Practical 2 - G6PD determination Flashcards
Principle of methemoglobin reduction test
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.
Tube used in experiment
heparin/EDTA bottle
saline used in this experiment
NaCl
Precautions in Methemoglobin reduction test experiment
use of a clean dry bottle
Volume of bllod required from patient in Methemoglobin reduction test
3mls of anticoagulated blood
Reagents required in methemoglobin reduction test
- 25% Sodium nitrite
0. 15% methylene blue
What does sodium nitrite do?
Oxidizes oxyhemoglobin to methemoglobin
What does methylene blue do
reduced methemoglobin to oxyhemoglobin
Addition of both sodium nitrite and methylene blue to blood sample where change of color from red to brown PERSISTS denotes
NO G6PD present. You require G6PD to convert metHb back to oxyHb and since this did not happen,G6PD is absent
Addition of both sodium nitrite and methylene blue to a solution where the final colour is red denotes
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
Color of methemoglobin
Brown
Color of oxyhemoglobin
Red
If final test colour matches with red oxyHb color the person is said to be
normal
If final test color matches with methemoglobin color of brown the patient is said to be
in full effect
A patient is said to have partial effect when
Final test results produce a lighter shade of RED compared to the control
Controls in the experiment are
Tubes where only either of sodium nitrate OR methylene blue is added
Partial effect usually occurs in what sex and why
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
What does it mean if a male presents with partial effect
He has been transfused
What could cause a false normal presentation.
G6PD deficiency —> Hb hemolysis—–> Bone marrow hyperplasia ….>pushing out young RBCs which have 3 times Hb concentration—> Masking of G6PD condition
What could cause a false negative presentation
low RBC—> low Hb—-> low G6Pd PRESENTATION THOUGH IT MAY NOT BE SO
Test to investigate HS, HE, HbS and thalassemia’s
Osmotic fragility test
What is the osmotic fragility test
A test that measures the ability of RBC to take up fluid without lysing
Factors affecting RBC fragility or ease of lysing
Functional state of RBC. i.e age of membrane
Volume
Surface area
The graph plotted from osmotic fragility test are
3 backwards S shape with normal RBC in the middle, less fragile RBC on the left and more fragile RBC on the right
Diseases that will show a curve at left side of normal range are
not fragile. They didnt lyse even at a low concentration of 0.1
Diseases at left side of normal curve include
Thalassemia tested by PCR
Iron deficiency tested by iron studies
Sickle cell Anaemia tested by electrophoresis
Curve at the right side of normal range denotes
weak RBCs. They lysed even at a high concentration even thought it is not expected to be so.
Diseases that will present with the right side curve
Hereditory spherocytosis
Hereditory elliptocytosis
Hereditory Leiomyomatosis
Normocytic RBCs have a mean cell fragility range of
0.4-0.45
Mean cell fragility range for sickle cell etc
0.3-0.35
Mean cell fragility range for H. spherocytosis etc
0.5-0.6
Mean cell fragility is
The concentration at which there is 50% hemolysis
