This document summarizes information about glucose-6-phosphate dehydrogenase (G6PD) deficiency. It discusses how G6PD is important for generating NADPH and reducing power in red blood cells. G6PD deficiency results in oxidative damage and hemolysis of red blood cells due to a lack of reducing equivalents. Clinical manifestations range from asymptomatic to acute hemolytic anemia triggered by oxidative drugs, infections, or fava beans. Diagnosis involves screening tests to detect low G6PD enzyme activity in red blood cells.

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2. 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

3. 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

4. ATP provides energy for :
1:maintenance of red cell volume
2: red cell shape
3: flexibility
Sodium potassium ATPase pump

5. HMP shunt pathway

6. 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

7. COMPOUNDS ASSOCIATED WITH G6PD DEFICIENCY
Drugs
Favism
infection

8. 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).

9. 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

10. 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.

12. PATHOPHYSIOLOGY

13. 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

14. 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

15. 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
The G6PD gene is 18 kilobases (kb) long with 13 exons, and the G6PD
enzyme has 515 amino acids.

17. 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?
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Signs of favism are malaise, nausea, vomiting, abdominal
pain

18. 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

19. 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

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21. 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

22. 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

23. 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

24. 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

26. 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

28. 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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