HEMOGLOBIN DETERMINATION PHYSICAL METHOD (RULE OF THREE)

PRE-ANALYTICAL PHASE  Applicable only to patients that have normocytic,

 Hemoglobin is the main content of the red blood normochromic RBCs
cells which is responsible for transporting oxygen  Hemoglobin: RBC count is multiplied to 3
from the lungs to the tissues and CO2 from the  Hematocrit: Hemoglobin is multiplied to 3
tissues to the lungs to be eliminated to the PHYSICAL METHOD (RULE OF THREE)
outside  RBC COUNT = 5.15 X 10^12/ L
 Hemoglobin is a conjugate protein that consists  HGB = 15.45 g/dL or 154.5 g/L
of 2 portions, the globin (a simple protein) and  HCT = 46.35% or 0.46 L/L
heme (organic compound in iron). Each COPPER SULFATE METHOD
hemoglobin molecule consists of the one  Principle: Gravimetric
molecule of globin (four globin chains) and four
 Based on the specific gravity of copper sulfate
molecules of heme
(CuSO4) – 1.053
 Hemoglobin may be found in the blood plasma;  Semi-quantitative; for mass assays
such a condition is referred to as
 Drop of blood is placed into the copper sulfate
hemoglobinemia. When the free hemoglobin in
solution and observed whether it will sink within
the plasma reaches a concentration between 30
15 seconds
and 300 mg/100mL of blood, hemoglobin is
 Sink: hemoglobin is the same as the specific
detected in the urine; such a condition is called
gravity of copper sulfate and has a value of
hemoglobinuria
 12.5g/dl
 In the normal adult, a hemoglobin concentration
 Float: hemoglobin value is <12.5 g/dl
is as follows:
DIRECT METHODS
o MALE: 13.5-17.5 g/dL
o FEMALE: 12.0-16.0 g/dL  Principle: Colorimetric
 Value may vary according to age, sex, and locality  Uses comparator block with an end color of
 Abnormal value as in hyperchromia (increased yellow brown
value) is found in polycythemia, dehydration and  Acid hematin method: 0.1N HCl (Sahli-Hellige
in changing from low to high altitude method)
 Hypochromia (decreased value) is seen in anemia  Alkali hematic method: 0.1N NaOH
METHODS FOR HEMOGLOBIN DETERMINATION: INDIRECT METHODS
 VAN SLYKE METHOD  Principle: Spectrophotometric
 KENNEDY’S AND WONG; ASSENDELT METHOD  Oxyhemoglobin method: aqueous ammonium
 PHYSICAL METHOD hydroxide
 COPPER SULFATE METHOD  Cyanmethemoglobin method: gold standard for
hemoglobin determination
 POCT (POINT OF CARE TESTING DEVICES)
ACID HEMATIN METHOD
 DIRECT
o ACID HEMATIN  Hemoglobin is converted to acid hematin with
o ALKALI HEMATIN dilute 0.1 N HCl and resulting brownish yellow
 INDIRECT color produced upon addition of distilled water is
o OXYHEMOGLOBIN METHOD matched with the color standard in the
o CYANMETHEMOLGOBIN METHOD comparator block
VAN SLYKE METHOD  MATERIALS:
o EDTA whole blood specimen
 Principle: Gasometric
o Distilled water
 Determines hemoglobin and oxygen saturation
o 0.1N HCl solution
 1 gram of hemoglobin = carries 1.34 mL of
o Hemoglobinometer
oxygen
 Comparator block
 Uses Van slyke apparatus or Natelson  Sahli pipette
microgasometer  Sahli graduated tube
KENNEDY’S AND WONG’S/ASSENDELT METHOD  Stirrer
 Principle: Chemical method  Dropper
 Measures iron bounded to hemoglobin
 1 gram of hemoglobin = 3.47mg of iron
 PROCEDURE (ACID HEMATIN METHOD)
1. Introduced 0.1 N HCl solution up to 2 mark of the
Sahli graduated tube
2. Using the Sahli pipette, draw blood up to 0.02 ml MATERIALS AND REAGENTS
or 20 uL mark. Observe the same technique as in  EDTA whole blood specimen
the use of WBC or RBC pipette.  Test tubes
3. Dispense the measured volume of the blood into  Pipettes
the graduated tube. Gently draw up and down to  Spectrophotometer
rinse the pipette and also to mix the resulting  Modified Drabkin's reagent
colloidal suspension.  Cyanmethemoglobin standard
4. Thoroughly mix with the use of the stirrer and o Contains 80 mg/dl of hgb; only standard used
allow to stand for 5 minutes. in hematology
5. Add distilled water drop by drop, stirring after  Parafilm
each addition and compare with the comparator COMPONENTS OF THE MODIFIED DRABKIN’S
block. REAGENT
6. When the resulting solution compares with the
COMPONENT USE/FUNCTION
color standard in the comparator block, get the
reading either from the grams or percentage Potassium ferricyanide converts to ferrous to
scale. ferric
7. Write your result. Potassium cyanide combines with
CYANMETHEMOGLOBIN (HiCN METHOD) methemoglobin
Dihydrogen potassium replaces potassium
 The cyanmethemoglobin is said to be the method
phosphate carbonate for faster
of choice for hemoglobin determination because:
reaction (3 mins)
o Cyanmethemoglobin is stable in dilutions
o Cyanmethemoglobin standard are readily Non-ionic improves cell lysis and
available detergents/surfactant decreases turbidity by
o All hemoglobin derivatives except CHONs
sulfhemoglobin are measured with their PREPARING THE STANDARD/CALIBRATION CURVE
conversion to cyanmethemoglobin
o The spectrum of cyanmethemoglobin is such
that other types of spectrophotometers can
be used for reading color intensity produced
 Hemoglobin iron is converted from ferrous to
ferric state to form methemoglobin by the action
of ferricyanide. Methemoglobin then combines
with potassium cyanide to produce the stable PROCEDURE
cyanmethemoglobin which is measured 1. Place 5 cc or 5 mL of Drabkin’s reagent into a test
spectrophotometrically at 540 nm tube.
 % Transmittance: proportional to hemoglobin 2. Using the Sahli pipette, draw blood to 0.02 cc or
concentration 20 uL mark. Make sure to wipe the outer wall of
the pipette, and then dispense the blood to the
test tube.
3. Gently draw up and down to rinse the pipette
and also to mix the resulting colloidal suspension.
4. Cover and mix well by inversion. Let it stand for 5
minutes.
5. Transfer the mixture to a cuvette.
6. Set the spectrophotometer to 100%
transmittance at the wavelength of 540 nm,
using a cyanmethemoglobin reagent as a blank
7. Continue reading the patient’s sample, and
record the percentage transmittance.
8. The HEMOGLOBIN CONCENTRATION IS
OBTAINED FROM A CALIBRATION CURVE
PREPARED WITH THE USE OF STANDARDS.
(Stable standard cyanmethemoglobin solution of
concentration representing 1:250 dilution of
whole blood containing 5, 10, and 15 grams of
hemoglobin/mL are available commercially for
calibration purposes and for periodic check-up on
the accuracy of the colorimeter
ERRORS IN CYANMETHEMOGLOBIN METHOD
 Usually caused by turbidity
 Drabkin’s reagent is light sensitive
 Contains cyanide which is highly toxic
ERROR SOLUTION
 Increased WBC  Sample must be
count: >20 x 10 9/L centrifuged and
Increased Platelet measure supernatant
count: >700x 10 9/L
 Lipemia  Add 0.01 mL of
patient’s plasma and
add to 5 mL of
reagent and use
solution as blank
 Hgb S or C (resistant  Make 1:2 dilution
to lysis) with distilled water
and multiply result
from the standard
curve by 2
 Abnormal globulins  Add 0.1 gram of
potassium carbonate
(only done in original
drabkin’s reagent)