9th week of SIP - Immunology

Hi everyone! Is my turn again! These few weeks I am attached to the Immunology department of my laboratory. Sound fun but practically nothing much to do as mainly the tests is done automatically. My lab is using a system called ADVIA Workcell whereby it employ a unique sample handling process that integrates the ADVIA Centaur Immunoassay System and the ADVIA 1650 Chemistry System to manage the patients' samples. In Immunology, the main machine we are using is the ADVIA Centaur Immunoassay System.

Taken from

http://diagnostics.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1009257~a_langId~e_-111~a_productId~e_172958~a_storeId~e_10001.htm

The tests which are done in the Immunology Department are for infectious diseases (e.g. HIV, Hepatitis, HCV, etc.), fertility (e.g. Prolactin, Progesterone, Follicle Stimulating Hormone, etc.) Thyroid Function (e.g. Free T4, Free T3, TSH, etc.), and more (can't possibly name everything out.). Some are scheduled tests; some are tests that are run everyday. And of course, my department also does Dengue Testing. But I will more focus on the HIV testing using ADVIA Centaur Immunoassay System as the samples are the most in the department and it has it own Centaur machine (just for running HIV).

IMMUNOLOGY - HIV TESTING

Human Immunodeficiency Virus (HIV) is a retrovirus that can lead to Acquired Immunodeficiency Syndrome (AIDS), a condition whereby the human's immune system begins to fail, leading to life threatening opportunistic infections. HIV primary infects vital cells in the human immune system such as T-helper cells, macrophages and dendritic cells. It will lead to al low level of CD4+ cells number to decrease below critical through direct viral killing of infected cells, increase rate of apoptosis in infected cells and killing of infected cells by lymphocytes that recognizes the infected cells. This will cause the cell mediated immunity will lost and the body will increase susceptibility to infection. Infection can occur through several routes such as unprotected sexual intercourse, contaminated needles, transmission from infected mother to her baby at birth or through feeding breast milk. Treatment is now available but it will only prolong life span of the person but cannot fully cure the person.

The assay used for testing HIV is HIV 1/O/2 enhanced assay (EHIV). The samples we need to test for HIV is the patients' serum (preferably from plain or EDTA tube). This assay is an in vitro diagnostic immunoassay for qualitative determination of antibodies to HIV Type 1, Group O, and/or Type 2 in serum. The assay is an antigen bridging microparticle cheminluminescence immunoassay used for the detection of antibodies to the HIV in serum.

Principle of the Assay:

It is a two wash antigen sandwiched immunoassay in which antigen are bridge by the antibody present in the patient sample. The Solid Phase contains a preformed complex microparticles and the HIV antigen. This reagent is used to capture anti-HIV Type 1 and Type 2 recombinant antigen that is used to detect anti-HIV antibodies bound to the Solid Phase in the patient's sample.

Before Automation Steps are performed by the machine, some steps need to done manually:

• Order the test by batch (by rack number). Note that each rack can only contain 5 sample tubes.
• Remove the caps of the sample tubes and make sure the appropriate barcode is pasted on it and the volume of the serum is sufficient. If not, the serum need to be aliquot out into a new test-tube with the patient barcode pasted on it.
• Place the sample tubes on the 1st rack ordered and place in the machine. Make sure the tubes sit in properly to prevent prop crashed.
• Place the last few tubes on the last rack ordered. In between any racks can be used, meaning that the racks that are used in between the two racks ordered will be tested for HIV.
• Then the machines will take in the racks one by one.

Automatic steps that are performed by the machine:

• 50ul of patient's serum will be aliquot out form the sample tube into a cuvette and incubate for 6 min at 37°C.
• Reagents will be aliquot into the cuvette and incubate again for 18min at 37°C.
• The cuvette will then wash with Wash 1.
• Another reagent will then be aliquot into the cuvette and incubate for 18min at 37°C.
• The cuvette will then wash with Wash 1 again.
• Acid Reagent and Base Reagent will then aliquot into the cuvette to initiate cheminluminescence reaction.

Reference Range:

• Reactive is more that 0.9
• Non-reactive is less that 0.9

If Reactive:

• Repeat the testing again. Spin down the sample tube at 10000rpm for 10 min, aliquot the serum out into a new test tube with appropriate barcode, then rerun in another machine.
• If repeat test is negative, report as "Not Detected".
• If repeat test is positive, call clinic to inform Doctor that Western Blot need to be done for confirmation.
• If Western Blot is negative, attached the report to screening report and send directly to Doctor.
• If Western Blot is positive, reports need to be notified to referring Doctor via phone and the report will be sent directly to the Doctor in a confidential envelope and CDC will be informed.
• The reactive samples will be sent to hospital for Western Blot test.

No wonder Ye Tun, you tell me that you received samples from my lab. You all should know where the Western Blot is done by now.... Hope my post helps you all to understand HIV testing. Feel free to ask me questions.

Lizzie Chew, TG01, 0503194C

8th week of SIP - Flow Cytometry

sorry for this late entry...

Flow Cytometry
Flow cytometry is a technology that simultaneously measures and analyses physical or chemical characteristics of single particles, usually cells, delivered in a flowing stream to the laser beam. The properties measured include the particle's relative size, granularity and fluorescence intensity. The flow cytometer I'm currently using is known as FACSCalibur from Becton Dickson.

Fig 1: FACSCalibur. Taken from http://igc-wiki.igc.gulbenkian.pt/lib/exe/fetch.php?w=&h=&cache=cache&media=uic:flowcytometry:facscalibur.jpg


Mechanism of FACSCalibur
The cytometer consists of 3 main subsystems: fluidics, optics and electronics. The fluidics systems is responsible for the transportation of cells, from 0.2-150µm in size, in a sheath stream to the observation point where the laser intercepts the cells (refer to Fig 2).. Only 1 cell should move through the laser beam at a given moment. To achieve this, the sample is injected into a stream of sheath fluid within the flow chamber (flow cell). The flow of sheath fluid accelerates the cells and restricts them to the center of the sample core, a process known as hydrodynamic focusing. Priming of the instrument to remove air bubbles and debris in the fluidics system is necessary to ensure proper interception of cells at the laser beam.

Fig 2: Inside the flow cell. Taken from http://users.path.ox.ac.uk/~nrust/diag1.gif


In the optics system (refer to Fig 5), the laser will illuminate the cells in the sample stream and light signals are generated, which in turn will be processed by the light detectors. Forward scattered light (FSC), light scattered just off the axis of the incident laser beam in the forward direction, is detected by a photodiode. A photodiode is a mechanical obscuration in the path of the laser beam that prevents the laser light itself from reaching the FSC detector, thereby allowing the detection of the FSC. The FSC signal is proportional to the cell-surface area/size (refer to Fig 3). Therefore, the larger the cells, the more intense is the FSC signal. Side scattered light (SSC) is collected at approximately 90° to the laser beam by a collection lens and then redirected by a beam splitter to the SSC detector (photomultiplier tube). It is proportional to the cellular granularity (refer to Fig 3). Correlated measurements of FSC and SSC are useful in distinguishing the different cell populations in blood sample (refer to Fig 4).

Fig 3: FSC and SSC. Taken from http://www.uams.edu/flowcytometry/Images/FSC&SSC.jpg


Fig 4: Cell popultions based on FSC vs SSC. Taken from http://www.flowlab.ucalgary.ca/flowlab/images/FSC.gif


Emission of fluorescence by the fluorochromes conjugated to a monoclonal antibody is also collected by the same lens that collects the SSC. The fluorescence signals are directed to their respective detectors via a system of mirrors and optical filters. The first mirror that collects light encounters is a 560SP (short pass) mirror, which allows light shorter than 560nm to pass and reflects light longer than that to other optical filters. The light that passes through this mirror then reach a beam splitter that reflects 10% of the light to SSC detector and lets the remaining 90% pass, which will reach the FL1 detector for short wavelength (green) fluorescence. A 530/30 bandpass filter is placed in front of FL1 detector to allow only light of wavelength 530 ± 30nm to reach the detector, eg FITC. The bandpass filter serves to optimise the specificity of the detector. Light reflected by 560SP mirror will reach a 640LP (long pass) dichroic, a beam splitter. Wavelengths shorter than 640nm are reflected to the FL2 detector for medium wavelength (yellow/orange) fluorescence, and longer wavelengths are passed and will eventually reach the FL3 detector through a 670LP filter. The amount of fluorescent signal detected is proportional to the number of fluorochrome molecules on the particle.

PS: Sorry guys.. it's kinda hard to digest at first, but hope u'll understand.. From Fig 5, you'll notice a half mirror placed just after the 640LP filter rite? dun risk asking me its purpose.. it's more confusing.. It just basically for FL4 detector.. wad's FL4 detector purpose? also pls dun take risk.. just pretend it's not there.. thanx..

Fig 5 (below): Optics System in FACSCalibur. Taken from http://www.sickkids.ca/fcf/images/FACSlayout.gif


Fig 6 (below): Examples of fluorochromes detected by different detectors. Taken from http://images.google.com.sg/url?q=http://facs.scripps.edu/images/facscolors.jpg&usg=AFQjCNGHeSQIyJ8hHvjT2spGEVX5e-ZWRg


All the signals detected are converted into electronic signals by the electronic system, and then processed by the computer. Analysis of the cell population can be displayed in several formats. The format I'm using is dot plot, which provides a two-parameter display of data. Each dot represents 1 or more events. Gating is required to restrict the analysis to only the cell population of interest. In Fig 7, the lymphocytes population is gated (in red colour) on the left graph, and therefore the resulting display on the right would only reflect the fluorescent properties of only the lymphocytes. A quadrant marker is used to divide the two parameter plots into four sections to distinguish populations that are considered -ve, single +ve or double +ve. The lower-left (LL) quadrant display events that are -ve for both parameters. The upper-left (UL) quadrant display events that are +ve for the y-axis parameter (CD4) and the lower-right (LR) quadrant are events that are +ve for the x-axis parameter (CD8). The upper-right (UR) quadrant display events that are positive for both parameters (CD4/CD8), or double +ve. Gated events are expressed in the form of percentage.

Fig 7: Dot plot with a gate emcompassing the lymphocyte population. Taken from http://www.bio.umass.edu/micro/immunology/facs542/hdots.gif


If you're still reading, thanx so much for ur time! distinction for ur patience! tried to minimise the information load.. but it still seems like... haix.. took a month to understand this flow cytometer.. I should say its inventor must be real free and nth else better to do.. but nevertheless, it's a veri much useful instrument anyway (ability to perform 10 000 events, shorter turnaround time).. Should you have any qn.. (erm.. can try not to ask?) ask simple qn will do.. and not too many pls.. thanx so much!

Joan
TG01

7th week of SIP - Virology

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.

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

6th week of SIP - Biochemistry

Hey everyone,

This week, I was posted to the biochemistry lab. The lab consists of 3 sections.

1^st section

Consist of the main analyzer Beckman Coulter Synchron LX20 PRO Clinical System. Test for levels of Urea, creatinine, glucose, total protein, AST, GGT, etc. in blood serum.

2^nd section

Consist of analyzers to conduct tests such as

• Blood gas
• Serum lactate and ammonia test
• Serum bilirubin
• HbA1c to test for diabetes
• G6PD testing
• Serum and urine osmolality.

3^rd section

Also called the ‘urine bench’. Conduct tests such as

• Urine FEME
• Fungal test
• Occult blood

Everyday, we receive the specimens, which comes together with a request form and a sticker label. We check the name and NRIC of the patient and the tests requested on the form. We then centrifuge the specimens for 10mins. However, some specimens for tests like HbA1c and Urine FEME does not require centrifugation. Blood specimens come in a plain tube (contain silicon gel), EDTA tube, or fluoride tube. After centrifugation, we will then process the specimens according to the tests requested.

I will explain more on G6PD testing.

G6PD Testing

• Blood specimens come in EDTA tube to prevent clotting.

• Prepare empty tubes for the samples and controls.

• 3 controls. Deficient control (commercial), intermediate control (commercial), and positive control (patient sample)

• 100µl of buffer (to lyse the cells) is aliquoted into each tube.

• Aliquote 5µl of samples into the tubes containing the buffer.

• Shake the tubes vigorously to mix, then incubate for 10mins to fully lyse the red cells, thus releasing the G6PD enzymes.

• Prepare the cards to test for fluorescence. On each card, there are blank circles for each sample. Label the sample number and controls on the card, then proceed to aliquot 10µl of the sample solution and controls onto their appropriate circles.

• Place the cards in an incubator for 10mins to dry the samples, as it will be difficult to see fluorescence if the samples are wet.

• After incubation, place the cards in a UV illuminator, ‘Model CX-21 Ultraviolet Fluorescence Analysis Cabinet”.

• The positive control should show fluorescence. Intermediate control should show slight fluorescence, and the deficient control should show NO fluorescence.

• Check for fluorescence for the samples, to determine if the patient has G6PD deficiency. If they are deficient for G6PD, their sample will show no fluorescence, and these samples will be tested again just to confirm the results.

Theory behind G6PD Test

G6PD is an enzyme found in RBCs. G6PD deficiency is due to labile G6PD enzyme that is present in young cells but rapidly disappears with cell aging. It results in hemolysis after exposure to certain oxidant drugs such as anti-malarials, and also during infections. The disease is transmitted as an X-linked recessive, and is more prone in males.

Alright, that is all for now. Feel free to ask any question. Hope you guys are enjoying yourselves just like me! =D

Out.

Adrian Tan TG01
0503205G