Sunday, October 25, 2009

G6PD screening & Quantitative assays

hello hello hello!
stella here for the final blog posting!
and i apologize for the late entry.

G6PD (Glucose-6-Phosphate Dehydrogenase) test is performed mainly on patients who develop symptoms of hemolytic anemia and neonatal jaundice. Hemolytic anemia takes place when the bone marrow is not able to compensate for the destruction by elevating the RBC production. G6PD is necessary for the maintenance of an intact red cell membrane because G6PD deficiency may lead to hemolysis of red cells. During the abnormal state, the RBCs will not be able to regenerate NADPH, a reaction that will be catalysed by G6PD in normal situations.

Both G6PD Quantitative and Qualitative (screening) assays are performed to enable differential diagnosis in G6PD deficiency to be made.
The principle of both G6PD assays is based on this reaction:

Glucose-6-Phosphate + NADP (no fluorescence) ---(G6PD)---> Gluconate-6-Phosphate + NADPH (fluorescence)

The NADPH produced will fluoresce under long-wave UV light during the reaction.
There is maerked deficiency or lack of enzyme G6PD when no fluorescnece is observed.
If Glucose-6-Phosphate is oxidised to Gluconate-6-Phosphate, the coenzyme NADP is reduced to NADPH with a corresponding elevation in fluorescence.

The procedures for both assays are different, with the screening assay much simpler as compared to the quantitative assay.

Steps for screening assay:
1) The EDTA consisting of patient's blood sample is inverted gently for several times for thorough mixing
2) 5ul of whole blood is added, 100ul of working reagent and 100ul of reagent blank into the wells of the titration plates
3) Working reagent is added and start timing
4) After 10 minutes, a small amount of reaction is taken and i spotted onto filter paper
5) The spots are dried for 5 mins using a hair-dryer
6) The spots are then viewed under long-wave UV light

Steps for quantitative assay:
1) The EDTA blood is washed 1 time with 0.9% saline
2) It is then sent for centrifugation to pack the cells at 3000rpm for 10 mins
3) The saline and the buffer coat are removed completely
4) 200ul of washed packed cells are pipetted into 200ul normal saline to obtain 1:1 rbc suspension
5) 50ul of this 1:1 suspension is pipetted into 250ul of !% saponin for lysis
6) It is then mixed well and left to stand for 10 mins at 2-8 degree celsius
7) The hemolysate is ready for testing


Reporting of results for Qualitative (screening) assay:
When fluorescence is observed, enzyme activity is indicated. This explains the presence of G6PD.


Reporting of results for Quantitative assay:
Reference ranges for adults/children = 7.2-17.4 ug/Hb
newborns/cord blood = 13.4-25.4 ug/Hb

Thursday, October 15, 2009

Culturing Of Cord Blood

Hey!
This is Hakim (0703555C) doing my 4th and final blog posting.

Culturing of cord blood is essential for those who want to store their baby's cord blood.

Cord blood is the blood that is in the umbilical cord and placenta after the birth of the baby. The blood can be used to treat haematopoietic diseases such as leukemia, as the cord blood contains stem cells. The cord blood can be stored for up to 20 years for transfusion for future use to the baby or their family members. Therefore, it is essential that the cord blood is sterile and is free of any microorganisms. In addition, storage and maintenance of cord blood is very expensive.

The cord blood that is received for culturing is just a small amount of blood (2 eppendorf tubes).

The cord blood is cultured on 4 different agar plates:
1. TSA (Trypticase Soy Agar) with sheep blood stored in aerobic conditions - an enriched media to support a wide variety of microorganisms
2. TSA (Trypticase Soy Agar) with sheep blood stored in anaerobic conditions - an enriched media to support a wide variety of anaerobic microorganisms
3. MacConkey Agar- selective media for gram negative bacteria and also to differentiate between lactose fermenters and non-lactose fermenters
4. Sabouraud Dextrose Agar - selective media for yeast cells

The barcode number of the baby must be written on the underside of the media plate. A sterile swab is inserted into the cord blood and is innoculated onto the media plates. Then, a streaker is used to streak the plate.

The anaerobic TSA is inserted into an anaerobic jar. An anaerobic pack which absorbs oxygen and releases carbon dioxide is added into the jar before incubation. In addition, a control plate of a Mueller Hinton Agar innoculated with pseudomonas aeruginosa is put inside the anaerobic jar. The reason for this is because pseudomonas aeruginosa is a strict aerobe and it is the only organism that grows green colonies on Mueller Hinton. Therefore, if there isn't any green colonies on the agar after incubation, we can conclude that the conditions is an anaerobic condition.

After 24 hours of incubation, the agar plates are checked for any signs of bacterial growth. If there is no bacterial growth, the plates are incubated further for another 24 hours. If there is presence of bacterial growth, we must report the findings so that the cord blood will not be stored.

Please feel free to ask any questions!
Thanks!