Star Team

Can I ask how is the blood sample processed? Will the blood be cultured and how are the leukocytes isolated? And also, is the pp65 antigen only found on leukocytes? Will they be found on RBCs?

Ming Boon
 

Hey i got a lame question but just curious, the indirect IF test rite, is it automated or done manually?

and can the CMV be tranmitted to human upon contact with the blood?

Nisha Bte Mohd Rafiq
0503254E
TG02
 

Blood sample processing:

Separation solution is added to blood sample at a ratio of 1:4 (sep. sol to bld sample and incubated in order to separate RBC from WBC. You’ll see a layer of yellowish WBC suspension atop of RBC suspension in the test tube after 20 mins. The leukocytes suspension is transferred using a dropper into another test tube, and centrifuged for 10 mins. The supernatant is discarded, and the cell pellet (mostly WBC together with a thin layer of RBC) is resuspended with cold deionised water (4°C) for around 10-30 sec to lyse the remaining RBC, PBS is then added and centrifuged. The supernatant is discarded and pellet resuspended with PBS again; a coulter counter will measure the WBC concentration and respective measurements recorded on each test tubes. Then 0.2ml of suspension is added into a glass cytocentrifuge slide and centrifuged. Now, the leukocytes are settled onto the slide, and will be fixed and permeabilized. (Fixed so that cells remain on the slide after washing off excess antibody in the later steps. Permeabilized so that monoclonal antibody can bind to the pp65 antigen in the infected cells)

The leukocytes are isolated because CMV infects the leukocyte but not the RBC, thus CMV pp65 antigens can be identified within the infected leukocytes. The blood will not be cultured, since infected leukocytes can be separated readily for indirect IF assay.

The indirect IF is done manually. CMV can be transmitted to humans through infected blood via blood transfusion.

Yong Yang
 

Hi, you said control slides are prepared from known positive and negative blood samples, are these controls from patients or commercialised ones?

Andre, TG01
 

hi yongyang,

you say that negative and positive controls cannot be clearly distinguished, the test is considered invalid. is there any further test to confirm whether is it positive or negative.

Lizzie
 

Hi Y square! XD

Since you mentioned that there will be a colour change either red or green, is it possible that the results can be obtained by spectrophotometer?

Charmaine Tan, TG01
 

Hey

What are the other limitations of this test?

Are there any other tests to detect CMV in blood leukocytes?

Thanks! :)

Adrian TG01
 

To andre,
The control samples are different cell lines infected with specific viruses such as CMV, HSV-1 or HSV-2 etc. These are commercialised cell lines, while the viruses are isolated from patient samples and kept in the lab to infect cells for control samples.

To Lizzie,
If there is invalid result for the control samples, we would have do troubleshooting, such as to check if there's any contamination to the antibodies, or if we missed any steps etc. Afterthat, we'll have to repeat the test on the patient samples again.

To Charmaine,
A spectrophotometer will detect fluorescence, however, it doesn't verify whether the conjugated antibodies are bound to the slides or bound to the infected cells (dull red). Thus, we'll have to look under the IF microscope to verify the result.

To Adrian,
The test requires 2-3 tubes of EDTA blood. Patient samples with 1 tube of blood will be rejected, since we can't obtain enough leukocytes for the test.
The test is labour intensive, since it's done manually and consists of many steps, we'll have to make sure we don't miss any of the steps. There are other tests for the diagnosis of CMV, but they are either more time consuming or more expensive.
 

Hi Yong Yang

I have got a quest.
YOu said monoclonal antibody is added and bound to CMV pp65 antigen on formalin fixed leukocytes. why do have to fix the leukocytes?? U can only use formalin as a fixative? can we use other fixative?

Vinodhini
TGO1
 

To Vino,
The cells are fixed so that they remain on the slide after washing off excess antibody in the later steps. Other fixatives can be used, however, the lab uses formalin since it's already provided in the antibody kit.