CHAPTER 9:

HEMOGLOBIN STRUCTURE,
SYNTHESIS, FUNCTION AND
MEASUREMENT

1
 Objectives
 At the end of this chapter the
students will be able to:
 Define hemoglobin (Hgb).
 Diagrammatically illustrate the structure of Hgb.
 Describe the biosynthesis of heme and globin
moieties of hemoglobin.
 Explain the functions of Hgb.
 State the principles of Hgb estimation in clinical
practice.
 Explain the principle of the cyanmethemoglobin
method of Hgb determination.

2
 Objective cont’d...
 Carry out calibration for the cyanmethemoglobin
method of Hgb determination.
 Perform Hgb quantization on a sample of blood using
the cyanmethemoglobin method.
 List the advantages of the cyanmethemoglobin
method of Hgb determination.
 Explain the principle of the Hemocue method of Hgb
quantization.
 Describe the advantages and disadvantages of the
Hemocue method.
 Perform Hgb quantization on a sample of blood using
the Sahli-Hellige method.
3
 Objective cont’d…
 Explain why the Sahli-Hellige method is considered
unreliable.
 Describe sources of specimen collection errors (pre-
analytic) that can cause inaccurate results.
 List the sources of error in various hemoglobin
determination techniques.
 Discuss reference ranges and the significance of
hemoglobin values on the basis of age and sex.
 Explain the correlation between red blood cell count,
hemoglobin and hematocrit results.
 Discuss quality control and checks utilized to establish
test validity and prevent erroneous results.
4
 9.1. Structure of Hemoglobin
 Hgb is normally present in red cells.
 The two primary structures are: -
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 ( Fe2+ ) iron attached
(ferroprotoporphyrin). 5
Basic structure of Hgb molecule showing
one of the four heme chains that bind
together to form the Hgb molecule

6
 General chemical composition
and configuration of Hgb
 Normal adult Hgb (Hgb-A) consists of four
heme groups and four polypeptide chains
with a total of 574 amino acids.
 The polypeptide chains are organized into
2-alpha chains and 2-beta chains.
 Each of the chains has an attached heme
group.
 Normal adult Hgb has 141 amino acids in
each of the alpha chains and 146 amino
acids in each of the beta chains. 7
 General Cont`d…
 The native configuration of the Hgb
molecule, the four hemes and four
polypeptide chains are assembled in a very
specific spatial (special) configuration.
 Each of the four chains in the molecule
coils into eight helices, forming an egg-
shaped molecule with a central cavity.
 In the process of the binding of the first
heme group to a molecule of oxygen, a
change in the overall configuration of the
Hgb molecule occurs. 8
 9.2. 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 of globin and lack of
heme reduces globin synthesis.

9
 Hemoglobin synthesis Cont`d..
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.
 Heme synthesis occurs largely in the
mitochondria by a series of biochemical
reactions involving a number of enzymes
and co-factors. 10
The Hgb molecule: Note:- four heme
molecules tucked (fold) inside globin chains
β2
Heme β1

α1
α1
11
 Heme synthesis Cont`d..
 The process begins in the mitochondrion with the
condensation of succinyl-CoA and (+) glycine to
form (Delta) 5-aminolevulinic acid.
 This initial condensation reaction occurs in the
mitochondria and requires vitamin B6.
 After a series of steps in the cytoplasm produce
coproporphyrinogen III, which reenters the
mitochondrion.
 In the final enzymatic steps, iron is inserted into
the ring structure of protoporphyrin IX to
produce heme. (clinical hemato. Theory & practice 5 ed.2011)
th
12
 Protoporphyrin
 Site of synthesis of HEME in the Protoporphyrin
mitochondria of RBC cytoplasm. III (9)

HAEME 13
 Globin
 Globin chains are composed of amino
acids arranged in a specific pattern.
 Site of synthesis is the ribosome's.
 4 normal chain types are produced.
Alpha chain composed of 141
amino acid chains.
Beta chain146 amino acid chains.
Gamma.
 Delta.
14
Hgb synthesis Cont’d..

15
 Hgb 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 (α2 β2) Hgb- A2 (α2 δ2) Hgb- F (α2 ϒ2

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 Iron
 Iron is an essential component of Hgb.
The decreased of tissue iron = cellular
dysfunction.
The increased of tissue iron = cellular
destruction.
Regulated by absorption, not excretion.
 The liver and reticuloendothelial
macrophages function as major iron stores.
 Iron circulates in the plasma bound to
transferrin. 17
Iron Compartments/Pools

18
 9.3 Function of Hgb
 Oxygen binds to central iron atom in heme.
Iron must be Fe2+ (ferrous) state to transport
oxygen.
Each Hgb molecule can carry up to 4
oxygen molecules.

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 Function of Hgb Cont’d..
 Two normal Hgb forms: -
Deoxyhemoglobin (Fe2+ without
oxygen), in tissues.
Oxyhemoglobin (Fe2+ with oxygen), in
lungs.
 Two abnormal Hgb forms: -
Methemoglobin (Fe3+ ,oxidized).
Carboxyhemoglobin (Fe2+ with CO).
Both are reversible.
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 Hgb/RBC Breakdown
 Aged RBC (1% lost daily) or defective RBC are
mainly removed by splenic macrophages [by
reticuloendothelial system (RES)].

21
 9.4. Methods of Hgb 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. c) Hemo-Cue.
(b)Oxyhemoglobin. d) Direct Read- Out.
(2.)Visual comparative methods
a)Sahli - Hellinge method.
b)BMS Hemoglobinometr.
(3) Cu SO4 specific gravity 22
 I. Spectrophotometric
1. Cyanmethemoglobin method
 ICSH recommended reference method.
EDTA anticoagulated whole blood or capillary
samples.
 All Hgb forms are measured.

EDTA
whole
blood

23
 Spectrophotometric Cont`d..
Principle: -
 Blood is diluted in a solution of potassium
ferricyanide and potassium cyanide
(Drabkins solution).
The potassium ferricyanide oxidizes

hemoglobin's to hemoglobin (Hi:

Methemoglobin) and the potassium
cyanide provides CN -- ions to form
hemoglobin cyanide (HiCN) which has
a maximum absorption at 540nm. 24
 Spectrophotometric Cont`d..
 Finally 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. 25
 Procedure
1. Pipette 20l of well mixed anticoagulated
blood into 5ml Drabkins solution (1:251).
2. Mix well and allow to stand at room temperature
for at least 5-10 minutes in the dark.
3. The absorbance is measured against
reagent blank at 540nm.
4. The absorbance of an aliquot of HiCN standard
is measured at the same wavelength.
Hgb (g/dl) = At  Cst  DF
Ast  1000
26
 Calibration
 The Hgb Standard is offered as a dry vial
containing a standardized amount of
Methemoglobin prepared from human Hgb.
 Reconstituting the Hgb Standard yields the
Cyanmethemoglobin Standard solution.
 The solution will yield an absorbance equivalent
to that of whole blood sample containing a Hgb
level of 18g/dl that has been diluted 1:251 with
Drabkins solution.
 Dilutions of the Cyanmethemoglobin Standard
Solution with Drabkins solution are used to
prepare a calibration curve as follows : 27
Calibration curve

28
 Reference range
 Adult males: 13-18g/dl
 Adult females: 11-16g/dl
 Newborns: 14-23g/dl
 Note: Reference values vary with age,
sex, physiologic condition, altitude, etc.
Thus local reference values should
established.

29
 Advantages:
 Stable Hemoglobin cyanide 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
sulfohemoglobin (S-Hgb) are readily converted
to HiCN.
30
 Sources of error in measuring
Hgb photo metrically
 Not measuring the correct volume of blood
due to poor technique or using a wet or
chipped pipette.
 When using anticoagulated venous blood,
not mixing the sample sufficiently.
 Not ensuring that the optical surfaces of a
cuvet are clean and dry.
 Air bubbles in the solution to be measured.
31
 Sources of error cont’d…
 Wrong wavelength.
 Improper instrument calibration.
 Reagent exposed to light.
 Extremely high WBC count
causes cloudiness.

32
 Sources of error cont’d…
 Lipemia: - causes an increase in the Hgb
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 Hgb or proteins are not lyses by
the reagent, so again the solution is cloudier
which makes the instrument read the Hgb
result higher than it is. 33
Hemoglobin Interferences

Hemolyzes Lipemic Icteric

plasma plasma plasma
Normal
(hemolysis) (lipids) (Bilirubin)
plasma

34
 2. Hemoglobin - HemoCue®
HemoCue® photometer.
 Uses dry reagent system (cuvetes).
 Determines concentration of azide
Methemoglobin photo metrically.
 Electronic check and whole blood control
samples must be run to monitor instrument
function and reagent.

35
 Principle
 The Hgb concentration in a fresh capillary
or anticoagulated blood sample (EDTA
preferred) is determined photo metrically
using a dry reagent system.
The red cells are lyzed and Hgb is
converted to azidemethemoglobin by
sodium nitrite and sodium azide.
This method of Hgb measurement is a
widely used point-of-care test.
36
 HemoCue® cont’d..
Procedure
1.Turn on HemoCue® instrument.
2. Run electronic calibration check (red
control cuvette).
3. Fill specimen cuvette with EDTA or
capillary blood in a continuous process
without bubbles.
4. Place cuvette in instrument, insert to
‘measure’ position.
 Hgb result will be displayed in g/dl.
37
Hemoglobin - HemoCue®

Specimen cuvette

Electronic
calibration red
cuvette
38
 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)
Result is reported directly by eliminating
errors in reading from a calibration chart.
High accuracy.
No expensive instrument needed.
39
 Hemocue Cont’d..
Disadvantage:
 Test cuvetes 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. 40
 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.
41
 3.Oxyhemoglobin Method
 The simplest and quickest method for general use
with a photoelectric.
 Colorimeter but no longer widely used.

Method:
 A 1:251 dilution of blood is made with 0.007N
NH4OH with thorough shaking to ensure mixing
and oxygenation of Hgb.
 The absorbance of the solution is read at 540nm
in a photo-/spectrophotometer against a 0.007N
NH4OH solution as a blank.
42
 Oxyhemoglobin Cont`d..
Disadvantage:
 Lack of a stable Oxyhemoglobin standard.
 Does not measure Carboxyhemoglobin,
hemoglobin or sulphohemoglobin.
II. Visual comparative method
1. Sahli-Hellige
 Is not recommended because of its
unacceptable imprecision and inaccuracy.

43
 Sahli-Hellige method Cont`d..
 Materials and Reagent
Sahli Hemoglobinometr .
Stirring glass rod.

Sahli pipette.

Dropping pipette.

Absorbent cotton.

0.1N HCl.

Principle: -20 L of blood is mixed in a tube

containing 0.1mol/l HCl which lyses the RBC
and converts the Hgb to acid hematin. 44
 Visual comparative Cont`d..
 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 & the concentration
is read from the graduated scale on the dilution
tube.
 Procedure
1. 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. 45
 Procedure Cont`d..
2. Draw venous or capillary blood to the 20μl
mark of the Sahli pipette. Do not allow air
bubbles to enter into the Sahli pipette.
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.

46
 Procedure Cont..
3. Wipe the outside of the pipette with
absorbent paper.
Check that the blood is still on the “20” mark.
4. Blow the blood from the pipette into the
graduated tube containing the 0.1N
hydrochloric acid solution.
5. Rinse the pipette by drawing and blowing
out the acid solution 3 times.
6. Place the graduated tube in the
Hemoglobinometr stand facing a window.
47
 Procedure Cont..
7. Allow 10 minutes for RBC lysis and formation
of acid hematin
8. 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.
9. Stir with the glass rod after adding each drop.
10. Remove the rod and compare the color of the
tube with the standard columns.
48
 Procedure Cont..
11. Stop when the colors match.
12. Note the mark reached.
 Depending on the type of Hemoglobinometr,
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.
Disadvantage of Sahli-Hellige method
 Fading of the color glass standard and
difficulty in matching it to the acid hematin
solution. 49
 Disadvantage Cont`d..
 Hgb-F is not converted to acid hematin and
therefore the Sahli method is not suitable for
measuring hemoglobin levels in infants up to 3
months.
 Conversion to acid –hematin is slow.
Because of this, all red cells may not lyses and
Hgb may not converted to Acid Hematin in
specified lesser time resulting a falsely
decreased Hgb value.

50
 Disadvantage cont’d…
 Acid Hematin is unstable.
 Interpersonal difference in
reading the endpoint of
dilution.
 As a result the Sahli-Hellige
method is not recommended
for Hgb determination.

51
 2. Alkaline Hematin Method
 A useful ancillary method under special
circumstances as it gives a true estimate of
total Hgb even if Hgb-CO, Hi or S-Hgb is
present.
Disadvantage:
 Certain forms of Hgb are resistant to alkali
denaturation, in particular, Hgb-F and Hgb
Bart.
 More cumbersome and less accurate than the
HiCN or HbO2 methods and thus is unsuitable
52
 3. BMS Hemoglobinometr
 Requires no dilution of blood.
 Use in clinics with a few Hgb tests are performed.
 Principle: - A drop of blood is mixed with a
saponin impregnated stick. This lyses the red cells
giving a clear solution of Hgb.
 The absorption of light by the haemolysed patient
blood sample ( existing in one half of the field of
view of the meter ) is matched with a scale on the
meter (that of the standard in the other half).
 The Hgb value in g/dl is obtained from a scale on
the meter. 53
 WHO Hgb colour scale
 The method is based on comparing the
colour of a drop of blood absorbed on a
particular type of chromatographic paper
against a printed scale of colours
corresponding to different levels of Hgb.

54
 III. Copper Sulphate Densitometry
 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. 55
 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.
56
 Sources of Error Cont`d..
 Clotted sample.
 Hemoconcentration.
 Hemodilution.
 Hemolysis does not cause hemoglobin
error.
 Technical errors (failure to adhere to SOP).
 Post analytic errors.

Do controls detect specimen

collection errors? 57
 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. 58
 Tips
 RBC count – total # of
red cells in millions/μl.
 Hemoglobin –
concentration of Hgb in
red cells reported in g/dl. X3=15.1

X3=45.0
 Hematocrit – percentage
(%) of red cells in a
known volume of whole /3=15.1

blood.
 RBC count, Hgb and
HCT values are correlated
to each other.
59
 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.
 Critical values: Hgb <7.0 or >18.5 g/dl.
60
 Exercise: Result Evaluation
3 adults

 Patient # 1: All results are normal.

 Patient #2: Anemia (and Leucocytosis).
 Patient # 3: Polycythemia/Erythrocytosis.

61
 RBC Count, Hgb, HCT Correlation
 Relationship: Hgb x 3 = HCT + 3%.
RBC x 3 = Hgb 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/mm3.
Hgb = 14.0 g/dl.
HCT = 36.0%.

 Hgb error; RBC and HCT 62

 Summary
 Biochemistry of the Hgb molecule.
 Cyanmethemoglobin method (or
modifications) of hemoglobin
determination, including specimen
requirements.
 Sources of specimen collection error (pre-
analytic) that can cause inaccurate results.
 Potential sources of error when measuring
hemoglobin photo metrically (i.e., substances that
interfere with photometric measurements).
 Quality control and checks utilized to establish
test validity and prevent erroneous results. 63
 Review Questions
1. Describe synthesis of the heme and globin moieties of
hemoglobin?
2. Summarize the functions of hemoglobin in the body.
3. What are the two most commonly applied color
comparison methods for measurement of hemoglobin in
a sample of blood? Write the test principle of each of
these methods.
4. Compare and contrast (in terms of accuracy, advantage,
drawbacks, etc.) the two routine methods of hemoglobin
quantization.

64
 Review Questions cont’d..

5. What is the clinical implication of hemoglobin

measurement in a sample of blood?
6. List at least five pre analytic errors and their
remedies in Hgb determination?
7. List at least five possible sources of error and
their remedies in Hgb determination using
photometric methods?

65