- LRN Meeting
- New Instrument
I enjoy sitting quietly at my desk, arranging and re-arranging words like a child with building blocks. This creative process gives me a deep sense of satisfaction. However, every now and then I have to renew my energy. Motivation is only a few steps away. Observing the daily work of the Laboratory is impressive. For this issue, I had fun touring the HIV/STD laboratory. The laboratorians in the area were helpful and knowledgeable as I asked questions and marveled at the highly technical equipment. They even managed to make sense of my drafts as I wrote the article “Lab Tour: HIV/STD.”
We have touched on the Laboratory Response Network (LRN) in past issues: “Laboratory Response Network: Texas Style” and “Emergency Preparedness Branch: ‘Travelling Road Show’ Wet Workshops.” The preparatory work of the LRN is ongoing. They meet yearly to plan responses to biological and chemical threats and learn from their colleagues. The article on the “Texas Laboratory Response Network Meeting” is a portal into this important work.
Laboratorians in the DSHS Laboratory are always working to improve our processes. “Don’t Be Snow White – Test Your Apples First” is a heady look at a new instrument being used for food testing. Stick with the article through the tongue-twisting introduction; the technology is fascinating and certainly offers food for thought.
by Jimi Ripley-Black
Did you know?
The DSHS Tuberculosis (TB) Laboratory has been testing TB specimens for the Louisiana Office of Public Health Laboratory since March 7, 2014, while the Shreveport regional laboratory is moved to Baton Rouge. This adds 300-500 specimens to the existing workload of more than 1,425 specimens per month. Our laboratory was asked to assume the testing for six months to a year.
The large room takes up the entire end of the fourth floor in the DSHS Laboratory building, allowing the HIV/STD and Serology teams to share the space. Scientific machines around the periphery beat with a deep hum that is as much felt as heard. Five long benches, placed in parallel across the brightly-lit room, hold testing equipment ranging from pipettes and Microtiter plates to sinks. The air smells faintly of chemicals with the metallic tang of moving machinery. Laboratorians in white coats monitor the large machines and measure fluids into tiny receptacles. To a visitor, the activity is nebulous and vaguely mesmerizing.
The HIV/STD team consists of five laboratorians who perform testing to identify HIV, AIDS and Sexually Transmitted Diseases (STDs) in Texas. Specimens for HIV testing are oral fluid, serum and dried blood spots. Specimens for syphilis testing are serum. Specimens for Chlamydia trachomatis (CT) and Neisseria Gonorrhoeae (GC) testing are urine, vaginal, endocervical and urethral specimens.
Approximately 38,000 tests per year are done to identify HIV-1, HIV-2 and the p24 antigen using a type of testing called HIV COMBO Ag/Ab EIA. “The exciting thing is that we went to the HIV COMBO, which allows us to catch HIV quicker,” said Team Lead Dawn Dutelle. HIV COMBO is the most up-to-date testing method that allows for detection earlier in the disease process. Both BioRad ELITE and EVOLIS are used to perform this testing.
This Multispot Rapid Test is used to confirm an initial reactive (positive) for HIV serum or plasma using a qualitative assay based on immune concentration. At a bench on the far side of the room, Microbiologist Sofia Madinaveitia observes an array of small, gray boxes (cartridges). Dark purple dots gradually develop, spaced equally in the four corners. The dot in the top, left corner is the control to confirm that the test is working. Two dots on the right indicate HIV-1 while an additional dot in the lower, left corner would indicate HIV-2. Madinaveitia carefully records the results.
Chlamydia and Gonorrhoeae (CT/GC) testing is done on approximately 24,000 specimens per year using the GenProbe/Hologic Tigris DTC instrument. In 2012, testing for gonorrhea and chlamydia was moved from the Women’s Health Laboratory in San Antonio. Integrating this kind of testing into the work of the Austin Laboratory, while improving the accuracy of results, is an accomplishment that makes the HIV/STD Team justifiably proud.
Syphilis is screened for on the BioPlex 2200 instrument. "This is a new instrument to our area that we started using in September ," said Dutelle.
||The Bioplex 2200, which sits to the right of the Tigris, uses a multiplex flow immunoassay for qualitative detection of T. pallidum IgG antibodies. (Treponema pallidum is the bacterial spirochete responsible for syphilis infections.) Using the antibody index to measure, the reagent contains beads for quality control:
- Internal Standard Bead (detector response)
- Reagent Blank Bead (absence of significant non-specific binding in serum or plasma)
- Serum Verification Bead (addition of serum or plasma)
- The different populations of dyed beads coated with recombinant proteins associated with T. palladium
The instrument takes 15-200 measurements per bead in just 19 seconds. The first result is returned 45 minutes from the start of testing and additional results are reported in just 36 seconds for each succeeding test. The Bioplex 2200 measures luminescent beads to determine results using Relative Light Units. This technology has allowed for higher accuracy in reading results. “The positivity rate has gone from 6 percent to 3 ½ percent,” said Microbiologist Martin Morales. He explains that the machine is an automated nucleic acid testing system that can process “five different results in 45 minutes.”
Dutelle points out the part of the machine where reagents are automatically diluted, which is a huge time-saver. Results are also reported directly into the computer system. The Tigris uses four assay technologies:
- Target Capture (isolation and purification of sample)
- Transcription mediated amplification of nucleic acid particles produces multiple copies of RNA for easier detection.
- Hybridization Protection (HPA - detection of light-emitting nucleic acid probes)
- Dual Kinetic Assay (analysis of chemiluminescent data to differentiate the presence of two signals in a single tube).
|All reactive Syphillis IgG results are further tested with the Rapid Plasma Reagin (RPR) test and the Treponemal Particle Agglutination Test (TPPA), according to CDC guidelines. The RPR test is a qualitative and semi-quantitative non-treponemal test that detects reagin – the body’s response to inflammation.
This non-specific test for syphilis is then followed by the TPPA (hemaglutination test specific for T. pallidum). Serum containing specific antibodies will react with the antigen sensitized colored particles to form a smooth mat of agglutinated particles in the microtitration plate.
|On a nearby bench is a 96-well Microtiter plate suspended over a mirror. Dutelle explains that TPPA is a confirmatory test for syphilis. Small amounts of serum are added to the Microtiter plate in different dilutions and the results develop over two hours or an overnight incubation and are read for agglutination.
||The mirror makes it easier to see the different reaction patterns in each of the wells. She points out the controls and different dilutions. Some of the wells look like bright pink dots, which correlates with a non-reactive test result. The reactive TPPA result is more spread out over the bottom of the microtiter plate.
In 2013, the HIV/STD team processed the following specimens:
- HCV-Hepatitis C virus = 1,720
- HIV (Human Immunodeficiency Virus) SERUM COMBO = 38,494
- DBS (Dried Blood Spot) - EIA (Enzyme Immunoassay) = 224
- ORAL Fluid EIA (Enzyme Immunoassay) = 594
- Serum WB (Western Blot) = 511
- Oral Fluid WB (Western Blot) = 122
- DBS (Dried Blood Spot) WB (Western Blot) = 124
- SYPH IgG (Syphillis IgG) = 9,011 since start of testing in September 2013
- RPR (Rapid Plasma Reagin) = 22,944
- TP-PA (Treponal pallidum Particle Agglutination Test) = 1,683
Even children below the age of 14 are now being diagnosed with STDs. The HIV/STD team at the DSHS Laboratory does crucial work in identifying cases in Texas. “The work that we do is very important,” said Dutelle. “That is why I come to work each day.”
by Jimi Ripley-Black
Every year, the Emergency Preparedness Branch hosts the Texas Laboratory Response Network (LRN) Meeting in Bandera, Texas. This meeting provides a forum for scientists to share current information on new technologies and best practices for detection of threats. It also facilitates networking among local, state, federal and non-governmental partners.
The LRN was established by the Department of Health and Human Services and the Centers for Disease Control and Prevention (CDC) as a result of the Presidential Decision Directive 39 to outline national anti-terrorism policies and assign specific missions to federal departments and agencies.
This directive became effective in August 1999 (http://www.bt.cdc.gov/lrn/).
The LRN laboratories are prepared to respond to biological and chemical threats and other public health emergencies. In the DSHS Laboratory, the LRN is made up of the Biothreat Team and the Chemical Threat Team that are a part of the Emergency Preparedness Branch. As one of the largest states, Texas is composed of 10 LRN sentinel Laboratories, strategically located across the state based on population and geography.
Attendance at the Texas LRN Meeting is composed of chemists, microbiologists and first responders who are responsible for preparing for, responding to and detecting chemical, biological and radiological threats. Typically, at least one representative from each of the sentinel labs attends.
The first day is a closed session during which representatives from the labs present updates, issues or challenges, and any goals and projects they are working toward for the year. This gives representatives the opportunity to ask questions of people from other LRN laboratories and brainstorm any issues. Day two opens up attendance to include various local, state and federal partners.
In 2014, presentations ranged from a West Nile Virus Overview in Tarrant County to a presentation on the field work that goes into biosurveillance, to an overview of FBI and Hazmat roles and how they relate to the LRN. Representatives from the Association of Public Health Laboratories (APHL) also attend this conference to present and assist the Emergency Preparedness Branch. This year, the APHL’s presentation was about first responder outreach activities across the nation – an important topic for all LRN labs.
For further information on the topics discussed, selected presentations can be found on the Emergency Preparedness webpage. As you will see, the conference covers a wide range of topics. Beyond being a great learning opportunity, the meetings are also a chance for people to put a face to the name, improving communication outside their own organization.
by Vanessa Telles
The GC/MS Group in the Organic Chemistry Department has a new instrument, and the name is a mouthful. The Triple Quadrupole Liquid Chromatography Mass Spectrometer (LCMSMS) is a tandem mass spectrometer with two quadrupole mass spectrometers, in series, with a quadrupole separating them for ion fragmentation.
Organic Chemistry will use the LCMSMS to test for pesticide residues in food products, such as fruits and vegetables, where pesticides are commonly used. Another use for this instrument is drug toxicology on human and animal specimens such as urine, blood, or plasma.
To prepare the samples, the food must be broken down and extracted in a useable, instrument-friendly form. The “QuEChERS” method, which stands for “Quick, Easy, Cheap, Effective, Rugged, and Safe," will be used for preparation. In general, the analyst homogenizes the sample in a blender. The sample is then placed in a centrifuge tube with reagents that are determined by the type of target analytes to be extracted. The sample is agitated for a few minutes then added to a cleanup column that separates the organic solvent extracts from the bulk mass debris of the sample. The extracts are ready for analysis by the LCMSMS instrument.
Prepared liquid sample extracts are injected into the Liquid Chromatography (LC) column where the analytes begin to separate. This is caused by their affinity to the stationary phase. The stationary phase is inside a column composed of either irregularly or spherically shaped particles. The affinity between the stationary phase and the mobile phase (containing the analytes) differs based on their polarity. The polarity of the analytes usually matchs the polarity of the stationary phase in the column to increase resolution and separation while reducing run time per injection. Once separated, the analytes are detected by the Mass Spectrometer (MS) based on mass/charge ratio. The LCMSMS instrument can be set up many different ways to detect particular ions or classes of ions.
In general, the first quadrupole scans all incoming analytes, which are then passed to the second quadrupole where ion fragmentation occurs. Finally, the ion fragments are scanned in the third quadrupole. This step looks for particular product ions from the fragmentation with unique identifiable functional groups. The LCMSMS instrument is very sensitive and selective so that a wide range of target analytes can be tested. The technology has a very low signal to noise background, allowing for clear and definitive identification and analysis. If the LCMSMS had existed in 1812, the Brothers Grimm might have written the story differently and saved Snow White from a long sleep.
Chemist Xiufeng Ji has been spearheading the method development project since November 2013. She has established the sensitivity limit ranges for the LCMSMS and is very excited about the prospect of getting the instrument in production sometime in 2014.
by Brandon Flammang
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