Hemoglobin DiseasesGROUP 10

Reporters:Yang, Sheryl RayZagada, TimothyZamora, MarvinZapanta, Patricia Joyce

Hemoglobin 101Yang, Sheryl Ray

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

1 HEME = 1 MOL OF O21 HEMOGLOBIN = 4 MOL OF O2

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

Heme Synthesis

Protoporhyrin IX

Fe+2

Ferroprotoporphyrin IX

(HEME)

Heme Synthesis

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

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

Hemoglobin

Molecular structure

Stage of life

ProportionNewborns

ProportionAdults

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

Hemoglobin MoleculeHb A1

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

Tensed and Relaxed State

O2- Dissociation Curve

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

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

Effect of IDA

Protoporhyrin IX

Fe+2

Ferroprotoporphyrin IX

(HEME)

Hemoglobin

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

Thalassemias Conditions caused by quantitative

abnormality in globin chain (i.e., reduced or no production).

Hemoglobinopathies and Thalassemias

Sickle Cell AnemiaZamora, Marvin

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

Sickle cell anemia

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

Sickle cell trait

Is the heterozygous state of SCD One sickle gene and one normal hemoglobin

gene (Hb AS) Usually have no symptoms

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.

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

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.

Pathophysiology Sickling occurs when oxygen decreases at the

tissue level – dissociation of oxygen from RBC Polymerization of Hgb molecules to crystals

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

Sickle cell crisesInfectious crises

- Abnormal splenic function- Depressed immune function- Streptococcus Pneumoniae is the major infectious agent among children

Sickle cell crises

Aplastic crises- caused by infection and fever- Parvovirus B19- Folate deficiency

Sickle cell crises

Bone, joint and other crises:- Hand-foot syndrome or Dactylitis- Priapism- Gallstones

Sickle cell anemia

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

Diagnosis Blood film appearance Screening tests for

sickling Hemoglobin

electrophoresis

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

Treatment

*Blood transfusion of PRBC*Administration of antisickling agents

-Cyanate-Urea-Nitrogen mustard-zinc procaine hydrochloride, Citiedil, and

piracetam

*Bone marrow transplantation

ThalassemiaZapanta, Patricia Joyce

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

THALASSEMIA

DEMOGRAPHIC Southeast Asia and Meditteranian regionCLINICAL PRESENTATION MINOR – mild anemia confused with iron

deffeciancy INTERMEDIATE-moderate anemia MAJOR- severe anemia – hydrops fetalis

intrauterine death

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

ALPHA THALASSEMIA

Involves the genes HBA1 and HBA2 Located at chromosome 16 Severity of disease depends on the number

of genes defective or missing

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

3 normal gene (-a/aa) Normal patient

Silent Carrier/ Alpha thalassemia minima/ alpha

thalassemia – 2 trait

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

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

All 4 alpha globin gene is deleted (--/--) Most severe case Hydrops fetalis

Bart’s Hemoglobin/ alpha (0) thalassemia

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

BETA THALASSEMIATYPE 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

THALASSEMIA LAB DIAGNOSTICS

Peripheral Blood Smear -Target cells, Heinze bodies, basophilic

strippling, nucleated RBC Reticulocyte – increased Decreased osmotic fragility Iron storage disease

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

HbA1c and Diabetes

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

HbA1c and Diabetes

Laboratory Experiment: Hgb Determination

Zagada, Timothy

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.

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)6Methemoglobin(Fe+++)

KCNCyanmethemoglobin

Procedure

Hgb Reagent (5ml)

Mix well. Stand for 3 min

Read at spectrophotometer

+20ul blood sample (EDTA whole blood)

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.

RESULTS

Group 6 Group 7 Group 8 Group 9 Group 10

Hgb g/dL

17.9 15.57 19.5 17.74 22

2.5

7.5

12.5

17.5

22.5

Hgb concentration per sample

Hg

b c

on

cen

trati

on

Normal:Male: 13-18 g/dLFemale: 12-16 g/dL

SOURCES OF ERRORS

Technical Error- Pipeting- Use of dirty, scratched or unmatched

cuvettes- Use of deteriorated reagents- Incorrectly calibrated spectrophotometer

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

Factors that affect Hemoglobin

Increased Hemoglobin kidney releases too much erythropoietin People living in high altitudes Anabolic steroid Smoking DehydrationPolycythemia vera

Factors that affect Hemoglobin

Decreased Hemoglobin-Vitamin-deficiency Anemiadeficiency of vitamin B12 or folate-BleedingBlood volume is replaced more quickly than red blood cells, leading to a lower concentration of hemoglobin-Kidney Diseaseresults in lower levels of erythropoietin-Pregnancy-Blood Disorders