Platelet cell count and hematocrit determination methods

1. Platelet count and hematocrit determination methods
Author: Negash Alamin
Profession: Clinical laboratory scientist
E-mail: drnegash@gmail.com
Date: 6/12/17

2. Platelet count
Principle: A platelet count is the enumeration of platelets which are tiny cells smaller than RBC
which help the blood clot. It may be included in the CBC or complete blood count if the
physician sees it as necessary. An anti-coagulated blood sample is drawn from a patient which
needs to be venous blood due to the nature of the test; then, it is mixed with a reagent
(ammonium oxalate) in 1:20 ratio i.e. 0.2 ml blood made up with 380 ml dilution fluid just like
WBC count dilution and charged into an improved Neubauer chamber on both sides; then, the
counting is performed under 40x objective in the area provided for RBC counting.
Materials:
1- Gloves as PPE
2- Improved Neubauer chamber.
3- Micropippette.
4- Venous blood
5- Reagent ammonium oxalate 10gram/ L preferably with a filter paper.
6- Compound microscope.
Procedure:
1- Once you prepare the ammonium oxalate by mixing it from a still stand point because
the reagent has a tendency to settle down to the bottom of the flask; then you can use
it to dilute your blood which will destroy the red blood cells so that you can view the
platelets as tiny shimmering fragments under a microscope.
2- Next charge your chamber slowly via 45° angle.
3- Place the chamber inside a petridish with wet cotton and cover it up and cover it with
the lid and wait for 15-20minutes.
4- Count the cells in the 5Rs if there is no thrombocytopenia and multiply with 1000.
5- If the cell count is less than 100,000 then you need to count all the 25 boxes in the
RBC counting area and multiply what you find by 200.
Calculations: In case Platelets count the dilution is the same as WBC count but the counting is
performed in the RBC area; thus the VCF is 50 and DCF is 20; thus giving us a multiplying
factor for the cells counted as 1000.

3. To see the math in detail observe carefully the picture below and the RBC counting areas. The
entire box has an area of 1mm by 1 mm; when you divide that by 25 small boxes you get an
area0.04 mm square. Multiply that with 0.1 then you get a volume of 0.004 mm cubed for one R
for 5R we get 0.02 mm cubed (volume used).
Clinical significance: The rise of fall of platelet cells indicate several conditions especially the
ones related with clotting disorders it may be requested in conjunction with other tests like PT
and PTT1
. The test may be ordered when a bleeding disorder manifests; such as prolong
bleeding, numerous nose bleeds (epistaxis), G.I bleeding, petechiae (small red spots on skin from
bleeding capillaries in the skin), heavy menstrual bleeding, purpura (small purple spots from
bleeding in the skin.)2
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4. Factors which decrease the count include:
 Disorders of the bone marrow like idiopathic thrombocytopenia (ITP) which is also
called immune thrombocytopenic purpura which a result of antibody attack against
platelets
 Viral infection like EBV (Epstein bar virus), and HIV.
 Drugs like Ibuprofen, antibiotics, hydralazine etc.
 Heparin induced thrombocytopenia i.e. a man receiving heparin therapy will produce
antibodies.
 Aplastic anemia.
 Lupus
 DIC (Disseminated intravascular coagulation) reduces the number of cells available for
clotting elsewhere where they are needed.
Factors the augment the count include:
 Malignancy
 TB
 Rheumatoid arthritis3
 Birth control medications
 Recovery from VB12 deficiency
 Mutation of JAK2 gene which causes thrombocythemia which is process where the bone
marrow produces high number of platelets4
Other pathological anomalies related with platelets include:
 Glanzmann’s thrombasthenia
 Bernard-Soulier disease
 Chediak-Higashi syndrome
 Wiskott-Aldrich syndrome
 May-Hegglin syndrome5
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5. Hematocrit determination
Principle: First of all the term ‘Hematocrit’ literally means ‘the judgment of blood’ in Greek and
is a term coined by Swedish professor of physiology Magus blix. It is also called erythrocyte
volume fraction (EVF) which is a procedure in which anticoagulated blood of the patient is
drawn into a capillary tube until its ¾ volume is filled with blood 6
and then sealed by a sealant
clay on the opposite side on the tube where blood has been drawn; then inserted into a special
kind of centrifuge for hematocrit and spun at 10,000 to 15,000 for 5 minutes to separate the
formed elements from the plasma; then the volume of packed RBC is measured either through
integral spiral reader, hand-held PCV reader or via a measuring ruler.
Figure 1 Professor Magnus Gustaf blix
Materials:
1- Patients venous blood (anticoagulated)
2- Capillary tube
3- Microhematocrit centrifuge
6
The capillary tube is red band when it is heparinized; blue band when it is plain; measuring 75 mm long with an
internal diameter of 1 mm; which means that 56.25 mm of its length has to be filled with blood.

6. 4- Appropriate sealant clay
5- Gloves and other PPE
6- One of the measuring apparatus as explained in the principle above
Method:
1- Fill the tube with blood ¾ of its volume
2- Seal the opposite side of the tube which is not contaminated with blood.
3- Prepare a balance for it in the centrifuge and insert it into the slot where the sealed clay
part faces outside.
4- Centrifuge as stated above and read using one of the methods stated; in case you are
using a hand held PCV reader the 0 line is adjusted just above the sealant (between the
sealant and the beginning of the RBC packed cells) and the beginning of the plasma line
is set on the hundred line mark. Then, read the size of the packed cell volume and report
in percent. In case you are using a simple ruler the length of the red cell column in mm is
divided by the length of the total column of the tube and reported in percentage.
Clinical significance:
Most routinely a hematocrit is often incorporated into the CBC count by physicians to diagnose
ailments like several types of anemia, polycythemia, to help in making blood transfusions. The
measurement of RBC status does not indicate a full diagnosis―it is a kind of red flag stating
there is something wrong with the RBC index. Thus, additional tests like blood smear, and
maybe even a bone marrow biopsy will reveal the exact issue with our patient.7
Generally the
RBC volume make up around 45% of the whole blood volume; but might be lowered in case of
excessive bleeding in either trauma or chronic internal conditions like G.I ulcers, Vitamin B12
deficiency, radiation, myelodysplastic syndromes of the bone marrow, kidney failure and
erythropoietin production stoppage, pregnancy (due to high amount of fluid in the body) 8
and
uterus bleeding in case of women.
A high hematocrit count may manifest due to Kidney tumor which leads to excessive
erythropoietin production, polycythemia vera, dehydration9
, high altitude abodement, dengue
shock syndrome (blood plasma leakage), congenital heart diseases or malformations which
reduce the total ejected volume of blood into the periphery blood vessels― which is
compensated by the body by more blood production.
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9
This is the most common reason for high hematocrit because in a situation when the body fluid plummets the
number of formed elements rises.