Hematology I –Hemoglobin  Physiologic forms

Hemoglobin  OXYHEMOGLOBIN- bright red; collected from

arteries. 
 Hemoglobin or haemoglobin, abbreviated Hb or  DEOXYHEMOGLOBIN- dark red; collected from
Hgb, is the iron-containing oxygen-transport veins. 
metalloprotein in the red blood cells 
 Synthesized from proerythroblast stage up to the
reticulocyte stage. 
 RBC = 33% volume, 97% dry weight. 
 Alpha chain – 141 amino acids (16th chromosome) 
 Beta chain – 146 amino acids (11th chromosome) 
 Hgb only appears visually in polychromatic
erythroblast stage.
Hemoglobin synthesis

 HEME - mitochondria 
 GLOBIN – RIBOSOMES IN THE ROUGH
  ENDOPLASMIC RETICULUM 
 Proerthroblast to reticulocyte stage. 

 Molecular weight: 64,500kda 

 Normal range:
 13.5 – 18.0g/dl 
   12.0 – 15.0g/dl
 Composition: 
 4 globin chains (2 alpha & 2 beta) 
TYPES OF GLOBIN CHAINS
 4 heme groups 
 1 2,3-diphosphoglycerate   Alpha (α) - Encoded in the 16th chromosome and
 Alpha chain – 141 amino acids (16th chromosome)  is composed of 141 amino acids. 
 Beta chain – 146 amino acids (11th chromosome)    Beta (β) –Encoded in the 11th chromosome and is
composed of 146 amino acids. 
Hemoglobin - function
 Zeta (Z) – Embryonic analogue of the Alpha chain. 
 Major function is to transport oxygen to the  Epsilon (ε) – Embryonic analogue of Beta, Delta(δ),
tissues.  and Gamma(γ) chains. 
 2,3 –dpg – only present in deoxygenated blood.    Heme doesn’t have variations, only the globin
 Each molecule of hemoglobin is capable of binding has. 
to up to 4 oxygen molecules. 
Normal hemoglobin
 The attachment of the 2,3DPG between the 2 beta
globin chains (center) will cause conformational  Embryonic hemoglobin 
changes on of the globin chains. Those changes will  Hb-gower 1 – 2 zeta & 2 epsilon 
reduce the affinity of hemoglobin to oxygen.   Hb-gower 2 – 2 alpha & 2 epsilon 
 Hb-Portland  1 – 2 zeta & 2 gamma 
 
 Fetal hemoglobin (Hb-F) -2 ALPHA & 2 GAMMA 
 Normal Adult hemoglobin (hba or hba1) – 2 alpha &
2 beta 
 Embryonic - Found in normal human embryo and Abnormal hemoglobin
foetuses with a gestational age of less than 3
1. Hemoglobin S – Causes sickling of RBC under
months (absent at birth) 
conditions of reduced O2 concentration. In
 Fetal – major HB found in foetuses and newborns;
homozygous state, it causes sickle cell anemia.
Has more affinity to oxygen than adult hemoglobin;
Heterozygous shows sickle cell trait. The glutamic
present in adults (0.1 to 0.5%) 
acid is replaced by valine in the 6th position of beta
 At birth (HbF=80%, HbA=20%) 
chain.
  2. Hemoglobin C – Often seen in combination with
HbS. Glutamic acid is replaced by lysine in the
 Hemoglobin a2 (hba2) – 2 alpha & 2 delta  6th position of beta chain.
 Hemoglobin a2 (hba3) – 2 alpha & 2 delta  3. Hemoglobin D – Glutamic acid in the 121st beta
 Glycosylated hemoglobin – hba1 with attached chain is replaced by glutamine.
sugar on its terminal end.  4. Hemoglobin G-Philadephia – The 68th amino acid
 Hba1a (1%) - Fructose 1,6 phosphate is in the alpha chain is replaced by Lysine
attached to the terminal end of the  globin 5. Hemoglobin E – Ressemble thalassemia trait. Most
chain (Valine)  common hemoglobinopathy in the Philippines.
 hba1b (2%) - Unknown carbohydrate is Glutamic acid is replaced by lysine in the position 26
attached to the terminal end of the globin of beta chains.
chain (Valine)  6. Hemoglobin H – Consists of 4 beta chains;
 Hba1c (3%) - Glucose is attached to the Associated with alpha thalassemia.
terminal amino acid valine of the chain of 7. Hemoglobin Bart’s – Abnormal variant of HbF;
hemoglobin.   Consists of 4 gamma chains.
 When sugars are present in the blood in high 8. Hemoglobin Sydney – Valine is replaced by alanine
concentration (400mg/dL), the glucose may bind to at position 11 of the beta chain.
the terminal end of HbA1  9. Hemoglobin Milwaukee – Valine is replaced by
 HbA2 – A normal variant of hemoglobin A that glutamic acid at position 11 of the beta chain.
consists of two alpha and two delta chains and is 10. Hemoglobin Bristol – Valine is replaced with
found at low levels in normal human 1.5-2.5%.  aspartic acid at the terminal end of the beta chain.
 HbA3 – Degradation product of HbA2  11. Hemoglobin M – Associated with
 HbA1c – Used to monitor diabetes.   methemoglobinemia.
Normal hemoglobin  Hemoglobin with an iron in the ferric state rather
than ferrous. 

Hemoglobin pigments
 
1. Carboxyhemoglobin (HbCO) – Formed by the
combination of hemoglobin with carbon monoxide
which is not capable of binding with oxygen.
2. Methemoglobin (Hi) – Also known as hemiglobin or
ferrihemoglobin; Hemoglobin with iron in the ferric
state rather than ferrous state. May be acquired or
inherited. Most of the time, it is acquired and
primarily due to exposure to certain drugs and
chemicals.
3. Sulfhemoglobin (SHb) – Not normally found in
blood but when present, its formation is irreversible
and remains for the life of carrier red blood cell.
Exact nature is still unknown but is thought to be
caused be exposure to certain drugs and chemicals
such as sulfonamides and aromatic amines.
 If hemoglobin is converted to an abnormal pigment, PROTEINS ASSOCIATED WITH HEMOGLOBIN
it is no longer capable of oxygen transport, and if breakdown
impaired severe enough, a condition called hypoxia
PROTEIN FUNCTION
or cyanosis occurs. 
 Carboxy - Hemoglobin has an affinity of more than
Haptoglobin Transport protein for
200 times to carbon monoxide than with oxygen.  
hemoglobin
 Methemoglobin - Nitrates, nitrites, quinines, and
Hemopexin Transport protein for heme
chlorates. 
 Sulfhemoglobin -  Incapable of transporting oxygen
Transferrin Transport protein for iron
but can combine with carbon monoxide to form
(ferric state)
carboxysulfhemoglobin (SHbCO). 
Albumin Protein that can bind to
Degradation of hemoglobin hemoglobin, heme, and iron
Ferritin and Protein that can bind to free
 After the removal of red blood cell from the Hemosiderin iron for storage
circulation, hemoglobin is broken down by the
 
macrophage of the mononuclear phagocyte
system into 3 constituents, iron, protoporphyrin, Measurement of hemoglobin breakdown
and globin.  
1. Estimation of exhaled carbon monoxide
 The iron goes into the storage (as ferritin or
2. Estimation of carboxyhemoglobin
hemosiderin) or sent back to the bone marrow to
3. Estimation of fecal bilirubin
be reused. 
 Globin may be degraded and returned to the
 CO is one of the byproducts of hemoglobin
amino acid pool of the body (In the liver or bone
breakdown. Severity of hemolysis is directly
marrow) to be reused. 
proportional to the amount of carbon monoxide
 The protoporphyrin ring is split, converted to
exhaled. 
biliverdin, heme, and co and sent to the liver to be
 Before exhaling the carbon monoxide, it will first be
excreted. 
transported to the lungs by the hemoglobin
 Ferritin and hemosiderin – Storage forms of iron
(carboxyhemoglobin) 
found in the bone marrow, liver, spleen and
 The biliverdin that turned to bilirubin can also be
skeletal muscle. 
measured. 
 Ferritin – Protein that binds to iron in the ferric
state.  Tests for abnormal hemoglobin derivatives and
 Hemosiderin – More complex storage form of iron abnormal hemoglobin
but only exists if the storage capacity of ferritin is
 Shaking test
exceeded. 
 blood is shaken for 15 minutes and the color
  formed will be measured. 
 Bright red=Oxyhemoglobin, Chocolate
brown=Hemiglobin/Methemoglobin, Cherry
  red=Carboxyhemoglobin, Mauve
lavender=Sulfhemoglobin. 
 Katayama’s test – Method for detecting
carboxyhemoglobin. 
 Blood + ammonium sulfide 
 Can detect as little as 10% saturation. (Result:
Rose red: + for Carboxyhb, Greenish brown= -
for Carboxyhb) 

 
 Spectrophotometric method – based on the  technique used to separate hemoglobins in order to
principle that hemoglobin pigments have a identify them. 
characteristic absorption band.   Based on the principle that hemoglobin molecules
 Cyanmethemoglobin (HiCN) method – in an alkaline solution have a negative charge and
Considered as best method for hemoglobin move toward the anode in an electrophoretic
determination  system. 
o Drabkins reagent + 20uL of blood then  Slowest to fastest: a2 > e > o > c, g > d > s, f, a, k, j,
read spectrophotometrically at 540nm  bart’s, n > j, k 
 Spectro – If the sample is run through the  Specimen is hemolysate. 
machine and there is an increased absorption  Proteins alone do not move through the electric
at: 555nm=CarboxyHB, field since their net charge is zero (Amphoteric).  
630nm=Methemoglobin, 620=Sulfhemoglobin.   Uses agarose gel (pH=8.6) or Citrate agar (pH=6.2 
 Methemalbumin – Heme that is bound to an
albumin instead of hemopexin.   
 Schumms test – for the determination of
methemalbumin (fairley’s pigment)  
 Plama/serum + ether + yellow ammonium
sulphide >Spectroscope 
 Schumm’s test - +for methemalbumin if there
is a narrow absorption band seen at 558nm. 
 Drabkin’s reagent = K ferricyanide + K cyanide 
 Note: Most forms of hemoglobin are measure
except Sulfhemoglobin (SHb)   Methods: 
 Determination of fetal hemoglobin   Cellulose acetate– pH 8.4  
 Alkali denaturation test – Based on the  CITRATE AGAR - pH 6.2 
principle that HBF resists alkali denaturation
while HBA does not.  
o Alkali denaturation = Sample is
hemolysate; Result is measured 
o Sample + ammonium sulphate-      
 hba = will be Denatured             
hbf = will resist denaturation 
o NORMAL VALUE = 0.2 – 1.0% 
 ACID ELUTION TEST – Based on the principle that
cells with hbf will resist acid elution to a greater
extent than  normal cells do. 
 Acid elution = hematoxylin and eosin B 
 Sample + citric acid phosphate then stained      
hba = elution                          hbf = resist elution 
  NORMAL VALUE = 1-5% OF CELLS HAVE
RESIDUAL HEMOGLOBIN. 

Hemoglobin ELECTROPHORESIS