CHAPTER 4: HEMOGLOBIN

STRUCTURE, SYNTHESIS,
FUNCTION AND
MEASUREMENT

6/26/2024 BSc, MPH, Mustafe M. Ahmed 1

 9.1 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).

6/26/2024 BSc, MPH, Mustafe M. Ahmed 2

Basic structure of hemoglobin molecule showing one
of the four heme chains that bind together to form the
Hb molecule

6/26/2024 BSc, MPH, Mustafe M. Ahmed 3

 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

6/26/2024 BSc, MPH, Mustafe M. Ahmed 4

6/26/2024 BSc, MPH, Mustafe M. Ahmed 5
 9.2. Hemoglobin synthesis

 Synthesis of heme and globin 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)

6/26/2024 BSc, MPH, Mustafe M. Ahmed 6

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

6/26/2024 BSc, MPH, Mustafe M. Ahmed 7

Protoporphyrin
Protoporphyrin III (9)
 Site of synthesis is the mitochondria in
RBC cytoplasm

6/26/2024 BSc, MPH, Mustafe M. Ahmed HAEME 8

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
 Beta chain
 Gamma chain
 delta chain

6/26/2024 BSc, MPH, Mustafe M. Ahmed 9

Globin

6/26/2024 BSc, MPH, Mustafe M. Ahmed 10

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

6/26/2024 BSc, MPH, Mustafe M. Ahmed 11

9.3 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

6/26/2024 BSc, MPH, Mustafe M. Ahmed 12

9.3 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 reversible

6/26/2024 BSc, MPH, Mustafe M. Ahmed 13

HGB/RBC Breakdown
 Aged (1% lost daily) or defective red cells are mainly
removed by splenic macrophages [by reticuloendothelial
system (RES)]

6/26/2024 BSc, MPH, Mustafe M. Ahmed 14

9.4. 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
d) Direct Read- Out
(2.)Visual comparative method
a)Sahli - Hellinge method
b)BMS Hemoglobinometr
( 3) Cu SO4 specific gravity
6/26/2024 BSc, MPH, Mustafe M. Ahmed 15
I. Spectrophotometric
1. Cyanmethemoglobin method
 EDTA whole blood or capillary samples is required
 Photometric semi or fully automated instruments used
 Drabkin’s reagent causes red cell lysis, release of
hemoglobin and conversion to cyanmethemoglobin
 Its pigment is measured photometrically at 540 nm
which is proportional to Hgb concentration
 All hemoglobin forms measured

EDTA
whole
blood

6/26/2024 BSc, MPH, Mustafe M. Ahmed 16

 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

6/26/2024 BSc, MPH, Mustafe M. Ahmed 17

6/26/2024 BSc, MPH, Mustafe M. Ahmed 18
6/26/2024 BSc, MPH, Mustafe M. Ahmed 19
6/26/2024 BSc, MPH, Mustafe M. Ahmed 20
 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.

6/26/2024 BSc, MPH, Mustafe M. Ahmed 21

 Advantages:
 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.

6/26/2024 BSc, MPH, Mustafe M. Ahmed 22

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
6/26/2024 BSc, MPH, Mustafe M. Ahmed 23
 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 higher than it is.

6/26/2024 BSc, MPH, Mustafe M. Ahmed 24

Hemoglobin Interferences

Haemolyzed plasma Lipemic plasma Icteric plasma

(haemolysis) (lipids) (bilirubin)
Normal plasma

6/26/2024 BSc, MPH, Mustafe M. Ahmed 25

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.

6/26/2024 BSc, MPH, Mustafe M. Ahmed 26

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.

6/26/2024 BSc, MPH, Mustafe M. Ahmed 27

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.

6/26/2024 BSc, MPH, Mustafe M. Ahmed 28

Hemoglobin - HemoCue®

Specimen cuvette

Electronic
calibration
red cuvette
6/26/2024 BSc, MPH, Mustafe M. Ahmed 29
Hemocue Cont’d
 Advantage HemoCue® system
 No dilution necessary
 the instrument reads the result when it is ready and
result is reported directly
 High accuracy
 No expensive instrument needed
 Disadvantage:
 Test cuvettes are expensive
 Finger prick technique must be good

6/26/2024 BSc, MPH, Mustafe M. Ahmed 30

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

6/26/2024 BSc, MPH, Mustafe M. Ahmed 31

Tips

 RBC count – total # of red

cells in millions/uL
 Hemoglobin –
concentration of Hb in red
X3=15.1
cells reported in 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
6/26/2024 BSc, MPH, Mustafe M. Ahmed 32
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

6/26/2024 BSc, MPH, Mustafe M. Ahmed 33

Exercise: Result Evaluation
3 adults

 Patient # 1: All results are normal

 Patient #2: Anemia (and leukocytosis)
 Patient # 3: Polycythemia/erythrocytosis
6/26/2024 BSc, MPH, Mustafe M. Ahmed 34
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

6/26/2024 BSc, MPH, Mustafe M. Ahmed 35
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
quantitation.
5. What is the clinical implication of hemoglobin
measurement in a sample of blood?

6/26/2024 BSc, MPH, Mustafe M. Ahmed 36

 Review Questions cont’d
6. List at least five preanalytic errors and their remedies in Hb
determination
7. List at least five possible sources of error and their
remedies in Hb determination using photomeric methods

6/26/2024 BSc, MPH, Mustafe M. Ahmed 37