provide, for the actual count, the following
articles. Microscope with mechanical stage, iris diaphragm
and condenser may be of the type used in ordinary laboratory work.
The binocular microscope is far better for the eyes. Have
the table, the microscope and the seat to be used by the worker
correctly placed so that the worker sits perfectly squarely and
at a comfortable height. Counting blood takes considerable
time; if the head of the worker is held at a lateral angle, or if
the microscope is too high or too low there is a strain upon the
neck muscles; the cervical vertebrae are held in an abnormal position
and the circulation through the eyes is affected. Strain
upon the intrinsic muscles of the eyes is caused by the attempt
to see plainly if the eyes are not placed at right angles to the
planes of the lenses of the eyepieces. In this connection
it should be stated very emphatically that the condition called
“eye-strain” due to the use of the microscope for considerable periods
of time is much more frequently “neck-strain” and is due to carelessness
in the position assumed while the work is being done. There
is no reason why the use of the microscope should injure the eyes
any more than reading, except that a microscope occupies a fixed
position and the worker tends to accommodate his position to the
needs of vision without being careful to consider the comfort of
his position. In order to avoid fatigue and injury to the
eyes it is only necessary to be very careful to sit comfortably,
squarely facing the microscope and to have suitable lighting and
proper arrangement of the iris diaphragm and the lenses.
The lighting is important. In our laboratories
a small electric light placed squarely beneath the condenser is
used for ordinary work. A larger light placed in front of
the small round reflecting mirror is good; this permits variations
in lighting for low-power or high-power magnification in addition
to the modifications secured by regulating the iris diaphragm.
Other articles needed are a lancet for pricking
the skin, various solutions and containers, a counting chamber,
pipettes for diluting the blood and sterilizers and other articles
ordinarily present in a laboratory for clinical diagnosis.
Several types of counting chamber are in use.
The best arrangement is made of a single piece of glass which is
so cut as to form the chamber. Lesss useful and accurate is
the chamber made by cementing pieces of glass together. The
chamber in which the moat is H-shaped is more convenient than the
older style in which the moat is circular. In any event the
counting chamber is a glass slide which has upon its upper surface
several parts. In the center there is a ruled area upon which
the counting is done. Several arrangements of the lines are
in use but all contain lines crossing at right angles, dividing
the chamber into spaces of varying sizes. The “small square”
is the unit space. This is formed by lines 1-20 millimeter
apart, thus squares are formed 1-20 x 1-20 millimeter in size.
In order to secure ease in counting an extra line is placed midway
between these lines at regular intervals thus forming smaller squares
1-40 x 1-40 millimeter; these are of no practical value in ordinary
work except that they help to keep the area being counted in easy
vision. The central area of the counting chamber is occupied
by 144 of the squares of 1-20 millimeter. Many of these are
subdivided. Around this central area many chambers are provided
with larger squares for the counting of the white cells; these are
made by lines 1-5 millimeter apart, with lines 1-20 millimeter apart
outlining the larger spaces; sometimes three lines 1-40 millimeter
apart take the place of the two lines 1-20 millimeter apart, in
the formation of the larger squares.
This ruled area is upon a part of the slide
which is elevated above the rest of the slide; the exact amount
of the elevation is of no consequence, and varies for different
counting chambers. Around this ruled glass area, which is,
properly, the chamber, there is a moat. This is a depression
around the ruled area, itself surrounded by another elevation of
glass. The glass around the moat is always 1-10 millimeter
higher than the counting chamber, so that when the cover-glass is
placed upon the slide a space exists between the ruled area and
the cover-glass; this space is 1-10 millimeter in depth in all chambers
in ordinary use.
The counting chamber requires care in cleaning
because the ruled lines are delicate and t he glass can be scratched
easily. If the chamber is made by cementing different pieces
of glass together, the chamber must never be washed or rinsed with
anything but warm water. This is really all that is necessary,
anyway, in ordinary cases. If the counting chamber is made
of a single piece of glass, cut to form the various parts, it may
be washed as any delicate glass-ware would be.
The ruled area must be treated with especial
care, it must never be rubbed in drying, but only very gently mopped,
or, better, left to dry after a final rinsing in warm distilled
PIPETTES FOR DILUTING THE BLOOD
These are commonly of the Thoma-Zeiss types.
They are composed of a glass bulb which receives the blood and the
diluting fluid. Within this bulb is a small glass bead which
facilitates mixing the blood and the fluid, and which also serve
to remove deposits from the inside of the bulb in case of accident.
A capillary tube receives the blood, and this is divided into ten
equal parts. The entire content of the capillary tube of the
erythrocyte pipette is one hundredth of the content of the bulb,
and the content of one of the ten divisions is 1-1000 the content
of the bulb. The fifth of these divisions is marked .5 and
the division next the bulb is marked 1. At the top of the
bulb another mark 101, is placed. Since the 101 includes the
content of the capillary tube, and since the diluting fluid within
the capillary tube is not mixed with the contents of the bulb, the
dilution in the bulb can be made anything between 1 to 1,000 and
1 to 100. The most common dilutions for red cells are 1 to
100, or 1 to 200.
The tube frequently used for counting the white
cells has a smaller bulb, or a larger bore in its capillary tube,
so the bulb contains only ten times as much fluid as the capillary
tube. This tube also is divided into ten equal parts.
The fifth division is marked .5 and the upper division is marked
1 as in the red cell pipette, while the upper limit of the bulb
is marked 11. Thus it is possible to secure dilution of 1-10,
1020 or even 1-100 by drawing the blood to the 1 mark, to the .5
mark, or to the lowest mark, which is 12-10 the content of the bore
of the capillary tube. Other markings are employed for the white
cell pipettes occasionally; their significance is easily understood
by a comparison of the markings with one another and with the red
For both pipettes there is a dilated area above
the constriction which marks the upper limit of the bulb; this receives
any superfluous amount which may be drawn up beyond the proper limit,
by accident. There is a rubber tube with a glass mouth-piece
for each blood pipette, and it is best to have these rubber tubes
a foot or more in length. The rubber tubes which come with
the pipettes are rarely long enough to permit accuracy in vision
or delicacy in manipulation of ascending columns. By having
the longer rubber tubes it is easier to see clearly the ascending
column of blood, and the increased elasticity due to the longer
rubber tube allows greater accuracy in manipulating the columns
as they are drawn upward.
CLEANING BLOOD PIPETTES
After the pipettes have been used rinse them
in clean water immediately. Rinse thoroughly in cold water,
then in hot water, then run air through them until they are perfectly
dry. When no water is visible, and the glass bead rolls around
freely within the bulb, the bulb is dry. It must be remembered
that while the bulb is still very wet the bead rolls around freely
also, but in that case the water is easily visible. If there
has been some staining of the inside of the bulb with the diluting
fluid, this may be removed with sulphuric, hydrochloric or nitric
acid, using the acid first in a very weak solution, then increasing
the strength of the solution until the stain is gone.
If coagulation of blood should occur within
the capillary bore or the bulb, this must be digested out.
An artificial gastric juice containing both free hydrochloric acid
and pepsin, or an artificial pancreatic juice containing trypsin
and mildly alkaline in reaction, should be drawn into the bulb and
the pipette left in an incubator over night. If the clot is
not dissolved the process must be repeated, perhaps using some other
artificial digestive fluid. After the clot has been digested the
pipette is rinsed with cool water, then with hot water, then is
dried in the usual manner.
A filter pump attached to the water tap cleans
the tube very easily and efficiently. Attach the pipette to
the intake of the filter pump, place the other end of the pipette
in cold or hot water, or in the acid solution if this is to be used.
Turn on the water and allow the cleaning solution or water to flow
through the pipette as long as seems desirable. Then remove
the lower end of the pipette from the water and allow air to flow
through until the pipette is perfectly dry.
If the filter pump is not at hand, the pipettes
must be cleaned by hand. Cold water, then hot water, then
95% alcohol, then ether are used in turn and then air is blown through
the pipette until the tube is dry. Draw the fluids into the
pipettes by suction through the rubber tube, then expel them by
using a rubber bulb. Air is drawn into the pipettes and then
is expelled by the rubber bulb. The final rinsing with alcohol
and ether is intended to hasten the drying process because these
fluids evaporate rapidly. (They are not necessary when the
filter pump is used because the time question is not important to
the filter pump, and hot water is not usually limited.) The
cleaned pipettes may be kept in any place which is free from dust
Several types of blood lancet are on the market.
A glove needle, which is triangular in form, makes an excellent
lancet for the purpose of securing blood. K The triangular needle
makes a cleaner puncture than does the ordinary round needle, though
any ordinary sewing needle can be used in emergencies, after it
has been sterilized. A surgeon’s needle is convenient and
easily cleaned. Half a steel pen, properly cleaned and sterilized,
is very good.
Lancets which are provided with a spring are
rather less easily cleaned. They have the advantages of producing
wounds of equal depth, and they can be set to make deeper wounds
if the skin shows marked pallor, or shallower wounds if the patient
is a bleeder or if the skin is very red. The needles without
a spring depend upon the skill of the operator for the accuracy
of gauging the depth of the wound.
Needles and lancets are best sterilized by rinsing
in warm water, then dipping them into carbolic acid. After
another rinsing in sterile water they may be dried on sterile cotton
or left to dry.
In order to count the blood cells it is necessary
that the blood be diluted. For the white cell count acetic
acid, in solutions of 3% to 8% is commonly employed. The red
cells are destroyed by the acetic acid, which also causes the nuclei
of the white cells to show more distinctly. Various stains
may be added to the acetic acid solution and these stain the white
cells. In our laboratories no stain is used; this is partly
in order to allow any pigmented granules of the blood to be visible.
For the red cells many solutions are employed.
In our laboratories normal salt solution is tinged with a few drops
of methylene blue and this is used for diluting the blood for the
red cell count. The cells remain in fairly normal condition
for several hours, which is all that is required for ordinary cases.
If the blood must be kept in the pipette for some time before it
can be counted, Hayem’s solution is excellent, and this is the one
most used. The following is the formula;
Distilled water, 100 c.c.
CLEANSING SOLUTIONS FOR THE SKIN
Sodium chloride, 1 gm.
Mercuric chloride 0.5 gm.
Sodium sulphate 5. gms.
Another solution which is preferred by many workers
is Toisson’s fluid. Its formula is:
Distilled water, 160 c.c.
Neutral glycerine, 30 c.c.
Sodium sulphate, 8. gms.
Sodium chloride, 1. gm.
Methyl violet, 25 mgs., or just enough to give
a faint purplish tinge to the solution. This fluid stains
the white cells. It does not keep so well s Hayem’s fluid,
does not fix the cells appreciably, and we have found it less satisfactory
than Hayem’s fluid for keeping the blood for a long time (a day
or two or three days)under experimental conditions.
For ordinary cases, a very mild soap solution
at room temperature followed by sterile tap water for rinsing is
preferred as mode of cleansing. These are provided in convenient
For cases requiring efficient sterilization
the solutions to be employed are selected according to the nature
of the infectious agent suspected. The methods of surgical
procedure are followed, and this sterilization must precede the
taking of the blood by an hour or by several hours, according to
the manner in which the skin reacts to the sterilization process.
The use of alcohol, ether or any solution which reddens or which
pales the skin, or which causes any sensory irritation worthy the
name is bad and interferes with the accuracy of the count.
Cotton or gauze pads for applying the cleansing
solutions should be sterile and at least five pads of gauze or bits
of cotton should be ready.
TAKING BLOOD FOR COUNTING
Very little blood is required for making ordinary
cell counts, and this is, preferably, capillary blood. Capillary
blood is easily secured, is alike all over the body, is modified
only by vasomotor activity except as the entire blood picture changes,
and the tiny wound made by securing the blood from the capillaries
The side of a toe is the selected site for taking
blood from babies or small children, for the following reasons:
Vaso-constrictor control is not abundant; the skin is thin; the
foot is easily held, if the child is awake, and the toe is not apt
to be irritated or infected afterward. If the child is asleep
the process may not awaken him.
The lobe of the ear is selected for adults for
the following reasons: The lobe of the ear is not visible
and the patient cannot see the blood; the vaso-constrictor control
is not abundant; the skin is thin, sensory nerves are scanty, and
the lobe of the ear is not subject to later irritation.
In the case of an adult the worker stands at
the left side and to the rear of the patient. The patient
sits comfortably by the side of a small table upon which the necessary
equipment is placed conveniently for the worker. The patient
cannot see the blood being taken. If the patient is in bed
he should turn the face away from the worker, thus permitting an
ear to be accessible.
If really efficient sterilization of the skin
should be necessary, this should have been done at least an hour
before the blood is to be taken, and the skin protected by cotton
or gauze in the interim. In most cases the washing which removes
dust and some desquamating epithelium is all that is necessary.
The ear lobe should be gently mopped with sterile water, dried,
and protected against dust if any delay should occur. The
lobe must not be rubbed or handled or caused to redden perceptibly
by the cleansing. Such cleansing agents as alcohol are to
be avoided because they dilate the capillaries and modify the counts.
If the patient has come in from the street a
mild soap solution may be used for cleaning, then the ear washed
with sterile water and dried with sterile cotton. If the cleansing
should cause visible reddening of the skin the ear should be protected
with sterile cotton and the patient allowed to rest for ten minutes
or more, until the normal circulation has been well established
in the ear.
Prick the skin with lancet or needle.
Notice the emergency of the first drop, its size, color, and the
manner in which it flows upward into a round drop or spreads around
over the skin. Wipe away this first drop. Take the blood
for the white cell count first. This is partly because the
white cells stand longer without being modified, partly because
the white cells are more quickly modified by the faint vasomotor
change due to the slight irritation of the prick, and partly because
a larger drop is used for the white cell count. (The first
drops are usually rather larger than later droops of blood from
so small a prick.) If the white blood cell pipette is used,
draw the blood to the .5 mark, then very quickly draw the acetic
acid solution to the 11 mark. Rotate the pipette gently while
the solution is being drawn into the bulb. Close both ends
of the pipette with thumb and finger and shake gently from side
to side (not endways) to mix the blood with the solution sufficiently
to prevent clotting. Lay the tube aside, or give it to an
assistant who will continue the shaking, gently from side to side.
If the tube is shaken vigorously the cells may be fragmented and
if it is shaken endways the cells may be forced into the capillary
tube, or the mixture of cells and solution may be forced into the
dilatation above the bulb; if the contents of the bulb were well
mixed the latter accident would be of negligible importance, but
if part of the contents of the bulb were forced into the upper capillary
tube of the pipette and the upper dilatation before the mixing is
completed serious error would be present in the count.
In our laboratories an erythrocyte pipette is
used for the white cell count. Draw the blood to the 1 mark
on the pipette, then draw the acetic acid solution to the 101 mark;
mix the contents of the bulb by gentle shaking in a sidewise direction,
as already directed.
The advantage of this greater dilution lies
in the absence of the debris caused by the destruction of the red
cells; they are all completely destroyed by the greater amount of
acetic acid and completely dissolved in the greater amount of fluid.
The white cells are more easily recognizable. In cases of
leukemia and leucocytosis, this method is necessary for accuracy,
and we think it preferable in all cases.
Next, fill the red cell pipette for the red
cell count. Draw the blood to the .5 mark on the capillary
tube, then draw the diluting fluid for red cells to the 101 mark.
Shake as directed for the white cell pipette. Lay the tube
aside until the count is to be made.
The most frequent errors, in making the dilution,
are these: The end of the pipette may be allowed to reach
the air, either by being raised too high or by being pushed through
the drop of blood. The entrance of air into the tube causes
the “breaking” of the column of blood, or, if the air is admitted
with the diluting fluid, bubbles are formed. In either condition
a fresh drop of blood must be taken in a clean pipette and greater
care observed. Sometimes the drop of blood is too small; if
the blood has been secured with difficulty and if the column of
blood reaches to the first mark below the .5, the count may be completed,
and the correction made in the final computation. If it is
practicable to secure another drop of blood this is a more satisfactory
method. Sometimes the pipette is pressed against the skin
too firmly, and the blood does not ascend into the tube no matter
how hard the breath is drawn. In such a case, the tip is apt
to be slightly lifted, and the blood is apt to ascend suddenly through
the tube and into the bulb. When too great force is employed
in the inspiration which draws the blood into the tube, the blood
is apt to rush too rapidly upward, fill the tube, and sometimes
the bulb itself. The pipettes must be rinsed immediately else
the blood may coagulate within them and cleaning becomes an extremely
Take next the blood for differential counts
and for such other tests as may be indicated. The pipettes
of diluted ordinary blood may rest for a time, even for an hour
or two, without harm, but in cases of unusual fragility of the cells
changes may occur more quickly. It is best in all cases to
make the actual counts as quickly after the blood is taken as is
After the blood has been taken for the various
tests indicated cleanse the ear lobe of the patient and note whether
there is any indication of persistent bleeding. The wound
should be closed by this time, if it is not handled. It may
be kept open for five minutes or more by the slight manipulations
necessary for wiping away the preceding drops of blood in preparation
for further tests.
USE OF THE COUNTING CHAMBER
If the counting chamber has a circular moat,
the following technique is to be employed.
Place the counting chamber on a perfectly level
surface, and the cover-glass by the chamber. Close the ends
of the pipette containing the red cells and shake again, using a
side-to-side staccato movement, rather gently, about one hundred
times. Avoid the endwise movement in shaking. Remove
the rubber tube and attach a stiff rubber bulb to the upper end
of the tube, force out about four drops of the mixture and discard
this. This is in order that the diluting fluid in the capillary
tube, which contains no cells or, at most, only a very few cells,
may not be used for counting. Let the fifth drop begin to
form at the end of the capillary tube, and touch this to the surface
of the counting chamber, near the rule area. Avoid allowing
the end of the pipette to touch the surface of the counting chamber;
if the glass should touch the ruled area the markings might be scratched.
The amount of fluid necessary must be learned by experience; it
is about half as much as would drop from the end of the capillary
tube of the pipette if the pressure on the bulb should be increased.
Holding the cover glass by its edges, lower it slowly over the counting
chamber; if a bubble of air happens to be caught in the fluid clean
the counting chamber and take another drop from the pipette.
If the counting chamber itself is not filled with the fluid, clean
the chamber and take another drop of fluid. If the amount
of fluid is too great, so that the moat is filled across, the cover
glass cannot fit accurately and the count will be too high.
In this case clean the counting chamber and take another drop of
the fluid. Only experience can win accuracy.
If the counting chamber has an H-shaped moat,
the procedure is somewhat less difficult. Place the cover glass
on the slide first, and note that the cover glass fits the outer
raised part of the chamber accurately. The test of this accurate
fitting is best made by means of the phenomenon known as “Newton’s
rings.” These are concentric bands of rainbow colors which
appear when two glass surfaces are so closely in contact that the
difference between their surfaces is not more than the distance
measured by wave-lengths of light. When the slide containing
the counting chamber, covered by the perfectly plane cover glass,
is held slantingly to the axis of vision, in changing position,
these rainbow colors should be visible. This means that the
fitting is fairly accurate. If the rings are not visible press
firmly upon the cover glass; this may cause the necessary approximation
of the two surfaces. If the rings are then visible, and remain so
when the pressure is removed, the cover-glass fits accurately.
If the rings disappear when the pressure is removed, there is some
dust or moisture present; clean the cover-glass and the counting
chamber and repeat the process. Occasionally a counting chamber
is found upon which it is impossible to secure the rings.
Such a chamber may still be accurate so far as the counting is concerned.
Another test for accuracy lies in the fact that
two perfectly dry plane glass surfaces closely approximated adhere
firmly. Having placed the cover glass I position turn the
slide over gently; if the cover-glass adheres, the fitting is probably
good. If the cover-glass falls off, the fitting is not good.
Cleanse and dry the cover-glass and the slide and try again.
This method of testing is less accurate than the finding of Newton’s
rings but it is useful in the use of certain chambers. A trace
of moisture causes adhesion, separates the surfaces and seriously
increases the count.
If Newton’s rings cannot be produced on a certain
counting chamber, the chamber may be tested by another in which
these rings are produced. Take the same pipette of blood and
make counts of the red cells on both chambers, in one of which Newton’s
rings have been produced, and in the other only the adhesion of
the cover glass has been found well marked. If the counts
made on the two chambers agree within the limits of accuracy permissible
for the method (not more than 3% of the total number of cells counted)
then the second chamber is accurate enough for all clinical purposes
and its use can e continued. If the two counts do not agree, the
imperfect counting chamber should be returned to the maker.
Having secured the proper fitting of cover glass
and counting chamber, shake the red cell pipette as before using
at least 100 vibrations. Discard the first four drops, and touch
the fifth drop to the edge of the chamber at the edge of the slide.
The fluid runs beneath the cover glass and fills the ruled area
at once, by capillary attraction. If any fluid runs out and
fills the moat, thus lifting the cover glass and increasing the
depth of the counting chamber, clean the cover glass and the counting
chamber and take another drop from the pipette, after shaking it
With both types of counting chamber, the later
processes are alike. Allow the filled chamber to rest for
three minutes or so in order that the cells may settle to the bottom
of the counting chamber. The cells of normal blood settle
quickly; those of anemic blood and blood which is from persons with
certain diseases settle slowly. If any cells are still floating
when the count is begun, this must be deferred for a time, until
all the cells rest upon the bottom of the chamber. Accurate
counts are not possible when any cells are floating, because they
are not in focus at the same level.
Use a 1/6 objective and a one inch eyepiece
on the microscope. Find the ruled area and select a large
square containing five rows of five each of the small squares.
Count all the cells in the upper row of five small squares and note
the number found. In this count include every cell which touches
the upper line and the left hand line, and the intersection of these;
also at the intersection of the right line and the upper line; exclude
form the count all the cells which touch the right line and the
lower line, the intersection of these, and the intersection of the
lower line and the left line.
Repeat for each of the five rows of small cells.
This makes a column consisting of five numbers, each of which indicates
the number of cells found in five small squares. The sum of
this column indicates the number of cells in 25 small squares.
Select another area containing twenty-five small squares arranged
in the same way, and repeat. For ordinary work four such columns,
indicating the number of cells in 100 small squares, is sufficiently
accurate. In cases of anemia, count the cells in 400 to 800
small squares, using two or more counting chambers of diluted blood.
Compute as follows: Multiply the total
number of cells counted by the dilution, and this by 4,000 (which
is the cubic content of each square in terms of a cubic millimeter,
since each small square is 1/20 by 1/20 by 1/10 millimeter in size).
Divide by the number of square counted. Example, 625 cells
were found in 100 small squares, the blood being diluted 200 times.
The computation is:
625 X 200 X 4,000 divided by 100=5,000,000 cells
per cubic millimeter.
Or, the average number of cells in each square
is 6.25. Each square is 1/4000 cubic millimeter in content,
so that there must be 25,000 cells in each cubic millimeter of the
diluted blood. Since the blood was diluted 200 times, the
number of cells I one cubic millimeter of undiluted blood is 5,000,000.
The counting of the white cells follows a similar
process. The same tests for accuracy of fitting the cover
glass and for placing the drop of fluid in the counting chamber
If the white cell pipette was used, count the
number of cells present in at least 800 small squares. If
the red cell pipette is used count the number of cells present in
4,000 small squares. In either case the computation follows
the same method,--total number of cells counted, multiplied by the
dilution, multiplied by 4,000 and divided by the number of squares
included in the count. For example, if the white cells pipette
has been used, with a dilution of 1:10, and if 300 cells were found
while counting 800 squares, the computation is:
300 X 10 X 4,000 divided by 15,000 white cells
per cubic millimeter.
If the red cell pipette was used, with a dilution
of 1:100, and if 120 cells were counted within 4,000 small squares,
the computation is:
120 X 100 X 4,000 divided by 4,000= 12,000 ells
per cubic millimeter of blood.
Always count at least 100 cells, even if it
is necessary to cover 16,000 or more small squares in the counting.
In doubtful cases count 1,000 to 5,000 cells, using several pipettes
Many counting chambers have large squares around
the area of rulings for the small squares; the area of these large
squares is easily understood by following the lines which form the
small squares outward. By using these larger squares the white
cell count is easily and quickly made, even though 8,000 small squares
are necessary for accuracy. The computations are made upon
a basis of the small square in all cases. This avoids any
possibility of error due to the use of different units.
This actual count gives the number of red cells
and the number of white cells per cubic millimeter of blood.
Attempts have been made to substitute estimations of the blood cell
volume for studies of the blood cell count, since it is often thought
more important to know the total mass of hemoglobin-containing protoplasm
than the manner in which this hemoglobin is arranged in cells. On
the other hand, the manner in which the hemoglobin is divided into
cells is an important factor in the oxygen carrying function of
the hemoglobin, since hemoglobin arranged in a comparatively large
mass with relatively small surface area is exposed to the air in
the lungs less efficiently than an equal mass of hemoglobin divided
up into smaller masse with relatively greater surface area.
The hematocrit is a form of centrifuge in which
small tubes are placed in opposite arms; each tube has 100 equal
divisions. The technique is simple:
Secure a large drop of capillary blood by the
method outlined for taking the blood for counting. Place a
rubber tube over one end of the glass tube; draw the glass tube
perfectly full of blood. Cover a finger with Vaseline, and
cover the free end of the glass tube immediately. Remove the
rubber tube; place the glass tube in the arm of the hematocrit.
Place the other glass tube, filled with water, in the arm of the
hematocrit, to balance the machine. If the blood of two patients
is to be centrifugalized at the same time, make marks with a grease
pencil upon the outside of each glass tube for identification.
Start the centrifuge, gradually attaining the high speed within
half a minute. Stop the centrifuge and note the height of
the column of red cells at one minute intervals. When two
successive examinations give identical findings, stop the centrifuge.
Each division of the glass tube represents 100,000 cells, if the
cells are approximately normal in size and in hemoglobin content.
The column of cells is approximately equal to the column of plasma
in normal blood. The blood plasma can be used for the determination
of bile and other pigments. A very thin layer of fatty globules
is occasionally seen at the upper end of the tube, in lipemia.
In the anemias the number of cells in each division
of the tube may vary very greatly, so that in cases in which accuracy
of cell count is important the method has no value. As a method
of determining the actual volume of hemoglobin-carrying protoplasm
the method is of value.
The time required for complete settling of the
red cells has been studied by many workers, and this has been shown
to vary greatly in different diseases.
The differential count is made in order to determine
the relative numbers of different types of white blood cells.
These cannot conveniently be differentiated in the process of making
the actual count of white cells. An attempt to use diluting
fluids which give a differential stain in the counting chamber is
not satisfactory, and in order to secure accuracy by thus combining
the actual and the differential count it would be necessary to count
the cells in several hundred chambers. The differential count
is made of thin smears of undiluted blood, stained in some manner
which affects different types of cells variably, according to their
chemical constitution. By this means it is easy to recognize
several different classes of white cells. Slides used for
the differential count must be clean but they need not be sterile.
Rather thick slides are more convenient.
New slides are greasy and must be well washed,
first in warm soap solution, then in hot water. A second soapy
washing is often necessary. They can be kept in acid bichromate
solution made approximately as follows: exact proportions
are not necessary:
Commercial sulphuric acid
In routine work take the drops which flow after
the blood has been taken for an actual count. If only the
differential count is to be made, prepare and prick the skin as
directed for the actual count.
Have ready microscope slides which are perfectly
clean and perfectly dry, at least eight slides for each patient.
Touch one end of a slide to the top of a drop of blood, then touch
this blood to the end of another slide. Allow the drop of
blood to flow along the angle between the two slides for a second
or two. Push the first slide along the surface of second slide,
away from the drop of blood. The blood follows the moving
slide and leaves a thin, even smear upon the second slide.
Never push or pull the first slide along after the drop of blood,
because thus many cells are injured and there is a tendency for
some cells to cling to the first slide and thus to accumulate in
groups. Repeat this process for each slide. If the amount
of blood is scanty it may be necessary to take only six slides;
if these cannot be secured prick the skin again. Never take
less than six slides of blood for a differential count. If
the condition suggests leukemia, severe anemia or the need for any
special study it is best to take twenty slides or more.
As the smears are made lay the slides, blood
side up, upon a flat surface until they are perfectly dry.
Then put them into an envelope already marked with the name of the
patient and the physician and the date and the hours of taking the
blood. These smears keep almost indefinitely and they can
be stained by different methods for special study of different structures
If the blood arranges itself in circular areas
or rings, the slide was greasy. If the blood forms stripes
or bands, the motion was jerky. It is necessary that the second
slide be moved along the first in a steady, even, deliberate manner.
If there are threads of fibrin or small thick places in the smear,
the blood was partly coagulated. If the smear is too narrow
and thick, the second slide was moved before the blood spread along
the edge. If the smear is too thick and spreads over the entire
slide, the drop of blood was too large. If it is too thin
and spreads over too small an area, the blood drop was too small.
If two or more specimens are under observation
at the same time it is necessary to mark each slide for identification.
This is best done with a lead pencil. Using a pencil with
a rather soft lead, write an initial or an identifying number upon
one end of the smear, near the end of the slide, before the slide
is stained. The pencil ruins the blood cells over which it
passes and leaves a small amount of the lead; fixing the blood on
the slide makes the lines as produced permanent. By making
the marks at the end of the slide they do not interfere with the
counting, since this is done in the central area.
Many different methods of staining blood smears
for the differential count are in use. They include so many
stains, each with so many modifications, that any satisfactory description
of them all would be too long for this book. For example,
about twenty different methods of using the eosin-methylene blue
stain devised by Romanowsky have been described, and each method
has its advocates. The method used in our laboratories is
different from any of those described elsewhere, but it gives accurate
and delicate staining of the structures included in an ordinary
differential count, and it is easily modified so that good pictures
can be secured of atypical blood.
Solutions required are:
Eosin yellowish, 0.5 gram in 100 c.c. methyl
alcohol. This fixes the blood and stains the acidophile structures.
Methylene blue, 1.0 gram in 100 c.c. tap water,
if the tap water is clearn and reasonably pure, or methylene blue,
The methylene blue stains the nuclei and the
basophilic structures of the protoplasm. It stains also the
malarial and certain other parasites.
Take one of the slides already prepared and
dried. Place the slide on a level surface, smear side up,
and drop upon it several drops of the eosin solution. Let
stand fifteen or a few more seconds; rinse gently in tap water.
Drop upon it a few drops of methylene blue solution, enough to cover
the smear very abundantly; let stand a minute or a little more;
add a few drops of tap water and allow to stand on the slide about
two minutes, rinse with tap water. Drain, allow to dry in
air thoroughly, and examine, using oil immersion objective, and
one inch eyepiece. Or, after rinsing with water, mount in
water under a thin cover glass and examine, using a dry one-tenth
objective and one inch eyepiece. The one-eighth objective
does not magnify sufficiently for the finer details of cell structure
to be visible. For careful study of the cell structures, a
one-eighteenth objective, oil immersion, is useful. In our laboratories
the dry one-tenth objective is used for ordinary work and the one-eighteenth
objective for careful study of selected cells in unusual cases.
See also “Other Staining Methods,” Page 325.
Make a general survey of the smear in order
to note the type of blood cells present.
Have ready a sheet of paper with columns arranged
for each lass of blood cells,--large hyaline, small hyaline, mononuclear
neutrophiles, polymorphonuclear neutrophiles, eosinophiles, basophiles,
for ordinary blood. For abnormal blood other columns are required
for normoblasts, megaloblasts, poikiloblasts, microblasts, reticular
red cells, malarial parasites, and other peculiarities of the red
cells which may be of interest in the particular case, and for myelocytes
of each type found in the blood being examined. The general
survey has indicated the columns required. As the count progresses
other columns may be added at any time if other cell types are found.
Begin the counting at one edge of the smear,
move the slide, by means of the mechanical stage, so that the field
is brought toward the observer (apparently) as far as the edge of
the blood smear, and is carried as far to the right as the edge
of the blood smear, or as far to the right as the limit of the mechanical
stage permits. Then move the slide toward the left, noting
each cell and making the notation in the column devoted to that
cell type. When the slide cannot be moved further to the left,
or when the limit of the smear in that direction has been reached,
move the slide away from the eye the diameter of one field, so that
some selected red cell which is barely in vision at the lower edge
of the field is moved just beyond vision at the upper edge of the
field. Then move the slide toward the right, counting and
listing the cells as they appear in successive fields. Continue
in this way, moving back and forth across the slide, until all the
cells have been counted on the slide, or until the desired number
of cells has been listed.
With practice it becomes easy to carry the counts
in mind, and to make the notations in groups of twenty, t en or
five, as the case may be. In our laboratories, neutrophiles
are counted in groups of twenty cells, small hyalines I groups of
ten, and other cells as units. These habits are made for the
sake of accuracy, convenience and speed of counting. Each
worker develops his own customs.
Count until the total number of cells counted
is at least five hundred, in cases which present no marked variations
from the normal and which show no marked irregularity of distribution
of the cells. In abnormal cases at least one thousand cells
should be counted, while in cases used for special study, in unusual
cases, and in all the leukemias two thousand to twenty thousand
cells or more should be counted. In one of our cases of leukemia,
with an actual count of 250,000 leucocytes of which 80% were myelocytes
of different forms, it was necessary to make a differential count
of 50,000 cells in order to secure satisfactory accuracy.
The number of cells to be examined depends upon
the fact that successive counts of any selected number give almost
or quite identical results. In normal blood successive counts
of one hundred cells each give approximately identical figures for
lymphocytes and for neutrophiles, but may give very different figures
for eosinophiles, while basophiles may not be found at all.
Successive counts of 100 cells may give eosinophiles of ten per
cent in one 100, and no eosinophiles at all for another 100.
(Eosinophiles have a tendency to be in groups even in the best smears
of blood.) Counts of successive five hundreds of approximately
normal blood give satisfactory accuracy for such blood.
In acute cases in which diagnosis must be made
quickly, as in suspected pyogenic processes probably requiring speedy
surgical interference, a differential count of two hundred cells
may serve the necessary purpose and enable treatment to be initiated
quickly. But unless there is urgent need of haste, every count
should include at least five hundred cells. Even when this
haste is imperative, the count of five to ten hundred cells should
be carried on later in order that accurate findings may be kept
on record for later study and comparisons.
COMPUTATION OF DIFFERENTIAL COUNT
When the total number of cells in all columns
reaches one thousand, if this is the number counted, add each column
and divide by ten. This gives the percentages of each column.
For example, if the neutrophile column has in it 678 cells, then
there is 67.8% of neutrophiles in the patient’s blood. If
his actual count was 5,000 white cells per cubic millimeter, then
he has 3,390 neutrophiles per cubic millimeter of blood. If
there are 11 eosinophiles in that column, he has 1.1% eosinophiles,
or 55 eosinophiles per cubic millimeter of blood. If the number
secured by determining the actual number of any cell type from the
percentage and the actual count gives a fraction of a cell, the
nearest number is taken. For example, if a patient has an
actual count of 5,700 leucocytes, and his large hyaline cells make
up 4.3% of these, the computation gives 245.1 large hyaline.
The report should indicate 245 large hyaline cells, because the
limits of unavoidable error in this work are too large for us to
report finding a difference of one cell in ten cubic millimeters.
OTHER STRUCTURES COUNTED WITH THE DIFFERENTIAL LEUCOCYTE COUNT
While making the differential count of the leucocytes,
certain other structures may also be counted. Columns may
be arranged for red cells containing malarial parasites, for example,
or for normoblasts, megaloblasts and other atypical red cells.
With careful staining the reticulated red cells may also be enumerated.
A column must be arranged for each structure to be counted.
As the leucocytes are counted, such other structures are also counted
and the figures placed in the column allotted to them. They
are not included in the sum of cells to be counted, however.
Only leucocytes are to be counted in making the total of five hundred
or a thousand or more upon which the percentages are to be computed.
The total leucocytes must be 100%, and the other structures, not
being leucocytes, must not be included. After the leucocyte
count has been completed, as already directed, those other structures
are considered. For example, in a patient with a total blood
count of 3,000, there was 10% of large hyaline cells, that is, 300
per cubic millimeter. The differential count was based on
the examination of 1,000 cells, so that 100 cells were in the column
devoted to large hyaline cells. While these cells were being
counted, 25 red cells were found which contained a malarial parasite,
hence there were 75 malarial parasites within red blood cells per
cubic millimeter of blood. That is, the amount of blood which
contained 300 large hyaline cells also contained 75 cells containing
malarial parasites within red cells. Extracellular malarial
parasites were not included in this count.
In another case, with an actual leucocyte count
of 2,500 cells, the neutrophiles included one half, or 50% of the
total count. The differential count was based on 1,000 cells
examined. While making the differential count thirty-four
megaloblasts and twenty normoblasts were noted. That is, there
were fifty normoblasts and eighty-five megaloblasts per cubic millimeter
in this blood from a pernicious anemia patient, taken three days
before his death.
In this same way, the number of several other
structures can be computed on the basis of the differential leucocyte
count, and much useful information gained thereby.
SPECIAL METHODS OF COUNTING
In making a differential count of the blood in
certain leukemias and leucocytosis, when sometimes a great predominance
of one type of cell is present, it may facilitate the process and
increase the accuracy of the differential counting if the work is
done in two stages. First, make a differential count of two
groups only, the predominant type and all others. Examine
and list 1,000 cells or more in this first stage. Stain another
slide and make a differential count of all cells except the predominant
type. Examine and list 500 or more of the cells for this count.
Determine the percentage of the predominant type of cell by the
first stage of counting, and of the other cells by the second counting.
For example, in one of our cases of lymphatic leukemia the small
hyaline cells made up 97% of the total blood count. The first
stage of counting gave 970 small hyaline cells and 30 cells of all
the others together. It is evident that this differential
count of 1,000 cells could not give any accurate differential count
of the 30 cells. The second stage disregarded the small hyaline
cells altogether, and 500 cells of the remaining types gave accurate
percentages of the granular cells and the large hyaline cells.
Of the 500 cells examined in the second count, there were 40 large
hyaline, 52 mononuclear neutrophiles, 200 polymorphonuclear neutrophiles,
120 eosinophiles, 88 basophiles, and when these percentages are
taken for the 3% of “other cells” of the first stage of counting
the final results were as follows (omitting the third decimal):
Total white cell count, 120,000
288 per cu.mm.
97.00% 116,400 per
372 per cu.mm.
1,440 per cu.mm.
864 per cu.mm.
636 per cu.mm.
One megaloblast and three normoblasts were found
in making the second st age of the count; these are too few to serve
as a basis for accurate computation but they indicate that there
is some beginning injury to the red bone marrow. They would
not have been found at all in making an ordinary differential count.
The figures thus secured are more nearly accurate than could be
secured by making a differential count based on an examination of
30,000 cells using the ordinary technique and the time required
for the counting was much less.
In cases of marked neutrophilic leucocytosis
and in cases of monocytic angina this two-stage method of counting
is very much more accurate and more convenient than the ordinary
Iodophilia is of little significance when taken
alone. When employed with other clinical and laboratory findings,
it may give very useful information.
The older method of staining with iodine-gum
preparations has been superseded by the staining with the vapor
of iodine. A wide-mouthed, closely stoppered jar is kept for
this purpose. About one gram of iodine crystals is placed
in this jar.
Blood smears freshly made after the manner already
described for the differential count are placed in the jar, smear
side exposed to the vapor of the iodine, and allowed to remain for
five hours or more. The slides do not over-stain, and they
may be left for several days without harm. One hundred leucocytes
should be examined, and if iodophilic granules are not found, the
reaction is negative.
Note whether the protoplasm of the white cells
is diffusely stained and list such stained cells as iodophilic.
Note whether granules are free in the plasma or are within white
cells; if so, whether they are most abundant within the hyaline
cells or the granular cells.
One of the slides prepared for the differential
count can be employed. If the nuclei are not perfectly distinct,
the slide should be floated with a watery solution of methylene
blue for two minutes, then washed and again mounted in water.
If the nuclei are still not distinct, the smear may be washed in
N/100 solution of sodium bicarbonate, then the methylene blue stain
repeated. The smear should be quite thin for accurate and
Have a sheet of paper with columns numbered
from 1 to 5. Rarely columns 6 and 7 will be required.
Begin at one edge of the smear, as in the differential count, noting
the number of nuclei in each neutrophile, but disregarding all other
blood cells. Count in this way the nuclei in 100 neutrophiles.
If a cell contains two nuclei, place a mark under column 2, if it
has four nuclei, place a mark under column 4, and so on, until 100
cells have been counted. (Plate XIV).
In counting the nuclei, a ring-shaped nucleus,
even if slightly beaded in appearance, counts as a single nucleus.
If the nuclear masses are united by a band, they should be counted
as one. If they are united by a very thin filament of nuclear
substance, they should be counted as two. If any cell has
its nuclei piled one above another, so that it is impossible to
determine the number of nuclei within it, it may be passed without
counting; but if more than two or three such cells are found, the
count must be repeated, using a thinner smear, for the higher counts
will be those most often passed, under such circumstances, and the
findings will thus be lower than the correct number.
Add each column. The sum of the cells
of column 1, plus twice the cells in column 2, plus three times
the cells in column 3, plus four times the cells in column 4, plus
five times the cells in column 5 and six times the cells in column
6, if any, equal the sum of the nuclei in 100 cells. This divided
by 100 gives the average number of nuclei for each neutrophile.
For example, in a certain specimen of blood
10 cells having 1 nucleus;
or 10 nuclei in all;
38 cells having 2 nuclei
; or 76 nuclei;
41 cells having 3 nuclei
; or 123 nuclei;
7 cells having
4 nuclei ; or 28 nuclei;
4 cells having
5 nuclei ; or 20 nuclei.
These 100 cells have, altogether, 157 nuclei;
or an average of 2.57 nuclei per cell.
The neutrophilic nuclear average of this blood
is 2.57. The nuclear average in normal adult human
blood is between 2.45 and 2.55. In normal children the nuclear
average varies from 2.00 to 2.4, according to age.
SPECIAL METHODS OF STAINING
Endothelial cells. A stain for differentiating
between mononuclear neutrophiles and monocytes supposed to be from
the reticulo-endothelial system outside of the bone marrow is as
Solution: 80 c.c. 100 alcohol
20 c.c. water, triple distilled
Warm gently to about 40 degrees C.
Add: .2 gram alphanapthol (Merck)
.15 methyl violet 5 B
.2 c.c. hydrogen peroxide
(must contain 3% of the gas)
Uses dried blood films prepared as directed
for the differential count. The films should not be more than
a few hours old. Place the slide on a level surface and cover
with six to eight drops of the solution. Allow to stain and
fix for half a minute. Add an equal amount of distilled water
and allow to stain for five minutes. Rinse several times with
water. Cover the slide with basic fuchsin solution (0.01%)
to counter-stain for 2 minutes. Rinse with water; remove water
with filter paper; dry in air; examine with oil immersion lens or
mount in balsam.
Basophilic elements, including nuclei, basophilic
granules, basophilic protoplasm, erythrocytes and platelets take
various shades of red and pink. Eosinophile granules show
a peculiar circular staining so that the granules look like rings.
Neutrophilic granules and the finer granules of the endothelial
cells show bluish tints. The difference between the neutrophiles
and the endothelial cells lies in the characteristic nuclear structure
and larger granules of the neutrophiles, and the characteristic
nucleus and the smaller blue granules in the protoplasm of the endothelial
cells. The stain is useful for its purpose. Pappenheim’s solution
is adapted especially to a study of nucleated red cells.
Solution:--Take a saturated solution of methyl
Add saturated solution
This stain will keep
for several days, in the dark.
Fix smears with heat,
Flood slides with stain
for five minutes.
Wash in water, dry, examine
with oil immersion.
The nuclei of the normoblasts and nuclear fragments
stain a clear blue, while basophilic granules within the red cells
stain bright red.
Ehrlich’s traced stain is now little used.
It consists of equal parts of saturated solutions of indulin, nigrosin
and aurantia, mixed together after a difficult and tedious technique.
It can be purchased in powdered form.
Ehrlich’s triple stain also is somewhat difficult
to prepare. It may be purchased ready made up, though the
commercial preparations are not usually very successful. Grubler’s
stains are commonly used. The solution is made as follows:
Take a 100 c.c. graduate and measure the ingredients
in the order given; do not rinse the graduate at all during the
process. As each substance is measured pour it into a 500
c.c. flask and shake vigorously for one or two minutes.
Saturated aqueous solution orange G
Saturated aqueous solution acid fuchsin
Triple distilled water
Saturated solution of methyl green (added drop by drop with frequent
shaking of the flask)
Absolute alcohol, (added drop by drop, with frequent shaking of
Glycerin (added drop by drop, with frequent shaking of flask)
This mixture can be used at once, but it seems
to improve during the few days following preparation. It deteriorates
within a few weeks, more rapidly in the light or if the bottle is
To stain—Fix slides with heat and place on a
level surface. Cover with solution taken from about the center
of the bottle containing the stain, using a glass pipette for the
purpose. Never shake the bottle. Leave stain on slide
for three minutes or more; the slides do not over-stain if left
twenty minutes. Rinse with water, remove excess water with
filter or blotting paper, dry, examine with oil immersion or mount
Erythrocytes stain yellow or buff. Normoblast
nuclei stain a very dark green, almost black. Nuclei of leucocytes
stain dark green but not so deep a color as the normoblast nuclei.
Fine granules of the neutrophiles and the endothelial cells stain
lilac or pale purple. Coarse granules of these cells and of
the eosinophiles stain crimson. Basophilic granules do not
stain. This stain is useful for distinguishing certain types
of granules but it is not useful for general work. It is very
difficult to secure good stains in cases of leukemia. Occasionally
a patient appears whose blood refuses to take the Ehrlich triple
stain, for no perceptible reason.
Leishman’s stain is best purchased in powder
form. For use make a solution of 150 mgs. of the powder in
100 c.c. of pure methyl alcohol. Smears are best made on cover
glasses for this stain. Use the technique given for making
smears on slides. Place a cover glass, smear side down, in
a watchglass. Drop the stain into the watchglass until the
cover glass floats. Allow to fix and stain for three minutes.
Add an equal amount of distilled water, and allow to stain further
for one minute. Remove the coverglass and wash in water, drain
on edge until dry, examine with oil immersion lens. Or, mount
in water and examine with dry one-tenth objective. The stain
gives a fairly good picture when freshly made. After about
ten days standing it gives a differential stain for the azur granules
Red cells take a coppery tint; in polychromasia
some cells are pinkish. Nuclei are in shades of reddish purple
or purplish red. Cytoplasm is bluish or blue. Eosinophile
granules are coppery red. Neutrophile granules take a pinkish
color. Basophile granules stain purplish or reddish purple.
Azur granules are cherry red. The stain is fairly good for
general differentiation. Leishman’s stain has bee simplified
and modified in many ways.
Wright’s stain has been developed from Leishman’s
stain. Its preparation is rather difficult and the resulting
powder not always successful. The powdered stain, which is
a precipitate formed by combining eosin yellowish with methylene
blue under certain conditions, can best be purchased. This
powder is to be dissolved in methyl alcohol, 1.5 gm. powder to 100
c.c. methyl alcohol. The solution keeps for a month or more.
Wright’s stain is useful for general differentiation. The
technique of staining is:
Place the dried slide on a level surface.
Flood with the methyl alcohol solution, which fixes and stains the
slide at the same time. Allow the stain to stand on the slide
one minute. Add an equal amount of distilled water, and allow
to stand for two or three minutes,--the longer period giving a deeper
blue stain, but eosinophilic granules are more deeply stained in
the shorter period of time. Longer standing than three minutes
may cause a precipitate to be formed. Rinse in water for about
half a minute. The thinner areas should be pinkish or yellowish
in tint. Experience is necessary to determine the exact degree
of differentiation which gives best results for each blood specimen.
Mount in water and examine by means of a dry one-tenth objective,
or dry and examine by means of an oil immersion lens. Red
cells show pinkish or yellowish. All nuclei are blue or purplish
blue, varying in shading for different types of cells. Neutrophile
granules are pinkish or pale purplish in color. Eosinophiles
are brilliant reddish pink or cerise. Hyaline cells show blue
protoplasm which may be very dark or rather pale. Platelets
are blue or purplish. Malarial parasites are blue with darker
purplish, reddish or bright red chromatin. Mast cells show
deep blue granules. The stain is fairly useful in general
differentiation. Tap water used for differentiation increases
the blue tints and this is often desirable.
Giemsa stain is much simpler than the ;methods
described, and it gives excellent differentiation. The formula
is simple and the stain constant in quality. The powdered
stain may be purchased or it may be made up as follows:
Azur II eosin
Methyl alcohol, c.p.,
Grind up the stains in the alcohol, using a
small amount of the alcohol first When thoroughly mixed add:
The solution keeps for several months, and sometimes
The technique of staining is simple also.
Place slides on a level surface, flood with
methyl alcohol for five minutes, drain but do not rinse. Put
fifteen drops of the stain on the slide, then add ten drops of distilled
water; stain for fifteen minutes. Rinse, drain, mount in water
and examine s usual. Or dry and examine with oil immersion
lens. They fade quickly in cedar oil; paraffin oil may be
used instead. One drop of half-saturated sodium carbonate
increases the staining of the basophilic elements. The use
of tap water instead of distilled water gives better differentiation
in our laboratories.
Determination of the
neutrophile nuclear average. Nuclei only are shown.
I. Five single nuclei.
II. Six double nuclei.
III. Six triple nuclei.
IV. Five quadruple nuclei.
V. Six nuclei of five lobes each.
VI. Six nuclei of six lobes each.
This method of staining differentiates cells
derived from lymphoid tissue from those derived from the red bone
marrow. Three solutions are required:
A. 100 c.c. distilled
5 drops saturated aqueous solution sodium hydroxide
1 gram alpha-naphthol
Boil this solution, cool, decant fluid from
any residue which may be present. Allow to stand three days
or more before using. This solution will keep a month or more.
B. 100 c.c. distilled
.5 gram basic paraphenylenediamine
Mix without heat. Allow to stand at least
twenty-four hours before using. This solution will keep a
month or more.
C. 100 c.c. distilled
5 c.c. formalin
Technique of staining. First mix together
equal parts of A and B and filter. This mixture must be used
within an hour or so.
Fix dried blood smear in solution C for five
Stain with the mixture of A and B for five minutes.
Rinse, mount in water and examine, using dry
one-tenth lens, or dry and use oil immersion lens.
Cells derived from lymphoid tissue show no granules.
Cells derived from bone marrow show blue granules.
The reagents are difficult to secure and preparation
of the solution is cumbersome. It is rarely of value in diagnosis
but it has given some good results in research work.
Merck offers simpler reagents; beta-naphthol-sodium
is sold in sealed glass ampoules as Mikrozidin. Solution A
is a 2% solution of Mikrozidin in distilled water. He supplies
also dimethylyaraphenylene-hydrochloride in similar ampoules.
Solution B is a 1% solution of this in distilled water. Equal
parts of the two solutions are mixed and the resulting greenish
precipitate filtered off. The further technique is the same
as in the original method. The oxidase granules appear brownish
or blackish by this method, instead of blue; the significance is
THE BONE MARROW
Examination of the cells from bone marrow is
easily made. Take a piece of rib or of any bone containing
red marrow; make a fresh break if the bone has not been removed
immediately before the smear is to be made. With forceps press
upon the bone just beyond the break until a drop exudes from the
broken end. Very quickly make smears from this drop upon slides,
following the method used for making blood smears. Dry in
air, and stain after any of the methods used in the study of blood
smears. Vital,--or supra-vital,--staining methods are employed
in the same way. If much fat happens to be present it may
be necessary to remove this by flooding the slide several times
with warm alcohol, ether and alcohol, and ether alone until the
fatty globules are washed away. Bone marrow from adults is
usually very fatty, while bone marrow from still-born babies, human
fetuses and certain laboratory animals is usually free enough from
fat to stain readily and easily.
To demonstrate the nerve endings in bone marrow
it is best to use histological methods, for which see any text-book
on histology. The methods employed are too long for discussion
in this chapter.
TECHNIQUE OF WARM SLIDE STUDIES
Very useful information can be secured from a
study of the blood in the vital state; that is, during the lifetime
of the blood cells on a warm slide. The conditions of the
warm slide approach those of normal blood in the capillaries and
the behavior of the cells and the formation of fibrin threads present
pathognomonic variations in many instances. This study requires
only a small amount of time and no expensive or complicated apparatus.
The only difficulty is that the microscope and the patient must
be brought together and that the examination must be made immediately
after the blood is taken. If the patient is too ill to go
to the laboratory the microscope must be taken to his bedside.
Since there is no noise, odor or confusion associated with the work
of warm-slide examinations it is not often annoying to the patient
or to anyone else to have the work done at a small table beside
The technique is simple but considerable practice
is necessary to attain skill in securing the correct amount of blood,
speed in beginning the observations, accuracy in watching several
factors at the same time and ability to distinguish between important
and unimportant variations from the normal conditions.
Have ready a microscope with a one inch eye piece
and a dry objective of high power, preferably a one-tenth, though
the one-eighth can be used fairly well. The light must be
strong but the field must be well limited by the iris diaphragm.
Electric lighting is steady and is usually accessible. Gas
light or daylight can be used efficiently, and the person who makes
these examinations should be constantly in the habit of using these
lights interchangeably whenever there is any probability that it
may be necessary to rely upon them for emergency work.
Several types of warm stage are on the market.
These are commonly kept at the selected temperature by electricity.
If electricity is not available at the bedside a heavy slide can
be selected, warmed by water of the selected temperature, dried
quickly and placed upon the stage of the microscope. The stage
of the microscope can be warmed by placing any heated object on
it for a few minutes before the examination is made. The cover-glass
is best warmed by allowing it to lie upon the warmed stage of the
microscope or by holding it between the palms for a few minuets.
In using these make-shift methods it is necessary to be very careful
to avoid too great heat.
Connect the warm stage and place it on the microscope
stage several minutes before the blood is taken. Have the
slide and the cover-glass perfectly clean; put them on the warm
stage. There is some difference in the time required to reach
the select ed heat in different warm stages. Be sure that
the slide and the cover glass are thoroughly warmed before the blood
is taken. Have the light in place and be sure that the iris
diaphragm is correctly adjusted for the study. The iris diaphragm
requires delicate adjustment, because these living blood structures
are not stained and are visible only on account of the variations
in their refrangibility.
Cleanse the lobe of the ear of the patient,
using only sterile water without any unnecessary handling.
Prick the skin with the usual sterile needle and wipe away the first
drop of blood.
Touch the flat side of the warm; cover-glass
very quickly to the top of a drop of blood just exuding from the
wound, place this on the warm slide, blood side down, put it on
the warm stage and examine immediately. Let another person
attend to the wound in the ear; if no other person is at hand allow
the wound to remain untended until the first examination of the
warm slide has been made. After a minute or so the observer
may leave the microscope for the few seconds necessary to wipe off
the lobe of the ear and thereafter the observations may be
interrupted for a few seconds at a time if necessary.
In observing the changes on the warm slide many
factors are to be kept in mind and all of these must be watched
all the time.
Note the presence or absence of fibrin threads
when the slide is first seen. These are lines of highly refractive
material, best seen in the spaces between blood cells. If
they are present when the slide is first examined note that fact
and note any later time at which new threads are first visible.
Note the amount of fibrin, the size and length of the threads and
their contour. They may be even and regular, or swollen at
long intervals or swollen at shorter intervals, presenting a beaded
appearance. Note whether the threads lie in straight lines,
apparently unrelated, or whether they radiate from cells or from
groups of platelets. Note whether the fibrin is formed in
net-like masses or in the normal straight threads. Note when
the fibrin ceases to be increased in amount. With practice
all these factors can be seen at a glance. Continue the observations
until no further fibrin appears.
Normal blood shows a few fibrin threads within
four to six minutes, and the fibrin continues to form, slowly, for
about five minutes longer. The condition characterized by
very scanty or absent fibrin is called hypinosis, and this is present
during starvation and in certain forms of malnutrition. Excessive
fibrin formation is called hyperinosis, and this condition is present
during pneumonia, acute rheumatism and in certain other inflammatory
states. Hyperinosis is usually marked in malignancy, and in
this case the threads are formed almost at once, are very irregular,
often beaded, radiate and arranged in irregular net-like tangles.
In pneumonia the threads are very speedily formed, are heavy, long
and abundant, and are regular in contour; they may or may not be
radiating, and they do not form nets in typical cases. The
test is very useful in making an earlier diagnosis of pneumonia
than is possible by any other means.
Note the time at which the first movement is
seen in a white blood cell, and note also the classification of
the cell which first moves. Usually an eosinophile or a neutrophile
moves first; rarely hyaline cells are first active. Eosinophiles
are recognizable by their very large granules; neutrophiles by their
fine granules and their irregular nuclei; hyaline cells by their
glassy protoplasm and their round, central nuclei. No attempt
should be made to distinguish between the different classes of hyaline
cells in ordinary cases.
Note the manner of movements. Protoplasm
may flow steadily or it may flow rapidly for a few seconds, then
slowly or may cease moving for a time; note these conditions.
Granules or intergranular protoplasm may seem to be the most active
part of the cell; note these relations. Pseudopodia may be
extruded slowly or rapidly; may flow from two or from several sides
or angles of the cells and may seem to be purposive or purposeless
and even antagonistic in their activity; note these conditions.
The pseudopodia may be extruded and the protoplasm flow into them
thus causing the cell to change its location on the slide.
The pseudopodia may be extruded, some protoplasm may flow into them,
then the protoplasm may flow back into the cell and the pseudopodia
be retracted, or pseudopodia may remain present and again the protoplasm
flow into them; these various activities may seem purposive or may
seem erratic. Pseudopodia may vary in form being long, short,
slender, broad, heavy, flat, active or inert, and may present many
peculiarities of structure and of activity. All such peculiarities
should be noted. The activity of the cells may change during
the period the slide is under observation; cells at first normally
active, inactive or excessively active may show increased or diminished
activity within a few minutes, and all such variations should be
Note the nuclear reactions. The nucleus
may follow the protoplasm in its activity, or it may remain almost
or quite immovable. Hyaline cell nuclei rarely move at all
in normal blood and if they do move this is of interest. Neutrophile
and eosinophile nuclei tend to follow the pseudopodia fairly quickly
in normal blood; if they do not move at all or if they follow the
protoplasm very quickly these facts should be noted.
The time when the leucocytes show diminished
activity and the time when the first inactive cells appear should
be noted. Pay no attention to inactive hyaline cells, since
these very often fail to show activity at any time, in normal blood.
Abnormally they may die with pseudopodia still visible.
Normal leucocytes live at least an hour and
they may live several hours on the warm slide. Keep them under
occasional observation at least forty minutes. If none, or
only a few, are dead after forty minutes on the warm slide they
may be said to “live well on the warm slide,” which means that they
have at least moderate vitality.
It is not necessary to watch the individual
red cells. These undergo various changes on the warm slide.
With evaporation of the watery content of the blood the r ed cells
crenate. First there appears a bright spot on one edge of
the cell, then another, then several and many such bright spots
until the surface seems covered with thorns. Crenation may
occur without evaporation, but it always occurs when the osmotic
tension of the fluid surrounding erythrocytes is increased.
The stroma and the hemoglobin of the red cells
may undergo various degenerative changes. Parasitic and bacterial
inclusions may thus be imitated. Suitable staining methods
quickly explain these structures, and it is best to disregard them
while making the warm slide examinations.
SPECIAL TESTS FOR THE WARM SLIDE
Supravital staining. Interesting facts
can sometimes be learned from a study of the blood cells which have
received some stain which does not visibly affect their life and
which have certain affinities for living granules or other cell
The same technique is employed for all these
stains. A few crystals of the stain may be mixed with the
fresh blood, and smears made from this mixture, or a solution of
the stain is allowed to dry upon a slide and the blood smear made
upon this stained surface. The following stains are employed
for supravital staining: Brilliant cresyl blue, Janus green,
methylene blue, toluidin blue, thionin, Capri blue, Nile blue, paraphenyl
blue, neutral violet, neutral red, pyronin, fuchsin and safranin.
Each of these stains gives some particular reaction which may be
of value in the study of cell structure or cell inclusions, but
each stain is characterized by some inefficiency or some source
of error for other studies than that for which it is most useful.
In other words, there is no known stain which renders all cellular
structures and inclusions simultaneously visible.
Brilliant cresyl blue and methylene blue give
useful information in certain anemias and one of these should be
used in routine blood examinations in order that immature red cells
may be recognized. Either of these stains demonstrates the
basophilic reticulation present in immature red cells, and the number
of these is an important factor in the study of anemic bloods.
In our laboratories the following technique
Prepare the slides in advance. Put a drop
or a few drops of a solution of the selected stain on about the
middle of a glass slide and allow it to dry, thus leaving a precipitate
of the stain on the slide. If the stain is left in unequal
masses or if there are clear spots left within the stained area
the stain was not completely and perfectly dissolved or the slide
was not quite clean; such slides should be cleaned, the solution
investigated and the preparation repeated. These slides with
the stained centers should e prepared in advance; they can be kept
in slide boxes, well covered, for several days or indefinitely if
carefully protected against dust and moisture. Just before
the examination is to be made place the stained slide on the warm
stage and take the blood as before, making such observations as
are desirable in connection with the special stain being used.
Brilliant cresyl blue shows the reticulation
of the red blood cells vividly and its chief value lies in this
fact. The reticulum of the younger forms of white blood cells
shows more plainly than is the case with older cells.
Methylene blue is useful occasionally.
Very thin deposits are necessary for satisfactory work. Nuclear
structures and basophilic granules are shown very clearly, and the
cells retain life for a long time after being stained with methylene
blue. Methylene blue may be decolorized and its color may
be regained as successive oxidation and reduction of the stain occurs,
and thus variations in the physiological activities of the cell
may be studied. As the cells die the nuclei take the methylene
blue present in small amounts with avidity not shown during life.
If too heavy a deposit of methylene blue is present the cells do
not show these reactions but the nuclei are deeply stained at once
and the cells die rather quickly.
The study of the fibrinolytic ferment is one
of the newest and most interesting methods of hematological technique.
This study in its present form is limited to osteopathic laboratories.
The method is as follows:
Prepare fibrinolysis pipettes in lots of one
to several hundred at a time. Have ready glass tubing of three
millimeters diameter and about forty centimeters long. Heat
to redness an area about five centimeters from the end of the tube
and draw out to a capillary tube five to ten centimeters in length.
Repeat this heating and drawing to a capillary tube, each time leaving
a space of about three centimeters of the tube unchanged in size.
Continue until the entire length of glass tubing is changed into
a series of small glass bulbs about three centimeters long and the
diameter of the glass tubing, separated from one another by capillary
tubes of five or more centimeters in length. Break the capillary
tubes about half way between the bulbs. Seal the ends of the
capillary tubes in flame and put into a clean dry box to keep ready
for use. The interior of the pipettes is sterile, the glass
having been heated to redness.
The exterior is easily sterilized by passing
through a flame, when the pipettes are to be used.
Have ready also a supply of small vials or glass
tubes. The tubes used for Wassermann tests are convenient.
Small baskets in which these tubes may be supported are necessary.
We use the small perforated aluminum baskets sold as coffee balls;
the lids being removed they are of convenient size and shape.
Each of these small baskets holds several vials or tubes, and also
the identifying cards upon which notes can be written at each examination.
Incubator, microscope, slides, cover-glasses
and ordinary laboratory equipment, cotton and boiled water for washing
the skin and a blood lancet are also required for the test.
A supply of tap water which has been sterilized
by boiling in a closed vessel on successive days is best for this
use. Distilled water can be used but fibrinolysis proceeds
best in water of the type ordinarily used for drinking,--either
spring water, tap water or well water. If distilled water
must be used, it should have added to it for each liter of water
the following formula:
When ready to take the blood pass three pipettes
through a flame, break off both sealed ends of the capillary tubes,
leaving about one centimeter of the capillary tube at each end of
the bulb of the pipette, cleanse the skin of the lobe of the ear
if the patient is an adult or a large child, or the skin of the
side of a toe or of the heel, if the patient is a baby or a small
child. Prick the skin deeply enough to secure several drops
of blood. Wipe away the first drop, using sterile cotton (or
gauze), and touch the end of a capillary tube to the next drop which
oozes from the skin. Fill the bulb about half full.
Repeat until three pipettes have been filled in this way.
Lay the pipettes upon a sterile plate and allow to rest for an hour.
Fill three of the small vials or Wassermann
tubes with sterile tap water and boil for three minutes, allow to
cool to about room temperature. Note on slip of paper the
name of the patient, of the doctor, the date and the hour the blood
was taken, the hour the blood clot was placed in the vials and into
An hour after the pipettes have been filled
the blood should be thoroughly coagulated and the clot should be
separated from the serum. If coagulation is not complete,
let the pipettes remain longer at rest. Break off the capillary
tubes at the clear end of the bulb and break the bulb-like part
which is filled with blood, just at its junction with the capillary
tube which is filled with blood. This leaves both ends of
the blood-clot free. Catch the serum on filter paper as it
oozes from the end of the broken tube. Drop the clots, still within
the bulb-like part of the pipette, into the vials or Wassermann
tubes already filled with sterile tap water. Close the tops
of the vials and shake them until the clots drop out of the pipettes.
Ordinarily three to five fairly vigorous shakes are enough to allow
the clots to fall out of the pipettes. If longer shaking is
necessary to free the cots, or if the clots do not fall out of the
pipettes completely, the clot is said to be adherent to the tubes.
Place the vials in a small basket or on a rack, and incubate until
the clots are dissolved, or until three days have passed without
beginning digestion. Examine the vials at twelve hour intervals
until fraying of the clot is noted, then at six hour intervals until
the clot is almost digested, and after that watch the progress of
digestion at shorter intervals until the clot is completely digested
and dissolved in the water. Note the time of beginning digestion
and of completed digestion.
When digestion begins in 20 to 30 hours, and
is complete in 45 to 55 hours, fibrinolysis is normal.
Pour the contents of two of the vials into a
centrifuge tube, fill another centrifuge tube with water to balance,
and centrifuge at moderate speed for ten minutes. Pour off
the supernatant fluid, and examine the sediment under a 1/6 or a
1/10 objective with 1 inch eyepiece. Note whether bacteria
are present; if so, contamination has occurred and the findings
while not actually negligible are less accurate. If any peculiarity
in the process of digestion has occurred, the test should be repeated.
A few bacteria do not militate against the accuracy of the test,
but absolute sterility is much to be desired. It must be remembered
that the skin cannot be completely sterilized; that some bacteria
are ready always present in the deeper layers of the skin and that
the mixture of blood and water, at body temperature, give excellent
opportunities for bacterial growth.
Normally, the sediment contains debris derived
from the digested fibrin; this is usually present in minute rounded
masses. Normally many blood cells, both red and white, appear
in the debris. When the blood cells are completely digested
there is some undifferentiated proteolytic ferment present.
Since this digests fibrin as well as blood cells, the repeat must
e “Fibrinolysis masked by action of some undifferentiated proteolytic
If three days pass without recognizable fraying
of the clot, the report must be “Fibrinolysis absent.”
If the clots dissolve completely, but more rapidly
or more slowly than normal, by six hours or more, the reports must
e, “Fibrinolysis delayed” or “Fibrinolysis hastened,” giving the
hours as noted at the different examinations of the clots.
One to three pipettes filled with the blood
of a person known to show normal fibrinolysis should be used as
controls for every test until the technique has been standardized.
The theory of fibrinolysis is discussed in chapter
V of this book. Briefly, it may be said that, in terms of
this theory, any person who has normal fibrinolysis has at least
one factor in protection against the growth of malignant neoplasms;
that any person lacking this ferment lacks this factor of protection;
that non-differentiated ferments often occur during the progress
of certain disease, especially during the growth of cancer in the
body, an these ferments which are not differentiated digest fibrin
and blood cells together.
A report on the fibrinolysis test should include
the following items.
Name of patient and doctor in charge of the
case; date and hour of taking the blood and of placing in incubator;
or time of last examination if no digestion occurs. The report
should state also either “Fibrinolysis normal,” “Fibrinolysis absent,”
“Fibrinolysis delayed,” Fibrinolysis hastened,” or Fibrinolysis
masked by some undifferentiated proteolytic ferment.
TESTS NOT COMMONLY USED IN ROUTINE EXAMINATIONS
Several tests are useful under certain circumstances,
but are not necessary for all cases. These are not included in routine
examinations unless statistics are being accumulated for research
purposes. In our laboratories, these special tests are made whenever
the condition of the patient indicates that useful information might
be secured thereby. Whenever special studies are being made in which
the information to be secured by any special test is thought to
be useful, that test is added to the routine work for all patients
for as long a time s is necessary to secure the statistics desired.
To secure all possible information from the
study of every blood specimen examined in our laboratories is impossible.
Some of the special tests require a considerable amount of blood,
and it is not good for sick people to yield so much blood. The time
required for making so many tests precludes their routine use. Many
of them must be made very soon after the blood is taken, so that
several persons must work at the same time if several of the special
tests are done for the same patient on the same day. For these reasons
it is best to limit the special tests to those which are indicated
in each case, plus those which can be made for research purposes
without too great demands upon the time of the laboratory staff
and without any unnecessary demands upon the blood or the strength
of the patient.
THE SPECIFIC GRAVITY OF THE BLOOD
Before the invention of the instruments which
depend upon color changes for the estimation of hemoglobin, the
determination of the specific gravity of the blood was the most
important method of estimating the amount of iron in the blood.
Modern hemoglobinometers give more accurate findings and the estimation
of the specific gravity is now considered of little value in routine
laboratory diagnosis. In research work it still have a place, and
it occasionally occurs that useful information is secured by a study
of the changes in the specific gravity of the blood under experimental
or pathological conditions.
The specific gravity of normal blood varies
almost exactly with the hemoglobin and the red cell count. Daily
variations in red cell count, specific gravity and hemoglobin content
of the blood form practically parallel curves in normal individuals;
variations in the curves fall within the percentages of unavoidable
error in technique.
Various methods and different students give
somewhat different findings but all agree that the variations follow
the blood count and the hemoglobin under normal conditions. Figures
varying from 1,052 to 1,063 for healthy young people are given by
For normal young women in Southern California
the specific gravity of the blood varies between 1,053 and 1,055;
for normal young men the specific gravity varies between 1,055 and
1059. Persons who seem perfectly normal occasionally show specific
gravity as low as 1,052 or as high as 1,060. The blood of normal
children has lower specific gravity than the blood of adults and
may be as low as 1,048. At birth the specific gravity is much higher,
corresponding to the high cell count and high hemoglobin present
at birth and during the first few days of life.
Pathologically the specific gravity varies considerably,
though this fact has not been found useful in diagnosis. In severe
anemias the specific gravity may be as low s 1,025, and in high
fevers or in jaundice as high s 1,070.
In certain forms of cachesia the specific gravity
does not vary wit the hemoglobin and the cell count; these may remain
almost normal while the specific gravity of the plasma may be considerably
lowered. In this case lowered osmotic tension is indicated by the
changes which occur in the red cells during the actual count.
The specific gravity of the red cells is higher than that of the
plasma. The specific gravity of plasma and serum vary together in
health and in disease.
The higher specific gravity of the red cells
in the plasma of lower specific gravity is associated with a common
osmotic tension because the hemoglobin molecule is heavy and of
large size. Since the osmotic tension varies with the number of
molecules in a solution, the red cells, with heavy, iron-containing
hemoglobin molecules of large size, are still isotonic with the
plasma with smaller, lighter molecules of inorganic salts. When
the osmotic tension of the plasma varies, the red cells imbibe or
give off enough water to equalize the tension within and without
Accurate determination of the specific gravity
is rarely needed in diagnosis. An estimation based upon the hemoglobin
percentage, the color index and the osmotic tension of the red cells
is sufficient for all ordinary clinical purposes. The specific gravity
of the red cells varies with the hemoglobin, almost exactly, and
the specific gravity of the plasma varies with its osmotic tension.
By comparing the hemoglobin, the color index and the osmotic tension
it is possible to determine whether the specific gravity is normal
or is increased or diminished from the normal, which is all that
is useful in diagnosis.
The gravimetric method is quite accurate. This
requires five cubic centimeters of blood, and the blood must be
taken from a vein. This is measured accurately, weighed upon delicate
chemical balances, and the weight and volume compared with the weight
and the volume of distilled water, with corrections for temperature
and height above sea level. The gravimetric method is of value only
for scientific precision in research.
By using a pycnometer the specific gravity can
be taken with accuracy, but a considerable amount of blood is necessary.
In certain diseases the removal of ten to fifty cubic centimeters
dos no harm, and normal persons can give much more than this amount
without recognizable effect. The technique is as follows:
Have ready the pycnometer and delicate chemical
balances, also a syringe of twenty cubic centimeters capacity fitted
with a medium needle, both sterile. Cleanse the skin over the vein
of the patient’s elbow, washing first with water and soap, then
with alcohol, they dry with sterile cotton. Leave the cotton in
place until the vein is to be pricked.
Weigh the perfectly dry, clean pycnometer. Fill
it with distilled water at a temperature of 99 degrees F., and weigh
again. Dry the pycnometer
Place a firm elastic band around upper arm of
the patient. Remove the cotton and take blood from the median basilic
vein, enough to fill the pycnometer. Cover the wound with sterile
cotton. Immediately fill the pycnometer with the blood and weigh
Cleanse the skin of the arm from which the blood
was drawn. If bleeding persists apply ice, or cotton moistened with
alcohol, and keep under gentle pressure.
Usually the wound is closed by the time the weighing
Computation: Subtract the weight of the empty
pycnometer from its weight filled with distilled water, also from
its weight filled with blood. Thus the actual weight of the water
and of the blood are determined. Divide the weight of the blood
by the weight of the distilled water of the same temperature (and,
of course, of the same height above sea level). This gives the specific
gravity of the blood.
Aremetrical methods require considerable practice
but only a little blood. Have ready several perfectly clean, dry
glass tubes; those used for estimating the specific gravity of urine
are convenient. Fill these with varying mixtures of chloroform and
benzine, or of other suitable fluids. These have the following qualities,--they
must not be miscible with blood but must be freely miscible with
each other; and one must have a specific gravity above and the other
below the specific gravity of any blood apt to be encountered.
Take five of the urinometers or other convenient
tubes and fill them with mixtures of the selected fluids in proportions
to produce specific gravities of 1,058, which is that of average
normal blood; 1,068, which is the highest reading for normal blood;
1,051, which is the lowest reading for normal blood; 1,025, which
is extremely low and 1,072, which is extremely high. Have these
tubes properly marked and arranged in a row.
Take a glass tube of about three microns bore
and about ten centimeters long, bent at right angles about one centimeter
from its lower end. Attach a rubber tube at its upper end and draw
blood from a capillary puncture upward into the tube for about two
centimeters. Vary quickly blow one drop of the blood into each of
the urinometers at about one half the depth of the liquid. The drop
will sink in the fluid whole specific gravity is lower than that
of the blood and will rise in the fluid whose specific gravity is
higher than that of the blood, and will remain stationary in the
fluid that equals the specific gravity of the blood. If the drop
does not remain stationary in any one of the fluids, prepare three
other urinometers and fill with mixtures of intermediate specific
gravities between that which causes the drop of blood to sink and
that which causes it to rise. For example, if the drop of blood
rises in the fluid with a specific gravity of 1,058 and sinks in
the fluid with a specific gravity of 1,051, the fluids next selected
should be of a specific gravity of 1,053, 1,055 and 1,057. If the
drop sinks very slowly in the fluid with a specific gravity of 1,051,
and rises rapidly in the fluid of 1,058, it may be best to prepare
only one extra tube, at 1,053, for the second test. Practice in
this work is necessary in order to determine what specific gravities
to select for the second test, and to avoid unnecessary manipulations.
If the drop of blood remains quiet in any fluid, that fluid has
the specific gravity of the blood. If the blood rises in one fluid
and sinks in another, some intermediate point is the specific gravity
of the blood; that is, if it rises slowly at 1,055 and sinks slowly
at 1,053 the specific gravity of the blood is 1,054.
Exton’s immiscible balance is more convenient
than the series of tubes This instrument consists of a standard
which supports a glass tube supplied with a tap at the bottom through
which a fluid may be added to the contents of the tube from the
bottom. The top of the tube is open. The tube is large enough to
permit the use of a gloat for taking the specific gravity. The tube
is filled with a mixture of varnolene (petroleum ether) and carbon
tetrachloride in such proportions that the specific gravity of the
mixture is that to be expected in the blood to be tested. The bulb
which is connected with the bottom of the tube is filled with lighter
fluid (varnolene) and a supply of the carbon tetrachloride is in
a dropping bottle. The lighter fluid thus can be added from the
bottom of the tube, the heavier at the top. Since the two fluids
are freely miscible this process causes speedy mixing of the two
fluids in the tube.
A drop of the blood to be tested is forced into
the tube from the end of a glass tube, as in the method previously
described. If the drop sinks, a drop or a few drops of varnolene
are allowed to flow into the tube at the bottom. The proportions
are thus varied until the drop of blood remains at about the middle
of the tube. The fluids are not miscible with blood and the blood
drop should remain spherical and distinct during the small amount
of time necessary for the manipulation of the fluids. The specific
gravity of the fluid in which the drop of blood rests is then determined
by means of a float. This float must be the one provided with the
instrument because this is standardized for the surface tension
of the fluids mentioned.
If considerable handling of the two fluids is
necessary n order to attain stability of the drop of the blood,
another mixture should be provided, of the specific gravity finally
determined upon, and a fresh drop of blood added to this. Frequently
this second drop of blood does not float freely in the mixture,
because there is some slight change in the blood when it is allowed
to remain for too long a time in the mixture of fluids. The second
determination is more quickly made, for obvious reasons. Sometimes,
of course the specific gravity first determined is found to be accurate.
There are several conditions in which the enumeration
of the blood platelets give useful information in diagnosis. These
structures are so fragile that it is difficult to make counts which
are as nearly accurate as are those made of the red and the white
blood cells, but even so the counts are useful.
The simplest method is that of Wright and Knnicutt.
Prepare two solutions as follows:
1. Brilliant cresyl blue 1 gram
Triple distilled water
This may be kept in the
refrigerator for some weeks.
2. Potassium cyanide .1 gram
Triple distilled water
This must be freshly
Immediately before using mix one part of solution
1 with three parts of solution 2, and filter.
Take the blood as in making ordinary red cell
counts, preferably making a 1 to 200 dilution, and place in the
counting chamber. Allow the diluted blood to stand in the chamber
for ten or fifteen minutes, in order that the platelets may settle.
Platelets are stained a lavender or lilac tine; red blood cells
are almost invisible and the leucocytes have dark blue nuclei. Count
the platelets in 200 small squares or more. The calculations are
the same as in the case of the red cell count.
Because the platelets are so small it is desirable
to use a higher power objective. In our work, however, an ordinary
one-sixth objective with a one-inch eyepiece has been found quite
satisfactory. By using a specially ground, very thin coverglass
it is possible to use a one-eighth eyepiece. There is a coverglass
sold which is of ordinary thickness but which has a thin area in
the center; this is satisfactory but it is very easily broken.
Several indirect methods are in use, and these
preserve the platelets rather better than is the case with the Wright
and Kinnicut method. Different solutions are employed by different
workers. The simplest is a 10% solution of sodium metaphosphate
in water. This preserves the platelets but does not stain them.
Water 200 c.c.
Sodium chloride 1 gram
Sodium sulphate 5 grams
Potassium iodide solution 35 grams
(Potassium iodide solution is made of a 5% potassium
iodide solution in water, to which have been added iodine crystals
Distilled water 266 c.c.
Mercuric chloride 2 grams
Sodium chloride 4 grams
Glycerin 26 grams
Kemp’s fluid is .9% sodium chloride in 2.5% formalin.
Distilled water 160 c.c.
Glycerin 30 grams
Sodium chloride 1 gram
Sodium sulphate 8 grams
Methyl violet 25 milligrams
Cleanse the skin as for any prick to secure blood.
Place one drop of the selected fluid upon the
surface of the skin. Prick the skin through this drop, so that the
blood mixes with the fluid as it emerges from the wound. When the
drop is definitely a pink color make smears on glass slides. Dry
in air, and stain by any of the usual methods for making a differential
count. Count the red cells and the platelets on this smear and determine
the ratio of platelets to red cells. By means of this ratio and
the actual count of the red cells determine the actual number of
platelets per cubic millimeter of blood.
There is little practical value in accurate estimations
of the viscidity of the blood. Variations in viscidity follow variations
in hemoglobin quite closely, and this is true for physiological
changes and for abnormal conditions of the blood. Occasionally there
is some need for the accurate determination of viscidity, and in
routine reports the relative viscidity should be noted.
The hemoglobinometer slides may be employed
for a rough estimation of the viscidity of the blood. When the slides
are separated, one slide is drawn across the other, and the amount
of stickiness noted. In normal blood this stickiness is enough to
delay, slightly, the separation of the slides, but not to cause
any appearance of threading. Blood which has increased viscidity
shows a tendency toward thread-formation when the slides are separated.
Blood which is subnormal in viscidity shows no recognizable stickiness.
If the viscidity is in any way abnormal, more exact methods should
Viscidity is indicated by the manner in which
the drop stands up as it emerges through the puncture. A normal
drop of blood is practically hemispherical; blood which is deficient
in viscidity spreads out over the skin; blood which is abnormally
viscid stands up much higher.
A more nearly exact and very satisfactory method
depends upon the size and rate of dropping. For this, a pipette
of certain diameter is employed; the blood taken into the pipette
and allowed to drop; the number of drops falling in five seconds
or ten seconds is the measure of viscidity; as 1/5 or 3/10. The
normal for the method must always be noted.
There is considerable variation in the viscidity
of the same blood at different times of the day and as a result
of dietetic changes.
A number of methods of determining the coagulation
time of the blood have been employed, but none has been altogether
satisfactory. The following methods have been employed in the laboratories
of the Research Institute.
The blood taken in the slides of Dare’s hemoglobinometer
coagulates in about five minutes, normally. The slides are allowed
to remain quietly for five minutes, and are then separated. If the
coagulation time is normal, the blood is found completely coagulated
but the serum has not yet separated. If the blood is not coagulated
the coagulation time is increased, and more exact methods should
be employed. If the blood clot has begun to separate from the serum,
the coagulation time is diminished, and more exact methods may or
may not be employed, according to the other blood findings and the
general condition of the patient.
For more exact determination of the coagulation
time, capillary tubes are used. These are made by drawing ordinary
glass tubing out into fine capillary tubes, which should have a
caliber of not more than the size of a dark hair and should be uniform
in every respect. These tubes are broken into lengths of one inch.
Ten of these tubes are filled for about half their length with blood,
and allowed to lie for a length of time dependent upon the results
of the observations made with the hemoglobinometer slides. If these
have shown delayed coagulation, the capillary tubes are allowed
to lie for six or even ten minutes; then one tube is broken. If
the blood falls out in a cast of the tube, coagulation is complete.
If it is perfectly fluid, two minutes should elapse, when another
tube should be broken. If it is still perfectly fluid, two or perhaps
three minutes may elapse before another tube is broken. The coagulation
time is given in the number of minutes required for complete coagulation
as shown by the formation of a cast of the tube when the glass is
broken. This is the most accurate and satisfactory method now known.
Howell’s method requires more blood. Four cubic
centimeters of blood are taken from the vein at the elbow, as usual,
and immediately mixed with an oxalate solution (5 cubic centimeters
of 1% sodium oxalate in ten cubic centimeters of 0.9% NaCl). This
prevents coagulation. The mixture is centrifugalized until the corpuscles
have been thrown down. Three small tubes are provided, which contain
one, two and three drops of 0.5% calcium chloride. Into each of
these tubes five drops of plasma are placed; the time accurately
noted, and the time of complete coagulation noted. By this method,
Howell found normal coagulation time in nearly all conditions except
hemophilia. Normal blood coagulates in nine to twelve minutes; hemophilic
blood coagulate in one to five hours. Purpuric blood gave normal
coagulation time by this test. It is evident that this method rules
out all delayed coagulation due to disturbances of the calcium-content
of the blood.
By the method of Lee and White about 1 cubic
centimeter is drawn from the bend of the elbow, as usual, using
a glass hypodermic syringe which has just been rinsed with .9 NaCl
solution. The blood is quickly placed in a small glass tube, about
eight millimeters in diameter, which also has been rinsed in .9
NaCl solution. The tube is then turned back and forth once each
half-minute and the time noted at which the blood no longer flows.
This method has little to commend it, for the friction modifies
When the coagulation time is normal but the
clot is very soft, defective fibrinogen is indicated. This is usually
due to some abnormal condition of the liver; occasionally it is
due to deficient diet. These conditions are usually easily recognized
by clinical phenomena. Cases of hemophilia may be associated with
deficient platelet count, and this is easily determined by making
an actual count of the platelets. In other cases of hemophilia the
platelets fail to agglutinate properly, and this can usually be
determined by studying warm slide specimens made with rather thick
smears of blood. In other cases of hemophilia there is deficient
calcium in the blood, and this can be determined by the following
CALCIUM AND COAGULATION
Cleanse the skin of the elbow and cover with
cotton or gauze wet with normal salt solution. Rinse a sterile syringe
and needle with normal salt solution and leave all surfaces thoroughly
wet with the solution so that the blood does not come in contact
with air. Take two cubic centimeters of blood and place this in
a small test tube containing one-half cubic centimeter of one percent
of calcium chloride solution. Fill capillary tubes with this mixture
and determine the coagulation time.
Compare the coagulation time of untreated blood
with the coagulation time of the blood treated with the calcium
solution. If the blood of the hemophiliac coagulates more normally
after the addition of the calcium, the addition of calcium-containing
foods to his diet is indicated. In such cases the parathyroid glands
may be abnormal and this aspect of the case should be studied.
A study of the relations between the solids and
the water content of the blood is sometimes useful in research work;
it is not now known to have any value in diagnosis.
Have ready a perfectly clean and perfectly dry
weighing glass; weigh this accurately. Put into the glass about
five cubic centimeters of blood taken from a vein. Weigh accurately.
Tilt the weighing glass so that the blood is spread over its inner
surface, in order to increase the evaporation area. Replace the
cover at an angle so as to permit evaporation. Place the glass in
a thermostat at 60 degrees to 70 degrees C. for one or two days.
Weigh again. Compare the weight of the whole blood with the weight
of the dried blood. The dried solids make up about one-fifth the
total weight of the blood, and in men the solids are a little greater
than in women. That is, the water content of normal blood is about
four-fifths the total weight. Figures given by various investigators
vary from 19.58% to 20.53% for women and from 20.35% to 22.69% for
men. The figures given are increased by those conditions which abstract
water from the blood and decreased by hydremia. The test is not
of value in diagnosis but it is occasionally useful in research
The determination of the electrical resistance
of the blood has been suggested as a speedy and accurate method
of determining the blood cell count. It has been found that the
electrical resistance varies as the amount of hemoglobin-carrying
protoplasm plus the density of the blood plasma. It is a better
indication of the cell-volume than of the cell count, though not
very accurate at that. The technique is cumbersome and unsuited
to ordinary laboratory diagnosis work. It has not, so far, been
found useful in research work.
The rapidity with which the red cells settle
from blood whose coagulation has been prevented in some way varies
in many conditions. The cells settle more rapidly in tubes which
are placed at an angle than in tubes which are placed upright; no
doubt this peculiarity is due to the Brownian movement of the cells.
Variations in the globulin content of the blood,
the salt content, the relations between the electrolytes and the
non-electrolytes of the plasma and the temperature at which the
test is made all affect the sedimentation rate. Cells settle more
rapidly in the blood of patients with malignancy, pregnancy, fevers
and inflammatory states, after severe burns and, generally speaking,
whenever there is tissue destruction or increased katabolism.
The technique is simple. The method of determining
the blood volume by the hematocrit may be employed, except that
the centrifuge is not used. The hematocrit tubes are placed at an
angle of about 45 degrees in a rack and the sedimentation of the
red cells timed. Normally the cells settle within about an hour.
More nearly accurate findings are secured by
taking a larger amount of blood (5 cubic centimeters or more) from
a vein, citrating the blood or cooling it very quickly to 33 degrees
F., and placing it in very small tubes which are allowed to rest
at an angle of about 45 degrees until the cells have settled. D
If the cells settle within 20 minutes to 40 minutes, the sedimentation
rate is increased; if the cells settle within 50 minutes to 80 minutes,
the sedimentation rate is normal. We have not found any useful information
resulting form the test and it has been discarded in our laboratories.
TOTAL VOLUME OF THE BLOOD
Many attempts have been made to find some adequate
and practical method of determining the total amount of blood in
the body, but none has been altogether successful. It is evident
that only qualitative results can be secured from an examination
of a small portion removed for that purpose unless the total amount
of blood in the body is known.
The Haldane-Smith method consists in the inhalation
of a measured amount of carbon monoxid, the removal of a measured
amount of blood from a vein, and the determination of the amount
of carbon monoxid hemoglobin by means of a spectroscope. The fraction
of the carbon monoxid inhaled found as carbon monoxid hemoglobin
in the measured blood indicates the amount of total blood in the
body. This method is not suitable for ordinary work. Cells injured
by carbon monoxid begin to be removed from the body quickly, and
there is considerable unavoidable error in technique. Patients with
even slight chronic carbon monoxide poisoning would give very erroneous
Methods based upon the injection of non-poisonous
stains into a vein, and the removal of a measured amount of blood
from an opposite vein, have been employed. The relative amount of
the stain found in the measured blood indicates the total amount
of blood in the body. The accuracy is diminished by the fact that
such stains are partially removed from the blood by the liver, kidneys
and other tissues of the body, and that complete mixture of the
blood may not occur before this elimination has begun.
Rowntree’s reports give interesting findings
based on the use of Congo red and other harmless stains, injected
into the blood. For the details of technique the original reports
should be consulted.
Briefly, the method includes the following factors:
The probable plasma volume is determined for the patient by multiplying
his weight in kilograms by 50, because the average plasma volume
is about fifty cubic centimeters for each kilogram of body weight.
The figure thus secured is divided by 200, because a dilution of
one part of stain to 200 parts of plasma is practically adequate.
This quotient is the amount of the dye to be injected. The dye itself
is a 1.5% solution of Congo red in fresh, triple distilled water.
Ten cubic centimeters of blood are taken from
one elbow and the estimated amount of the dye injected into the
same vein through the needle used in withdrawing the blood. Three
to six minutes later 20 cubic centimeters of blood are taken from
the other elbow. Both specimens are oxalated and both are centrifugalized
and the hematocrit readings recorded. The plasma from the two specimens
is prepared for the colorimeter tubes and the readings give the
amount of the dye in the plasma taken after the injection. By comparing
the amount of dye injected with the amount recovered it is a simple
matter to determine the amount of blood plasma in the entire body.
By comparing the hematocrit readings with the total blood plasma
the total amount of blood in the body can be determined.
The findings secured by Browntree and his associates
have been uniform and have given valuable information. The method
is the simplest and the least harmful to the patient of the various
All of these methods have certain unavoidable
errors and all are rather dangerous in the hands of unskilled persons.
Many studies of the total volume have been made, for animals and
for human subjects, and various fractions have been given as the
relation between blood volume and body weight. These fractions vary
from 1/13 to 1/20 for both men and women. For laboratory animals
a fraction of 1/20, with a small increase during pregnancy, has
been found by nearly all those making careful tests. That is, about
5% of the total weight of the body consists of blood. In obese animals
the proportion is lower, as might be expected. In obese human subjects
the proportion of blood to body weight has also been found lower.
Pregnant women show some increase in the proportion of blood to
the body weight.
STUDY OF BLOOD PARASITES
The total plasma volume of the body remains remarkably constant
in health and in disease, and the variations in total blood volume
depend chiefly upon variations in the volume of the cells. In chlorosis,
however, there seems to be an actual increase in the plasma volume,
while in dehydration and also in edema, the plasma volume is diminished
in nearly all cases. In edema the water is held in the tissues,
not in the blood. It seems that so long as an adequate water intake
is present, the blood plasma tends to remain constant under ordinary
conditions. This being the case, ordinary methods of blood examination
give quite accurate information, and the lack of a practical method
of determining the total amount of blood in the body is probably
not a cause of diagnostic errors.
The presence of parasites in the blood may be
suspected when symptoms are reported such as follow such infections,
or when the blood picture is atypical and is associated with eosinophilia,
especially if the patient has been living in countries in which
such infections are endemic. In making a differential count, fragments
of parasites may be seen, or the large hyaline phagocytes may contain
inclusions which arouse suspicion of parasitic infections. Inclusions
found in the neutrophiles are not commonly noted but may be found
occasionally. The red blood cells may show the effects of erosion
due to blastomycetes, malaria or other infections. Fragments of
the parasites or large hyaline bodies suggesting spores may occasionally
be noted in the plasma while making a differential count, and these
suggest more careful study of the blood with reference to the possibility
of parasitic infection. Any findings which cannot be explained should
always lead to more careful study of the blood in order that the
puzzling factors may be interpreted, if possible.
SEARCH FOR UNKNOWN ORGANISMS
The first method to be employed in the search
for parasites is the study of a rather thick smear of fresh blood
on the warm slide.
Take a warm slide on the warm stage of the microscope,
cover with a warm cover glass, examine with the two-thirds objective
and one inch eyepiece. Look over the entire drop of blood. Note
whether the red cells show any unexplained movement; note whether
any living worm-like organisms are present. Look over all thin areas
with a one-sixth objective, then with one-eighth or one-tenth objective.
If no organisms can be found discard the method.
Use next a method of concentrating the blood.
Rub the lobe of the ear until it becomes distinctly reddened, using
some alcohol or rather hot water or some other aseptic fluid. Dilating
the blood vessels is, in this case, desirable. Have ready a centrifuge
tube containing five cubic centimeters of five per cent acetic acid
Prick the lobe of the ear rather deeply, and allow several drops
of blood from the ear to flow into the centrifuge tube containing
the acetic acid solution, mix thoroughly and centrifuge at about
five hundred revolutions per minute for six to ten minutes. If the
blood flows freely, two centrifuge tubes may be prepared in the
same way. If the blood does not flow freely, the second tube, for
balancing the first, should be filled with water to the same weight
as the blood mixture. On removing the tube from the centrifuge the
lower end will be filled with debris of the red cells amidst which
the white cells and any parasites which might be present will be
found. Thick smears from the upper part of this debris, and other
thick smears from the lower part of the tube should be prepared
and examined without staining. The parasites may be found in this
manner, or thinner smears should be made, dried and stained, using
Giemsa’s stain, thionin, some of the eosin-methylene-blue preparations
and hematoxylin for one slide each, if the nature of the parasite
is not indicated by the history or symptoms or by the findings on
If this method shows no parasites, venous blood
must be used.
Take ten cubic centimeters of blood from a vein
in the elbow, using the technique employed for determining blood
chemistry, and put four cubic centimeters of the blood into a centrifuge
tube containing six cubic centimeters of five per cent solution
of sodium citrate, and six cubic centimeters of blood into a tube
containing four cubic centimeters of the citrate solution. Each
tube then contains ten cubic centimeters of mixtures of blood and
citrate solution in varying proportions. Centrifuge ten minutes
at about a five hundred revolutions per minute.
The red cells then occupy the lower end of the
tube, the clear fluid the upper portion, while there is a thin layer
of white cells at the top of the red cell column. Take one thick
drop from the white cell layer form each tube, for immediate examination.
Make eight or ten smears from the same layer of each tube, lay these
aside to dry for staining. Examine both the thick smears, unstained,
using first the lower powers and then the high powers. Note evidences
of living organisms in both. Stain the thinner smears, using several
different stains, as before. Note parasites and also note whether
there are any cellular inclusions in the large hyaline phagocytes
or in the neutrophiles.
If these methods show no evidences of parasites
in the blood, repeat with blood taken at different times of the
day and night until the parasites have been found or until several
examinations several times repeated have given negative results
Take blood at about midnight, after the patient has been three or
more hours asleep at least two different times. Take blood immediately
after a heavy osteopathic treatment, especially planned to cause
increased rapidity of the circulation of the blood through the liver
and the spleen, at least two different times. Take blood in the
early morning, at about two o’clock in the afternoon and about two
hours after a heavy protein meal, at least two times each, unless
the parasites are found, or some explanation of the puzzling findings
secured, before the group of tests has been completed. Whenever
blood is taken for any of these tests, make two to six smears for
differential count, using the usual technique. Save two or three
of each of these smears, after they have been dried, for later study
after the nature of the disease has been determined.
If there are any sores upon the skin, wash away
the superficial debris and take smears from the granulation tissues
or from the deeper floor of the sore or the ulcer, and examine.
If there are tumors beneath the skin, remove one of these for tissue
examination. Make smears from scrapings of the cut surface of the
tumor. These scrapings often show an infectious agent which is not
shown by slides of the tissues. If there are enlarged lymph nodes,
remove one for tissue examination, and make smears from scrapings
from the cut surface of the node. Stain the scrapings with thionin,
Giemsa’s stain, hematoxylin and one or several of the eosin-methylene-blue
stains, using the technique employed for differential counting.
Make at least one thick smear and examine on a warm slide, for the
recognition of living or unstained parasites.
If there are enlarged lymph nodes and the surgical
removal of one for the sake of diagnosis does not seem desirable,
some of the juices of the gland may be taken for examination. Take
the syringe used for taking blood for chemical examination, have
the needle sterile but dry, plunge the needle into the substance
of the gland and withdraw the plunger enough to secure a drop or
a few drops of fluid. From this material make one thick smear and
several thin smears, on microscope slides. Examine the thick smear
at once, unstained, and preferably on the warm stage. Dry and stain
the thin smear using several different methods.
Inoculation of guinea pigs, white rats and rabbits
from the blood of the patient and from the glandular extracts often
determines or verifies the diagnosis. Use the technique employed
for bacterial inoculations.
SEARCH FOR MALARIAL ORGANISMS
Examination of the unstained smears for malarial
organisms is no longer a method in general use, which is, in some
ways, unfortunate. The manner in which the organism moves within
the cells, and the relations between cellular movements and the
presence of the parasite are interesting, and in some cases are
useful in the study of the efficiency of the parasiticidal activities
of the phagocytes. The pigment granules of adult malarial organisms
makes their recognition fairly easy. This pigment may not be present
in the younger forms.
Stained smears give the most rapid and accurate
diagnosis of malaria. Organisms may be noted in making an ordinary
differential count and their number per cubic millimeter determined.
The method is described in the paragraphs which give the method
of making the differential count.
When malaria is suspected a thick smear may
be used, in order to concentrate the organisms. Take three or four
drops of blood in the center of an ordinary slide, spread the blood
out over an area of about one square inch, making the smear as even
as is practicable. Allow to dry at room temperature; the slide must
be thoroughly dry before staining.
Solutions necessary for this special method
1. Fixing fluid
Glacial acetic acid 1
Formalin (40%) 5 centimeters
Distilled water 100 cubic
2. Manson’s fluid
Borax 5 grams
Methylene blue 2 grams
Tap water 100 cubic centimeters
Stand the dried thick slide in a jar containing
at least fifty cubic centimeters of the fixing fluid for ten minutes.
If the hemoglobin has not all disappeared by the end of ten minutes
stand in a jar of tap water until all of the red or pink color has
disappeared. Flood with Manson’s fluid for half a minute, rinse
gently in tap water. Mount in water and examine with dry one-tenth
objective, or dry and examine with oil immersion lens. It is possible
to make a fairly accurate determination of the number of parasites
per cubic millimeter by counting the number of parasites in several
fields, then counting the number of neutrophiles or the number of
small hyaline cells in the same fields. The number of neutrophiles
or of small hyaline cells per cubic millimeter having already been
determined by the ordinary blood counts, the number of malarial
parasites per cubic millimeter can be estimated quite easily.
In old cases of malaria the parasites can usually
be found at almost any time. In cases in which the chills and fever
follow a regular rhythm, as in tertian and quartan fevers, the smears
should be taken, if possible, just before the onset of the chill.
In autumnal fever the smears are most apt to show the parasite if
they are taken eight or ten hours after the beginning of the fever.
The patient should receive an energetic osteopathic
treatment planned to secure increased speed of circulation through
the spleen just before the smears are taken, if this can be done.
Such a treatment tends to diminish the severity of the attack, when
it is given just before the onset of the chill. In cases with irregular
attacks of fever such treatments increase the number of parasites
in the peripheral blood, and they also hasten recovery from the
The different parasites which cause malaria
may be distinguished as follows:
Plasmodium vivax (P. tertiana; Hemameba Laverani
tertiana; P. malariae tertianum) shows rather active ameboid movements.
The red cells infected are enlarged somewhat, are pale, and may
show basophilic degeneration, but the non-infected cells do not
show basophilic degeneration. The pigment is finely granular, yellowish
brown, abundant, and it remains fine throughout the life of the
parasites; it may be somewhat more abundant at the periphery but
some fine granules are scattered over the entire organism. The young
schizonts are ring-shaped at first, later they assume rather irregular
forms. They grow until they fill the entire red cell, and they may
become larger than the red cells of the infected blood. These segment
into fourteen to twenty or even twenty-four merozoites, and these
form a rosette which is often imperfect and of irregular arrangement.
The extracellular forms are not easily distinguished from those
of P. malariae but they do not resemble those of P. falciparum.
The macrogametocytes are large and round with abundant, finely granular
pigment which is scattered over the entire parasite. The protoplasm
stains deep blue with ordinary methylene blue or thionin stains;
the nucleus is poor in chromatin. The microgametocytes have less
abundant pigment, the cytoplasm stains rather a greenish light blue
and the nucleus is rich in chromatin.
Plasmodium malariae (Oscillaria malariae; P.
quartana; Hemamebae malariae; H. Laverani quartana; P. malariae
quartana) shows only sluggish ameboid movements and may not move
at all on the warm slide. The red cells infected do not swell nor
do they become paler than normal. They may assume a brownish or
brassy tint, apparently due to the change of hemoglobin into methemoglobin
or some related compound. Basophilic degeneration may appear in
no-infected as well as in infected red cells. Pigment granules are
coarse, not abundant, and are mostly arranged near the periphery
of the cell. Young schizonts are ring shaped, later becoming oval
or round. They have a chromatin granule which soon becomes band-shaped,
and these form equatorial bands. The schizonts do not fill the red
cell completely and they divide into eight to fourteen merozoites,
arranged in a rosette which is nearly always perfect. Extracellular
forms are somewhat smaller than is the case with P. vivax, but they
have almost or quite the same structure.
P. falciparum (Laverania malariae; tropical
parasite; plasmodium precox; Hemamebae malariae precox; P. immaculatum)
shows little ameboid motion but has a slight activity somewhat resembling
pulsation. The infected red cells do not swell but may shrink slightly,
and they show the peculiar brownish or brassy color noted in red
cells infected with P. vivax. The pigment granules are coarse and
dark in color. Young schizonts are small and ring-like and they
have one or two chromatin granules at the periphery. Occasionally
two or even three parasites may be found within one red cell. Larger
schizonts are not often seen in peripheral blood, and they rarely
occupy more than half or two-thirds the area of the red cell. In
larger forms the pigment granules are coarse, scanty and tend to
clump together. The grown schizonts divide into seven to twenty-four
merozoites, and these form rosettes which are not always perfect.
The extracellular macrogametes have a distinctive crescent-like
form, becoming oval and round or roundish as the parasite increases
in size. The cytoplasm stains deeply and the nucleus has scanty
chromatin. The pigment granules are coarse and clumped near the
center of the cell. Extracellular microgametocytes are also crescentic,
and become oval or round with growth. The cytoplasm stains feebly
and chromatin is fairly abundant in the nucleus. The pigment granules
are coarse and are scattered over all the cell.
SEARCH FOR FILARIA
If filaria are suspected, the blood should be
taken for examination at several different times of the day. Late
in the afternoon, after the patient has rested, or better, has slept
for several hours the blood may show the parasites of any variety.
If the ordinary nocturnal form is present it may be necessary to
take the blood at a time as near midnight as is convenient, after
the patient has slept for several hours. The diurnal forms are most
abundant at about noon. Other forms may be found at almost any time
of day. Fresh specimens of blood are best examined. If the cover
glass is ringed with vaseline the parasites may remain alive and
active for several days.
If the parasites are not found in ordinary thick
smears, the blood should be centrifuged in order to concentrate
the worms. The skin may be reddened by rubbing with the aseptic
solution, then pricked, and several drops, or even several cubic
centimeters, collected. This may be mixed with either 2% watery
solution of sodium citrate, or with 3% acetic acid, and the mixture
centrifugalized at about 500 revolutions per mite for five or ten
minutes. The worms accumulate in the leucocytic layer after centrifugalization
and thick smears made from this layer show them usually quite active.
The two-thirds objective is usually best for examining these preparations,
though the dry higher power lenses are best for studying the organisms
in thin smears.
The blood can be taken in small tubes and allowed
to clot. The serum which separates contains nearly all of the worms.
If a considerable amount of serum is collected this may be centrifuged
and the sediment examined.
For permanent mounts the smears should be thinner
and should be dried thoroughly in the air. They are then stained
with hematoxylin, Giemsa’s stain, or any of the ordinary blood stains.
The hematoxylin-stained slides keep the best.
In temperate zones this infection is rarely
seen. For the differentiation between the different forms a book
on tropical diseases should be consulted.
SEARCH FOR LEISHMANIA
These parasites are found within phagocytic cells
in the blood, especially within large hyaline phagocytes. In making
ordinary differential counts they may sometimes be noticed as inclusions
within the large hyaline endothelial cells. In these cells occasionally
great numbers are found. It is only very rarely that they are seen
free in the plasma or within neutrophiles. In slides stained with
Giemsa’s stain or with any of the eosin-methylene-blue preparations
the cytoplasm of the parasites takes a pinkish or bluish lavender
tint. The nucleus and the parabasal body stain dark blue. The parabasal
body, with its long axis lying at right angles to the long axis
of the cell is quite distinctive and makes the diagnosis clear when
the parasites are well stained.
If the organism is not found by ordinary methods
of examination the blood may be concentrated. Several cubic centimeters
of venous blood should be well mixed with 1% or 2% solution of sodium
citrate and the mixture centrifuged at about five hundred revolutions
per minute for five or ten minutes. The leucocytic layer should
then be collected and made into rather thin smears, and these stained
after any of the usual methods for staining blood smears. The organisms
are found within large hyaline cells.
Puncture of the spleen or of an enlarged lymphatic
gland for the purpose of securing a few drops of the fluid for examination
may be necessary for diagnosis. The removal of a small fragment
of rib may be necessary in order that the red marrow may be examined.
In either case the organism may be found within large hyaline cells
or free in the tissue juices.
Material may be secured from a local lesion
of the skin quite easily. If the skin has not yet become eroded
the lesion should be punctured and the fluid made into smears. If
the skin has become eroded the superficial debris and bacteria should
be washed away, the floor of the ulcer scraped, and deeper scrapings
made into thick smears for immediate examination, and thinner smears
for staining after several different methods.
The organisms which produce the different types
of Leishmania cannot be differentiated from direct study of the
smears, but cultures and animal inoculations may give differentiating
features. For the differential diagnosis a book on Tropical Diseases
should be consulted.
STUDIES OF THE FRAGILITY OF RED CELLS
In certain forms of anemia the red blood cells
are more resistant to lipolytic solutions and to hypotonic solutions.
In other forms of anemia and in certain other diseases the red blood
cells are less resistant to such solutions than are normal red cells.
For a study of the relations mentioned it is always necessary to
determine the concentration of solution which just barely destroys
normal red cells, then make a series of solutions of greater concentration
and of less concentration than this. The blood to be tested is t
hen placed in each of the varying solutions and the manner in which
the red cells behave is noted. The technique for all these solutions
is about the same, and may be illustrated by the method of determining
diminished resistance to hypotonic solutions.
Normal blood plasma has an osmotic tension about
equivalent to a 0.9% solution of sodium chloride in distilled water,
and normal red cells begin to lake at about 0.4% solution of sodium
chloride. In a 0.3% solution normal red cells are completely laked.
The technique of determining the resistance
of red cells to hypotonic salt solutions requires two racks, each
containing twelve graduated centrifuge tubes. One rack is for normal
blood, as control, the other for the blood to be tested. Mark the
tubes in each rack as follows:
.5%, .48%, .46%, .44%, .42%, .40%, .38%, .36%,
.34%, .32%, .30%, and .28%.
Fill the first tube to the line which indicates
five cubic centimeters with 0.5% sodium chloride solution.
Add distilled water to all the tubes except
the first, to fill them to the line indicating five cubic centimeters.
In this way the tubes all contain five cubic centimeters of solutions
of sodium chloride of the strength marked upon them.
Both racks are exactly alike. Mark one with
the name of the control and the other with the name of the patient.
Cleanse the lobe of the ear, if the patient is adult, and make a
rather deep prick, so that about two cubic centimeters of blood
can be taken into a graduated pipette. Drop one tenth cubic centimeter
into each of the twelve tubes of sale solution. Note the time. Repeat
for the control. Note this time. Allow the racks to stand for half
an hour, then observe. The red blood cells should have settled to
the bottoms of the tubes within a few minutes after the blood was
placed in them. After half an hour there may be a faint pink color
in one tube and all the tubes with weaker solutions show darker
tints. In one tube the red cells may have all disappeared, and in
all tubes with weaker solutions the cells also have disappeared.
If these conditions are not present at the end of half an hour leave
the racks for a longer time until hemolysis has begun in the tubes
on the rack of the control. This is usually within an hour, but
occasionally two hours may be necessary.
Note the strongest solution in which hemolysis
is just visible, in the control and in the patient’s blood. Note
the strongest solution in which hemolysis is complete (and the red
cells destroyed completely) in the control and in the patient’s
The report should give these four findings,
and also the time at which the blood was placed in the tubes and
the time at which the last observation was made. Since the fragility
occasionally varies during the day even in normal blood, the exact
time should always be given.
TESTS FOR BILE PIGMENTS IN SERUM
Tests for bile are not, properly, included with
these discussions of blood cells, except that it is often essential
that the source of toxemias, which affect the blood cells, may be
The tests devised by Van den Berg, depend upon
the fact that fluids containing bile give the diazo reaction, provided
the bile is not associated with the proteins of the plasma. The
first is called the “direct reaction” and it is so called because
of the bilirubin is free in the serum.
The patient must not eat green or colored vegetables
for two days beforehand.
Take five cubic centimeters of blood from a
vein in the elbow, after proper sterilization of the skin. Put the
blood into a suitable vessel until it clots and the serum becomes
separated. The serum must not be recognizably stained with hemoglobin;
it may be greenish, yellowish or brownish from the bile present.
Measure one cubic centimeter of the clear serum into a small tube,
add one cubic centimeter of Ehrlich’s diazo reagent. A brilliant
reddish or purplish color may appear immediately, in which case
there is a positive direct reaction. This indicates obstructive
jaundice. Sometimes the color appears only after the mixture has
been standing several minutes, in which case there is a delayed
positive direct reaction, and this has the same significance.
The indirect reaction is so called because the
bile pigment is combined with the proteins of the plasma in such
a way that the reaction does not occur until these have been precipitated.
Take one cubic centimeter of clear serum, add
two cubic centimeters of alcohol of about 96%. Place the mixture
in a centrifuge tube, balance with an equal weight of water, and
centrifuge at about eight hundred revolutions per minute for ten
minutes. Remove one cubic centimeter of the clear supernatant fluid
and to this add one-half cubic centimeter of alcohol and one-fourth
cubic centimeter of Ehrlich’s diazo reagent. The immediate appearance
of reddish or purplish color indicates the positive indirect reaction,
while the appearance of the same color after several minuets standing
indicates the delayed positive indirect reaction. When this reaction
is positive and the direct reaction negative, the jaundice is not
obstructive but is due to liver injury or to blood cell destruction.
Quantitative findings can be secured. One unit
of bilirubin is taken to be 0.5 milligrams in one hundred cubic
centimeters of blood. One-half to one-tenth of a bilirubin unit
is present in normal blood. For the quantitative estimation cobalt
sulphate is used for the standard. Prepare a solution of 3.92 grams
of crystalline cobalt sulphate in 100 cubic centimeters of distilled
water and place in one cup of the colorimeter. In the other cup
place the serum which shows the positive reaction. If the color
of the serum is the same as the standard solution, the blood of
the patient contains five units of bilirubin. If the color is lighter
or darker than that of the standard, the cups are raised or lowered
until the tints match, when the concentration can be read off on
the colorimeter scale.
The Van den Bergh reactions are useful for distinguishing
between obstructive jaundice and hemotogenous jaundice or the jaundice
due to liver injury, but the reaction is not delicate and it is
not useful for accurate determination of small amounts of bile.
Ehrlich’s diazo reagent is made as follows:
Sulphanilic acid 5 grams
Hydrochloric acid, (conc.) 50 grams
Distilled water 1,000 cubic centimeters
Sodium nitrate 1 gram
Distilled water 200 cubic centimeters
These solutions keep well in darkness.
When the test is to be made take fifty parts
of solution A and one part of solution B, mix and use immediately.
The icteric index is better for estimating small
amounts of bile. A solution of ten milligrams of potassium bichromate
in one liter distilled water is taken to have an index of 1, and
this solution is used for a standard. The solution keeps well in
Take about ten cubic centimeters of blood from
a vein of the elbow, place in a convenient vessel until the serum
has separated from the clot. Take two or three cubic centimeters
of serum, which must not be stained with hemoglobin, and mix with
an equal amount of 0.9% sodium chloride solution. Place the diluted
serum in one cup of the colorimeter, the standard solution of potassium
bichromate in the other cup, and compare. The standard solution
corresponds to an icteric index of 1., and normal blood has an icteric
index of about 5. Icteric index of about 15, which corresponds to
about two units in Van den Bergh’s reactions, is present in cases
of mild jaundice. An icteric index between about 6 and 15 is present
in mild cases of cholemia, in which case the blood cells show more
or less marked evidences of injury.
The Gmelin test is often positive for a small
amount of serum. Allow the serum which is formed when the blood
clot is ready to be placed in the incubator for the fibrinolysis
test to soak into filter paper. Allow one or two drops of nitric
acid which contains some nitrous acid to fall upon the filter paper
and to touch the blood serum. Normal serum shows a brownish tint
at the line of the acid, while cholemic blood gives a play of colors,
including purplish or violet shades.
A more delicate test is made from oxalated blood.
Take about five cubic centimeters of blood from a vein into a test
tube containing about ten milligrams powdered potassium oxalate.
Mix and centrifuge for about ten minutes. Remove the clear serum
into another tube, and underlay this with nitric acid which contains
a small amount of nitrous acid. A white coagulum will appear at
the junction of the two fluids. In this white band there will appear
a blue-green color at once in severe cholemia, or within half an
hour in less serious cases.
REPORTS OF BLOOD EXAMINATIONS
Reports of work done in the best clinical laboratories
are always given to the doctor who orders the work to be done. It
should be kept clearly in mind that laboratory work is done for
the doctor, and that only the doctor is responsible to the patient.
A carbon copy of the report is sent also, and the doctor usually
gives this to the patient, unless there is some reason why the patient
should not receive it. Reports are written in technical language
and the doctor should explain to the patient or to some member of
his family the significance of the various items. The laboratory
worker should not give such explanations, unless requested by the
doctor in charge of the case to do so. K The doctor who has made
the physical examinations and who has studied the symptoms and the
history is the only one who is able to interpret the findings in
a simple and practical manner, in the light of all the factors involved.
Patients receiving copies of examinations should always be advised
to keep them, and to show them to any other doctor who may be called
to give treatments for any disease at a later time. This is especially
important in cases of rare and chronic diseases. Much valuable time
may be saved to the patient if he has such records ready at a time
of later illness, in such cases.
With the reports of blood examinations there
is sent, upon another sheet of paper, such notes and explanations
as may be useful to the doctor in explaining the significance of
the findings and in determining the best methods of treatment. While
the laboratory worker is rarely in active practice, still there
is much useful information which he can give in selected cases,
provided he is doing his work upon a professional basis. The notes
are not intended for the patient and should never be given to him
or to his family.
The reports now being used in the clinical laboratory
of The A. T. Still Research Institute are the most useful we have
seen. The routine examination forms are printed upon pink paper
and the special-test forms are printed upon light brown paper. This
makes it easy to select from the files the reports which may be
needed for special studies. The forms used for other work are of
different colors; uranalyses forms are printed upon yellow paper,
blood chemistry forms on green paper, and so on.
The following forms are those used for blood reports in 1930:
ILLUSTRATIVE CASE REPORTS
Miss R., aged twenty-eight years, presented an
unusual history. Three years before coming to an osteopathic clinic
for examination she had shown symptoms of pulmonary tuberculosis,
and this had been treated by dietetic measures alone.
Blood examination showed the usual findings
in alkalosis, and after more explicit questioning a detailed account
of her diet for the tubercular infection was secured. She had been
given foods of the alkaline-ash type exclusively. Three times each
day she was given lemon juice and soda. Twice each day she was given
a soda enema. The urine was analyzed twice each month, and if it
was neutral or alkaline no change was made in the diet. If the urine
was acid at any time the alkalinization of the food intake was increased.
Within a few weeks after the intensive alkalinization
she began to complain of muscular cramps, most marked in the fingers.
During the next three years the spasmodic contractions increased
in frequency and in severity, and various types of paresthesia developed.
During this time she consulted several other doctors of medicine.
Without making any very careful study the condition was named either
hysteria or acidosis by these men. Alkalinizaton was advised by
every doctor consulted during this time. Colonic irrigations of
soda and water were advised. The condition became gradually more
When she came to the osteopathic clinic the
contractions were titanic in type. The muscles of the left hand
were first involved and the “obstetrical hand” position assumed.
The spasms then extended to the arm and shoulder, then to the neck,
trunk and legs, until the entire body was involved in titanic convulsions.
Attacks occurred two or three times a week.
By a specially devised method the total blood
alkalinity was found equivalent to 450 milligrams of sodium hydroxide
per 100 cubic centimeters of blood. The diffusible alkali was much
more noticeably increased than was the bound alkali.
The osteopathic examination showed the rigidity
of the lower thoracic region which is always present in tuberculosis,
and also a definite lesion of the fifth cervical vertebra. In order
that the effects of de-alkalinization might be studied, no osteopathic
treatments were given during the first month.
The patient acknowledge an intense craving for
hot white biscuits and beef-steak. These were given her for her
first meal, in moderate helpings. She was permitted other foods
with acid ash until the urine became normally acid, then a wholesome,
well-balanced diet was advised. The soda intake was stopped immediately.
The spasms were less severe the day after the
soda was stopped. By the end of the month they appeared rarely.
Some paresthesias of the hands were still noticed, and the relation
of the fifth cervical lesion to the innervation of the arms and
hands was explained to her. This explanation induced her to carry
on the treatment which had been advised.
During the fifth week after the diagnosis had
been made the cervical lesion was corrected and the spinal rigidity
relieved. The lesions did not occur.
Six weeks after the lesions were corrected a
second blood examination showed no abnormal findings. During the
subsequent nine years she has been healthy and comfortable, except
that she had colds several times, and was in an automobile accident
once which broke an arm and caused cerebral concussion of mild degree.
G., a boy of nine years, suffered fro attacks which somewhat resembled
those of Jacksonian epilepsy. He had suffered from an attack of
food poisoning at the age of seven years, and before that time had
been in excellent health all his life.
The attack of food poisoning was treated by
the administration of large doses of soda by mouth and he was given
soda enemas at frequent, though irregular intervals during the six
weeks following the attack. After this time he was troubled with
various gastric and intestinal disturbances, and for these increasing
amounts of soda were given him.
On making the routine blood examination the
findings characteristic of alkalosis were noted. The urine was found
to be alkaline at three examinations on successive days.
The soda was stopped immediately. The spasmodic
attacks disappeared within three days and never re-appeared. The
gastric and intestinal symptoms persisted.
Osteopathic treatments were not given until after the urine became
acid, because it was desired that the effects of de-alkalinization
alone should be studied. The spasms had ceased by the time the urine
became acid, and the osteopathic examination showed a definite lesion
of the sixth thoracic vertebra. This was corrected at the third
treatment. The gastric symptoms diminished gradually during the
next week, and the intestinal symptoms diminished during the two
weeks following the correction of the lesion. No further symptoms
ever appeared, and during the next ten years his health was excellent.
Mr. Q., thirty-nine years old, had suffered
from diabetes at the age of thirty-two years. He had been given
osteopathic treatment at that time, and had been given a diet list
of foods exclusively of the alkaline-ash type. He avoided all white
breads, all sugars, all plums, cranberries, prunes, meats, and,
indeed, every article of food said to have any tendency to cause
acid reactions. He did not use soda, but he did take alkaline laxative
and purgative drugs, on his own initiative, and he tested the urine
occasionally with litmus paper. When the urine was acid in reaction
he worried terribly and hastened to take some alkaline medicines.
The only common alkaline substance which he avoided was soda.
For two weeks before he came to the clinic for
treatment he suffered from cramps in the muscles of his legs. These
awoke him from sleep, and the pain was really quite severe. On making
a blood examination the characteristic staining reactions and nuclear
structure of alkalosis were recognized, and the blood was studied
with reference to its alkalinity. At that time the technique of
studying the reaction of blood was not so nearly accurate as is
the case at this time, and it was only possible to determine that
the alkalinity was considerably increased in the blood serum. In
the urine the alkalinity varied, sometimes being only just recognizable
sometimes definitely alkaline on voiding.
Certain lesions were found and these were corrected
before the blood study was completed. The lesions were not the cause
of the cramping and the corrections did not affect the leg muscles.
The lesions did cause some of the apathy and melancholy from which
he suffered, and these symptoms were considerably relieved by the
osteopathic treatments. The mental acuity was increased, and no
doubt this permitted a better understanding of the conditions than
might have been the case if he had not received those treatments
at that time. Having the most extreme faith in the doctor who had
prescribed the alkaline-ash diet, it might have been difficult to
persuade him of the error of persisting in such a diet indefinitely.
When the relation of his diet to his symptoms
was explained to him, he was willing to accept more nearly normal
foods. He confessed to a craving for candy and white bread, and
these foods were given him in moderation. As soon as the urine became
normally acid in reaction, a good wholesome diet was outlined and
this he employed for several weeks. The cramps diminished gradually
for ten days, then disappeared altogether. With this relief of his
symptoms he disappeared from observation for seven years. At the
end of that time he brought his little son for examination. He reported
excellent health during the interim. He had a good position, had
married a sensible wife and had not paid much attention to his diet
during the past five years, because his table was well supplied
with good, wholesome food from which he selected what he wanted
Miss T., aged twenty-five years, complained of
weakness, insomnia and occasional attacks of deep breathing associated
with air-hunger. Acidosis was suspected from these symptoms. The
routine blood examination showed the structures usually present
in acidosis. Miss T. had suffered from an attack of inflammatory
rheumatism, and the medical practitioner who attended her warned
her against the use of any acid fruits. He specified especially
that tomatoes, lemons, grapefruit and oranges were dangerous, and
he advised a diet chiefly of toasted white bread, good red meat
and whatever she liked, except sour things. She was very fond of
candy and pastries. With increasing weakness she avoided exertion,
but she did not gain in weight.
The alkalinity was diminished in the blood to
the equivalent of two hundred fifty milligrams of sodium hydroxide
per liter, and of this only fifteen per cent was of the diffusible
type. (By the methods used twenty per cent diffusible of a total
of three hundred milligrams is normal.)
The spinal column showed the irregularities
usually present in acidosis. These were corrected during two weeks,
with no change in the diet. The symptoms diminished considerably,
and the alkalinity of the blood increased to the equivalent of nearly
three hundred milligrams of sodium hydroxide.
The increase in the alkalinity of the blood
after the osteopathic treatments, while the diet remained unchanged,
was no doubt due to the fact that the correction of the lesions
permitted normal circulation and innervation of the viscera, with
resulting increased oxidation, the formation of katabolites more
nearly neutral or, in some cases, definitely acid, and more nearly
normal excretion of these from the body. In this case some exhaustive
and interesting studies of the urine were made, but these are too
long to be included in this report.
After this study of the effects of treatment
alone, upon the reaction of the blood, the diet was changed materially.
A good, wholesome diet which included rational proportions of fruits,
vegetables and other foods was outlined. The alkalinity of the blood
returned to normal and the symptoms disappeared completely. During
the three years which intervened since that time, she has been normal
CHRONIC CARBON MONOXIDE POISONINS
Mrs. N. 3. Symptoms included only persistent,
dull headache and weariness for which no adequate cause could be
found. On physical examination some vague tension of the cervical
and upper thoracic spinal muscles was found, but no definite lesions
and no recognizable evidences of visceral pathology.
On blood examination the peculiar cherry-like
tint suggested carbon monoxide poisoning, and by the spectroscopic
examination a small, but recognizable, amount of carbon monoxide
hemoglobin was determined.
The patient had a closed car but drove only
short distances and at intervals of several days. She used no gas
for heating or cooking. Her home was in an old house on a quiet
street. No manufacturing district or oil well was near. There seemed,
at first, no possibility of the inhalation of fumes.
On studying the plans of the house in which
she lived it was found that the house had been piped for gas. On
further investigation one of these old gas pipes was found beneath
her bedroom, and it was leaking steadily, though only slightly.
Mrs. N. was having a new house built, and in this house her bedroom
was on the second floor. As soon as she moved into the new home
the headaches and weariness passed away, gradually, and within a
few weeks her health was fairly good. She then received a few treatments
for the abnormal tension of the tissues of the neck and shoulders,
and she became perfectly well again. Several weeks later another
blood examination showed no evidence of carbon monoxide hemoglobin.
In such a case is this it would be easy to infer
that the worries inseparable from building a new home were the cause
of the symptoms, and that the mental relief following the successful
completion and occupancy of the new place were the cause of the
recovery. The error of such an inference is obvious form the results
of the blood tests.
Miss K,11. This young woman, aged eighteen years,
complained of languor with persistent dull headaches and some pain
in the eyeballs. Physical examination showed no cause for the symptoms.
No bony lesions were found, and only some slight but persistent
tension of the muscles of the neck could be discovered. Relief of
this tension was followed by some slight relief of the discomfort,
but the tension re-appeared, together with the aches, within a few
hours. She was a solicitor for a wholesale cracker house, and she
spent most of her time in her little, old, closed car, driving from
one hotel, grocery or eating-house to another, taking orders. She
rarely had the car windows open because the breeze disturbed her
papers. She lived in a house composed chiefly of wire screening,
on a quiet street, and no gas pipes were in the suburb anywhere.
Blood examination showed some evidence of chronic
carbon monoxide poisoning and a trace of carbon monoxide hemoglobin
was found on spectroscopic examination The treatment is obvious.
She exchanged the old, closed car for a new roadster, and she bought
some convenient cases for her papers. Within a few days the symptoms
diminished, and within a few weeks she was apparently as well as
could be. A blood examination made three months later showed no
evidences of carbon monoxide hemoglobin.
Mr. W., aged forty-three years, worked in a
Los Angeles office in which smoking was habitual. His home was an
hour’s ride distant, and he smoked all the time, riding with friends
in the smoker.
He had very severe headaches which did not yield
to any treatment, and he complained of marked fatigue for which
he could not find any cause. The blood showed the usual characteristics
of carbon monoxide poisoning. He was persuaded to ride in the non-smoking
compartment of the street-car and to have more fresh air in his
office. He was also advised to diminish smoking to the lowest comfortable
extent and to smoke in the open air. These changes were followed
by considerable relief of the headache and the feeling of fatigue.
There was recognizable improvement in the blood cells and in the
color. But the discomfort of postponing smoking until he could be
in the fresh air, and the lack of the usual conversations with his
friends in the smoker proved too great. He returned to his old habits
and accepted the headaches and other discomforts due to the bad
habits. A few months later the bad habits, headaches and friendly
relations were all terminated at once by a sudden attack of pneumonia.
Miss J. 2. This young woman was a student in
a business college. She complained of increasing weakness with dull
headache and apathy, and occasional insomnia. No adequate cause
for these conditions could be found on physical examination. No
bony lesions and no abnormal tension of tissues could be discovered
even after very careful examinations.
Her school-work was done in well-ventilated
rooms. No smoking was permitted in the school; she herself did not
smoke, and no member of her family smoked. The house did not have
gas pipes, and never had been piped for gas. She had a large, airy
room and slept on a porch. No gas wells or manufacturing district
was near her home. She had no automobile and rarely rode in a closed
She walked to and from school twice each day,
and sometimes she made an extra trip at night. The distance was
rather more than a mile. This walk took her through a tunnel in
which traffic was very heavy. The cars were forced to stop and start
at intervals of a few minutes. The air was full of the fumes due
to imperfect combustion of gasoline and oil. The walk through the
tunnel required about twenty minutes, which meant that she spent
from an hour and a half to two hours every day breathing bad air.
A small amount of carbon monoxide hemoglobin was shown by spectroscopic
examination of the blood, after the usual findings had been noted
in the routine blood tests.
The treatment was indicated by these facts.
The walk through the tunnel was discontinued. The symptoms diminished
gradually and two months later the blood showed no abnormal conditions.
Mrs. D. 4. This woman of thirty years complained
of being unable to live at high altitudes. Her home was in a city
nearly nine thousand feet above sea level and her husband’s business
compelled their residence there for at least ten months each year.
During the two months spent at sea level and for the two weeks or
so following her return home she was well. At other times she was
weak, inert, with dull headaches and constant fatigue.
Blood examinations made just before her return
to her home, after two months at sea level, showed no abnormal findings.
Blood examinations made just after her return to sea level, after
ten months at her home, showed the usual evidence of chronic carbon
monoxide poisoning. No abnormal findings could be discovered on
In her mountain home she enjoyed tinkering with
her car. At that altitude the use of a gasoline engine presents
certain problems and she enjoyed solving them in the most satisfactory
manner possible. At that altitude, also, there was much cold weather,
so that her work was done within the garage. She had some ventilation
in the garage, but this could not be very satisfactory.
On changing her habits of living, substituting
other interests for the car, and returning to her home after the
usual two months at sea level, she found herself able to go through
the winter with no ill health at all. It was not the high altitude
which affected her health, but the hours spent with her automobile
in a poorly ventilated garage. This case illustrates the danger
of superficial diagnosis. Changed environment forced changed habits,
and these caused the recovery. People who are fairly normal can
live where other normal people can live, but nobody can be well
who persistently breathes air which contains much carbon monoxide.
Patients who suffer from lesions of the eighth
to the tenth thoracic vertebrae or the corresponding ribs often
show moderate degrees of cholemia. This condition disappears with
a week or ten days after the lesions have been corrected.
Miss Y., aged twenty years, complained of dull
headaches nausea, some itching of the skin. She was very slightly
The blood cells showed the effects of some hemolytic
agent and the serum contained a moderate amount of bilirubin. The
surface tension of the serum was diminished. The urine contained
bile pigments but no recognizable amount of bile acids. There were
no other abnormal conditions of the blood or the urine.
A lesion of the eighth thoracic vertebra was present and the seventh
and eighth right ribs were approximated. No other cause for the
cholemia could be found. The lesions were corrected by three osteopathic
treatments given at three day intervals. Ten days after the last
treatment the blood and the urine were normal. The symptoms disappeared
after the third treatment.
Mr. F., aged twenty-nine years, a worker in
an osteopathic laboratory, was in excellent health. He used his
own blood in some experimental work in the technique of testing
blood serum for bile pigments and bile acids, and found the condition
normal. After three days of this work he helped to lift some heavy
boxes from beneath a table, and thus strained his back Thus he produced
a lesion of the seventh to the ninth thoracic vertebrae, which caused
pronounced discomfort but no really serious symptoms. He continued
his tests for bile acids and bile pigments, and on the third day
after the strain he found that his own blood contained five times
the normal amount of bilirubin, according to the method he was using,
and that the reactions for bile acids were definitely positive.
No bile pigments or acids were present in the urine.
The lesions were then corrected by means of
a single, rather heavy, osteopathic treatment. Three hours later
the bile acids and the bile pigments were perceptibly increased
in the blood, and both bile acids and bile pigments were present
abundantly in the urine. The next day, sixteen hours after the lesions
had been corrected, no bile acids were present in the blood serum,
the bile pigments were within normal range, and the urine was normal.
The lesions did not recur and no further ill
effects were noticed.
Miss J., a teacher, aged thirty-nine years, complained
of increasing weakness and persistent, though slight, loss of weight.
Pulmonary tuberculosis was suspected.
On blood examination no evidence of tubercular or other infection
could be found. There was no excess of hyaline, eosinophilic or
endothelial cells. The neutrophilic cells showed the changes associated
with fatigue and moderate acidosis. Immature red cells and white
cells of all classes were numerous, though no anemia was present.
Myelocytoid forms were also abundant, especially among the neutrophiles.
A diagnosis of fatigue plus bony lesions affecting
a considerable area of red bone marrow was made. This diagnosis
was accepted by the osteopathic physician in charge of the case.
When she was told the findings she acknowledge
that her hours of work were too long. She was trying to care for
an invalid mother during the nights, and was writing short stories
to earn more money, while still teaching every day.
She had an unusually rigid thorax; both ribs
and vertebrae were involved. The treatment was evident when these
facts were known. In order to study the effects of rest alone, and
partly because her circumstances prevented her receiving osteopathic
treatments for six weeks, she was persuaded to cease the extra writing,
to have help with the invalid mother by night as well as by day,
and to secure as much rest as possible in every other way. He diet
was already wholesome and well balanced.
With rest alone the symptoms diminished considerably
The blood cells lost the evidences of toxemia but the immature forms
Six weeks later she was able to have good osteopathic
treatments. With increasing flexibility of the thorax, better breathing
habits, plus the continued rest already begun as soon as the diagnosis
was definite, she began to gain in weight and to regain her normal
physical condition. A blood examination made six months later showed
no abnormal cells.
Mrs. L. 7, aged sixty-four years, suffered from
what seemed to be amild attack of influenza. Fever was slight but
she seemed weaker than she should be if this were the correct diagnosis.
A blood examination was made for this reason. There was a moderate
neutrophilic leucocytosis with low nuclear average and many endothelial
cells were present. The fibrin was formed abundantly and immediately
upon the warm slide, and the threads were long, coarse, regular
in outline, arranged in a dense felt-like mass. A diagnosis of early
pneumonia was made upon these findings, though clinical symptoms
were negative. Treatment for pneumonia was initiated promptly and
she made a good recovery. During her convalescence the sputum was
scanty and contained rusty streaks; there were abundant pneumococci
in the sputum. There was only a small recognizable area of consolidation,
and the speedy recovery was no doubt due to the early diagnosis.
Mr. N. 5, aged seventy-three years, suffered
from an attack of influenza and pneumonia was feared. No symptoms
of cardiac involvement had been noted. Blood examination showed
a moderate leucocytosis with low nuclear average; abundant endothelial
cells were found. The blood was moderately concentrated and the
leucocytes were grouped on the warm slide and in the smears for
the differential counts. Splenocytes were abundant. The serum contained
a trace of bilirubin. Fibrin was formed abundantly and immediately.
The probability that pneumonia was complicated
by cardiac inefficiency was mentioned in the notes which were sent
with the report Treatment was planned for pneumonia plus cardiac
inefficiency, and the patient recovered, though rather slowly.
Two months later he was killed in an accident.
The autopsy showed some remaining hepatization in the lower right
lung, and the mitral and the tricuspid valves of the heart showed
the effects of an old, severe endocarditis
Miss F. 11, aged seventeen years, was brought
for examination because she was losing weight, and because she was
becoming more and more irritable. The blood examination showed moderate
secondary anemia with very slight evidences of toxemia of the type
usually associated with malnutrition. The blood platelets were very
low; 50,000 per cubic millimeter.
The possibility that there was a lack of Vitamin
A in the diet was suggested to the doctor in charge of her case.
On investigation it was found that the patient displayed a marked
aversion to eggs, and that other foods containing Vitamin A were
Correction of the diet was followed by moderately
increased weight and by the disappearance of the nervous symptoms
and the irritability.
The osteopathic physician reported that no definite
bony lesions were present but that there was some abnormal tension
of the spinal tissues in the upper lumbar region. This disappeared
with the correction of the diet, with no further attention.
Mrs. R., aged thirty-nine years, married twenty
years, no children, had been advised to submit to an operation for
a rapidly growing tumor. The ordinary symptoms of pregnancy were
absent. Certain atypical cells were found in the blood smear during
the progress of the differential count, and on further study these
appeared to be derived from the placenta. Surgical work was postponed,
and a few days later an X-ray plate showed fetal vertebrae and other
bones. The boy born after a rather stormy pregnancy and labor was
normal, and is now nearly fifteen years old.
It is rare that the placental cells are useful
in diagnosis, yet such cases have been found several times during
twenty-eight years of blood cell study.
Mr. L. 22, aged fifty-two years, complained of
certain vague gastric symptoms. Gastric analysis showed absent hydrochloric
acid but no other important findings. Roentgenologist’s report showed
some delay in the emptying time of the stomach but no evidence of
gastric ulcer or cancer.
Blood examination showed immediate and abundant
formation of fibrin on the warm stage, with threads of irregular
length and contour, often beaded, often arranged in radiating lines
with a group of platelets or a lymphocyte at the center. Refractive
granules were abundant, and these included iodophilic, Sudanophilic
and unstained particles. Rouleaux were scanty and the red cells
arranged themselves in masses.
Leucocytes showed the evidences of toxemia of
the type associated with disturbances of protein katabolism. Hyaline
cells and eosinophiles were increased, both relatively and absolutely.
The eosinophiles often showed abundant, basophilic, hyaline, intergranular
Fibrinolysis was masked by undifferentiated
proteolysis. The probability that late malignancy was present was
noted in the extra report sent to the doctor. Surgical interference
was decided upon, and an inoperable cancer was found around the
pyloric region of the stomach. At autopsy, two months later, this
was found to be completely surrounding the pyloric antrum. It was
of the scirrhous variety, and there was no gross ulceration of the
gastric mucosa. These facts explained the erroneous roentgenological
Mr. O. 2, aged sixty years, suffered from severe
pain in the stomach with occasional nausea; neither the pain nor
the nausea seemed to bear any relation to the taking of food nor
Blood examination showed the cell findings reported
in the case of Mr. L, 22. Gastric analysis showed absent hydrochloric
acid and also a few small masses of cells showing abundant and often
Fibrinolysis was absent and no undifferentiated
proteolytic ferment was found.
Surgical interference was based upon these findings.
A small cancer was found upon the anterior aspect of the pyloric
antrum. Gastro-jejunostomy was performed, and the patient is alive
and well at this time, twelve years later.
Mrs. W. 17, aged forty-three years, showed a
small, hard tumor in the left breast. This was associated with vague
pain in the same general region, radiating along the intercostals
nerves to the spinal column.
There was a history of an abscess in the place
occupied by the tumor, which was present about twenty years before
the tumor was noted. She was not in habit of paying very much attention
to her own body, and had always been an unusually busy and active
Blood examination gave a normal blood picture.
Fibrinolysis was normal and no abnormal findings were reported for
any of the special tests.
Surgical interference was postponed, on these
findings. The rib lesion was corrected and the pain disappeared.
The tumor seemed unaffected by the treatment. It was kept under
observation for several years, but no increase in size ever occurred.
After her death, fifteen years later, this tumor was removed for
histological examination, and it was found to be composed of scar-like
threads of connective tissue with no evidence whatever of malignancy.
Miss C. 5, aged fifty-three years, noted slight
and repeated uterine hemorrhages. On blood examination the findings
characteristic of malignancy were reported, and fibrinolysis was
absent. There was no evidence of an undifferentiated proteolytic
Pelvic examination discovered a myofibroma of
the uterus but no evidence of cancer. Uterine curettings and a bit
of tissue from the region of a small cyst on the cervix were removed
for microscopic examination but no evidence of malignancy was found.
The uterine hemorrhage was repeated, and a second blood examination
was made five weeks after the first examination. The evidences of
malignancy were somewhat more marked and there was present some
undifferentiated proteolytic ferment. At this time the patient complained
of pain in the lower abdomen and the pelvis.
Because of the myofibroma and the pain it was
decided to perform a hysterectomy. A tumor of the right ovary was
found, and on microscopical examination this proved to be a papillary
adenocarcinoma. There were several small metastatic tumors upon
the adjacent peritoneum. Radium treatment followed the removal of
the cancer and the uterus. She made an excellent recovery, and is
still in good health, eleven years later.
NON-MALIGNANT UTERINE BLEEDING
Mrs. T. 4, aged fifty-eight years, suffered from
slight but frequent uterine hemorrhage. Diagnosis of uterine cancer
was made by an eminent medical surgeon, who advised immediate hysterectomy.
This she refused, for the time being. She consulted an osteopathic
surgeon, who found a lesion of the fifth lumbar vertebra. The patient
explained this by a fall she had had about ten days before the first
uterine hemorrhage. This osteopathic surgeon advised the correction
of the lesion before operating. The pelvic examination discovered
a heavy, edematous, congested cervix and uterus but no definitely
Blood examination showed normal fibrinolysis
with no evidences of malignancy. The lumbar lesion was corrected,
the uterine hemorrhage ceased Pelvic examination three weeks later
showed normal cervix and uterus, and no reason for surgery.