Practical 2 - G6PD determination

Question 1

Principle of methemoglobin reduction test

Answer 1

This test consists of oxidation of OXYHb to MetHb by sodium nitrite and the subsequent enzymatic reconversion to OXYHb in the prescence of methylene blue by stimulation of the PPP pathway in that the conversion of methemoglobin back to oxyhemoglobin requires G6PD TO WORK.

Question 2

Tube used in experiment

Answer 2

heparin/EDTA bottle

Question 3

saline used in this experiment

Answer 3

NaCl

Question 4

Precautions in Methemoglobin reduction test experiment

Answer 4

use of a clean dry bottle

Question 5

Volume of bllod required from patient in Methemoglobin reduction test

Answer 5

3mls of anticoagulated blood

Question 6

Reagents required in methemoglobin reduction test

Answer 6

1. 25% Sodium nitrite

0. 15% methylene blue

Question 7

What does sodium nitrite do?

Answer 7

Oxidizes oxyhemoglobin to methemoglobin

Question 8

What does methylene blue do

Answer 8

reduced methemoglobin to oxyhemoglobin

Question 9

Addition of both sodium nitrite and methylene blue to blood sample where change of color from red to brown PERSISTS denotes

Answer 9

NO G6PD present. You require G6PD to convert metHb back to oxyHb and since this did not happen,G6PD is absent

Question 10

Addition of both sodium nitrite and methylene blue to a solution where the final colour is red denotes

Answer 10

G6PD present. Methylene blue was able to convert brown coloured methemoglobim back to red coloured oxyhemoglobin using G6PD, an important requirement. since change in colour occured, G6PD is present

Question 11

Color of methemoglobin

Answer 11

Brown

Question 12

Color of oxyhemoglobin

Answer 12

Red

Question 13

If final test colour matches with red oxyHb color the person is said to be

Answer 13

normal

Question 14

If final test color matches with methemoglobin color of brown the patient is said to be

Answer 14

in full effect

Question 15

A patient is said to have partial effect when

Answer 15

Final test results produce a lighter shade of RED compared to the control

Question 16

Controls in the experiment are

Answer 16

Tubes where only either of sodium nitrate OR methylene blue is added

Question 17

Partial effect usually occurs in what sex and why

Answer 17

Usually occurs in females.
The X chromosome has full control in determining G6PD. G6PD gene is located on long arm of X chromosome
A defective x chromosomes in females will not dominate since both will have equal dominance and effect isnt in full swing

Males have XY chromosomes, X has full control over Y, effect is greatly seen

Question 18

What does it mean if a male presents with partial effect

Answer 18

He has been transfused

Question 19

What could cause a false normal presentation.

Answer 19

G6PD deficiency —> Hb hemolysis—–> Bone marrow hyperplasia ….>pushing out young RBCs which have 3 times Hb concentration—> Masking of G6PD condition

Question 20

What could cause a false negative presentation

Answer 20

low RBC—> low Hb—-> low G6Pd PRESENTATION THOUGH IT MAY NOT BE SO

Question 21

Test to investigate HS, HE, HbS and thalassemia’s

Answer 21

Osmotic fragility test

Question 22

What is the osmotic fragility test

Answer 22

A test that measures the ability of RBC to take up fluid without lysing

Question 23

Factors affecting RBC fragility or ease of lysing

Answer 23

Functional state of RBC. i.e age of membrane
Volume
Surface area

Question 24

The graph plotted from osmotic fragility test are

Answer 24

3 backwards S shape with normal RBC in the middle, less fragile RBC on the left and more fragile RBC on the right

Question 25

Diseases that will show a curve at left side of normal range are

Answer 25

not fragile. They didnt lyse even at a low concentration of 0.1

Question 26

Diseases at left side of normal curve include

Answer 26

Thalassemia tested by PCR

Iron deficiency tested by iron studies

Sickle cell Anaemia tested by electrophoresis

Question 27

Curve at the right side of normal range denotes

Answer 27

weak RBCs. They lysed even at a high concentration even thought it is not expected to be so.

Question 28

Diseases that will present with the right side curve

Answer 28

Hereditory spherocytosis

Hereditory elliptocytosis

Hereditory Leiomyomatosis

Question 29

Normocytic RBCs have a mean cell fragility range of

Answer 29

0.4-0.45

Question 30

Mean cell fragility range for sickle cell etc

Answer 30

0.3-0.35

Question 31

Mean cell fragility range for H. spherocytosis etc

Answer 31

0.5-0.6

Question 32

Mean cell fragility is

Answer 32

The concentration at which there is 50% hemolysis