Hi Everyone!
I am happy to be here to share my experience in SIP and some of the techniques/tests I learnt. Basically virology has 4 sections: Viral Serology lab; Viral Immunofluorescence (VIF) lab; Viral Isolation (VI) lab & Tissue Culture lab. *BSL2 lab.
As virology deals with lived viruses (especially viral isolation), potentially biohazardous materials and potentially dangerous chemicals (carcinogenic, cytotoxic, etc), safety procedures must be strictly followed. BSL2 safety cabinets are used and PPE are necessary.
I have gone to viral immunofluorescence and I am now in the tissue culture and viral isolation. I found that all the sections are related to each other, e.g. tissue culture grows cells for viral isolation. Immunofluoresecence is used as a confirmatory test for viral isolation and also used for mycoplasma testing of cell-lines.
::Medical Microbiology
I am sharing about flu surveillance which needs tissue culture, viral isolation and immunofluorescence techniques. Specifically I am sharing about flu surveillance shall-viral samples.
Clinical significance
When we receive the specimens for flu surveillance, we are screening for whether the sample has flu A or flu B or no flu virus. If the sample is found flu A positive, viral identification needs to be done to see whether it is H1, H3 or H5. If the sample is found H5, it can be H5N1(avian flu) and the further testing must be done in the Virology BSL-3 lab. Results are then recorded and reported to the public-health regulatory authorities.
Sample receiving/processing
Flu surveillance samples are usually respiratory samples like BAL (bronchial lavage), NPA (nasal pharyngeal aspirate), throat swab, lung autopsy specimens. They are received by viral isolation lab and the specimens are processed accordingly.
When receiving the sample, BAL, NPA and swabs should come in Hanks' transport media (prepared by tissue culture lab). Hanks' media is to let the virus survive before the specimens is processed and the viruses are isolated. I have observed the processing of BAL and swabs. BAL has to be centrifuged at 3500 rpm for 20 min at 4C. After that supernatant is used for inoculation into the cell culture tube. Swabs just need to be vortex and they are ready for inoculation.
Principles of the tests
Shell vial assay (viral isolation)
MDCK is the most sensitive cell lines for influenza A and B. MDCK cells are grown in the shell-vials. Shell-vials are the sterile yellow-cap 5ml tubes with flat bottom with a round sterile coverslip in each tube. MDCK cells grow on the surface of the coverslip. Then the shell vials are inoculated with processed specimens and they are centrifuged to allow the cells and viral antigens stick to the coverslip. After 48 hours of incubation at 33C, shell-vials are ready to be tested by indirect immunofluoresence assay (IFA).
NOTE: All the viral procedures are carried out in class2 biosafety cabinet.
Indirect immunofluoresence assay (IFA) for viral identification
Monoclonal antibodies specific to flu A and flu B are used to detect the presence of flu A or flu B antigens in the specimen. The principle is that if the viral antigens are present in the cells, monoclonal antibodies (MAb) specific to that antigen will bind to the antigens. Then secondary antibodies containing FITC (fluorescence substrate) conjugate will bind to monoclonal antibodies. When viewed under the fluorescence microscope, FITC substrate was exicted by UV light and give apple-green coloured fluorescence. Therefore a positive test is indicated by the presence of apple-green coloured fluorescence. Conjugate also contains a counter stain, Evan's Blue, which stains the cells dull red. Hence, positive test is seen as dull-red background with green fluorescence while negative test is seen as dull-red without any fluorescence.
I am happy to be here to share my experience in SIP and some of the techniques/tests I learnt. Basically virology has 4 sections: Viral Serology lab; Viral Immunofluorescence (VIF) lab; Viral Isolation (VI) lab & Tissue Culture lab. *BSL2 lab.
As virology deals with lived viruses (especially viral isolation), potentially biohazardous materials and potentially dangerous chemicals (carcinogenic, cytotoxic, etc), safety procedures must be strictly followed. BSL2 safety cabinets are used and PPE are necessary.
I have gone to viral immunofluorescence and I am now in the tissue culture and viral isolation. I found that all the sections are related to each other, e.g. tissue culture grows cells for viral isolation. Immunofluoresecence is used as a confirmatory test for viral isolation and also used for mycoplasma testing of cell-lines.
::Medical Microbiology
I am sharing about flu surveillance which needs tissue culture, viral isolation and immunofluorescence techniques. Specifically I am sharing about flu surveillance shall-viral samples.
Clinical significance
When we receive the specimens for flu surveillance, we are screening for whether the sample has flu A or flu B or no flu virus. If the sample is found flu A positive, viral identification needs to be done to see whether it is H1, H3 or H5. If the sample is found H5, it can be H5N1(avian flu) and the further testing must be done in the Virology BSL-3 lab. Results are then recorded and reported to the public-health regulatory authorities.
Sample receiving/processing
Flu surveillance samples are usually respiratory samples like BAL (bronchial lavage), NPA (nasal pharyngeal aspirate), throat swab, lung autopsy specimens. They are received by viral isolation lab and the specimens are processed accordingly.
When receiving the sample, BAL, NPA and swabs should come in Hanks' transport media (prepared by tissue culture lab). Hanks' media is to let the virus survive before the specimens is processed and the viruses are isolated. I have observed the processing of BAL and swabs. BAL has to be centrifuged at 3500 rpm for 20 min at 4C. After that supernatant is used for inoculation into the cell culture tube. Swabs just need to be vortex and they are ready for inoculation.
Principles of the tests
Shell vial assay (viral isolation)
MDCK is the most sensitive cell lines for influenza A and B. MDCK cells are grown in the shell-vials. Shell-vials are the sterile yellow-cap 5ml tubes with flat bottom with a round sterile coverslip in each tube. MDCK cells grow on the surface of the coverslip. Then the shell vials are inoculated with processed specimens and they are centrifuged to allow the cells and viral antigens stick to the coverslip. After 48 hours of incubation at 33C, shell-vials are ready to be tested by indirect immunofluoresence assay (IFA).
NOTE: All the viral procedures are carried out in class2 biosafety cabinet.
Indirect immunofluoresence assay (IFA) for viral identification
Monoclonal antibodies specific to flu A and flu B are used to detect the presence of flu A or flu B antigens in the specimen. The principle is that if the viral antigens are present in the cells, monoclonal antibodies (MAb) specific to that antigen will bind to the antigens. Then secondary antibodies containing FITC (fluorescence substrate) conjugate will bind to monoclonal antibodies. When viewed under the fluorescence microscope, FITC substrate was exicted by UV light and give apple-green coloured fluorescence. Therefore a positive test is indicated by the presence of apple-green coloured fluorescence. Conjugate also contains a counter stain, Evan's Blue, which stains the cells dull red. Hence, positive test is seen as dull-red background with green fluorescence while negative test is seen as dull-red without any fluorescence.
Firstly cells grown in shall vials are scraped and cells are resuspend in phosphate buffer saline (PBS) just to get adequate concentration of cells to be spotted on the wells of the slide. The cell suspension is then spotted on the wells. Two wells (one for flu A and the other for flu B) will be used for each specimen. Then the slide is air dry. When the slide is dry, the slide is fixed in cold acetone for 10 mins, monoclonal antibodies (MAb) specific to flu A and flu B are added to respective wells and the slide is then put inside the humidity chamber and incubated at 37C for 30 minutes. After incubation, the slide is rinsed with PBS and then washed with PBS for 10 minutes (immerse in PBS and use the shaker). This is to remove excess and unbound monoclonal antibodies in the wells. Then the conjugate specific to flu A and flu B MAb is added to respective wells and the slide is then put inside the humidity chamber and incubated at 37C for 30 minutes. After incubation, the slide is rinsed with PBS and then washed with PBS for 10 minutes (immerse in PBS and use the shaker). This is to remove excess and unbound conjugate in the wells. After that, the slide is air dry, mount and observed under the fluorescence microscope.
If the specimen is flu A positive, the cell suspension of that specimen is again spotted on the three wells labelled H1, H3 & H5. The procedure is same as mention above, it's just that monoclonal antibodies specific to H1, H3 & H5 of flu A are used. H1 is reported as H1N1, H3 is H3N2 and H5 is sent to BSL3 lab for further testing.
I think I covered basic principles, procedures and significance of the tests. If you have any question, feel free to ask, regarding to viral isolation, tissue culture, immunofluorescence and other things. Actually viological tests (Viral Immunofluorescence (VIF); Viral Isolation (VI) & Tissue Culture) are quite time-consuming, labour-intensive and need special facilities and techniques and everything is done manually. Viral serology is 70% automated.
All the best for your SIP and MP!
Cheers
Ye Tun
TG01
13 comments:
Hey Ye tun,
For your IFA tests, do you guys also test for parainfluenza strains? and if so, would the procedures for flu A and parainfluenza be alike?
Hi Star Team
Yes. We also do viral identification for parainfluenza strains. IFA procedures for parainfluenza is alike to flu A. Monoclonal antibodies specific for paraflu strains 1, 2, 3 are used. Usually the doctor requests for respiratory virus panel by immunofluorescence, which include RSV,FluA, FluB,Paraflu, Adeno & respiratory pool. If paraflu or fluA is detected positive, viral identification has to be done.
Ye Tun
TG01
hey ye tun...
is flu A H1 or H3 harmful in any sense? or is it just normal flu virus?
~Jeremy~
Hi Jeremy
H1N1 and H3N2 are the influenza A viruses circulating among people and cause sesonal flu nowadays. But H1N1 (spanish flu) was a pandemic in 1989 and killed more than 50 million people. H3N2 caused Hongkong flu pandemic in 1968.
Since those strains were experienced before, vaccines and treatments have been developed and are widely available. Most of the people nowadays also have antibodies againsts those strains and so the cases are not very severe unless there are other conditions or complications which can increase the severity.
Usually, for clinical/diagnostic specimens, we just report to the doctor that that patient is fluA or fluB positive or negative, without mentioning the strain. But strains detected are reported to regulatory bodies for surveilliance.
Cheers
Ye Tun, TG01
Hi man,
Good to see u keeping that smile on your face despite all the stress! =D
Anyway, u mentioned that "Hanks' media is to let the virus survive before the specimens is processed". What are the components of this media and how do they aid in viral survival?
Also, u said that "MDCK is the most sensitive cell lines for influenza A and B." I guess there are other antigens can be found in the specimens? Does that mean that many different cell lines will have to be used for different viral isolation?
Finally, I think it will be good to include some pictures or diagrams (e.g. antibody binding) to facilitate better understanding.
Keep Smiling!
Kent
TG01
Hi Kent
Thanks for your comment.
Basically Hanks' tansport media is the Hanks' balanced salt solution mixed with mantainance media for cell culture. Salt solution provides right environment for the cells and it contains CaCl2, MgSO4-7H2O, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4-2H2O, and d-glucose. The media contains essential nutrients for cells. As we can see from its composition,it has salts/ions to give right osmotic environment, buffers to give the right pH and energy source for the cells so that the cells are in their normal physiological conditions which allow the viruses to grow inside them.
If we are testing viral antigens (viral infected cells, not necessarily lived virus), we can accept the swabs immersed in normal saline. But for viral isolation, we need the lived virus in the specimens, therefore the cells must be in a condition that it can maintain the virus inside them. That's why Hanks' media is needed for tansportation of specimens for viral isolation.
Different cell lines have sensitivity to different cell lines. One cell line may be sensitive to a number of viruses and one virus may also be isolated in more than one cell-lines. Therefore a number of cell-lines are used to isolate a virus
Usually cytopathic effects (CPE) are observed in the viral isolation in tube culture (not in shell-vials). But CPE alone cannot confirm the virus in the specimens, therefore, confirmatory test by immunofluorescence must be done for very cases.
I will post some pictures if I can find them.
Cheers
Ye Tun
TG01
Hi Casper.......
You mentioned that 70% of the work is automated, how come it's still labour intensive? Interesting sharing! Thanks.
Pei Shan
TG02
Hello!
I am dealing with indirect immunofluorescence too =) However, i have been seeing loads of artefacts which are showing positive results after FITC is added. Did u encounter that problem too? If so, is there like a specific way to see if the bright green fluorescence is the flu virus or simply non-specific binding of the artefacts?
Chen kangting
0503331A
TG02
Hi Pei Shan
Hi. I have yet to go to viral serology section, but from what supervisor told me and what I observed, it is 70% automated. But automation is not a total automation because technicians still need to process the samples, if necessary, and load the samples in the machines. There are also a lot of manual tests such as hemeagglutination test. The person also need to give lab no manually (using stamps/ no stickers/ no barcode)and key into the system manually.
Heavy quality control also has to be done and reporting of results must really be careful as the lab is the reference lab for HIV.
But from what I've observed in other sections like viral immunofluorescence, isolation and culture, there is little oppotunity for automation.
Cheers
Ye Tun
TG01
Hi Kangting
Thanks for the comment. It's a good question.
Yes. I often encountered artefacts and non-specific bindings. But to determine the fluorescence is true positive, there are some factors to considered.
A positive immunofluorescence result (virus antigen present) was dependent upon observation of specific intracellular fluorescence patterns.
E.g. in detecting Cytomegalovirus (CMV)by IFA,the nucleus of the cell should be stained with apple-green fluorescence; the fluorescence shouldn't be seen in cytoplasm. So it really depends on the nature of the virus. For respiratory virus panel, cytoplasmic for RSV and the paraflu, nuclear for flu A and flu B, and cytoplasmic and nuclear for Adeno.
As we are using Evan's blue as a counterstain to stain the cells dull-red, it is easier to see the patterns and distriubution of the fluorescence.
For respiratory panel, we include respiratory pool, for which we use a mixture of all the specific monoclonal antibodies to the respiratory viruses. So, if one of the respiratory viruses is positive, respiratory pool should be a positive too. That's how we ensure the true positive.
Sometimes, we need to repeat the test to trouble-shoot and make sure the result is correct. If it is very difficult to determine the result, we need to seek advice from the senior consultant virologist. Of course, standard and validated lab procedures and quality control with the skill of the lab personal will help to get a true result.
Cheers
Ye Tun
TG01
Hey ye tun!
What's the difference between Flu A and Flu B? Thanks!
Take care!
Charmaine Tan TG01
Hi Charmine
Actually both flu A and flu B are influenza viruses and they have properties such as genome, stucture, size, etc. The difference is that flu B is only found in human, there's no other animal source and therefore no antigenic shift occured and it is not divided into different strains. But flu A can potentially found in other animals like birds, chickens, pigs. So flu A undergo antigenic shift and there are different strains of flu A.
Actually H5N1 or avian flu we are so afraid of today, is just a flu virus for birds, but when antigenic shift occurs, that strain of virus gains property to infect other animals like human. Since human never experiences H5N1 before and has little immunity to it, it causes severe consequences.
Antigenic drift can occur in both flu A and flu B and it causes common/sesonal flu.
For info about flu strains can be found out in the URL below:
http://www.cdc.gov/flu/avian/gen-info/flu-viruses.htm
Cheers
Ye Tun
TG01
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