HEMA311 01

HEMATOLOGY 1 LABORATORY TERM

WEEK 8: HEMOGLOBIN DETERMINATION (ACID HEMATIN AND CYANMETHEMOGLOBIN)

HEMOGLOBIN o Kasi ‘yong amount ng gases na kayang

dalhin ng hemoglobin is correlated to the
• Hb is the red iron-bearing protein contained within the hemoglobin concentration.
erythrocytes in normal blood. o Mas maraming gases na kayang dalhin
o Composed of heme molecule and globin ‘yong dugo ng patient, mas maraming
chain. hemoglobin.
o Heme molecules ➔ iron
o Hemoglobin gives the blood a red color 3. Chemical Method (Iron Content Method)
➢ Iron content of the blood.
• Function:
➢ It is the carrier of oxygen from the lungs to the 4. Colorimetric Methods
tissue where it readily releases this oxygen to A. Visual
the tissues and then returns to the lungs to a. Tallquist
combine with more oxygen. ▪ Old method
o Carry oxygen and carbon dioxide that is to ▪ Ipapatak ‘yong dugo ng patient sa isang
facilitate gas exchange. blotting paper. May standard or
• Four heme molecules are conjugated with 2 pairs of comparator na nasa paper din, ‘yong paper
globin chains. na standard may kulay. So, ‘yong dugo ng
• Heme represents the colored component. patient na ipinatak sa papel ico-compare
• Globin is a complex protein. sa standard na nasa papel din.
• The molecular weight of one Hb molecule is 64,458 b. Dare
Daltons. ▪ Blood in glass column (parang capillary
tube). Ilalawan ‘yong capillary tube and
Hemoglobin results are affected by the following factors: then ico-compare ‘yong kulay nong blood
na nasa capillary tube with those
standards.
1. Age
2. Sex
5. Acid hematin
3. Altitude of the locality
➢ Reagent ➔ 0.1 NHCL
▪ Converts hemoglobin to acid hematin
METHODS
➢ Sahlis Hellige
➢ Haldene
1. Copper Sulfate or Specific Gravity Method
➢ Haden – Hausser
➢ Blood Bank
➢ Newcomer
o Magtitimpla ng copper sulfate solution and
➢ Sahli- adams
then ipi-prick ‘yong patient and ipapatak
➢ Osgood
mo ‘yong blood doon sa copper sulfate
solution.
6. Alkali Hematin
o Titingnan mo kung lulutang ‘yong blood or
➢ Reagent ➔ Alkali ➔ NAOH
hindi. Kapag ‘yong drop of blood nag sink
▪ NAOH reagents convert hemoglobin to
to the bottom of the copper sulfate solution
alkali hematin.
pwede siya mag donate ng dugo, ibig
▪ But it cannot determine hemoglobin F
sabihin mataas ‘yong hemoglobin. Pero
a. Clegg and King
kapag ‘yong drop ng blood lumutang sa
copper sulfate solution and hindi nag sink,
7. Photoelectric
ibig sabihin mababa ang hemoglobin ng
1. Oxyhemoglobin method – best method for
donor.
measuring plasma hemoglobin.
➢ 20 ul blood + N/10 ammonia soln
2. Gasometric Method (Oxygen Capacity Method)
2. Cyanmethemoglobin or HiCN method the best
➢ Take advantage of the ability of the hemoglobin
method considered by hematologist
to carry gases.
➢ Drabkin’s reagent +20 ul blood read
➢ Gases that can bind to hemoglobin ➔ oxygen,
540nm
carbon dioxide, carbon monoxide.
➢ We do not determine hemoglobin itself directly.
Instead, we are determining gases.

BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE | HEMA311 | MIDTERM | LAYAN, JAYVHIE 1

 EXPLANATION OF THE TEST

• The hemoglobin determination test is used to:

1. Screen for disease associated with anemia
2. Determine the severity of anemia
3. Follow the response to treatment for anemia
4. Evaluate polycythemia
2. Using the Sahli pipette, draw blood up to 20 ul
CLINICAL SIGNIFICANCE

• Increased (hyperchromia) in:

▪ Polycythemia
▪ Dehydration
▪ in poorly compensated heart disease with
cyanosis changing from high to low altitudes.
• Decreased (oligochromia) in anemias.
3. Dispense the measured volume of the blood into the
Hemoglobinemia is the presence of free hemoglobin in graduated tube. Gently draw up and down to rinse the
the plasma. pipette.

• This is found:
a. severe infection
b. severe burns
c. poisoning with potassium chlorate and mushroom
d. paroxysmal hemoglobinuria
e. HTR ➔ Hemolytic Transfusion Reactions
4. Thoroughly mix with the use of the stirrer and allow to
stand for 5 minutes.
HEMOGLOBIN DETERMINATION
(ACID HEMATIN)

• Materials and Instrument

➢ Specimen
➢ Peripheral or unclotted venous blood

5. Add distilled water drop by drop, stirring after each

addition and compare with the comparator block.

➢ Hemoglobinometer

6. When the resulting solution compares with color standard

in the comparator block, get the reading either from the
PRINCIPLE OF ACID HEMATIN METHOD grams or percentage.

• Hemoglobin is converted to acid with dilute 0.1 N

HCL and resulting brownish yellow color produced
upon the addition of distilled water with the color
standard in the comparator block.

Procedure

1. Introduce 0.1 N HCL solution up to mark 2 of the Sahli

graduated tube.

BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE | HEMA311 | MIDTERM | LAYAN, JAYVHIE 2

 Patient Preparation

1. Explain the test purpose.

2. If your blood sample is being tested only for hemoglobin,
you can eat and drink normally before the test

Materials

• Anticoagulated blood
REFERENCE RANGES • Test tubes
(WORLD HEALTH ORGANIZATION) • Pipette
• Spectrophotometer
• Adult males: 13.0 - 17.0 gm/dl. • Drabkin’s Reagent
• Adult females (non-pregnant): 12.0 –15.0 gm/dl. • Cuvette
• Adult females (pregnant): 11.0 - 14.0 gm/dl.
• Children, 6-12 years: 11.5 - 15.5 gm/dl.
• Children, 6 months to 6 years: 11.0 –14.0 gm/dl.
• Children, 2 –6 months: 9.5 –14.0 gm/dl.
• At birth (full term): 13.6 –19.6 gm/dl.
• CRITICAL VALUES
▪ < 7 gm/dl (severe anemia)
▪ > 20 gm/dl (hyperviscosity)

CYANMETHEMOGLOBIN METHOD
Procedure
Hemoglobin
1. Place 5 ml of Drabkin’s reagent into a test tube
• Hb is the red iron-bearing protein contained within the 2. Using Sahli pipette, draw blood to 0.02 cc mark. Make
erythrocytes in normal blood. sure to wipe the outer wall of the pipette, and then
dispense the blood to the test tube.
Functions o 20 ul

• It is the carrier of oxygen from the lungs to the tissue

where it readily releases this oxygen to the tissues and
then returns to the lungs to combine with more oxygen.

The cyanmethemoglobin is said to be the method of

choice for hemoglobin determination because:
3. Gently draw up and down to rinse the pipette and also to
1. Cyanmethemoglobin is stable in dilutions. mix the resulting colloidal suspension.
2. Cyanmethemoglobin standard are readily available.
3. All hemoglobin derivatives except sulfhemoglobin are
measured with their conversion to cyanmethemoglobin.

Principle

• Hemoglobin iron is converted from ferrous to

ferric state to form methemoglobin by the action of
ferricyanide. 4. Cover and mix well by inversion. Let it stand for 5
➢ Normal hemoglobin charge ➔ 2+ minutes.
➢ Methemoglobin ➔ 3+ 5. Transfer the mixture to a cuvette
▪ Methemoglobin 3+ iron ➔ renders the
hemoglobin molecules useless.
Because oxygen can’t carry
methemoglobin.
➢ We deliberately convert hemoglobin into
methemoglobin from the ferrous state of F2+ to
ferric state FC3+.
➢ Facilitated by ➔ ferricyanide
• Methemoglobin then combines with potassium 6. Set the spectro to 100% transmittance at the
cyanide to produce the stable cyanmethemoglobin wavelength of 540 nm. Using a cyanmethemoglobin
which is measured spectrophotometrically. reagent blank.

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7. Continue reading the patient’s sample and record the
percentage transmittance.

• Transmittance: Abs = 2 - log (% T)

• Conc of unknown: abs of unknown x value of std
abs of std

Reference Value

• Male: 13.5 – 18.0 g/l

• Female: 12 15

Clinical Implications

1. Decreased hemoglobin levels are found in anemia

2. Increased Hgb levels are found in:
a. PV
b. CHF
c. COPD

Sources of Errors

1. Errors inherent in the sample

a. Blood sample collected through improper
venipuncture
b. Blood sample collected through improper skin or
capillary puncture technique
2. Errors inherent in the method
3. Errors inherent in the equipment
a. The accuracy of the equipment is not uniform
b. Unmatched cuvettes
c. Improper standardization of the photometer or
colorimeter
4. Operator’s error – human errors

• Human errors can be reduced by good training,

understanding the clinical significance of the test and the
necessity for a dependable method, adherence to
oral and written instructions and familiarity with the
equipment and with the sources of error.
• The technologist who is interested in the work will be less
prone to make errors than others.

BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE | HEMA311 | MIDTERM | LAYAN, JAYVHIE 4