Anna Lemira Gibson.

Clinical laboratory technic for nurses online

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of the bone marrow. There is some evidence that in
shed blood they take part in the process of coagulation.
Normally they average about 350,000 per cubic milli-
meter. They are absent in purpura, lymphatic leukemia,
and acute aplastic anemia. There is a marked increase
in myelogenous leukemia and early Hodgkins, and large,
abnormally granular platelets and large protoplasm plate-
let masses are often seen.



The Blood



Blood Platelets. Counting. (Journal A. M. A.,
May 20, 1911)

The blood is diluted i : 100 in the pipette used for count-
ing red blood corpuscles. The diluting solution consists
of 2 parts of an aqueous solution of brilliant cresyl blue
(1:300) and 3 parts of aqueous solution of potassium
cyanide ( i : 1400), and these solutions are kept in separate
bottles and mixed immediately before using. The cresyl
blue solution should be kept on ice and the cyanide solu-
tion should be made up every ten days ; the mixed solution
should be filtered before using.

The ordinary blood-counting chamber is used ; count
with the high dry objective. After the chamber is filled,
it is left at rest for 10 minutes, in order that the platelets
may settle.

Agglutination Test

One of the properties of the blood is the power of
agglutinating, or clumping and rendering immobile the
bacteria which it may be
called upon to attack.
This property also,
though present at all
times, is capable of de-
velopment and increase,
as is seen in the course WIDAL REACTION

of various diseases ; as, A B. typhosus- before adding ty-

for example, in typhoid phoid blood. B-K typical reaction ;
.,, the bacilli collect in clumps, 1. e., be-

fever, where the specific come u agglutinated .
agglutinins of the pa-
tient's blood are so developed as to show a clumping of
typhoid bacteria when the patient's serum and a culture
of typhoid bacteria are brought together.




Ii6 Clinical Laboratory Technic

First cleanse the lobe of the patient's ear with alcohol,
then make a small incision with a needle or stilette, letting
the blood flow into a capillary pipette. When this clots, the
serum is blown out on the loop of a sterile platinum wire
and added to the fluid culture. To obtain a fluid culture
of typhoid bacilli, make a transplant from a culture on
agar-agar to bouillon, and place in the incubator over
night.

Paratyphoid Bacilli

These bacilli differ from typhoid bacilli by producing
gas in glucose media and by showing different agglutina-
tion reactions. The two types generally recognized are
known as "A" and "B."

Type "A," in all other respects, is like the typhoid
bacillus.

Type "B" makes milk alkaline, but does not coagulate
it, and after eight or ten days it becomes translucent.

Bacillus Coli Communis

This bacillus differs .from typhoid as follows :

1. It is not motile.

2. Produces gas in media containing glucose.

3. Changes the blue color of litmus milk media to a
pink color, and usually coagulates the milk.

4. Does not show the clump reaction.

Agglutination Test. Widal Reaction

Take 9 drops of the fluid culture with a sterile platinum
loop and place at separate points on a glass slide. Add a
loopful of patient's serum and mix all together. Cover
with a cover glass and note the time (this makes a dilu-



The Blood 117

tion of T V)- Then take i loopful of the T V and add to
the 4 drops which have been placed on the opposite end
of the slide, and mix. This makes a dilution of ^. Label
and note the time.

If before the end of I hour the great majority of the
bacilli are both clumped and motionless in the T V dilution,
the reaction is only suggestive; but if in the ^ dilution



A 9 drops of bouillon culture
12-48 hours old.



B 4 drops from the same bou- "I I o



illon.



o



o



:



B



agglutination occurs, it is practically conclusive evidence
of typhoid infection. It takes from 4 to 6 hours for the
complete change to occur, and no less than six bacteria
must become agglutinated to constitute a positive reaction.

Hanging-drop Slide

Place drops of oil at the four corners of the cover glass.
Take i loopful of the T V or ^V dilution, place in center




HANGING-DROP SLIDE

of the cover glass, and then place the glass drop-side
down over the depression of the hanging-drop slide.

As a control, take 3 drops of the bouillon culture and
place on a slide free from serum, keeping it under observa-
tion during the experiment. The bacilli must not clump
and must remain motile.



n8 Clinical Laboratory Technic

Detecting B. Typhoid in Small Amounts of Blood.
Liebermann's Method. (Journal A. M. A.,
March, 1915)

Draw 2 drops of blood into i c.c. of distilled water.
One drop represents 0.05 c.c. of serum and the dilution
is equal to ^V

To this add I c.c. of saline and continue diluting with
different amounts of saline, making up to i c.c. each time.

Koenigsfeld's Method. (J. A. M. A., March, 1915)

Two kinds of medium are used, Endo's and Drigalski-
Conradi's ; mannite is used in the latter instead of lactose.
Bile is added to each tube, and this, on top of the slanting
solid medium, is inoculated with 3 to 4 drops of blood.

The bile has a tendency to check the development of
all except typhoid bacilli. The colonies develop in 10 to
14 hours.

The colon bacillus turns both tubes red ; acid- forming
cocci turn the Endo media red, but leaves the mannite
tube colorless.

B. typhosus develops rapidly and permits an early
agglutination test.

Malarial Parasites

Three species are associated with malarial fever in
man.

i. TERTIAN MALARIA (Plasmodium Vivax). A
small circle or ring-like body which stains blue. Within
the circle, usually near the circumference, is a round dot
which stains red. The ring becomes larger and more
irregular in outline, and pigmented granules appear.
The red blood cell becomes swollen. The blue and red



The Blood



119



elements of the parasite both divide into 15 to 20 seg-
ments. Cycle is completed in 48 hours.







Tertian malarial parasites in red blood corpuscles (Wright's
stain) : I, Young parasites (in the corpuscle on the right two
or three parasites) ; 2, young parasites; 3, half-grown parasites;
4, half-grown parasite (on the left, a blood-plate and near the
center another blood-plate lying on a red corpuscle) ; 5, half-
grown parasites (in the corpuscle on the left two parasites) ;
6, full-grown parasite (the nucleus lies in a clear space). All
the infected blood corpuscles in the foregoing figures contain
minute granules that stain red (granular degeneration) (photos
by L. S. Brown). From "Pathologic Technique," Mallory and
Wright.

2. QUARTAN MALARIA (Plasmodium Malaria). This
form is much the same as the tertian form in the early
stages. The pigment granules are larger and are usually



I2O



Clinical Laboratory Technic



arranged around the periphery of the organism; in the
tertian they appear to be distributed all through it. The
red blood cells remain about the normal size or a little
larger. In the later stages they become shrunken. They
undergo segmentation, dividing into 6 to 12 segments.
The full cycle is completed in 72 hours. This form is
not common in America.




i




1 F~



Estivo-autumnal malarial parasites in red blood corpuscles
(i, 2, 3, and 4, Wright's stain) ; i, 2, and 4, young parasites ; 3, on
the left, a "crescent," on the concave side of which is shown a
portion of the periphery of the red corpuscle, which it distends ;
on the right, a young parasite ; 5 and 7, " Crescents " in red blood
corpuscles ; 6, ovoid form of parasite in a red blood corpuscle
(photos by L. S. Brown). From "Pathologic Technique"
Mallory and Wright.



The Blood 121

3. ^STIVO- AUTUMNAL MALARIA (Plasmodium Fal-
ciparum). In the beginning it is very much like the
two other forms, only smaller. The ring becomes larger
and has the appearance of a signet ring, narrow and of
uniform thickness except at one section, which is thicker.
Average number of segments is 15. The characteristic
"crescent" shape occurs in the last stages of this form.
Length of development cycle is 24 to 48 hours.



Blood Parasites

(a) Trichinella, found in cases of trichinosis.
(&) Filaria bancrofti.

( c ) Filaria sanguinis hominis. This
parasite is found in the blood during
the night, and causes dilatation of
the lymphatics, chyluria, abscesses, and
elephantiasis.

(d) Trypanosomes, found in cases
of sleeping sickness.



Wassermann Reaction. Obtaining the Blood

The arm is scrubbed with alcohol 2 inches above and
2 inches below the elbow. Apply the tourniquet, but do
not obliterate the pulse. Insert the needle in the basilic
vein and withdraw 5 c.c. of blood. Place this in the ice
chest to coagulate.

Pipette off 2 c.c. of the clear serum and incubate at
56 C. for i hour.

It has been found that if the blood serum of a case of




122 Clinical Laboratory Technic

syphilis is mixed in the presence of complement with
extracts of syphilitic liver or alcoholic extracts of normal
organs, the complement will be fixed and prevented from
taking part in the subsequent hemolitic reaction.

Three distinct substances are required to form a com-
plete reaction :

1. A cell to be destroyed or a poison to be neutralized.

2. A substance capable of destroying the cell or neutral-
izing the poison (amboceptor or antibody).

3. A completing substance or the complement, with-
out which the cell cannot be destroyed or the poison
neutralized.

Wassermann -Test. (Noguchi's Serum Diagnosis of

Syphilis.) Apparatus Needed
6 pipettes graduated o.i c.c.
2 pipettes, 10 c.c.; graduated o.i c.c.
6 pipettes, i c.c. ; graduated o.oi c.c.
24 small test tubes, 10 x i cm.

2 flasks (100 c.c.).

Test tube racks with parallel rows of holes.
Glass tubing, f-inch bore.

METHOD. Place 8 c.c. of normal salt solution in a flask ;
allow 2 drops of blood from the ear or finger of a normal
person to drop into the flask (i drop of blood to every
4 c.c. of salt solution). Place in the refrigerator over
night. The cells gravitate to the bottom and are washed
with an excess of salt solution. The supernatant fluid is
poured off and replaced with fresh salt solution. Centri-
fuge 4 times, shaking the tube after each addition of fresh
salt solution. A i% suspension of washed corpuscles
is used.



The Blood 123

THE TEST. Place i tube for each test in the front row
and i tube for its control in the rear row.

Add i drop of serum to be tested (0.02 c.c.) from a
capillary pipette, o.i c.c. of 40% fresh Guinea-pig serum,
made by adding i part of complement to i^ parts of
normal salt solution, is added to each tube. Where fresh
complement cannot be obtained, dried slips of paper, each
containing 2 units of complement, may be substituted.
To the first tube add the slip bearing the antigen.

i c.c. of a i% suspension of human corpuscles is added
to both tubes. Shake thoroughly at intervals.

CONTROL. To each tube of the positive control add
i capillary drop of a syphilitic serum known to give a
positive reaction.

To each tube of the negative control add i capillary
drop of normal serum known to give a negative reaction.

Add complement to each tube.

Antigen only is placed in the tubes of the front row.

i c.c. of the corpuscle suspension is now added to each
tube and the rack is placed in the incubator.

An hour is allowed for the antibody to combine with
the antigen and for the complement to be fixed.

When the dried paper is used, a longer period of in-
cubation is necessary.

On the opposite page the plate shows the dLTerent
stages of the test.

The top rack shows:

a. Two tubes for diagnosis.

b. Two tubes for positive control.

c. Two tubes for negative control.

d. Antigen.

The contents of the tubes are as follows :



124 Clinical Laboratory Technic

Front: Test serum plus complement (2 units) plus
antigen plus corpuscle suspension (i c.c.).

Rear: Test serum plus complement (2 units) plus o
plus corpuscles (i c.c.).

Appearance of the tubes after the first incubation
(i hour at 37 C).

The middle rack shows :

d. Antigen.

e. Amboceptor.

The two pieces of paper in the front tube represent
antigen and amboceptor. In the rear tube the one piece
represents amboceptor.

Appearance of the tubes after the second incubation,
with the addition of the anti-human amboceptor slips.

Hemolysis occurred in both tubes of the negative set.
In the positive control tubes it took place in the rear tube
only, the same as in the two tubes for diagnosis ; there-
fore the reaction is positive and the serum is syphilitic.

The bottom rack shows :

The appearance of the tubes after several hours.

The absence of hemolysis in the front tubes for diag-
nosis and positive control means positive reaction.

Antigen

Free human heart muscle from excess of fat and put
in meat grinder, and mix with nine times its weight of
cubic centimeters of 95% ethyl alcohol.

Place in the incubator for 10 days, shaking the mixture
from time to time. Filter and add 0.4% of cholesterin.
Return to the incubator for 3 days, shaking occasionally.

When the cholesterin is all dissolved, the mixture is
ready for use. Dilute with 4 parts of normal salt solu-



The Blood 125

tion, adding slowly and in small quantities at a time.
The antigen must not possess any appreciable hemolytic
or anticomplementary qualities. It must possess suffi-
cient antigenic strength to allow its use in small quantities.

Noguchi recommends the following tests:
TUBE I

0.4 c.c. antigen emulsion.

o.i c.c. 10% human corpuscles.

Incubate 2 hours.

Complete absence of hemolysis at the end of this time
indicates that the antigen emulsion does not possess any
appreciable hemolytic property.
TUBE II

0.4 c.c. antigen emulsion.

o.i c.c. 40% guinea-pig serum.

Make volume i c.c. with saline.

Incubate I hour. Then add 2 units of amboceptor
and o.i c.c. 10% corpuscle suspension. Incubate 2 hours.
Complete hemolysis indicates that the antigen is not in-
herently anticomplementary.
TUBE III

i unit of syphilitic antibody.

0.02 c.c. antigen emulsion.

o.i c.c. 40% guinea-pig serum.

Make up to i c.c. Incubate i hour. Then add 2 units
of amboceptor and o.i c.c. 10% corpuscle suspension.
Incubate 2 hours. Absence of hemolysis shows that
0.02 c.c. of the emulsion has sufficient antigenic strength
to fix completely 2 units of complement. If the antigen
emulsion fulfills these requirements, it is suitable.

The amount used in the Noguchi test is o.i c.c. (at
least 5 units).



JL^O Clinical Laboratory Technlc

Preparation of Complement Slips

Serum is" obtained from large guinea pigs. Cut the
carotid artery and collect the blood in a large, flat dish.
Cover this and leave at room temperature for 4 hours,
then place in the refrigerator. Place squares of thick blot-
ting paper in a sterile, flat dish, and pour the serum over
it until the paper is soaked and an excess remains.

Remove the paper to another dish and quickly dry in
a current of air at a temperature not above 10 C.

Standardize in the following manner: In a series of
tubes, each containing i c.c. of erythrocyte suspension
and i unit of amboceptor, add bits of complement paper
(5 mm. width strips) of increasing length, 2, 3, 5, 7, 10,
and 15 mm.

Incubate at 37 C. for 2 hours.

The tube in which hemolysis is just complete contains
i unit of complement.

The fixation test requires 2 units, therefore the remain-
ing paper is measured off into squares having twice the
dimension of that bit found for i unit.

These slips should not be used if it is possible to obtain
the fresh guinea-pig serum; and if the fresh serum is
used, titrate the complement, using for. the test double
the quantity necessary to produce complete hemolysis in
i c.c. of a 5% emulsion of blood cells in the presence
of 2 units of amboceptor.

Amboceptor

This is made by immunizing rabbits against human
blood cells. The human blood corpuscles are collected in
a sterile centrifuge tube. Fill the tube two-thirds full of
0.85% salt solution. Centrifugalize 3 minutes, then decant



The Blood 127

the salt solution ; add more, and repeat the process 3 times.
Increasing amounts are injected in large rabbits intraperi-
toneally in the following manner :

ist injection 5 c.c. washed human corpuscles.

2d " 8 cc., "

3d " 12 c.c.

4th " 15 c.c.

5th " 20 c.c.

Injections are made at 4 or 5-day intervals. Nine or
10 days after last injection bleed the rabbit from the
carotid artery. Place the blood collected at room temper-
ature for 4 hours. Collect the clear serum by decantation
and leave the clot for another 24 hours. Repeat this for
3 days, or until no more serum is given out by the clot.
Mix the portions of serum collected, o.ooi c.c. of serum
or less, which will cause complete hemolysis of I c.c. of
i% suspension of human erythrocytes plus 0.02 c.c.
of guinea-pig serum, will equal I unit.

Amboceptor Slips

Cut thin filter paper (Schleicher & Schull, No. 590-
597) into squares, 10 x 10 cm., and soak in the serum;
absorb the excess with another sheet of paper. Dry at
room temperature (place the squares on a sheet of un-
bleached muslin).

When thoroughly dry, cut into 5 mm. widths and
standardize.

METHOD. Take 6 tubes and add i c.c. of the erythro-
cyte suspension, 0.02 c.c. complement, and then add in-
creasing lengths of the amboceptor strip, i. e., i mm.,
2 mm., 3 mm., 4 mm., 5 mm., 6 mm. Incubate 2 hours.



128 Clinical Laboratory Technic

The smallest strip which causes complete hemolysis at
the end of the 2 hours contains I amboceptor unit.

Mark the strip in sections of twice this length and cut
off when doing the test. Keep the strips dry and in a
'sealed receptacle.

Definition of Terms
Agglutinins

When bacteria or foreign blood corpuscles are injected
Jnto an animal, a new property is developed in the serum
of that animal ; and this serum, when deprived of its
own complement, by inactivation or dilution, is capable
of clumping the bacteria or corpuscles used for im-
munization.

n
Amboceptor

One of two active principles necessary to cause hemol-
ysis, bacteriolysis, or any cytolysis caused by serum, the
other active principle being complement.

Antibodies

Immune body is a synonym of antibody. Antibodies
possess specific affinity for the antigens which are used
for their production. A group of antibodies is capable
of producing antibodies when injected into another animal,
thus forming anti-antibodies.

Anti-complementary Action

Substances capable of reducing or removing the action
of the complement.

Antigen

Substances which have the power of producing specific
antibodies, as bacteria and blood corpuscles, which are



The Blood 129

antigens because they produce specific antibodies called
amboceptors and agglutinins. Diphtheria toxin is an anti-
gen, and injections, of it are followed by a specific anti-
toxin.

Complement

One of the two active principles necessary for hemol-
ysis. The other principle is called amboceptor, and is
unable to cause a dissolution of cells without the first;
hence the term complement.

Cytolysis

Dissolution of cells by specific amboceptors and com-
plement.

Hemolysis

Dissolution of blood corpuscles by various forces, set-
ting the hemoglobin free into the medium in which cor-
puscles are suspended. Distilled water will cause hemol-
ysis, also acid and alkalies. Hemolysis by serum is some-
what different, and is caused by two distinct groups of
substances, complement and amboceptor, both contained
in the serum, and the one is inactive without the other.

Immune Bodies

Synonymous with antibodies.

Inactivation

When fresh serum, which contains both amboceptor and
complement, is heated at 55 for ^ hour, it becomes in-
active, because of the destruction of the complement. The
amboceptor is not affected materially by the process.



130



Clinical Laboratory Technic



Blood Pressure

Pressure is generally applied to the brachial artery by
means of the arm band.

The blood pressure is variable, especially that in the
arteries. This pressure is expressed as being equal to so





"Tvcos" BLOOD PRESSURE
APPARATUS

The gauge measures pressure from o to 300 millimeters of
mercury and the dial is movable. The needle should be adjusted
at the o mark before taking the pressure. The dial is graduated
to read in millimeters and fifths of millimeters.

many millimeters of mercury, and by this expression is
meant that the pressure within the artery is able to sup-
port a column of mercury that many millimeters in height.

Systolic Pressure

The systolic pressure is the maximum pressure caused
by the systole of the heart, or the apex of the pulse wave.

Diastolic Pressure

This is the minimum pressure in the artery the
pressure at the end of the diastole of the heart or at the
bottom of the pulse wave.

Under normal conditions the systolic pressure in the
adult, expressed in terms of a mercury column, equal



The Blood 131

110-115 millimeters, while the diastolic pressure is only
65-75 millimeters. Maximum pressure during the first
years of life varies from 75-90 mm. Hg. The pulse pres-
sure is the difference between the systolic and diastolic
pressure. The pulse pressure in the artery averages
45 millimeters of mercury.

The mean pressure is obtained by dividing the sum of
the systolic and diastolic pressure by 2, or by adding half
of the pulse pressure to the diastolic pressure. A blood
pressure determination should be a routine procedure in
the first examination of every patient.

Palpatory Method

Find the pulse at the wrist of the arm to which the arm
band has been applied, and while the pulse is under obser-
vation raise the pressure with the hand bellows or pump
until the pressure within the constricting band is sufficient
to prevent the im-pulse from reaching the wrist. The
position of the arrow on the face of the dial at the instant
when the pulse passes the compressing band will repre-
sent the systolic pressure. Readings should be made at the
return of the full pulse.

Auscultatory Method

This method is used where there is no diastolic fluctua-
tions. Instead of feeling the pulse, a pulse tone caused
by pressure of the constricting cuff is listened to through
the stethoscope. Raise the pressure to the obliteration
of the pulse, then place the stethoscope over the brachial
artery below the cuff. The pressure is gradually allowed
to fall and a pulse tone is heard as the circulation com-
mences, and this tone undergoes a number of changes



132 Clinical Laboratory Technic

p

until it becomes very faint and almost disappears. The

reading of the sphygmomanometer at this moment repre-
sents the diastolic pressure.

Factors Influencing Blood Pressure

Posture : Standing Sitting Supine Rt. Lateral L. Lateral
132 134 152 155 no

Age : During the first years it varies : 75-90.
15 years to 21 : 100-115.
Adults : 120-140.

Sex : Female sex is lower as a rule.

Time of day: In the early hours of sleep there is a de-
cided fall, which gradually rises towards morning.
Minimum A.M. Maximum P.M.

Exercise: Raises pressure, also emotion and excitement.

Clinical value: In the treatment of children, as a guide
to stimulation and other treatment; in obstetrics, in
diagnosis and treatment of toxemias; also in myo-
carditis, nephritis, typhoid fever, pneumonia, and
arterio-sclerosis.

Blood Chemistry

Blood chemical analysis surpasses in value qualitative
and quantitative urinary analysis.

It is preferable to take the blood in the morning before
breakfast. (See page 138, method of collecting.)

Determination of Non-Protein Nitrogen. Folin's
Method

Normally 25 to 30 milligrams of non-protein N in
100 c.c. blood. This is increased in chronic nephritis,
uremia, plumbism, acute intestinal obstruction, and pro-
static disease.



The Blood 133

Solutions Needed

i. ACID DIGESTION MIXTURE. Mix 300 c.c. of phos-
phoric acid with 100 c.c. concentrated H 2 SO 4 . Cover
and set aside for several days (not absolutely necessary).
To 100 c.c. of clear acid add 10 c.c. of 6% CuSO 4
solution and 100 c.c. ammonia free water. Use I c.c. in
the non-protein nitrogen digestion.

2. NESSLER'S SOLUTION (Stock Solution). Mix
150 grams K. I., no grams iodine, and 100 c.c. water;
add an excess of metallic mercury (140 to 150 grams).
Shake vigorously for 15 minutes. The solution becomes
quite hot. When the red iodine solution has begun to
pale, cool in running water and continue shaking until
the reddish color is replaced by the greenish color of the
double iodide. Separate the solution from the surplus


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