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INTRODUCTION Red cell require energy in order to fulfill function

Only source of energy is in form of ATP derived from

1: GLYCOLYTIC PATHWAY

2: PENTOSE PHOSPHATE PATHWAY

3: GLUTATHIONE CYCLE

Reducing power is required:

To reduce metHb back to its functional state

To counteract the strong oxidative stress that cell carries

Processes that reduce metHb utilizes:

1:NADH reduced from NAD+by glycolytic pathway

2:Reduction of free oxygen radicals produce during reactions to infection is provided by NADPH catalyzed by G6PD

3:NADPH drives glutathione cycle, major reducing agent in the red cell

ATP provides energy for :

1:maintenance of red cell volume

2: red cell shape

3: flexibility

Sodium potassium ATPase pump

HMP shunt pathway

Gl-6-P dehydrogenase deficiency Most common erythrocyte enzyme disorder

Disease is sex linked carried by gene on X chromosome Xq28 therefore common in males

Age dependent, decrease slightly

First recognized during Korean world war when African soldiers develop hemolytic anemia when given anti-malarial drugs

Normal cells use 0.1% of their G6PD

Females heterozygote for G6PD deficiency always contain two population of cells, one normal and one G6PD deficient as compared to males

COMPOUNDS ASSOCIATED WITH G6PD DEFICIENCY

Drugs

Favism

infection

Oxidant drugs

Commonly used drugs that produce hemolytic anemia in patients with G6PD deficiency anemia

Antibiotics (for example, sulfamethoxazole and chloramphenicol),

Antimalarials (for example, primaquine but not quinine),

Antipyretics (for example, acetanilid but not aceta minophen).

Favism: Some forms of G6PD deficiency, for example the

Mediterr anean variant, are particularly susceptible to the hemolytic effect of the fava (broad) bean, a dietary staple in the Mediterranean region.

Favism, the hemolytic effect of ingesting fava beans, is not observed in all individuals with G6PD deficiency, but all patients with favism have G6PD deficiency

Infection: Infection is the most common precipitating factor of

hemolysis in G6PD deficiency.

The inflammatory response to infection results in the generation of free radicals in macro phages, which

can diffuse into the red blood cells and cause oxidative damage.

PATHOPHYSIOLOGY

PATHOPHYSIOLOGY

G6PD is necessary for maintaining adequate levels of GSH

In case of deficiency generation is impaired results in

accumulation of cellular oxidants (reactive oxygen species (ROS))

Buildup of cellular oxidant leads to RBC injury and hemolysis

Hb is oxidized to methemoglobin, which precipitate in form of

Heinz bodies

Heinz bodies attach to erythrocyte membrane cause increase

cell rigidity, membrane permeability

CONT… Heinz bodies are removed by splenic macrophages producing bite

cells and blister cells

Membrane loss leads to formation of spherocytes

These cells ultimately hemolyzed in spleen

Hemolysis is self limited

Important to remember that under stress of severe oxidants(drugs,

chemicals) normal cells may experience oxidant damage

G6PD VARIANTS More than 400 variants (isoenzymes) have been identified

They differ in their activity, stability and electrophoretic mobility

Mutant enzymes have classified into 5 classes according to degree

of severity

WHO CLASSIFICATION OF G6PD DEFICIENCY

class Enzymatic activity

%

Clinical effects

I severe usually

<10

CNSHA, acute

exacerbations

II

e.g. med

<10 Favism, drug

induced),

Neonatal jaundice

III e.g. A- Moderate >10, < 60 drug induced),

neonatal jaundice

Iν >60 none

CLINICAL FINDINGS Mostly deficient patients have no clinical symptoms

Hemolysis is variable dependent on degree of oxidant

stress, and sex of the patient

Symptoms are same as of acute intravascular hemolysis

FAVISM is disorder in which some individual with G6PD

deficiency develop hemolytic episode after ingestion of

fava beans?

isouramil, divicine

Signs of favism are malaise, nausea, vomiting, abdominal

pain

LAB FINDINGS

G6PD deficiency is easily detected in hemizygous(male),

or homozygous individual but it is difficult to detect in

heterozygous female

In female heterozygotes two population of cells exist, one

with normal G6PD activity and other deficient

It occurs due to inactivation of one of the two X

chromosome in individuals cells early in development of

embryo

LAB FINDINGS During hemolytic attack, polychromasia, spherocytes, erythrocyte

fragments and bite cells may be seen on blood smear

Bite cells are thought to be typical of G6PD deficiency

Reticulocytosis is characteristic following hemolytic attack

A Peculiar cells referred to by variety of descriptive terms (irregularly contracted cell, RBC hemi ghost) is present after oxidant related hemolysis. These cells are rigid, decreased volume, Hb is confined to one side while other side is transparent. Transparent site mostly contain Heinz bodies

6

Cont… Leukocyte count may be increased during hemolysis

Platelet count is normal

Indirect bilirubin and serum LDH may raised

Heptoglobin is decreased

Definitive diagnosis depends upon the demonstration of

decrease in RBC G6PD enzyme activity

FLUORESCENT SPOT TEST Reliable and sensitive screening test

Add 10µl of Whole blood either anticoagulated (EDTA,heparin or

added before clotting) is added to 100 µl mixture of G6P,NADP, and

saponin

Drop of mixture is placed on piece of filter paper

Examined it under UV light of fluorescence

G6PD enzyme present in RBCs metabolizes G6P,producing

NADPH.NADPH fluoresces but NADP not, lack of fluorescence

indicate deficiency

The test can be carried out on whole blood stored in ACD for up to 21

days at 4oC

False normal:

If there is reticulocytosis,fluorescence may

be seen with G6PD deficient blood sample

False deficient:

If patient is anemic, very little fluorescence

may be seen despite the sample is normal

DYE REDUCTION TEST

Hemolysate of patients blood+G6P+NADP and dye

brilliant cresyl blue are incubated together

If hemolysate contain G6PD, NADP reduced to NADPH

which in turn reduces blue dye to its colorless form

RESULT:

Time take for change to occur is inversely

proportional to amount of G6PD present

DETECTION OF HETEROZYGOTES FOR G6PD DEFICIENCY Elution test Individual cells retaining Hb02 in metHb elusion test are stained, remaining

appear as ghost cells Method: incubated the sample with Nile blue sulphate 1:Oxygenate the sample during incubation by bubbling with O2 with help of pipette 2:After 2-3 h add 20µl of KCN to 1 ml of reaction mixture 3:Make blood films 4:Dry, wash and stain it with hematoxylin, counter stain with erythrosin for

2 mins Examine cells under microscope and count the proportion of stained HbO2 and unstained cells

ENZYMATIC ASSAY

Erythrocyte hemolysate is incubated with G6P and NADP

Rate of reduction of NADP to NADPH is measured at

340nm in spectrophotometer

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