COMPLETE BLOOD COUNT whole blood is what we measure in

Hematocrit levels.
1. Hgb/Hct
- Uses capillary tube
2. WBC Count
- Centrifugation: 10000 g to 15000 g
3. RBC Count for 5 min
- Length: 7 cm (7-7.5 cm)
4. Differential Count - Bore: 1 mm (1- 1.2 mm)
5. Red Blood Cell indices - Red Band: With Heparin
- 5 cm filled with blood (3/4 of the
6. Platelet Count capillary tube length)
- 4-6 mm clay seal/plug
NOTE: In manual counting, RBC Indices are
optional.

HEMATOCRIT
 Percentage of Packed RBCs in a
whole blood
 Percentage of the total volume of
whole blood that is occupied by
packed red cells when a known
PRINCIPLE:
A small amount of whole blood is
centrifuged to determine maximum packing
of erythrocytes, expressed as PCV (Packed
Cell Volume) or HCT (Hematocrit)
2 WAYS TO COLLECT THE SPECIMEN

volume of whole blood is centrifuged  Venipuncture

at a constant speed for a constant  Capillary Puncture

period of time. SPECIMEN REQUIREMENTS:

 Slightly decreased after 50 years old  Whole blood anti-coagulated with

EDTA or Heparin
 Decreased in anemia, over
 Capillary blood collected in
anticoagulation heparinized capillary tube (Hgb-Hct
 Increased in polycythemia, test request only)

dehydration, and prolonged standing * If the test requests for Hemoglobin

Hematocrit, that’s the time to use the
2 METHODS FOR DETERMINATION OF capillary blood collected in Heparinized
Capillary Tube.
HEMATOCRIT

MACROMETHOD: we use wintrobe tube

EQUIPMENTS
MICROHEMATOCRIT METHOD: uses
capillary tube  Hematocrit tubes (Red Band)
- We need to use the band with
Hemoglobin and Hematocrit test goes anticoagulant to avoid clotting
together since Hemoglobin are inside RBCs  Non-absorbent sealing clay
and the percentage of packed RBCs in a  Microhematocrit reader
  Microhematocrit centrifuge
(Hemafuge)
 Gloves
 Wax
How many centimeters of blood should be
collected in Microhematocrit test?
- 5 cm filled with blood which is 3/4 of
the capillary tube length) so that
there will be spaces for the clay and
wax
Clay
- 4-6 mm clay seal/plug
LAYERS:
1st layer: Fatty Layer
2nd Layer: Plasma
3rd Layer: Buffy coat (WBCs and platelets)
4th Layer: Packed red cells (the one that we
will measure)
5th Layer: Clay
Example:
Upon receiving test request form: CBC/
complete blood count
Tube: Violet top with EDTA
- Invert the EDTA Tube with blood to
properly mix the blood and the
anticoagulant and for the blood not
to clot.
- Invert the Violet Top 8 times
PROCEDURE
1. Get a capillary tube(heparinized) then
fill, fill at approximately three quarters
full. Prepare your clay and wax. Seal the
end with clay first then wax.
 Fill at least two-four plain capillary tubes
it from the EDTA tube sample collected
as it will automatically aspirate fill it at
approximately three quarters full or two-
thirds full. If using tubes with a colored
ring at one end, fill form on the opposite
end. Wipe the capillary tube after then
seal the end of the tube with the colored
ring using nonabsorbent clay. Hold the
filled tube horizontally and seal by
placing the dry end into the tray with
sealing compound at a 90-degree
angle.
- Wax can also be used to seal the tube,
you can put the wax in the oven for it to be
in its best condition for sealing
- If the capillary tube was broken while
sealing it with wax, dispose it in the sharp
container and recollect. However, if EDTA is
used, dispose, and get another sample in
the EDTA tube.
- If the test request is MALE: 40 – .50 L/L
Hematocrit/Hemoglobin only, capillary
FEMALE: .37 – .47 L/L
puncture can be performed but do not use
only 1 capillary tube, use 2 -3 tubes.

 Afterwards, label it. Label should be at

the bottom with covering the specimen.
Put a number and the number used will
also be used in the Hematocrit using a
micropore not obscuring the sample.

 Balance the tubes in a microhematocrit

centrifuge (put the capillary tube/s in a
test tube while balancing it using a
cotton put in the bottom of the test tube) Capillary Micro-Hematocrit Reader
with the clay ends facing the outside
away from the center, touching the SOURCES OF ERROR
rubber gasket. Falsely Increased Hematocrit

 Tighten the head cover on the centrifuge 1) Dehydration

and close the top. Centrifuge the tubes 2) Buffy Coat included
at 10,000 g to 15,000 g for 5 minutes 3) Hemoconcentration
to obtain maximum packing of red blood 4) Insufficient centrifugation or a delay
cells in reading results

 Determine the hematocrit by using a Falsely Decreased Hematocrit

microhematocrit reader. Read the level 1) Hemolysis
of red blood cell packing; do not include 2) Improper sealing
the buffy coat (WBCs and platelets) 3) Increased anticoagulant
when taking the reading concentration
4) Introduction of excess interstitial fluid
HOW TO READ: 5) Short draw in an evacuated tube
- Place the hematocrit tube vertically on
the reader. The bottom of the blood Not mixing properly can either cause either
should be on the 0% line (in between increase/decrease
the clay and the packed cell volume)
- Put the tube along the chart until the
meniscus of the plasma intersects the
100% line.
- Move the ruler up and down and
measure the packed cell volume at the
top of RBC column.

 The values of the duplicate hematocrits

should agree within ± 0.02L/L

NORMAL VALUES:

TOTAL WBC COUNT
- Hemocytometer and a close-up view of
the counting areas as seen under the
microscope.
- Has 4 large corner squares for WBC
count. The areas for the standard white
blood cell count are labeled W (16
squares), and the areas for the
standard red blood cell count are
labeled R (25 squares). The entire
center square, outlined in blue, is used
for counting platelets. The side view of
the hemacytometer shows a depth of
0.1 mm from the surface of the counting
grid to the coverslip
 For Total WBC count we will use
capillary blood, diluting fluid and we
will use the thoma pipette
 For differential WBC count we will
use PBS
HEMOCYTOMETER
- Is a specimen slide which is used to
determine the concentration of cells
in a liquid sample.
- It has a rectangular indentation that
creates a chamber.
- The device is carefully crafted so
that the area bounded by the lines is
known, and the depth (0.1mm) of
the chamber is also known.
PRINCIPLE:
Dilution of a sample of blood with a special
diluting fluid, and charging the
hemocytometer chamber with this resulting
solution, after which, it is focused under a
microscope and the cells are counted.
ACETIC ACID - most commonly used
Diluting Fluid for WBC count
PURPOSE:
- Used to lyse the Red Blood Cells
and Platelets
After using a blood sample with a diluting
fluid charge it into the:
IMPROVED NEUBAUER COUNTING
CHAMBER
✓ 2 ruled areas, each area divided into 9 big
squares, the side of every square is
bordered by a double line and measures
1mm. Thus, each has a volume of 0.1 cubic
mm and the whole ruled area measures 0.9 - 2% Acetic acid (Usual)
cubic mm. - Turk’s solution - Enhances nuclear
✓ Consist of a double line surrounding each definition
group of 16 small squares
- 1% HCl
✓ Depth= 0.1mm
DILUTION ratio: 1:20
✓ Each of the four large corner squares are
divided into 16 smaller squares, and each
smaller squares have a volume of 0.25 x
0.25 x .1= 0.00625 cubic mm.

✓ All corner squares are used for WBC

counting. Order of counting: upper right,
upper left, lower left, lower right

✓ The central large square is divided into 25

small squares, each with a volume of
0.004 cubic mm. These small squares are
each subdivided into 16 smaller squares,
each having a volume of 0.00025 cubic
mm.
WBC COUNT
 Free-flowing capillary or well-mixed
anticoagulated venous blood is
added to a diluent at a specific
volume in the thoma pipette.
 The diluent lyses the erythrocytes
but preserves leukocytes and
platelets and nucleated erythrocytes.
 The diluted blood is added to the
hemocytometer chamber.
- WBC counts should be performed
within 3 hours of dilution
Normal Values: (Lorma Value)
 (Adult) 5.0 to 10.0 x 10^9/L
 (At Birth) 10 to 30 x 10^9/L
MATERIALS:
1. Hemocytometer
2. Thoma Pipette
3. WBC Diluting Fluid
Calibrated stem with .5-mark, 1 mark, and
11 mark at the tip, with bulb and bead.
Purpose of BEAD - For mixing of diluting
fluid and blood.
TIPS:
If there is moist or detergent in the Pipette,
use the diluting fluid to wash it out.
PROCEDURE
 Obtain blood from EDTA tube or a
capillary tube until 0.5 mark of the
pipette. Use the tube to aspirate
 Wipe blood on the sides of pipet, be
careful not to touch the opening of the
pipet.
 Obtain diluting fluid to 11 mark, then
wipe excess diluting fluid.
 Shake the pipet to mix for 2-3 minutes.
* Let it stay for 10 minutes to completely
lyse RBCs.
 Discard few drops
- Make sure to use clean coverslips
 Charge the hemocytometer. Should not
flood the chamber
7. Wait for cells to settle
8. Count WBC in the 4 corner large squares
under LPO.
RULE:
 Cells that touch the top and left lines
should be counted
 Cells that touch the bottom and right
lines should be ignored.
FORMULA:
 Cells/ul = (No. of cells counted) x
(Dilution Factor) (Depth factor) / Area
counted (mm2)
 DILUTION FACTOR: 20
 DEPTH FACTOR: 10
- If its at the top: 10
- Bottom: 0.1
 AREA COUNTED: 1sq.mm x 4 = 4
sq.mm
 NO. OF CELLS COUNTED: “N”
Example: 300 WBCs counted in 4 large
corner squares. Blood is up to 0.5 mark.
Cells/ul = (No. of cells counted) x (Dilution
Factor) (Depth factor) / Area counted (mm2)
Cells/ul = (300) (20)(10)/4mm2 = 15,000 /ul
or 15.0 x 10^9/L
SHORTCUT FORMULA
1. Cells counted multiplied by 0.25 if 0.1
mark of blood
2. Cells counted multiplied by 0.125 if 0.2
mark of blood
3. Cells counted multiplied by 0.08 if 0.3
mark of blood
4. Cells counted multiplied by 0.06 if 0.4
mark of blood
5. Cells counted multiplied by 0.05 if 0.5
mark of blood
Example:
Blood until 0.5 mark
Number of leukocytes counted in 4 squares
= 200
Number of leukocytes per liter = (200 x
0.05) x 10^9
Result reported: 10.0 x 10^9/L
NOTES:
 No of cells counted = Add the cells
counted in large corner squares.
 Dilution factor: 20 (if 0.5)
 Depth Factor is 10
 Area counted: 4 mm2
 After use, the hemocytometer and the
cover glass are washed with distilled
water and detergent, after which they
 are dried with cloth and placed in
absolute alcohol. They are dried and
clean with alcohol swab before use.

 If the machine produced an abnormal

result, proceed to manual counting.

 Use 0.5 mark if <5x10^9/L

 use 0.3 mark if >10 X 10^9/L,
since the cells are too high in
order to balance diluting fluid and
the sample

HEMOGLOBIN
 Higher in morning and lower in evening

 Increased in STRENOUS EXERCISE,

HIGH ALTUTUDE, SMOKING,
POLYCYTHEMIA, CONGESTIVE
HEART DISEASE.

 Decreased when patient is LYING

DOWN DUE TO HEMODILUTION,
ANEMIA, RENAL DISEASE, LEAD
POISONING.

NORMAL VALUE: 127-183 g/L