Platelet count and hematocrit determination methods
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1. The document describes the principles, procedures, and clinical significance of platelet count and hematocrit determination methods. Platelet count involves diluting blood with ammonium oxalate and counting platelets under a microscope, while hematocrit involves filling a capillary tube with blood and centrifuging it to measure the ratio of red blood cells to plasma. 2. Both tests help diagnose bleeding, clotting, and anemia issues by checking platelet and red blood cell levels. Abnormally high or low counts can indicate conditions like blood cancers, blood loss, kidney disease, or dehydration. Precise methods and calculations are required to obtain accurate results.
Platelet 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 1 Labbtestsonline.org 2 Labtestsonline.org
- 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 3 Labtestsonline.org 4 Labtestsonline.org 5 Labtestsonline.org
- 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. 7 Labtestsonline.org 8 Labtestsonline.org 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.