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Method of Determining the Rate of Coagulation

Puncture the lobe of the ear, then take a piece of
capillary glass tubing and holding it downward from the
puncture let it fill for 3 or 4 inches. At intervals of
30 seconds scratch the capillary tubing at short distances
and break off between the fingers.

When coagulation has taken place, a long, worm-like
coagulum is formed.

Color Index

In normal blood this is approximately i. To obtain the
color index divide the percentage of the hemoglobin by
the percentage of red blood cells, 5,000,000 red cells being
considered as 100%. To obtain the percentage of red
cells, multiply the two extreme figures to the left by 2.
EXAMPLE. A count shows the presence of 2,400,000 red
cells; the percentage would be 48 (24 X 2 ) The hemo-
globin percentage is 72 ; then the color index would be
72 divided by 48, or 1.5.

Sahli's Hemoglobinometer for Obtaining the Percent-
age of Hemoglobin in the Blood. Method
First wash off the lobe of the ear with alcohol, then
take hold of the lower end with the thumb, first and second
fingers. x Insert a glover's needle quickly. Wipe off the
first drop of blood. The second drop is drawn up by
suction into the pipette to the 20 cmm. mark.

The Blood

97

Pour N/io HC1 into the graduated tube to the mark 10
on the scale of the tube, then add to this the 20 cmm. in
the pipette. When the mixture assumes a clear, bright
color, add distilled water, drop by drop, until the color
matches the color in the standard tube.

Talquist's Hemoglobin Scale

This is a book of specially prepared filter paper, with
a scale of ten shades of blood colors. These are so tinted
as to match blood taken up on a piece of filter paper, and
are graded from 10 to 100. The comparison should be
made as soon as the blood on the filter paper has lost its
humid gloss.

Oligochromemia

A diminution in the normal amount of hemoglobin.

Hemacytometers or Blood Counters

These instruments are used for determining the numer-
ical ratio between the red and white corpuscles contained

H EM ACYTOMETERS

in the human blood. Undiluted blood, owing to the ex-
cessively large numbers of blood discs contained in a
single cubic millimeter, is unsuitable for counting, and
it is necessary to dilute the blood in certain proportions.

98 Clinical Laboratory Technic

A definitely determinable dilution is obtained with the aid
of the mixing pipettes.

The melanger or large pipette is- used for counting the
red corpuscles, and gives a dilution of i : 200 when the
blood is drawn up to the mark 0.5.

Method

Cleanse the lobe of the ear with alcohol and insert a
sterile glover's needle or a straight Hagedorn needle by
a rapid puncture. The first drop of blood should be wiped
away, and as soon as the blood is flowing freely, put the
point of the pipette into the drop as it emerges from the
ear ; by sucking gently on the rubber attached to the other
end, draw up the blood to the mark 0.5 on the pipette.
Wipe the end of the pipette, then plunge the point into
Gower's solution and fill the bulb up to the mark 101.
Thoroughly mix the blood by rolling the pipette between
the palms.

The small pipette is used for counting the white cor-
puscles and gives a dilution of i : 20 when the blood is
drawn up to the mark 0.5. The bore of this tube being
large, it fills and empties more readily, therefore it
should be kept in a horizontal position. Draw the blood
up to the mark 0.5 on the pipette, wipe the end of the
pipette, then plunge the point into 0.3% acetic acid, and
fill the bulb up to the mark n.

When the white count is very high, the red counter
may be used, diluting the blood 200 times instead of 20.
This must be remembered in determining the final count.

Clean the pipettes with water, then alcohol and ether.
The mouthpieces attached to the rubber tubing should be
boiled often and kept in 95% alcohol.

The Blood 99

The Counting Chamber

The number of corpuscles contained in a cubic milli-
meter of the dilute solution of blood is determined by
counting in a cell of known depth and area. The count-
ing chamber consists of a plane glass plate with a circular
hole, which being cemented to a plane slide of stout glass
forms a well. The bottom of the latter is formed by a
disc, the thickness of which is exactly o.i mm. less than
that of the perforated glass.

The counting chamber consists of 16 squares of J mm.
side, each of which is again divided into 25 small squares
having sides -\$ mm. The area of each of the small
squares is -^fa square millimeter, and the distance between
the floor and the lower surface of the cover is ^ mm.;
therefore, each square represents 3^0 tf cubic millimeter.

To clean the counting chamber, rinse in cold water and
dry carefully.

1. Place the cover glass (an optically plane glass) in
position; then by slightly pressing with the rubber end
of a pencil, concentric rainbow rings will be seen. These
are known as Newton's rings, and must remain after
pressure is removed, otherwise there is dust under the
glass.

2. Blow out 3 drops of the diluted solution in the
pipette.

3. Place a drop upon the surface of the counting
chamber of such a size that when the cover glass is let
down over it the whole of the disc is covered without any
being spilled into the moat around it.

4. Place the cover glass over the drop before the cor-
puscles have time to settle.

IOO

Clinical Laboratory Tcchnic

5. After waiting a minute for the corpuscles to settle,
if the distribution seems uniform the counting is begun.

Register for Recording the Number of Blood Cells
Counted

Place the index finger of the left hand through the ring,
holding the Register in position so as to bring the pusher
directly under the thumb. In commencing to count, only
the o's should appear ; thus, ooo. The Register will count
to 999 ; the next count causes the ooo's to appear, making
i, ooo. To set the Register at o, press i-ie pusher down
the required number of times until the o appears at the
right, and then change the other figures at the left by
turning the thumb buttons on back of the Register till the
o's appear, always turning the thumb button of middle
dial first.
Counting the White Blood Corpuscles

Count all the corpuscles in the 400 small squares, ruling

out those touching the outside lines. Count four separate
drops, then divide by four to get an average; multiply
the result by the dilution ( ^ th en by 4,000 (each square
is equivalent to oVo of a cubic millimeter), and divide

The Blood 101

by 400 (the number of squares counted). The result
will be the number of corpuscles per cubic millimeter.

EXAMPLE. Average number of corpuscles in four
drops is 45.

45 X 20 X 4,000

= 0,000

400

Counting the Red Corpuscles

Count all the corpuscles in the 25 small squares, begin-
ning at the upper left-hand corner (a), counting the 25
small squares, then the upper right-hand corner (&), the

lower right-hand corner (c), and the lower left-hand
corner (d), making a total count of 100 small squares.

The sum of all the corpuscles in the 100 small squares
is multiplied by the dilution Gtro)> then by 4,000, and
divided by 100 (the number of squares counted).

EXAMPLE. The number of corpuscles counted in 100
squares is 685.

685 X 200 X 4,000

- = 5,480,000

IO2 Clinical Laboratory Technic

Vital Staining of Blood. (Journal A. M. A., March,

Stain fresh without fixation. The stains may be dis-

solved in physiological salt solution, Ringer-Locke solu-

tion, or even directly in the blood itself. The blood is

mixed with the stain and examined immediately, prefer-

^jably on. aj \Va;rin ;stage.

..The^staining 1 solution may be placed on the glass, the
rnai gin ;Hqing .sealed with paraffin or balsam, to prevent
evaporation.

In typical vital staining the nucleus is not stained, only
certain granules and fibers in the cytoplasm taking the
stain. As the cell begins to die, "post-vital staining of
whole or parts of the cells may occur. Lymphocytes are
recognized by the size and shape of the nuclei ; neutrophile
leucocytes, by their fine granulations; eosinophiles, by
their coarser granules.

Among the stains used in vital staining are methyl
violet, neutral red fuchsin, toluidine blue, thionine, and
Nile blue; o.i c.c. of any one of these stains to 250 c.c.
of water or salt solution.

In studying mitochondria, or small, rod-shaped gran-
ules in the cytoplasm of leucocytes, a ^-\$1^ solution of
Janus green is used. The Janus green (diethylsafrani-
nazo-dimethylanilin) is dissolved in 0.85% salt solution.

Modification of Ringer-Locke Solution

Sodium chloride 6. c.c.

Calcium chloride 0.2 c.c.

Potassium chloride 0.4 c.c.

Sodium carbonate 0.2 c.c.

Grape sugar i.oc.c.

Distilled water 1,000. c.c.

The Blood 103

Staining Reticulated Cells

Many of these cells are present in anemic blood.

METHOD. Place one small drop of i% aqueous solu-
tion of brilliant cresyl-blue upon a slide. Allow to dry
in such a manner that a thin film of the dye is obtained.
A small drop of blood on a cover-glass is placed upon
the dried dye. Allow the blood to spread, using gentle
pressure if necessary.

Examine with the oil immersion objective. A blue
staining reticulum of delicate, contorted filaments will
be seen within the red blood cell: Platelets appear as
irregular shaped hyaline bodies, with a round, blue-
staining, granular central structure. The white blood
cells stain blue.

Mechanical Stage

This stage is a great aid to exact work in differential
counting. It is attached to the stage of the microscope
by two set screws. By rack and pinion giving equal speed
in both movements, right and left scale reads 60 mm.;
backward and forward, no mm. It is provided with one
adjustable and one fixed stop, which is actuated by a
spring to hold the slide in place.

Smears for Differential Count

Cleanse the ear lobe with alcohol, and after puncturing
it with a sterile needle wipe off the first drop. A clean
slide is held without touching its surface, and touched to
the summit of the drop as soon as it emerges. This drop
is then drawn out smoothly over the glass with the edge
of another slide.

The slide should not come in contact with the skin
while it is being charged with the blood, and no pressure
should be used in spreading the drop.

IO4 Clinical Laboratory Technic

Dry quickly by waving the slide in the air.

Cover the smear with Wright's stain for i minute.

Add same amount of water to the stain, let this remain
3 minutes, then wash the slide with water.

A differential count of 400 cells should be counted to
secure reliable results. The percentage value of each
variety is then figured.

Normal varieties and figures in I cmm. of blood :

Polymorphonuclear neutrophiles 65-70%

Lymphocytes 20-35%

Transitionals large mononuclears 3~ 5%

Eosinophiles 2- 4%

Mast cells \%

While making the differential count, the variations in
size of the red blood cells, achromia, and polychromato-
philia, and the number of blood platelets should be noted.

Cover-glass Method for Smears

Touch the center of the cover-glass to drop of blood
from the ear, then drop immediately on another cover-
glass and allow to spread. Separate by sliding one cover-
glass from the other.

Erythrocytes. Wright's Stain

a. Normal red blood cells.

b. Microcytes, small red blood corpuscles.

c. Crenated cells. If the density of the plasma is in-
creased in any way, as by evaporation, many of the red
cells become shrunken and crenated by the passage of
water out of the corpuscle.

d. Macrocytes, large red blood corpuscles.

e

n

a

n

c

b

'*>

5B ^ ^I^Sf. <W

e f

*

The Blood 105

e. Poikilocytes, malformed, pear-shaped red cells.

/. Red cells showing degenerative changes.

g. Normoblast, a nucleated red cell of the ordinary
size.

h. Megaloblast, a large nucleated red cell.

i. Amitosis, or direct cell division.

/. Free nucleus.

k. Polychromatophilia, various stains or tints.

/. Achromia, absence of color.

m. Stippled cells, characteristic of lead poisoning.

n. Blood platelets.

o. Karyokinesis, mitotic or indirect cell division.

p. Malarial organisms,

Leucocytes. Wright's Stain

a. Lymphocytes.

b. Large mononuclears.

c. Polymorphonuclear neutrophiles.

d. Eosinophiles.

e. Mast cells.

/. Myelocytes, neutrophilic.
g. eosinophilic.

h. basophilic.

NOTE. Neutrophilic granules are purple ; basophilic, dark blue ;
acidophilic, red.

Leucocytes. Wright's Stain

Lymphocytes

These cells are about the size of a red blood cell, and
are derived from the lymph glands. They have a large
nucleus, with small margin of protoplasm. The nucleus
stains intensely, while the protoplasm is light blue and
is usually free from granules.

io6 Clinical Laboratory Technic

Large Mononuclears

Normally these cells have a round or oval nuclei which
does not stain so deeply as the lymphocytes, and they
possess more protoplasm. At a later stage in the decay
of these cells, the nuclei are more indented, frequently
horseshoe-shaped, stain less intensely, and have a very
granular protoplasm.

Polymorphonuclear Neutrophiles

These cells have a multiple, irregular-shaped nucleus.
The nucleus stains deeply and the protoplasm is dotted
with neutrophilic granules. They are derived from the
neutrophilic myelocytes of the bone marrow.

Eosinophiles

These cells have an irregular-shaped nucleus, and the
protoplasm is covered with coarse acidophilic granules
which are highly refractive. They are formed from the
acidophilic myelocytes of the bone marrow.

Mast Cell

The nucleus of this cell is multiple and irregular, and
the protoplasm is covered with large basophilic granules.
They are formed from the basophilic myelocytes.

Pathogenic Leucocytes

Myelocytes

NEUTROPHILIC. The nucleus is large and irregular,
and the protoplasm is covered with neutrophilic granules.

EOSINOPHILIC. This cell has a large oval nucleus,
and the protoplasm is covered with eosinophilic granules.

BASOPHILIC. The nucleus of this cell is large and the
protoplasm is covered with small basophilic granules.

The Blood 107

Myeloblasts

These cells are often mistaken for large lymphocytes.
They are the lymphoid cells of the bone marrow and are
the parent cells of the myelocytes. The nucleus stains
more deeply than that of the large mononuclears, and
the small amount of protoplasm is stained more deeply
blue. There are no granules. They also show three or
four nucleoli in the nucleus.

*

16 17

19 EO

LEUCOCYTES After Gibson

13, polymorphonuclear neutrophiles (abnormally granular) ; 14,
lymphocytes; 15, mononuclears; 16, myeloblast ; 17, young myelocyte;
18, megacaryocyte imbedded in platelets; 19, megacaryocyte-dividing
nucleus ; 20, megacaryocyte sending out pseudopodium.

Megacaryocyte

The giant cell of the bone marrow. These cells are
frequently found in myelogenous leukemia. The nucleus
has a gnarled appearance. There is considerable bluish
protoplasm. These cells are often seen imbedded in
platelets.

Blood Diseases
Anemia

A condition in which the quality and quantity of the
blood is deficient.

io8 , Clinical Laboratory Technic

Pernicious Anemia

Leukopenia. Color index is high. Red blood cells,
2,000,000 to 200,000. Marked anisocytosis. Megalo-
blasts increased. Decrease in blood platelets. Poikilo-
cytosis, polychromatophilia, and stippling, normoblasts,
myelocytes, and myeloblasts sometimes present.

Chlorosis. Leucocytes normal. Color index is low.
Slight anisocytosis. Normoblasts.

Splenomyelogenous Leukemia (Myeloid Leukemia)

The red blood cell count is about 3,000,000 and the
color index low. The leucocyte count is on the average
from 200,000 to 500,000, may go as high as 1,000,000.

Some types of myelogenous leukemia have a low white
count, although the spleen and liver are markedly en-
larged. Such cases show a large number of cells of
mononuclear type, but of bone marrow origin (abortive
myelocyte).

In acute cases and in exacerbations of the disease
there are many myeloblasts.

Lymphatic Leukemia. Red blood count about
3,500,000. Color index slightly below normal. Small
lymphocytes, 70-98%. White blood count, 8,000 to
250,000.

In acute lymphatic leukemia the large lymphocyte
predominates.

Pseudoleukemias

HODGKINS DISEASE. Increase in lymphocytes in early
stage with few abnormal mononuclears. Mononuclears
are generally very granular, and increase as the disease
progresses. Eosinophiles increased. Blood platelets are
increased. Marked decrease in last stages.

The Blood 109

LYMPHOSARCOMA. Marked anemia. Neutrophilic
leucocytosis. Decrease in lymphocytes.

LYMPHOID PSEUDOLEUKEMIA. 5,000 to 14,000 white
blood count, with 70% lymphocytes.

Multiple Myeloma

Increase of Tiirck's irritation forms. (Test urine for
Bence-Jones body.)

Granulomatoses

Neutrophilic leukocytosis.

Oxydase Reaction. Graham's Stain

METHOD. Fresh smears are preferable. Dry thor-
oughly in the air.

1. Fix smears for 2 minutes in a freshly prepared
mixture of 9 parts of 95% alcohol and I part of strong
formaldehyde solution.

2. Wash in water.

3. Flood with the following alphanaphthol solution:

Alphanaphthol (Merck's Reagent) I gm.

Alcohol (40%) i oo c.c.

Hydrogen peroxide 0.2 c.c.

4. Allow a reaction time of 5 minutes.

5. Wash and place in dish of running water 15
minutes.

6. Stain with the following solution:

Pyronin o.i gm.

Alcohol (40%) 96 c.c.

Aniline 4 c.c.

Dissolve the pyronin in the alcohol, then add the
aniline.

7. Wash in water.

no Clinical Laboratory Technic

8. Stain I minute with a 0.5% aqueous solution of
methylene-blue (Griibler's B. X.).

9. Wash in water, blot, and dry.

10. Mount in neutral balsam.

The neutrophilic granules stain a purplish-red in color,
and are usually very abundant. Old and degenerating
forms show fewer granules. The mast cells take a more
basic stain. The eosinophilic granules are larger and
lighter and more refractile, with lighter staining centers.
The granules in the myelocytes vary in number and size.
Nuclei of all cells appear blue, and cytoplasm, light blue.

Red blood cells appear greenish-yellow.

Platelets are blue.

This stain is of value in differentiating myelogenous
from lymphatic leukemia.

Fragility of Red Cells

There is less resistance to hypotonic salt solution in
cases of hemolytic jaundice, and an increased resist-
ance in obstructive jaundice (cancer of pancreas and
gallstones). Pernicious anemia shows an increased
resistance.

Fragility Test

Withdraw 6 c.c. of blood from a vein in the arm and
transfer to a test tube half full of 0.5% sodium citrate
solution in 0.9% sodium chloride. Mix by inverting
tube several times. Centrifuge, pour off supernatant
fluid, and wash the cells twice with 0.7% sodium chloride.
Draw off as much supernatant fluid as possible with a
pipette and use the remaining blood cells for the test.

Make hypotonic sodium chloride solutions from a i%
solution of chemically pure sodium chloride and distilled

The Blood HI

water. The solutions are kept in tightly corked 100 c.c.
bottles and run in strength from 0.70 to 0.175%. With
a pipette, draw off i c.c. and transfer to a series of small
test tubes (i c.c. for each tube), then add 0.05 c.c. of
blood cells to each tube. Invert each tube twice and
allow to stand two hours at room temperature. Make a
reading of the tubes. The point at which there is the
first tinge of pink in the salt solution is considered the
initial hemolysis, and the point at which there can no
longer be seen any sediment of blood is the complete
hemolysis.

The average points of hemolysis as obtained by this
method are (from Hill) :

Normal blood .457 (initial) 3.40 (complete)

Secondary anemia .475 (initial) .322 (complete)
Pernicious anemia .477 (initial) .322 (complete)
Chronic family

(hemolytic) jaundice .600 (initial) .400 (complete)
Obstructive jaundice .400 (initial) .225 (complete)

Matching Bloods for Transfusion. Minot's Modi-
fication of Moss' Method

1. Red Blood Cell Suspension. Puncture the ear of
each of the two persons whose blood is to be matched
and let a large drop of blood fall directly into a small
test tube containing i c.c. of a 1.5% solution of sodium
citrate in 0.9% salt solution. Mix by inverting several
times.

2. Serum. A few drops of blood are collected in a
small test tube or a Wright's capsule. Allow to clot, then
loosen clot from the wall of the tube. Let stand until
the serum has separated well.

112 Clinical Laboratory Technic

3. Make vaseline rings on 2 slides. In the ring mix
one drop of the patient's serum and one drop of the
donor's red blood cell suspension. In the other ring mix
one drop of the patient's red blood cell suspension and
one drop of the donor's serum.

Transfer to a cover glass, invert over a hanging-drop
slide so that the drop hangs freely within the hollow
space. Mix the corpuscles at intervals by tilting the
slide.

Examine with the low objective power.

When agglutination occurs, the corpuscles come to-
gether in clumps or masses. Clumping occurs within
a few minutes, but it is safe to wait one-half hour. In
case of rouleau remix the cells. The serum of the patient
must not agglutinate the corpuscles of the donor.

Hemolysis Test

It is advisable to do a Wassermann test on all donors.
The donor's red cells should be tried against the serum
of the recipient, and the patient's cells against the donor,
to prove the absence of hemolysing or agglutinating
bodies.

DONOR. Withdraw two samples of 5 c.c. each from
the arm vein. Place one in a test tube and allow to clot.
The clot is loosened from the walls of the tube and the
serum separated by centrifugation. Place the second
sample in a test tube half full of .5 sodium citrate in
90% sodium chloride. Centrifuge, then pipette off the
supernatant fluid. Wash three times with normal salt
solution.

RECIPIENT. Repeat the same procedure with the re-
cipient's blood. Place variable quantities of recipient's

The Blood 113

serum in three small test tubes. Add to each tube
0.25 c.c. of a 2% suspension in normal salt solution of the
blood cells of the donor. The same procedure is carried
out with the donor's serum and the blood cells of the
recipient.

Controls should be made with the donor's serum and
donor's cells, and recipient's serum and recipient's cells.
Controls are also made with donor's cells in normal salt
solution and recipient's cells in normal salt solution.
Add normal salt solution to each tube, raising the total
volume to 0.5 c.c.

Incubate the test tubes in a water bath for 2 hours.
Make readings. The controls of the patient's may be
omitted if the amount of blood taken from the patient
is small. It is advisable to place the tubes in the ice box
over night and make a second reading in the morning
unless the case is urgent.

Blood Groups 1

GROUP I (\$% of individuals). Serum agglutinates
cells of no other group. Cells are agglutinated by sera
of Groups II, III, IV. As donors may be used for
Group I only. As recipients may receive from Groups I,
II, III, IV.

GROUP II (40% of individuals). Serum agglutinates

cells of Groups I, III. Cells agglutinated by sera of

Groups III, IV. As donors may be used for Groups

I, II. As recipients may receive from Groups II, IV.

GROUP III (10% of individuals). Serum agglutinates

1 Tests have shown that individuals fall into 4 groups.

U4 Clinical Laboratory Technic

cells of Groups I, II. Cells are agglutinated by sera of
Groups II, IV. As donors may be used for Groups I, III.
As recipients may receive from Groups III, IV.

GROUP IV (45% of individuals). Serum agglutinates
cells of Groups I, II, III. Cells are agglutinated by no
group. As recipients may receive from Group IV only.

It is preferable to have donor and recipient of the
same group. Two known sera, Types II and III, re-
spectively, are sufficient for the determination of the
group to which any blood cells belong.

Macroscopic Blood Test

Place a drop of Serum II and a drop of Serum III
on the right and left ends of a slide, respectively ; add
Yz drop of blood from ear to each drop of serum, using
clean rod each time. Mix blood and serum. If agglu-
tination occurs there will be a granular or brick dust
appearance of the drop in about I minute.

Blood Platelets

These are circular or oval discs 2-3 microns in diam-
eter. They are punched-off projections of giant cells

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