For platelet count, the following materials will be

used: gloves, labgown, eyewear, detergent, bleach,

distilled water, 2 200mL beakers, 500mL beaker, wipes, 4
petri dishes, filter paper (cut in circles with a
diameter equal to the petri dish), capillary tubes, blood
samples in labeled vacutainers, rocker, unopette
reservoirs, 20L unopette capillary pipet, hemacytometer
with coverslip, microscope, alcohol pads, hand counters,
logbook and pen.
Platelet Count. The Unopette system will be used in
counting platelets with the use of a hemacytometer,
microscope, and hand counter. This methodology is broken
down into five parts, namely: (1) sample collection and
labelling, (2), moisture chamber construction (3)
hemacytometer cleaning, (4) specimen dilution and
transfer, (5) microscope examination and platelet
counting.
Sample Collection. With the remaining pure blood
samples extracted from donors, the researchers of
this study will first perform platelet counting on
the samples taken from the AB group, then the A
group, the B group, and lastly, the O group.
With the pure blood specimens divided into separate
labeled vacutainers each containing 1mL, those taken
from the donors with the blood type AB (that will be
labeled A to F) will be put into a rocker for 5 to
10 minutes to avoid coagulation. The first
vacutainer will be labeled A and will contain 1mL
blood with 1mL 5% leaf extract, the second
vacutainer will be labeled B and will contain 1mL
blood with 1mL 15% leaf extract, the third
vacutainer will be labeled C and will contain 1mL
blood with 1mL 25% leaf extract, the fourth
container will be labeled D and will contain 1mL
blood with 1mL 50% leaf extract and the fifth
container will be labeled E and will contain 1mL
of blood with 1mL 100% leaf extract, the sixth
container will be labeled F and will contain 1mL
blood only.
Those taken from donors with blood type A (that will
be labeled G to L) will be put into a rocker for 5
to 10 minutes to avoid coagulation. The first
vacutainer will be labeled G and will contain 1mL
blood with 1mL 5% leaf extract, the second
vacutainer will be labeled H and will contain 1mL
blood with 1mL 15% leaf extract, the third
vacutainer will be labeled I and will contain 1mL
blood with 1mL 25% leaf extract, the fourth
container will be labeled J and will contain 1mL
blood with 1mL 50% leaf extract and the fifth
container will be labeled K and will contain 1mL
of blood with 1mL 100% leaf extract, the sixth
container will be labeled L and will contain 1mL
blood only.
Those taken from donors with blood type B (that will
be labeled M to R) will be put into a rocker for 5
to 10 minutes to avoid coagulation. The first
vacutainer will be labeled M and will contain 1mL
blood with 1mL 5% leaf extract, the second
vacutainer will be labeled N and will contain 1mL
blood with 1mL 15% leaf extract, the third
vacutainer will be labeled O and will contain 1mL
blood with 1mL 25% leaf extract, the fourth
container will be labeled P and will contain 1mL
blood with 1mL 50% leaf extract and the fifth
container will be labeled Q and will contain 1mL
of blood with 1mL 100% leaf extract, the sixth
container will be labeled R and will contain 1mL
blood only.
Those taken from donors with blood type O (that will
be labeled S to X) will be put into a rocker for 5
to 10 minutes to avoid coagulation. The first
vacutainer will be labeled S and will contain 1mL
blood with 1mL 5% leaf extract, the second
vacutainer will be labeled T and will contain 1mL
blood with 1mL 15% leaf extract, the third
vacutainer will be labeled U and will contain 1mL
blood with 1mL 25% leaf extract, the fourth
container will be labeled V and will contain 1mL
blood with 1mL 50% leaf extract and the fifth
container will be labeled W and will contain 1mL
of blood with 1mL 100% leaf extract, the sixth
container will be labeled X and will contain 1mL
blood only.
Blood samples should not be allowed on the rocker
for more than 15 minutes to avoid cell break down.
Moisture chamber construction. Four petri dishes
will each have a filter paper cut out to the petris
diameter. The filter paper will be fitted inside the
petri dish and will be sprinkled with a minimal
amount of water to retain moisture. It should not be
soggy and very wet so as to avoid condensation and
cell overlapping. Two capillary tubings will be put
parallel to each other to serve as support when the
hemacytometer be placed inside the moisture chamber.
Hematocytometer cleaning. The hematocytometer and
its glass cover slip should be cleaned before use,
with diluted bleach, diluted detergent solution and
distilled water. Its glass cover slip is slightly
thicker and heavier than the normal plastic ones. It
automatically comes with the hematocytometer of use.
Household bleach will be poured into a 200mL beaker,
labeled diluted bleach just until the bleach coats
its bottom. It will then be diluted with tap water
until the 200mL mark. Dishwashing detergent will be
poured into a 500mL beaker, labeled detergent just
until it coats the bottom of the beaker. It will
then be diluted with tap water until the 400mL mark
and will be mixed thoroughly. Another 200mL beaker
will contain distilled water for final rinsing.
The hemacytometer and the cover slip will be dipped
in the detergent solution and will be scrubbed
gently with the fingers until the ends and the
corners are clean. Then it will be rinsed with tap
water. The hemacytometer and the cover slip will
then be dipped in the diluted bleach solution. The
fingers should not make contact with the surface of
the hematocytometer nor the cover slip to avoid
seeing artifacts when viewing the blood sample
microscopically. The hemacytometer and the coverslip
will then be rinsed with distilled water, and will
be dried using a lint-free wipe. Then it will be set
inside its moisture chamber to avoid contamination.
Specimen dilution and transfer. The diluent for
platelet counting will contain 11.45g ammonium
oxalate, 1.0g Sorensens phosphate buffer, and 0.1g
thimerosal; measured with the use of an analytical
balance. The measured solutes will then be diluted
to 1L in a 1.5L capacity beaker. This will be
tranferred to a clean, and sterilized glass
container with a cork cap, to be labeled as
Diluent. The reagent will be stored below 30C,
protected from sunlight with the use of carbon
paper. This diluent will induce RBC lysis but will
preserve leukocytes and platelets.
With the protective shield on the capillary pipette,
the diaphragm of a Unopette reservoir will be
punctured and will be added with the labeled blood
samples using a 20L capillary pipette provided with
the Unopette system. The shield from the pipette
assemble will be removed by twisting. The pipette
will be held almost horizontally, before touching
its tip to the blood sample. The pipet will fill by
capillary action and will cease automatically when
the blood reaches the end of the capillary bore in
the neck of the pipet.
Any excess blood outside the pipet will be wiped so
as not to interfere with the dilution factor. The
reservoir will be squeezed lightly to force out some
air while maintaining reservoir pressure.
The pressure on the reservoir will be released by
removing ones finger from the pipets opening. This
will then draw blood into the reservoir. The
reservoir will then be gently squeezed and released
to return the mixture into the reservoir and not out
of the capillary tubing. The reservoir will then be
inverted ten times to thoroughly mix blood sample
and diluent. the mixture will be left to stand for
10 minutes to ensure RBC lysis before charging the
hemacytometer.
To charge the hemacytometer, the pipet will be
withdrawn from the reservoir to be converted into a
dropper assembly by reseating the cap securely in
reverse position. The reservoir will then be
inverted and the first 3 drops of the mixture will
be discarded. The diluted blood will be charged into
the hemacytometer by gently squeezing its sides to
expel its contents until the chamber is properly
filled. The hemacytometer will then be placed inside
its moisture chamber for 10 to 20minutes to allow
platelet settling and avoid cell overlapping. The
moisture on the filter paper will retain the
evaporation of the diluted specimen while standing.
Microscope examination and platelet counting. The
hemacytometer will be mounted on a microscope and
will be viewed under the HPO lenses, with 40x
magnification. The condenser will be lowered, with
the diaphragm closed. The platelets will appear oval
or round, with one or more dendritic processes or,
they may appear as pinkish and bluish spots. The
platelets will be counted starting from the upper
left of the large middle square, until all the small
squares in the middle square have been counted.
Cells touching the upper and left lines will be
counted, but not any cells that touches the lower or
the right line of the hematocytometer as viewed on
the microscope. The number of platelets counted on
the 25 small squares in the middle square will be
multiplied by 1000 to get the number of platelets
per cubic millimeter.