Published on November 16, 2018
Determination of Hematocrit (Hct) (Packed Cell Volume; PCV): Determination of Hematocrit ( Hct ) (Packed Cell Volume; PCV) DR QAZI IMTIAZ RASOOL Objective: Objective To define haemocrit T o describe various methods using the centrifuge force. Discuss the normal value Clinical application. SIGNIFICANCE: SIGNIFICANCE most accurate and simplest of all tests in clinical hematology In detecting the presence and degree of ANEMIA OR POLYCYTHEMIA . In comparison, HB estimation is less accurate, and RBC count far less accurate. Hematocrit: Ancient Greek haima (αἷμα, "blood") kritēs (κριτής, "judge"), hematocrit means "to separate blood".: Hematocrit : Ancient Greek haima (α ἷμ α, "blood ") kritēs (κριτής, "judge"), hematocrit means "to separate blood". Defined % by volume of packed RBC in blood after centrifugation. Referred to as Packed Cell Volume (PCV) or erythrocyte volume fraction (EVF). Methods: : Methods : 3 methods, 1 . Macrohaematocrit A large volume of blood is required in this method. Approximately 2 to 4 ml is required. 2. Microhaematocrit This method requires small amount of blood, 2 to 3 drops only. The blood can be obtained by finger puncture 3. Automated method - the PCV is calculated by an automated analyzer and it is not directly measured . Macrometric method (Wintrobe) or macrohaematocrit : Macrometric method ( Wintrobe ) or macrohaematocrit ( low speed centrifugation takes place in tubes with a diameter of 3 mm) Principle Anticoagulated blood is taken in a Wintrobe tube. Fill upto the uppermost mark and then rotate for desired length of time. The packed cell volume (PCV) of red cells is directly read from the graduated tube as %. Requirement: : Requirement : 1 . Blood specimen: EDTA or double oxalated anti-coagulated blood is used. Determine P.C.V. within six hr. of blood collection . 2. Wintrobe Tube: 110 mm in length and 2.5 mm in diameter. The lower 100 mm are graduated or marked, from 100 at top and 0 (zero) at bottom for PCV. 3. Long necked pasture p ipette or a special type of syringes is used for filling Wintrobe tube . 4. Centrifuge machine with known speed. Procedure: : Procedure : Mix 0.4 ml of EDTA with 2 ml blood. Fill the Wintrobe tube upto upper most mark with the help of pasture pipette or syringe. Fill the another Wintrobe tube to balance first one. If the blood sample is not available, fill the tube with water. Place the Wintrobe tube in opposite side in centrifuge . Turn the centrifuge to slow speed, then slowly increase the speed to 3,000 rpm . Centrifuge for 30 min. at 3,000 rpm . . switch off the centrifuge and allow it to stop by itself. Take out the Wintrobe tube and read PCV directly with the help of graduation mark given on the tube. Microhaematocrit : Microhaematocrit Principle : Anti-coagulated blood is centrifuged in a sealed capillary tube, and then PCV is determined by a special haematocrit reader. Material and instruments: Material and instruments Microhaematocrit tube (capillary tube) 75mm in length and 1mm in diameter which contains heparin and show a red ring at the end of the tube. Microhaematocrit centrifuge device. Plastic seal to seal one end of Microhaematocrit capillary tube. Microhaematocrit reader. Slide11: 1. Fill 2 capillary tube s 75 mm in length - 3/4 TH with blood anti-coagulated with EDTA or heparin. 2. Blood for heparinized capillary tubes may be collected by capillary puncture. Wipe any excess blood from the outside of the tube. Seal the end of the tube with the colored ring with nonabsorbent clay Procedure: PROCEDURE: : PROCEDURE: 1. Clean your finger with 70% alcohol and let dry. 2. Prick finger with lancet, near the tip but not too close to the nail. Prick so that blood flows freely. Try squeezing up from your wrist if blood does not flow after pricking finger. Place the tip of a capillary tube onto a drop of blood on your finger. Call your instructor to seal 2mm of the tube. 5. The instructor will spin the tubes in a centrifuge ( 5 minutes at 10000 rpm), . 7. Using a special reading device(since the capilary tube is not graduated), Observations and Results:: Observations and Results: 3 LAYERS: T all upper layer of clear plasma—amber slightly yellow-colored. It should not be pink or red which would indicate hemolysis of red cells in the sample or within the body in hemolytic diseases. Greyish-white , thin layer (about 1 mm thick) the so-called “buffy layer”, consisting of platelets (thrombocytes) above and leukocytes below it. T all bottom layer of red cells which have been closely packed together. A greyish red line separates red cell layer from the layer of leukocytes above it. Slide15: The sealing should be about 2 mm deep.. Card Reader: The reader is used as, hold the tube against Scale Results: Results % of the volume of blood occupied by RBC Hct orPCV Hct % = Height of RBCs (mm) × 100 Height of RBCs and plasma (mm )} Microhematocrit method is preferred due to: 1 – Easier 2 – More accurate 3 – Uses less amount of blood Slide17: ■ Diagnosis of Anaemia (if H is less than normal) ■ Diagnosis of polycythaemia ( if H is more than normal ) ■ Calculation of blood volume from the following equation: Plasma volume Blood volume = 1 - H Clinical Significance : Slide18: ■ Calculation of renal blood flow from the following equation: True renal plasma flow Renal blood flow = 1 - H ■ Determination of blood indices : H MCV = x 10 RBCs count Hb content MCHC = x 100 H Normal values.: Normal values. The normal values of PCV vary according to the age and sex of the individuals. The normal ranges are: Males: 40 %–54 % Females: 36 %–47 % Newborns: 55-68 %. Clinical implications: : Clinical implications: PCV is affected by the shape, & the number of the RBCs & the plasma volume. High PCV either indicates either increase in number of circulating RBCs or decrease in plasma volume seen in cholera due to loss of water in the stool A low PCV indicates either decrease in RBC or increase in plasma volume hemodilution VARIATIONS 1) PHYSIOLOGICAL VARIATIONS: VARIATIONS 1) PHYSIOLOGICAL VARIATIONS a ) Hct has decreased values in capillaries ( hematocrit = 20% in skeletal muscle capillaries), spleen capillaries the value is 70-80%, because of the sequester function of the spleen; b ) N ew-born children have a physiological polycythemia c ) P regnancy - is decreased (there is a hemodilution because the plasma volume increases) d ) P eople who live at height (6000-7000 m) increased values. (hypoxia). Clinical implication Higher HcT: : Clinical implication H igher HcT : ( erythrocytosis ) POLYCYTHEMIA VERA as high as 70 % HIGHER ALTITUDE,may increase over time. HYPOXIA Low blood oxygen levels LUNG OR HEART DISEASE — low oxygen levels, make more RBC DEHYDRATION. BURN ( due to loss of plasma) Lower HcT: : Lower HcT : An insufficient supply of healthy red blood cells (anemia) A large number of white blood cells — usually a very small portion of your blood — due to long-term illness, infection, leukemia, lymphoma or other disorders of white blood cells. Acute kidney disease (lower Erythropoietin production lead to less RBCs production by the bone marrow). Pregnancy may lead to women having additional fluid in blood. This could potentially lead to a small drop in hematocrit levels PATHOLOGICAL POLYCYTHEMIA : PATHOLOGICAL POLYCYTHEMIA - polycythemia vera or primary polycythemia ; -secondary polycythemia occurs whenever the tissues become hypoxic (for instance heart failure, pulmonary sclerosis, intoxications with carbon oxide) in both polycythemia ( poliglobulia has the same meaning) increased (up to 80 %!) becomes more viscous and predisposes at thrombosis . b ) Patients with anemia have decreased haematocrit because of the decreased RBC number . c ) After bleeding at the beginning the hematocrit remains the same but after a period of time, the hematocrit decreases. This hemodilution is explained by the passage of water and micromolecules from the interstitium into the blood capillaries (this is an adaptative reaction Sources of error and comments: Sources of error and comments An increased amount of anti-coagulant decreases the Hct reading as a result of erythrocyte shrinking. Improper sealing of the capillary tube causes a decreased Hct reading as a result of loss of blood during centrifugation. a higher number of erythrocytes are lost in relation to the plasma. The microhematocrit centrifuge should never be forced to stop by applying pressure to the metal cover plate. This will cause the RBC layer to “sling” forward and results in a falsely elevated value. Slide26: The buffy coat of the specimen should not be included in the Hct reading, because its inclusion falsely elevates the result. A decrease or increase in the readings may be seen if the microhematocrit reader is not used properly. If too much time elapses between when the centrifuge stops and the capillary tube is removed, the red cells can begin to settle out and cause a false reading of the hematocrit.