Chapter-5

HEMOGLOBIN

1
 Structure of Hemoglobin
 Hemoglobin is normally present in red cells only
 Two primary structures
◦ Globin
◦ Heme which is composed of
 Protoporphyrin
 Iron
 The heme structure consists of a ring of C, H and N
atoms called Protoporphyrin IX with an atom of
Ferrous ( Fe 2+) iron attached ( ferroprotoporphyrin).
Basic structure of hemoglobin molecule showing
one of the four heme chains that bind together to
form the Hb molecule
 Iron
Compartments/Pools
 Iron is an essential component of hemoglobin
◦ Decreased tissue iron = cellular dysfunction
◦ Increased tissue iron = cellular destruction
◦ Regulated by absorption, not excretion

 Iron circulates in the plasma bound to

transferrin
Iron Compartments/Pools
 Hemoglobin synthesis

 Synthesis of heme and globin moieties proceeds

separately, though not entirely independently,
 the process is controlled by feedback mechanism
◦ e.g., formation of heme increases the synthesis globin
and lack of heme reduces globin synthesis.
 Heme molecule: a porphyrin ring with an iron atom at
its center (in a ferrous state)
◦ Porphyrins are tetrapyrroles

 the four pyrroles linked by a methane bridge

Hb synthesis cont’d
 Heme synthesis occurs largely in the
mitochondria by a series of biochemical reactions
involving a number of enzymes and co-factors.
Protoporphyrin
Protoporphyrin III
 Site of synthesis is the (9)
mitochondria in RBC cytoplasm

HAEME
 Globin
 Globin chains are composed of amino acids
arranged in a specific pattern
 Site of synthesis is the ribosomes
 4 normal chain types are produced
◦ Alpha chain composed of 141 aminoacid chains

◦ Beta 146 amino acid chains

◦ Gamma

◦ delta
Globin
Haemoglobin Molecule
 Consists of 4 globin chains + 4 heme groups
◦ heme groups are identical
◦ Different globin chains determine the hemoglobin type
 3 normal hemoglobin types (by 6 months of age)

Hgb A Hgb A2 Hgb F

alpha2beta2 alpha2 delta2 alpha2gamma2
Function of Hemoglobin
 Oxygen binds to central iron atom in heme
◦ Iron must be Fe+2 (ferrous) state to transport
oxygen
◦ Each hemoglobin molecule can carry up to 4
oxygen molecules
 Function of Hemoglobin
 Two normal Hgb forms
◦ Deoxyhemoglobin (Fe+2 without oxygen), in tissues
◦ Oxyhemoglobin (Fe+2 with oxygen), in lungs

 Two abnormal Hgb forms

◦ Methemoglobin (Fe+3, oxidized)
◦ Carboxyhemoglobin (Fe+2 with CO)
 Both are irreversible
 HGB/RBC Breakdown
 Aged (1% lost daily) or defective red cells are mainly removed
by splenic macrophages [by reticuloendothelial system (RES)]
 Methods of Hemoglobin
Measurement
• Is the measurement of concentration of Hgb in red
cells (whole blood)
• Hgb is reported in g/dL
• There are different methods

(1) Spectrophotometric
a) Cyanmethemoglobin
b) Hemo-Cue
c) Oxyhemoglobin(reading assignment)
(2.)Visual comparative method
a)Sahli - Hellinge method
b)BMS Hemoglobinometer(reading assignment)
( 3) Cu SO4 specific gravity
I. Spectrophotometric
1. Cyanmethemoglobin method
reference method
◦ EDTA whole blood or capillary samples
◦ Photometric semi or fully automated instruments
◦ Drabkin’s reagent causes red cell lysis, release of
hemoglobin and conversion to
cyanmethemoglobin
◦ Pigment is measured photometrically at 540 nm
◦ Proportional to Hgb concentration
◦ All hemoglobin forms measured EDTA
whole
blood
 Cont..
Principle:

 Blood is diluted in a solution of potassium ferricyanide

and potassium cyanide (Drabkin’s solution).

 The potassium ferricyanide oxidizes hemoglobins to

hemiglobin (Hi: methemoglobin) and the potassium
cyanide provides CN -- ions to form hemiglobin
cyanide (HiCN) which has a maximum absorption at
540nm.
 The absorbance of the solution is measured in a

photometer or spectrophotometer at 540nm and

compared with that of a standard HiCN solution
 Reagent
 Drabkins solution
◦ Potassium Ferricyanide -(Hexacyanoferrata)=K3Fe(CN)6
◦ Potassium cyanide
◦ Potassium dihydrogen phosphate
 Highly poisonous
◦ store securely in locked cupboard in light opaque
container wrapped in silver foil
 Procedure
 Pipet 20l of well mixed anticoagulated blood into
5ml Drapkin’s solution (1:251)
 Mix well and allow to stand at room temperature

for at least 5-10 minutes in the dark

 The absorbance is measured against reagent

blank at 540nm.
 the absorbance of an aliquot of HiCN standard is

measured at the same wavelength

Hb (g/dl) = At  Cst  DF
Ast  1000
 Calibration
 The Hemoglobin Standard is offered as a dry vial
containing a standardized amount of methemoglobin
prepared from human hemoglobin.
 Reconstituting the Hemoglobin Standard yields the
Cyanmethemoglobin Standard solution.
 The solution will yield an absorbance equivalent to
that of whole blood sample containing a hemoglobin
level of 18g/dl that has been diluted 1:251 with
Drabkin's solution.
 Dilutions of the Cyanmethemoglobin Standard
Solution with Drabkin's solution are used to prepare a
calibration curve as follows:
calibration curve
Expected Values:

 Adult males: 13-18g/dl

 Adult females: 11-16g/dl
 New borns: 14-23g/dl

 Note: reference values vary with age, sex,

physiologic condition, altitude, etc. Thus local
reference values should established.
 Advantages:
 Stable Hemiglobincyanide standard available to
calibrate instrument
 Convenient method
 Readily available and stable standard solution
(readings need not be made immediately after
dilution)
 All forms of hemoglobin except sulfhemoglobin (SHb)
are readily converted to HiCN.
Sources of error when measuring
Hemoglobin photometrically

 Not measuring the correct volume of blood due to

poor technique or using a wet or chipped pipette.
 When using anticoaglulated venous blood, not
mixing the sample sufficiently.
 Not ensuring that the optical surfaces of a cuvette
are clean and dry
 air bubbles in the solution to be measured
 Wrong wavelength
 Improper instrument calibration
 Reagent exposed to light
 Sources of error cont’d

 Lipemia
 Extremely high WBC count causes cloudiness
 Presence of abnormal Hemoglobins
 Presence of abnormal proteins

Note:
 Lipemia causes an increase in the Hb result due to

cloudiness in the solution read by the

spectrophotometer.
◦ In lipemia, centrifugation can clear the specimen and
the supernatant reading will be accurate.

 Abnormal Hemoglobins or proteins are not lysed by

the reagent, so again the solution is cloudier which
makes the instrument read the Hemoglobin result
Hemoglobin Interferences

Haemolyzed plasma Lipemic plasma Icteric plasma

(haemolysis) (lipids) (bilirubin)
Normal
plasma
2. Hemoglobin -
HemoCue®
 HemoCue® photometer
◦ Uses dry reagent system (cuvettes)
◦ Determines concentration of azide
methemoglobin photometrically
◦ Electronic check and whole blood control samples
must be run to monitor instrument function and
reagent
Principle

 The hemoglobin concentration in a fresh

capillary or anticoagulated blood sample (EDTA
preferred) is determined photometrically using a
dry reagent system. The red cells are lysed and
hemoglobin is converted to azidemethemoglobin
by sodium nitrite and sodium azide. This method
of HGB measurement is a widely used point-of-
care test.
HemoCue® cont’d

 Procedure
◦ Turn on HemoCue® instrument
◦ Run electronic calibration check (red control
cuvette)
◦ Fill specimen cuvette with EDTA or capillary blood
in a continuous process without bubbles.
◦ Place cuvette in instrument, insert to ‘measure’
position
◦ Hemoglobin result will be displayed in g/dL
Hemoglobin - HemoCue®

Specimen
cuvette

Electronic
calibration
red cuvette
Hemocue Cont’d
 Advantage HemoCue® system
◦ No dilution necessary
◦ the instrument reads the result when it is ready
(no need to let stand for lysing of RBCs) and result
is reported directly eliminating errors in reading
from a calibration chart
◦ High accuracy
◦ No expensive instrument needed
 Disadvantage:
◦ Test cuvettes are expensive
◦ Finger prick technique must be good
Sources of error HemoCue® method

◦ Failure to properly collect the blood sample if

done as a capillary collection
 Blood not collected from a free flowing finger
prick
◦ Failure to fill cuvette properly
◦ If not, read within 10 minutes
of collection
◦ Others?
Quality control

 Spectrophotometer/photometer
◦ Whole blood control must be performed
◦ Performed in duplicate; should match within
plus/minus 0.5 g/dl
◦ Calibrator for making a standard curve
 HemoCue®
◦ Calibrator cartridge
◦ Whole blood control
II. Visual comparative
method
1. Sahli-Hellige
 Is not recommended because of its unacceptable
imprecission and inaccuracy

Principle
 20 L of blood is mixed in a tube containing

0.1mol/l HCl which lyses the RBC and converts the

hemoglobin to Acid Hematin
 After 10 minutes ( or more ), 0.1mol/l HCl or water

is added drop by drop, with mixing , until the color

of the solution matches the color of the glass
standard positioned alongside the dilution tube
 Sahli-Hellige cont’d
 The concentration of Hemoglobin is read from the
graduated scale on the dilution tube

Disadvantage
 fading of the color glass standard and difficulty in
matching it to the acid hematin solution.
 Conversion to acid –hematin is slow
◦ Because of this, all red cells may not lyse and Hb
may not converted to Acid Hematin in specified
lesser time resulting a falsely decreased Hgb value
 Acid Hematin is unstable
.
 Disadvantage cont’d
 HbF is not converted to acid hematin and therefore
the Sahli method is not suitable for measuring
hemoglobin levels in infants up to 3 months.
 Difficulty in matching the acid hematin with the glass
standard (color matching is subjective/personal bias)
 Interpersonal difference in reading the endpoint of
dilution.
 as a result the Sahli-Hellige method is not
recommended for Hb determination
Materials:
 Sahli hemoglobinometer
 Sahli pipet
 Stirring glass rod
 Dropping pipet
 Absorbent cotton
 0.1N HCl
Procedure:

 Fill the graduated tube to the ''20'' mark of the

red graduation or to the 3g/l mark of the yellow
graduation with 0.1N HCl.
 Draw venous or capillary blood to the 0.02ml
mark of the Sahli pipet. Do not allow air bubbles
to enter into the Sahli pipet.
◦ With EDTA anticoagulated venous blood ensure
that it is well mixed by inverting the tube
repeatedly for about 1 minute immediately before
pipetting it.
◦ If using capillary blood, wipe away the first drop
of blood from the finger.
Cont..

 Wipe the outside of the pipet with absorbent

paper.
◦ Check that the blood is still on the “20” mark.
 Blow the blood from the pipet into the graduated
tube containing the 0.1 hydrochloricacid solution.
 Rinse the pipet by drawing and blowing out the
acid solution 3 times.
Cont..
 Place the graduated tube in the hemoglobinometer
stand facing a window.
 Allow 10 minutes for RBC lysis and formation of acid
hematin
 Compare the color of the tube containing diluted
blood with the color of the standard
 If the color of the diluted sample is darker than that
of the reference, continue to dilute by adding 0.1N
HCl or distilled water drop by drop.
 Stir with the glass rod after adding each drop.
 Remove the rod and compare the color of the tube
with the standard columns.
 Stop when the colors match.
Cont..
 Note the mark reached.
 Depending on the type of hemoglobinometer, this
gives the hemoglobin concentration either in g/dl
or as a percentage of ''normal''.
 To convert percentages to g/dl, multiply the
reading by 0.146.
III. Copper Sulphate Densitometery

 This is a qualitative method based on the

capacity of a standard solution of copper sulphate
to cause the suspension or sinking of a drop of
blood.
 The measurement of specific gravity of a sample
of blood corresponds to its hemoglobin
concentration.
 The method is routinely utilized in some blood
banking laboratories while screening blood
donors for the presence of anemia.
Sources of Error
 Pre-analytical errors are a common cause of
inaccurate results
wrong patient identification
improper venous blood sample collection
Wrong anticoagulant type and concentration
Failure to mix blood with anticoagulant
Mislabeling
improper capillary blood collection
Clotted sample
Hemoconcentration
Hemodilution

Do controls detect specimen collection errors?

Sources of Error
 Technical errors (failure to adhere to SOP)
 Post analytic errors
 Quality Control
 Control samples monitor the correct functioning of
equipment, stability of reagents and testing
technique
 Controls do not detect specimen collection errors
 Control samples with known assayed values are
used to check result reliability
◦ Controls detect invalid results caused by errors in
testing technique, reagents or instrument
malfunction
 Tips

 RBC count – total # of

red cells in millions/uL
 Hemoglobin –
concentration of Hb in
red cells reported in X3=15.1

g/dL X3=45.0

 Hematocrit –
percentage (%) of red
cells in a known
volume of whole blood
 RBC count, HGB and
HCT values parallel
each other
 RBC Count, HGB, HCT

 Each health institution should establish its own

reference ranges
 Significance
◦ Decreased RBC, HGB and/or HCT values….Anemia
 Decreased production, increased
loss/destruction
◦ Increased RBC, HGB and/or HCT
values….Polycythemia
 Increased production
 Critical values: HGB <7.0 or >18.5 g/dL
Exercise: Result Evaluation
3 adults

 Patient # 1: All results are normal

 Patient #2: Anemia (and leukocytosis)
 Patient # 3: Polycythemia/erythrocytosis
RBC Count, HGB, HCT
Correlation
 Relationship: Hb x 3 = HCT + 3%
RBC x 3 = Hb or RBC x 9 = HCT
 Used to estimate values or check data correlation
 ‘Rules’ only apply if red cells are normal in size
and hgb content
Which parameter does not correlate?
RBC = 4.00 million/cmm
Hb = 14.0 g/dl
HCT = 36.0%

 Hb error; RBC and HCT correlate