Hemoglobin disorders Biochemistry Report

1. Hemoglobin Diseases
GROUP 10
Reporters:
Yang, Sheryl Ray
Zagada, Timothy
Zamora, Marvin
Zapanta, Patricia Joyce

2. Hemoglobin 101
Yang, Sheryl Ray

3. Primary function of the Red blood cell is to
manufacture hemoglobin, which in turn, transports
oxygen to the tissues and carbon dioxide from tissues
to the lungs.
Hemoglobin molecule is composed of four subunits,
each containing heme and globin
Hemoglobin Synthesis

4. Protein component called globin
Four molecules of the nitrogenous substance
Protoporhpyrin IX
Four Iron atoms at Ferrous (Fe+2) state that combine
with Protoporphyrin IX to form four heme molecules
One 2,3 Diphosphoglycerate (2,3 DPG) molecule as a
sometime resident in the center of Hb unit
Hemoglobin Synthesis
Components of Hemoglobin

5. Heme Synthesis

7. Protoporhyrin
IX
Fe+2
Ferroprotoporphyrin
IX
(HEME)
Heme Synthesis

8. Produced on specific ribosomes in the cytoplasm of
red blood cells. The globin in each hemoglobin
molecule consists of four polypeptide chains which
determine the type of hemoglobin formed.
Globin Chains

9. Greek
Designation
Greek
Name
No. of
Amino
Acids
Chromosome
α Alpha 141 16
β Beta 146 11
δ Delta 146 11
γ Gamma 146 11
ε Epsilon 146 11
ζ Zeta 146 16
Globin Chains in Hemoglobin
• Consist of varied
sequences of amino
acids
– polypeptide chains

10. Hemoglobin Molecular
structure
Stage of life Proportion
Newborns
Proportion
Adults
Portland 2 Zeta +
2 Gamma
Embryonic 0 0
Gower I 2 Zeta +
2 Epsilon
Embryonic 0 0
Gower II 2 Alpha +
2 Epsilon
Embryonic 0 0
Hb A1 2 Alpha +
2 Beta
Newborn &
Adult
20 97
Hb A2 2 Alpha +
2 Delta
Newborn &
Adult
<0.5 2.5
Hb F
(Fetal)
2 Alpha +
2 Gamma
Newborn &
Adult
80 <1
Normal Human Hemoglobin

11. Hemoglobin Molecule
Hb A1

12. T and R states of Hemoglobin
• Hemoglobin exists in two major conformational
states: Relaxed (R ) and Tense (T)
• R state has a higher affinity for O2.
• In the absence of O2, T state is more stable;
when O2 binds, R state is more stable, so hemoglobin
undergoes a conformational change to the R state.
• The structural change involves readjustment of
interactions between subunits.
Tensed and Relaxed State

13. Tensed and Relaxed State

14. O2- Dissociation Curve

15. Iron in the ferrous state is
required to convert
protoporphyrin Ix to heme.
Circumstances that cause
reduction in the iron
available for Hb synthesis
or failure to incorporate
iron into heme will cause
anemia to develop.
Iron Metabolism for Heme Synthesis

16. Most common cause of anemia
Due to
INCREASED PHYSIOLOGIC DEMANDS
Rapid growth; infants, children
Pregnancy, lactation
Iron Deficiency Anemia
* INADEQUATE INTAKE
* Iron deficient diet
* Inadequate absorption
* CHRONIC BLOOD LOSS
* Menstrual flow
* Gastrointestinal bleeding
* Regular blood donation
* Chronic hemolysis

17. Effect of IDA
Protoporhyrin
IX
Fe+2
Ferroprotoporphyrin
IX
(HEME)
Hemoglobin

18. Hemoglobinopathy
Conditions caused by qualitative structural
abnormalities of the globin polypeptide chains that
result from alteration of the DNA genetic code for
those chains
Hemoglobinopathies and
Thalassemias

19. Thalassemias
Conditions caused by quantitative abnormality in
globin chain (i.e., reduced or no production).
Hemoglobinopathies and
Thalassemias

20. Sickle Cell Anemia
Zamora, Marvin

21. Hgb S – most common abnormal
hemoglobin
- Normal glutamic acid at 6th position in the β
chain is replaced by Valine
Results in:
- Altered solubility
- Altered ability to withstand oxidation
- Instability
- Increased propensity for methemoglobin
production
- Increased or decreased oxygen affinity
Sickle cell disease

22. Sickle cell anemia
- Sickle cell disease (SCD)
- Drepanocytosis
- Hb SS
- SS disease
- Hemoglobin S
- Homozygous

23. Sickle cell trait
Is the heterozygous state of SCD
One sickle gene and one normal hemoglobin gene
(Hb AS)
Usually have no symptoms

24. Overview
Sickle cell disease is a general term for a group of
genetic disorders caused by sickle hemoglobin (Hgb S
or Hb S)
Erythrocytes becomes elongated and sickle shaped
Removed from the circulation and destroyed at
increasing rates, leading to anemia.

25. Overview
An autosomal recessive inherited defect
The disease is chronic and lifelong
Lifespan average of 40 years.

26. Pathophysiology
caused by a point mutation in the β-globin chain of
hemoglobin
glutamic acid valine at the 6th position
*found on the short arm of chromosome 11.

31. Pathophysiology
Sickling occurs when oxygen decreases at the tissue level –
dissociation of oxygen from RBC
Polymerization of Hgb molecules to crystals

32. Sickle cell crises
Vasoocclusive crises
- Increase in blood viscosity
- restricts blood flow to an
organ
Hemolytic crises – acute
accelerated drops in Hgb
levels and RBCs break down
at a faster rate.
- common in patients with
G6PD deficiency

33. Sickle cell crises
Infectious crises
- Abnormal splenic function
- Depressed immune function
- Streptococcus Pneumoniae is
the major infectious agent
among children

34. Sickle cell crises
Aplastic crises
- caused by infection and fever
- Parvovirus B19
- Folate deficiency

35. Sickle cell crises
Bone, joint and other crises:
- Hand-foot syndrome or Dactylitis
- Priapism
- Gallstones

37. Sickle cell anemia
Laboratory findings
-severe anemia (Hgb 5-9 g/dl)
-normocytic, normochromic RBC
-aniso and poikilocytosis is present
-with leukocytosis and
thrombocytosis

38. Diagnosis
Blood film appearance
Screening tests for sickling
Hemoglobin electrophoresis

39. Hemoglobin electrophoresis
A
(%)
F
(%)
S
(%)
C
(%)
D
(%)
E
(%)
Degree of
clinical
abnormality
Hemoglobinopathy
Hb CC 1-
7
>90 Mild
Hb AC 50-60 <2 40-50 None
Hb SC 1-7 50 50 Mod - Severe
Hb SS 1-
10
80-90 Severe
Hb AS 55-70 <2 30-45 None/Mild
Hb DD <2 95 None
Hb AD 50-65 <2 35-50 None
Hb EE 1-5 95 Mild
Hb AE 60-80 20-
40
None

40. Treatment
*Blood transfusion of PRBC
*Administration of antisickling agents
-Cyanate
-Urea
-Nitrogen mustard
-zinc procaine hydrochloride, Citiedil, and
piracetam
*Bone marrow transplantation

41. Thalassemia
Zapanta, Patricia Joyce

42. THALASSEMIA SYNDROMES
Each individual has 4 genes of hemoglobin ( HBA1,
HBA2, HBB1,HBB2) (aa/aa)(bb/bb)
Thalassemia is characterized by partial or total
absence of one or more chains of hemoglobin (either
α chain or β chain). Resulting to abnormal form of
hemoglobin. Which leads to destruction of RBC leads
to anemia
It is a genetic disorder
TYPES OF THALASSEMIA
1. α - thalassemia
2.β - thalassemia

44. THALASSEMIA
DEMOGRAPHIC
Southeast Asia and Meditteranian region
CLINICAL PRESENTATION
MINOR – mild anemia confused with iron deffeciancy
INTERMEDIATE-moderate anemia
MAJOR- severe anemia – hydrops fetalis intrauterine
death

45. Alpha thalassemia
Deletion of alpha-
globin gene resuts
to
Children /Adult
Excess beta globin –
usually unstable and
precipitate in cell
Forms HEINZ BODY
Fetal / new born
Iincrease
HYPOCHROMIC and
MICROCYTIC RBC

46. ALPHA THALASSEMIA
Involves the genes HBA1 and HBA2
Located at chromosome 16
Severity of disease depends on the number of genes
defective or missing

47. Alpha (+) thalassemia:
-deletion of I or more alpha globin gene
If 1 gene = silent carrier
If 2 genes = alpha trait (thalassemia minor)
If 3 genes = H hemoglobin (thalassemia intermedia)
Alpha (0) thalassemia
If 4 genes = Bart hemoglobin (thalassemia major)
ALPHA THALASSEMIA

48. 3 normal gene (-a/aa)
Normal patient
Silent Carrier/ Alpha thalassemia
minima/ alpha thalassemia – 2 trait

49. 2 normal gene
(aa/--) – cis form
(-a/-a) – trans form
Clinically normal
Minimal anemia
Decrease MCV and MCH
Alpha thalassemia trait/ alpha
thalassemia minor/ alpha thalassemia
– 1 trait

50. Only 1 normal alpha-globin gene (-a/--)
Increase ratio beta globin : alpha globin
Sensitive to oxidative stress
RBC prone to hemolyze
Hemoglobin H / HbH disease

51. All 4 alpha globin gene is deleted (--/--)
Most severe case
Hydrops fetalis
Bart’s Hemoglobin/ alpha (0)
thalassemia

52. BETA THALASSEMIA
Β thalassemia will not manifest at birth since predominant is
Hgb F. production of B chain will occur only at 3 – 6 months
after birth
Involves the gene HBB1 and HBB2
Located at chromosome 11
Severe transfusion dependent anemia

53. BETA THALASSEMIA
TYPE OF BETA THALASEMIA
Homozygous beta thalassemia ( thalassemia major, cooley’s
anemia, Meditteranean anemia)
- severe life long
-all beta gene mutated
-severe anemia
Heterozygous beta thalassemia (thalassemia minor)
-one normal beta chain and 1 abnormal beta chain
-mild anemia

54. THALASSEMIA LAB DIAGNOSTICS
Peripheral Blood Smear
-Target cells, Heinze bodies, basophilic strippling,
nucleated RBC
Reticulocyte – increased
Decreased osmotic fragility
Iron storage disease

55. As blood glucose enters the
erythrocytes it glycosylates
the ε-amino group of lysine
residues and the amino
terminals of hemoglobin.
RBC life span – 120 days
HbA1c and Diabetes

56. HbA1c and Diabetes

57. HbA1c Normal/abnormal Blood glucose level via
meter
4.0 - 6.0% Normal for those
without diabetes
3 – 8mmol/L
6.1 – 7.0% Target range for those
with diabetes
4 – 8mmol/L
7.1 – 8.0% High 8 – 11mmol/L
8.1 – 9.0% Too high 11 – 14mmol/L
Greater than 9.1% Very high 15mmol/L and above
HbA1c and Diabetes

58. HbA1c and Diabetes

59. Laboratory Experiment: Hgb
Determination
Zagada, Timothy

60. Cyanmethemoglobin method
A method used for hemoglobin determination
The reagent hemolyzes the erythrocytes which
releases the hemoglobin into the solution.
REACTIVE INGREDIENTS:
-potassium cyanide and potassium ferricyanide.

61. Principle in Cyanmethemoglobin
method
When blood is mixed with a solution containing
potassium ferricyanide and potassium cyanide, the
potassium ferricyanide oxidizes iron to form
methemoglobin.
The potassium cyanide then combines with
methemoglobin to form cyanmethemoglobin
Hgb(Fe++)
K3Fe(CN)6
Methemoglobin(Fe+++)
KCN
Cyanmethemoglobin

62. Procedure
Hgb Reagent (5ml)
Mix well. Stand for 3 min
Read at spectrophotometer
+20ul blood sample (EDTA whole blood)

63. PURPOSE OF EDTA
EDTA (ethylenediaminetetraacetic acid) is the most
commonly used anticoagulant in evacuated tubes.
EDTA reduces platelet activation by protecting the
platelets during contact with the glass tube that may
initiate platelet activation.

64. RESULTS
0
5
10
15
20
25
Group 6 Group 7 Group 8 Group 9 Group 10
Hgb g/dL 17.9 15.57 19.5 17.74 22
Hgbconcentration
Hgb concentration per sample
Normal:
Male: 13-18 g/dL
Female: 12-16 g/dL

65. SOURCES OF ERRORS
Technical Error
- Pipeting
- Use of dirty, scratched or unmatched cuvettes
- Use of deteriorated reagents
- Incorrectly calibrated spectrophotometer

66. SOURCES OF ERRORS
Physiologic Error
- Turbidity in the mixture causes falsely elevated values
Turbidity maybe caused by:
 Lipemia
 Extremely high leukocyte counts
 Easily precipitated Globulins

67. Factors that affect Hemoglobin
Increased Hemoglobin
kidney releases too much erythropoietin
People living in high altitudes
Anabolic steroid
Smoking
Dehydration
Polycythemia vera

68. Factors that affect Hemoglobin
Decreased Hemoglobin
-Vitamin-deficiency Anemia
deficiency of vitamin B12 or folate
-Bleeding
Blood volume is replaced more quickly than red blood
cells, leading to a lower concentration of hemoglobin
-Kidney Disease
results in lower levels of erythropoietin
-Pregnancy
-Blood Disorders