Hematocrit Procedures PDF

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HEMATOCRIT PROCEDURES A centrifuged hematocrit is also called a packed cell volume (PCV). As in the automated hematocrit, a hematocrit is the volume of the red cells as compared to the volume of the whole blood sample and is reported in L/L or as a %. The packed cell volume is determined by centrifuging the specimen in capillary tubes and measuring the height of the red cell column. Back then, hematocrit is a name or the brand of the actual equipment or instrument used in order to determine the packed cell volume. However, at present, PCV and hematocrit are already interchangeable such that when we say hematocrit, it is already synonymous to PCV and when we say PCV, it is already synonymous to hematocrit Two categories in obtaining hematocrit 1) Direct method (manual method) 

Follows the principle of centrifugal force in order to obtain the different layers needed to measure the proportion of RBCs in the sample

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Disadvantage : result can be slightly affected by the plasma trapped in the RBC layer

a. Macromethod  EDTA-anticoagulated blood  

Wintrobe tube: a thick walled 11.5 cm glass with a uniform 3mm internal bore and a flattened bottom Centrifugal force: 2000-2300g for 30 minutes

b. Microhematocrit technique  Utilization of very small amount of patient sample 

Materials needed  Microhematocrit capillary tubes o

Heparinized microhematocrit tube -

o

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Plain microhematocrit tube -

Blue band on one end has no additive/anticoagulant

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Blood sample: venous blood that is already anticoagulated with EDTA

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It is preferred to prevent over anticoagulation which may lead to crenation affecting PCV results 

A short EDTA sample will have an increased anticoagulant-to-red cell ratio, which causes the red cells to shrink and the hematocrit to be falsely decreased. EDTA must not exceed 2 mg/ml of whole blood.

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Ideal anticoagulant to blood ratio: 2mg/mL

Sealing clay, paraffin wax Microhematocrit centrifuge aka microfuge o Relative Centrifugal Force: 10,000-15,000 x g or o

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Red band on one end Blood sample: free flowing capillary blood

Revolutions per minute: 10,000 – 12,000 rpm or 10,000-15,000 rpm

Microhematocrit reading device

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Procedure 1) Perform the capillary puncture procedure 2) Fill 2 heparinized capillary tubes up to 2/3 full or up to 5 cm full. Tilt each tube so that the blood is near the colored band. Hold the tube horizontally and wipe all of the excess blood off of the tubes before it dries. Be careful not to wipe across the end of the tube. Absorbent material will pull out more plasma than cells 3) Mix gently 4) Seal one end with sealing clay. Reinforce with paraffin wax. 

Filling and sealing with the clay perpendicular to the table keeps the clay from becoming contaminated with blood, helps prevent leakage and is safer. This is the fire-polished end. Add the sealant until it is just above or below the colored band. Filling and sealing this way keeps the clay from becoming contaminated with blood, helps prevent leakage, is safer for the person testing, and keeps the gasket in the centrifuge from being cut by the capillary tubes.

5) Label accordingly 6) Position each tubes in the microfuge head exactly opposite each other with the sealed end facing away from the center 7) Cover the microfuge. Centrifuge at 10,000 rpm for 5 minutes 8) Open the centrifuge after it has come to a complete stop. Read the results immediately after the centrifuge stops. If this is not possible, place the tubes upright until they are read. The red cells will slide if the tubes are left in a horizontal position and the hematocrit will be falsely increased. Remove the capillary tubes 9) Read the results When reading hematocrits, make sure the clay red cell interface is aligned with the 0% line and the bottom of the plasma meniscus is at the 100% line. The reading that corresponds to the top of the red cell column is the hematocrit

The 100 % line must bisect the bottom of the meniscus and the zero line must align with the red cell/clay interface. When both the 0% and 100% lines are positioned correctly, the hematocrit in per cent is the line that aligns with the top of the red cells. The buffy coat, which is comprised of white cells and platelets, is not included. Duplicate readings should match within 1 L/L and must be within 2 L/L. Readings that match can be averaged and reported in 0.5 L/L. Centrifuged hematocrits are always reported in wholenumbers or halves. Microhematocrit reader: i.

Line up the bottom layer of the packed red cells with the horizontal black line of the platform

ii.

Rotate the top plate using the small hole so that the black, spiral line will align with the top of the plasma membrane Hold the bottom plate until the black spiral line will align with the top portion of the packed red cell

iii. iv.

Read results

These cards are available from laboratory vendors and are inexpensive. i. Place the capillet ii.

Line up the bottom layer with the line indicating the 0 level.

iii.

Move capillary tube across the card until the very top portion of the plasma would be intersecting the line indicating the 100 level.

iv.

Determine the height of the red cells

Rule of three: used to check the accuracy of the hemoglobin, hematocrit and RBC counts: 

3xRBC=Hb

 Hbx3=Hct +/-3 For example RBC= 3.0x1012/L Hb= 9.2 g/L Hct= 26% Rule of three accuracy check: Hct: 3x 9.2 = 27% 26 +/- 3 = 23% to 29% Thus, 27% is acceptable 2) Indirect method 

Performed by automated analyzers or equipment

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HCT%=[RBCxMCV]÷10

Technical errors o

Excess anticoagulant

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Insufficient mixing of blood

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Improper sealing of capillary tube

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Inadequate centrifugation

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Allowing the tubes to stand too long

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Including the buffy coat

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Incorrect use of the hematocrit reader PRINCIPLES OF HEMOCYTOMETRY OR MANUAL CELL COUNT

Materials  Sample: venous blood with EDTA 1) Diluting pipettes: Thoma Pipettes o Thoma pipettes are commonly used in the laboratory  Two types 1. WBC pipette: has a white bead 2. RBC pipette: has a red bead  Graduated pipette with a capillary stem and a bulb  Calibrated markings:0.1 unit  Important markings:  0.5 and 1.0 mark  11 (WBC pipette) or 101 mark(RBC pipette)

2) Hemocytometer: rectangular glasswares with counting grids to allow counting of cells from a sample

a. Fuch’s Rosenthal Hemocytometer o Depth: 0.2 mm o Grid:4x4 mm o 16 large squares: 1x1 mm o 1 large square=16 small squares o 1 small square= 0.25x0.25 mm

b. Speirs-Levy Hemocytometer: for eo o Has 4 chambers o Depth: 0.2 mm o Grid:5x2 mm o 10 large squares: 1x1 mm o 1 large square=16 small squares o 1 small square= 0.25x0.25 mm

c. Levy-Hemocytometer with Improved Neubauer ruling: for routine manual cell count o has a standard depth of 0.1 mm o Dimension of the counting grid: 3x3 mm o Total area of the counting grid: 9mm2 o has 5 large squares o The 4 corner large squares: WBC count  divided into 16 small squares o Central square: RBC count  25 smaller squares  16 much smaller squares  Dimensions: 0.2mmx0.2mm

PRINCIPLES AND PROCEDURES OF HEMOCYTOMETRY o General Procedure 1) Collection of blood sample 2) Dilution of blood with appropriate diluting fluid using the Thoma pipette RBC DILUTING FLUIDS Gower’s solution Hayem’s solution Toison’s solution Dacie’s solution Eagles fluid (NSS) The following are isotonic with RBCs to preserve RBCs

WBC DILUTING FLUIDS 2% acetic acid Turk’s solution

PLATELET DILUTING FLUIDS Rees and Ecker Ammonium oxalate

Hypotonic to RBCs

Preserves morphology of platelets; together with stains, they would be refractile

3) Charging the hemocytometer or dispensing the sample in the hemocytometer a. Discard the 3-5 drops from the dilution in Thoma pipette because the first few drops simply containing diluting fluids b. Dispense the sample in the periphery of the coverslip in a manner that moves via capillary action to the edges of the platform completely filling them up i. Avoid having bubbles between or overflowing the counting area ii. capillary action and surface tension: important properties of dilution necessary to fill the counting area c. allow the cells to settle for 5 minutes 4) Manual cell count a. WBC count: manipulate the x and y axis feed knobs to move to next counting squares

b. RBC count

Inverted L Rule:

5) Computation and reporting

POISSON’S LAW OF DISTRIBUTION: all cells settle in a random manner o

Acceptable difference RBC...