July 2014 - Volume 6, Issue 1

Article Index

Editorial

This issue marks five years of publication for The Laboratorian. In that time, we have sometimes touched on similar themes, but there is always additional detail to be explored in the marvelously complex world of laboratory testing. In July 2009, we covered “DNA Analysis for Galactosemia.” The DNA screen is actually second-tier testing for the Newborn Screening Galactosemia and Biotinidase Screening program – our featured Lab Tour area.

Our most recent Newborn Screening test, implemented in December 2012, checks for Severe Combined Immune Deficiency (SCID). Read about the “First Annual Texas NBS SCID Pediatric Immunology Consultants Meeting” to learn how we are already soliciting feedback from medical professionals to improve the screening’s effectiveness.

Training is an important part of testing here at the DSHS Laboratory. In support of testing flu specimens, the Viral Isolation Team hosted training on influenza testing and subtyping. This kind of training promotes quick identification of flu outbreaks – increasing the chance of stopping the identified strains before they spread. Read “Influenza Detection and Subtyping Workshop” for more details.

Training also promotes effective response. On June 6, first Responders had an important training opportunity presented by the DSHS Laboratory Emergency Preparedness Branch. Eight people participated in the workshop covering biological and chemical threats, as detailed in “First Responder BioThreat and ChemThreat Training.”

Did you know?

The Texas Governor's Committee on People with Disabilities created online learning modules to help make Microsoft Office 2010 documents accessible to people with disabilities. You can view these tutorials on the Governor's accessible documents pages.

Lab Tour: Galactosemia and Biotinidase Screening

The Galactosemia (GALT) and Biotinidase Deficiency Screening Laboratory is tucked into a small area at the end of the third floor. The team of nine staff tests approximately 2,500 Newborn Screening (NBS) patient samples per test per day, six days a week, largely by hand. Test results are entered into the Laboratory Information Management System (LIMS) and checked for accuracy using computers in an adjoining cubicle area. However, the small work area does not mean that they are unimportant. Testing can literally mean life or death for babies with galactosemia. Babies with biotinidase deficiency may take longer to exhibit symptoms, but diagnosis can prevent life-long effects, including severe neurologic impairment.

Laboratorians in the area don’t often see the end result of their work. Instead they take pride in the accuracy of their results. Galactosemia and Biotinidase Deficiency Screening Team Manager, Chris Goben, assisted in a recent effort to track down demographic information for four patient samples. Due to a printing error, mismatched serial (kit) numbers had been printed in five different numbered areas on the Newborn Screening forms. All five areas should have the same number. “It was like solving a puzzle,” he said. “We were able to put the pieces back together within two hours. Preventing and solving problems like this is where I get my job satisfaction.”

Often, the first signs and symptoms of galactosemia include vomiting, diarrhea, lethargy, failure to thrive and jaundice, caused by hepatic failure. Without treatment,
	disease progression is rapid and death (typically due to E. coli septicemia) can be the tragic final outcome within 7-14 days of birth. The screening team races to beat the clock. Testing for galactosemia is completed within three business days, including the time from specimen accessioning in the laboratory to final release of results.

Treatment for galactosemia involves a change from feeding milk-based products to soy-based products and avoiding all foods containing lactose and galactose. This can have a larger impact on the family, particularly the mother and baby, as it negates the option of breast-feeding. This dietary change must be continued for life.

If untreated, symptoms of biotinidase deficiency include seizures, hypotonia, neurologic impairment such as lack of coordination (ataxia), hearing loss, severe skin rashes, hair loss (alopecia) and, in some rare instances, metabolic coma resulting in death. Some of these effects can be reversed with treatment.

The lack of available biotin caused by biotinidase deficiency is addressed by administering high doses of biotin as a supplement. Additional dietary restrictions may include avoiding consumption of certain foods such as raw eggs, which contain high levels of avidin, a compound that irreversibly binds to biotin and counteracts treatment. Like with galactosemia, treatment and dietary modifications are life-long.

NBS specimens are collected from all Texas babies 24-48 hours after birth and again at 7-14 days of age in the form of dried blood spots. All NBS areas use 96 well microtiter plates for specimen analysis. Each well holds either a small dot punched from a NBS bloodspot card or a dried spot control specific to the procedure.

The Galactosemia and Biotinidase Deficiency Screening Team runs two separate enzyme assays for these two metabolic disorders. The galactosemia assay detects the presence of the enzyme galactose-1-phosphate uridyl transferase (GALT) through use of a fluorometric, semi-quantitative method. A lack of fluorometric signal indicates an abnormal result and the possibility of classical galactosemia. This process involves a two-step reagent addition process interspersed with two incubation periods and is largely handled manually by technical staff:  add first reagent and incubate for three hours at 37°C, add second reagent and incubate for 30 minutes at room temperature, then analyze on a fluorometer.

The biotinidase assay detects the presence or absence of the biotinidase enzyme through use of a qualitative, colorimetric method – meaning that a series of reagents are added to test samples in order to produce a visible color change. Technicians visually examine each test well individually for the presence or absence of a color change and record results manually onto a standardized worksheet. This test requires a more elaborate five-step reagent addition process with a single six-hour incubation: add first reagent and incubate for six hours at 37°C with agitation at 900rpm, add remaining four reagents in specific, timed succession, then visually analyze. To ensure precision and minimize technical or transcription errors, test results are read and recorded by two technicians. These results are then entered and checked by two technicians into the laboratory information management system (LIMS).

“Results are time-sensitive and can have a significant impact on the success of treatment,” said Goben. “With regard to testing, we have enough emergency power outlets to continue testing in the event of a power outage.” The final assessment and reporting step requires an active connection to an internal computer network, but testing can continue on a limited basis until it is restored.

Test results for these two disorders are not impacted by patient feedings, age, birth weight or the typical course of treatment for the disorder. However, test results may be negatively affected if a specimen is exposed to excessive heat and/or humidity, which is a real concern during Texas summers. Certain chemicals, such as alcohols and polyurethane, can also skew results. More rarely, results can be skewed through administration of sulfonamides (not common for newborns), either orally or topically to the puncture site; drawing blood samples from peripherally inserted central catheter (PICC) lines; drawing patient samples post-transfusion; and administration of enzyme replacement therapy (ERT).

Galactosemia and biotinidase are both genetic disorders – the inheritance pattern is autosomal, recessive. An autosomal recessive disorder means two copies of an abnormal gene must be present in order for the disease or trait to develop; in other words, both parents have to carry the trait to pass it on to their children. Both genes in a pair must be defective to cause the disorder. People with only one defective gene are carriers who do not suffer from the disorder. Classical galactosemia impacts approximately one in 60,000 births. Biotinidase manifests as partial deficiency in approximately one out of 60,000 births and profound deficiency in approximately one out of 110,000 births. Given the Texas annual birth rate (approximately 350,000), roughly 5-7 critically effected patients may be detected per disorder annually. Actual detections can vary widely as these disorders are rare and dependent on the genetic makeup of the population.

by Jimi Ripley-Black

First Annual Texas NBS SCID Pediatric Immunology Consultants Meeting

The first annual Texas Newborn Screening (NBS) Severe Combined Immune Deficiency (SCID) Pediatric Immunology Consultants Meeting was held at the DSHS Laboratory on May 16, 2014. The DSHS Newborn Screening Clinical Care Coordination and Newborn Screening DNA Analysis Group SCID Screening Team hosted five Pediatric Immunology consultants. These consulting doctors came from cities across Texas, including Austin, San Antonio, Houston, Dallas and Corpus Christi.

On the agenda were NBS Laboratory updates, NBS Clinical Care Coordination updates, and a review of the first 17 months of screening data, from December 2012 through April 2014. (SCID screening was implemented at the DSHS Laboratory on December 1, 2012.) Three physicians also presented cases.

The consultants were impressed by the low false positive rate (0.08%) of the SCID screen and suggested that the DSHS provide the screening false positive rate to hospital neonatologists. This would help remove the current misconception that the false positive rate for SCID testing is high. During the first two months of SCID testing, before screening algorithms were adjusted, a higher number of presumptive positive specimens were initially reported. Knowing the false positive rate is actually low would encourage neonatologists, and other physicians, to pursue diagnostic testing with the assistance of an immunologist.

The SCID screening test identifies a reduction or absence of T cell receptor excision circles (TRECs), which are a byproduct of normal T cell development. From December 2012 through April 2014, a total of 14 SCID cases were diagnosed in Texas after an out-of-range newborn screen. In addition, another 247 cases of T-cell lymphopenias were identified through screening for SCID.

One of the 14 cases diagnosed with SCID was a child from India who received a polio vaccine before coming to the United States. The infant was immunocompromised and developed polio from the vaccine (SCID diagnosis was made due to a mutation in the Rag-1 gene). Because this case was discovered and referred to a Texas immunologist, it was diagnosed as a “Texas case” and will be reported in publication in the CDC’s Morbidity and Mortality Weekly Report.

The consultants felt that SCID screening and testing for TRECs brought about a “new day” in how doctors review patients and proceed with diagnosis. Because of NBS, patients are being referred to immunology specialists before symptoms appear, and that increases survival rates and makes the screen very useful.

by Lucindra "Cindy" Corrigan

Influenza Detection and Subtyping Workshop

On May 20 and 21, 2014, the DSHS Viral Isolation Team hosted a workshop at the DSHS Laboratory called “Influenza Detection and Subtyping: CDC Flu rRT-PCR Dx Panel.” The training, presented in partnership with the Association of Public Health Laboratories (APHL), Centers for Disease Control and Prevention (CDC) Division of Influenza, and National Center for Immunization and Respiratory Disease (NCIRD), was intended for public health laboratorians who work at the bench and perform influenza detection and subtyping using the CDC Flu rRT-PCR Dx Panel. The public health laboratories that participate in influenza surveillance in Texas use this flu testing kit, provided by the CDC.

Seven people attended. They came from local health departments around the state, including San Antonio, South Texas, Corpus Christi, Dallas, Houston, Tyler, and Laredo. The workshop consisted of both lectures and a hands-on laboratory component to cover all aspects of the assay performed. Information was also presented regarding influenza viruses, surveillance programs, how the assays fit into the influenza testing and reporting algorithm, and regulatory requirements.

Day one focused on the lecture portion of the training. Steve Lindstrom, from the CDC, provided an overview of the influenza virus, use of molecular typing and subtyping of influenza, and updates on the H5, H7 and H3v virus components. LaShondra Berman, also from the CDC, presented the various shipping requirements for submitting specimens for influenza surveillance and diagnostic testing. Virology Group Manager Martha Thompson presented on various aspects of influenza surveillance in Texas and the diagnostic testing performed in the DSHS Laboratory. The day concluded with an overview of the CDC Flu kit panel used for testing, including how to order reagents and where to go for technical assistance.

Day two was a mixture of lecture and hands-on time spent running the assay. The morning portion was spent in the laboratory learning the nucleic acid extraction process as well as the preparation reaction. After a break for lunch, the group reconvened to retrieve the results and attend a few more lectures. They discussed electronic reporting and surveillance, PCR performance evaluation and validation, and data analysis and reporting the results of the morning PCR run.

Hard work went into creating this training. Thompson worked with representatives at the APHL and the CDC to coordinate speakers and the logistics. DSHS Viral Isolation Team members Jennifer Gonzales and Peter So “…were very involved in setting up the lab and helping to facilitate the wet lab portion,” said Thompson.

In a normal flu season, the DSHS Laboratory Viral Isolation Team tests about 1,000 influenza specimens. These specimens come from across the state of Texas. Providers that participate in influenza surveillance include physicians, clinics, hospitals and local health department laboratories.

The DSHS Laboratory performs real time RT-PCR to detect and identify influenza A and B along with influenza subtypes H1, H3 and 2009 pandemic H1. This assay also allows detection of novel strains of influenza. Some of the specimens received in the laboratory are then sent to the Centers for Disease Control and Prevention (CDC) for further characterization of the virus. This information may contribute to the vaccine strain for the upcoming year.

by Vanessa Telles

In addition to testing, the Emergency Preparedness Branch Biological Threat and Chemical Threat teams provide specialized workshops and training opportunities. Most recently, they hosted their first pilot training on “Biological and Chemical Terrorism: A Laboratory Overview for First Responders and Law Enforcement.” This training was designed for first responders and law enforcement officials who may respond to suspicious mail or substances and then collect and submit these specimens for biological and/or chemical testing at the State Laboratory Response Network (LRN) Laboratory. While the actual training took place on one day, the planning and coordination that led up to the day was conducted over the course of eight months. The 6th Civil Support Team (CST) acted as a consultant to refine the finished product and documentation. Other laboratories that previously conducted the training were used as templates.

Eight participants from the Austin Fire Department, Killeen Fire Department, and Williamson County and Cities Health District were in attendance. The training consisted of presentations from the BioThreat and ChemThreat Teams detailing LRN capabilities. This was followed by a tour of each laboratory area that participates in sample receipt and processing. Topics of discussion included the Laboratory Response Network (LRN), specimen submission and processing, and laboratory testing capabilities and turnaround time for results.

After the presentations, participants were taken to the BioThreat Laboratory. There they observed a demonstration on how threat letters are processed and the different strains of bacteria such as Bacillus anthracis (anthrax) growing on agar plates. They then visited the Chemical Threat Laboratory where they gained an understanding of the instrumentation used for clinical samples for chemical warfare agents and

metabolites. The complexities of sample matrices and the interpretation of Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography/Mass Spectrometry (GC/MS) in making an identification of a substance or mixture were also discussed.

This type of training is important. It fosters better relationships and communication between the BioThreat and ChemThreat teams and their counterparts. The opportunity communicates what goes on behind the scenes and why certain requirements are in place.

The BioThreat team has received rather interesting specimens for testing. For example, a suspicious white powder was found in the packaging of a steam vacuum. Instead of sending the suspected white powder, the entire steam vacuum was shipped to the DSHS Laboratory. This poses an interesting dilemma. How do you fit the vacuum under a biosafety cabinet? Training like this helps minimize the arrival of inappropriate specimens – such as steam vacuums, large mail boxes, or loaded guns.

At the completion of the training, participants were given a paint can containing job aids related to specimen submission and collection. Each can contained biological and chemical testing contact numbers and provided a visual aid on the appearance of a appropriately sized specimen. Most importantly, participants gained knowledge, including the ability to describe chemical warfare agents and industrial chemicals of concern, the field screening requirements prior to specimen submission, an understanding of the incubation and sample processing times and how they affect turnaround times for results, and the types of equipment utilized in the different laboratories and their uses.

by Vanessa Telles

Photo Puzzle

Count the safety violations.

This photo was staged for entertainment purposes.

Click here to view the answer key.

Answer Key: Photo Puzzle

1. No open-toed shoes in the laboratory
2. Talking on a cellphone in the laboratory
3. Talking on a cellphone with gloves on
4. Not wearing a lab coat
5. Not wearing eye protection
6. Not wearing gloves
7. No food and drink in the laboratory
8. Unlabeled chemical containers

Note: External links to other sites are intended to be informational and do not have the endorsement of the Texas Department of State Health Services. These sites may not be accessible to people with disabilities.

THE LABORATORIAN
July 2014, Volume Six, Issue One   (Publication #E14-13156)
Published by DSHS Laboratory Services Section
PO Box 149347, MC 1947
Austin, TX 78714

512 776 7318
888 963 7111, ext 7318 Toll Free
email The Laboratorian

LABORATORY DIRECTOR
Grace Kubin, PhD
512 776 7318
email Grace

NEWSLETTER EDITOR
Jimi Ripley-Black
512 776 6505
email Jimi