platelet count methodology with human blood samples

8/11/2019 Platelet Count Methodology with human blood samples

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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


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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


container will be labeled Q and will contain 1mL


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


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vacutainer will be labeled Tand 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


container will be labeled W and will contain 1mL


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


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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 bleachjust 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.


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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


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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.


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platelet count methodology with human blood samples

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