The Laboratorian - Volume 2, Issue 4

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The Laboratorian - Volume 2, Issue 4
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April 2011 - Volume 2, Issue 4

Article Index

- Mumps Testing
- Lab History, part 2
- Correlating Results
- Container Preparation
- Student Programs

Editorial

You may have noticed the new look for the Laboratory website and this issue of The Laboratorian. On February 24, 2011, the DSHS launched a newly redesigned website with changes intended to provide a cleaner layout and make it faster and easier to find and share information and see featured items. The launch marked the culmination of an intensive project to assess needs, choose a product that addressed those needs, and implement changes. I was fortunate enough to serve in three roles for this project—as a member of the DSHS Web Change Control Board, the Prevention and Preparedness Liaison for the project, and the Laboratory Web Developer—and I can testify that a lot of people worked hard to realize the current improvements to the DSHS website. Work is still being done, so watch for further enhancements in the near future.

Read on for laboratory testing information related to the recent mumps outbreak and details on the correlation of Pap smear and cervical biopsy results. Also included is part two of our three-part series on “Laboratory History” and a look at the important work of our Container Preparation group.

by Jimi Ripley-Black


Mumps Outbreak Testing at the DSHS Laboratory

A laboratorian examines a cell culture under a microscope for signs of viral growth

A mumps outbreak was identified in Texas during the second week of December 2010. All outbreak-related specimens are sent to the DSHS Laboratory in Austin, which began testing mumps specimens on December 15, 2010.

Between December 2010 and March 2011, the Viral Isolation laboratory obtained 18 mumps positives from 42 specimens. In comparison, between December 2009 and March 2010, the laboratory obtained only 7 positives from 41 specimens. This year, mumps was isolated in 12 of the 18 positives, compared to only two in the preceding year.

Specimens—in the form of saliva, throat swabs, and urine—received in the Viral Isolation laboratory begin a 10-day observation period. Specimens are first processed and inoculated into cell culture tubes and then incubated and monitored daily. Each day, the cell culture is examined under a microscope for signs of viral growth. The state of viral growth in the culture is described as stages of cytopathic effect (CPE), which refers to degenerative changes in the cells. Once CPE is detected, the presence of the mumps virus can be confirmed with a mumps fluorescent antibody assay.

The Diagnostic Serology Section received 36 specimens for mumps IgM antibody testing between December 2010 and March 2011. Two specimens were positive with a 1:10 titer, and one specimen was positive with a 1:20 titer. Twenty-five of these specimens were also tested for mumps IgG, 4 of which were negative. A vaccinated, healthy individual will have a positive mumps IgG result and a negative mumps IgM result.

Serology specimens arrive at the laboratory in the form of serum in specialized tubes. Mumps Immunoglobulin M (IgM) antibody detection involves the testing of patient serum by Indirect Flourescent Antibody (IFA) assay. This procedure is performed using several dilutions of patient serum. A flourescein-labeled antibody is added, which adheres to the antigen-antibody complex. Any mumps IgM antibodies present bind to the antigen already present on the slide. Once the slides are rinsed and dried, the slides are read under a fluorescence microscope. A positive staining reaction displays a bright apple-green fluorescence, which highlights the presence of the mumps IgM antibody and indicates a recent mumps infection. If an antibody-antigen complex is not formed, the fluoroscein-labeled antibody will be washed away, exhibiting a negative stain result (no flourescence).

Mumps IgG antibody testing is performed by enzyme-linked immunosorbent assay (ELISA), which is designed to detect antibodies by producing an enzyme triggered color change in a microtiter plate. Positive specimens will trigger a color change, negative specimens will not. The presence of IgG antibody indicates prior exposure or vaccination to the mumps virus and immunity to mumps.

Mumps is a viral infection that causes fever, headache, and inflammation of the glands under the jaw. Once a common childhood illness, mumps has been largely contained through a nation-wide vaccination program that began with the first vaccine licensed in 1967. Further information on mumps can be found via the Centers for Disease Control and Prevention (CDC) Mumps Clinical Questions and Answers page.

by Jimi Ripley-Black

 

Laboratory History: Establishing a State Laboratory

Quarantine measures were the major concern during the early work of the Texas State Medical Officer. When Dr. Robert Rutherford was appointed to the role in 1879, the main concern was dealing with smallpox and yellow fever. Therefore, it is not surprising that no mention was made of the application of bacteriological or other laboratory procedures.

Texas 1890-1903. In 1896, then State Health Officer, Dr. R.M. Swearington, concluded his report of state quarantine by requesting "an appropriation of $2,000 per annum (or as much thereof as may be needed) to employ an expert in microscopy and a chemist to analyze drinking water and perform bacteriological examinations."

In 1902, then Governor Joseph Draper Sayers dispatched Dr. George W. Tabor, State Health Officer, to San Francisco to study bubonic plague. Shortly after Dr. Tabor's return to Austin, he learned that the U.S. Public Health Service had diagnosed yellow fever in Laredo and other points along the border. As Mexican officials insisted that cases of malaria were being mistaken for yellow fever, Dr. Tabor in the company of Dr. John T. Moore, a bacteriologist in the medical department at the University of Texas, and Dr. John N. Foster, assistant physician at the State Insane Asylum in Austin, went to Laredo "to make microscopic examination," and assist in application of laboratory knowledge in settling the dispute. Perhaps antedating Dr. Tabor's trip to Laredo was the use of the Negri test to identify these bodies in the test for rabies, performed by a physician from the Austin Lunatic Asylum.

Texas 1904-1927. In 1904, the Texas Pasteur Institute was opened as a branch of the Austin Lunatic Asylum. People came from far and near for diagnosis of rabies in pets and other animals and to take the renowned Pasteur treatment for emergency prevention of hydrophobia. The Pasteur vaccine was prepared from desiccated spinal cords of rabbits infected with fixed rabies virus until 1934. At the time, the manufacture of phenolyzed vaccine began and the vaccine was shipped to local health officials all over the state.

In his 1910 report to the Governor, the State Health Officer, Dr. W.M. Brumby, commented on the successful program in Texas that pioneered the testing of germicides. Since the Texas Health Department lacked a bacteriologist, the department had obtained the services of the pathologist at the University of Texas Medical Department for the testing of germicides. Dr. Brumby had also personally undertaken the bacteriological analysis of drinking water to arouse interest in the prevention of typhoid fever epidemics. Mr. "Vic" Ehlers related that when he joined the staff of the State Health Department as a sanitary engineer in 1915, he frequently collected samples of drinking water and performed the necessary bacteriological tests. In this work, Mr. Ehlers became acquainted with Dr. I.M. Lewis, Professor of Botany and Bacteriology at the University of Texas in Austin, who was interested in teaching his students how to perform bacteriological tests on samples of drinking water.

The law that created the Pure Food Commission in 1906 also provided that the state Dairy and Food Commissioner should be an analytical chemist and bacteriologist, and led to the establishment of a laboratory for food analysis. However, the Commissioners tended to be pharmacists or chemists, rather than bacteriologists. The first Commissioner worked without a salary for some months because of legislative omission of an appropriate salary. The Commission was located at the College of Industrial Arts at Denton until 1911, when the laboratory was relocated at the state Capitol in Austin, but it did not become a responsibility of the State Board of Health until 1921.

When the Legislature appropriated $3,600 to employ a bacteriologist and a chemist and for the maintenance of a Bacteriological Laboratory during the next two years, Dr. Brumby employed Dr. Mannie Graham. The Bacteriological Laboratory was opened in the State Capitol in 1912. Dr. Graham participated in the hook worm survey in Texas conducted by the International Health Division of the Rockefeller Foundation. He reported that the 1,082 diagnostic bacteriological tests in 1912 were done "chiefly on diphtheria, typhoid, malaria, blood, urine and feces." The laboratory began examining drinking water for use on railway trains and in cities, doing sputum tests for tuberculosis, testing stool specimens for hook worm, and performing spinal colloidal gold tests for syphilis. In 1922, the first blood Wassermann tests were being done and, by 1925, the laboratory was supplying silver nitrate drops for use in eyes of newborns.

For some years the Bacteriological Laboratory was located on the third floor of the Capitol, west of the galleries of the House of Representatives. Subsequently, the Bacteriological Laboratory was removed to more secluded quarters southeast of the Senate, sharing space with the Food and Drug Laboratory.

Bureau of Laboratories building in 1928

This building was erected in October 1928, at 412 East 5th Street, for the purpose of housing the Bureau of Laboratories of the Texas State Department of Health. A third story was later added.

Texas 1928-1942. Through a consolidation, effected in 1928, the Texas Pasteur Institute of the Austin State Hospital, the Food and Drug Laboratory, and the Bacteriological Laboratory became the Bureau of Laboratories of the State Health Department and were relocated in a small, new, two-story red brick building at the corner of Fifth and Neches Streets; the Legislature appropriated $12,500 for the building and equipment. Dr. J.W. Wilhite, who had directed the Pasteur Institute for the twenty-two years of its existence, was slated to be the first director of the consolidated laboratory, but he died of pneumonia early in 1928, before the move was made. The new building was dedicated to him, although it was called the State Hygienic Laboratory Building. Dr. S.W. Bohls, Assistant Director of the Pasteur Institute at the time, became the first director of the Bureau of Laboratories.

In 1934, a small addition to the two-story red brick building relieved some of the congestion resulting from the growing volume of work. Divisions of bacteriology, mycology, virology, syphilis serology, parasitology, entomology, biological production, and environmental chemistry were active. The laboratory was licensed by the National Institutes of Health to make bacterial biologics and smallpox vaccine in 1936.

Egg incubator at the Texas laboratory

 

Smallpox vaccine is produced in the chorio-allantoic membrane of the developing chick embryo. This is the egg incubator at the Texas laboratory.
A laboratorian harvesting virus-infected egg membranes An opening is cut into the eggs and the eggs are inoculated with the smallpox virus. To the left, a laboratorian is harvesting the virus-infected membranes.
The finished smallpox vaccine is sealed into glass capillaries The finished vaccine is sealed into glass capillaries.

This article is part two of a three-part series on the history of the DSHS Laboratory. Click here to view part one.

 

Correlation of Pap Smear and Cervical Biopsy Results

Squamous cell carcinoma is the most common form of invasive cervical cancer. The invasive stage of squamous cell carcinoma is preceded by a preinvasive stage during which abnormal cells develop within the squamous epithelium of the cervical mucosa. The abnormal cells lack the ability to spread beyond the cervical mucosa and present clinically as circumscribed grossly identifiable lesions. Preinvasive lesions do not invariably progress to invasive carcinoma. According to their propensity to develop into invasive cancer they can be stratified into two groups. The first group comprises low risk lesions with a high propensity of spontaneous regression and a low rate of progression to invasive carcinoma. The second group comprises high risk lesions with a high propensity of persistence and a high rate of progression to invasive carcinoma if left untreated. Preinvasive intraepithelial lesions as well as invasive carcinoma may shed abnormal cells that can be detected by examination of a pap smear.

Most patients with abnormal Pap smear results are referred to colposcopy for triage of further treatment. Colposcopy involves a visual examination of the cervix which allows the detection of low risk and high risk preinvasive intraepithelial lesions as well as invasive carcinomas. Biopsy samples can be obtained from these areas and submitted for histologic evaluation. Low risk as well as high risk preinvasive intraepithelial lesions and invasive carcinomas display rather unique histologic features that allow a fairly accurate distinction between these three groups. Preinvasive intraepithelial lesions identified in a cervical biopsy are graded according to a two-tiered system termed as CIN (cervical intraepithelial neoplasia). Low risk lesions are designated as CIN 1 and high risk lesions as CIN 2 or CIN 3 (referred to as CIN 2,3).

Abnormal cells identified on a Pap smear are classified according to the 2001 Bethesda System for reporting results of cervical cytology. The diagnostic categories in order of increasing severity of cytologic abnormalities are as follows: ASCUS (atypical squamous epithelial cells of undetermined significance), LSIL (low-grade squamous intraepithelial lesion), HSIL (high-grade squamous intraepithelial lesion), and squamous cell carcinoma.

The correlation of Pap smear results with cervical biopsies is an important component of quality assurance/improvement programs in the cytology laboratory because there is a close concordance between Pap smear and cervical biopsy results. Patients with a LSIL pap smear should have CIN 1, patients with a HSIL Pap smear should have CIN 2, CIN 3 and patients with a carcinoma pap smear should have invasive carcinoma on a biopsy. In that respect, however, it has to be emphasized that the Pap smear is merely a screening test with inherent limitations in sensitivity and specificity, resulting in a less than ideal correlation of Pap smear results with histologic biopsy reports.

CLIA regulations require a laboratory to compare all cytology reports with a diagnosis of HSIL or carcinoma with the histology report of the corresponding cervical biopsy, if available in the laboratory. Any discrepancy between the cytologic and histologic diagnosis has to be investigated and its cause has to be determined. CAP regulations mandate the correlation of all cytologic findings

A cytotechnologist at work
with findings in corresponding histologic specimens. Significant disparities between histological and cytologic findings have to be resolved in a confidential peer-reviewed quality management document and an addendum report has to be issued, if necessary. These regulations, however, include neither any specific detail regarding the correlation process nor an exact definition of what a discrepancy or significant disparity actually constitutes. Therefore, these two tasks are left up to the laboratory.

The correlation process starts with the identification of all Pap smears which have a corresponding cervical biopsy. A corresponding biopsy can be defined as one which was obtained either together with the Pap smear or within a certain time period after the Pap smear. This time period may be defined by the laboratory. The Pap smear diagnoses from all these cases are compared to the biopsy diagnoses and all cases with diagnostic discrepancies are identified.

A histotechnologist at work

What actually constitutes a discrepancy may again be defined by the laboratory. One commonly accepted way is outlined as follows. Certain cytologic diagnoses are grouped together with certain histologic diagnoses to form corresponding pairs. These pairs are ranked in order of increasing severity of disease, as shown in Table 1 below. It is commonly accepted that differences between cytologic and histologic diagnoses of one level of severity are acceptable and thus do not constitute a
discrepancy. A difference, however, of more than one level of severity would constitute a significant discrepancy. For example, a Pap smear diagnosis of ASCUS with a cervical biopsy diagnosis of CIN 1 would not represent a discrepancy. On the other hand, a Pap smear diagnosis of ASCUS with a cervical biopsy diagnosis of CIN 3 would represent a significant discrepancy.

After all cases with diagnostic discrepancies have been identified, all cytologic and histologic slides from these cases are reexamined. If the reexamination confirms both original diagnoses, the discrepancy is deemed to have been caused by one of two following scenarios. The first is spontaneous progression or regression of cervical lesions in the time interval between Pap smear and cervical biopsy. The second is sampling error during performance of the Pap smear or the cervical biopsy. If the reexamination confirms only the original histologic diagnosis, but not the original cytologic diagnosis, the discrepancy is due to a cytologic screening or diagnostic error. If the reexamination confirms only the original cytologic diagnosis, but not the original histologic diagnosis, the discrepancy is due to a histologic technical or interpretation error. The majority of discrepancies are caused by sampling errors or spontaneous progression/regression of cervical lesions. Histology interpretation errors constitute the second most common cause for discrepancies. Other causes for discrepancies are infrequently encountered. Although discrepancies between cervical cytology and biopsy results are not uncommon according to the published data, the rate varies widely among different laboratories and ranges from four to more than 20 percent. In this respect it is important to remember that the lack of correlation standardization impedes conclusive interlaboratory comparisons through a benchmarking process.

Table 1. Diagnostic levels of corresponding Pap smear and cervical biopsy results
Pap smear Cervical biopsy
negative negative
ASCUS  
LSIL CIN 1
HSIL, carcinoma< CIN 2, CIN 3, carcinoma

by Gregor Stransky, MD

 

Container Preparation Group: Going Beyond the Scope

Container Preparation group

 

Despite the multitude of tasks they do, not much is known about the Container Preparation Group (Container Prep) and the intricacies of their work. Manager Dawni Allen* calls her group the “unsung heroes” of the laboratory and rightfully so. This article aims to shed light on how Container Prep operates and, in so doing, how they contribute to improving the health and well-being of Texans.

Container Prep is part of the Specimen Acquisition Branch of the Texas Department of State Health Services (DSHS) Laboratory in Austin; it is one of the groups that support not only the testing laboratories but also programs of the DSHS agency. The group’s essential function is to supply every submitter with the testing supplies they need to submit specimens. This includes distributing Newborn Screening (NBS) test kits, supplies for blood collection, collection containers for water samples, and shipping containers for specimens. These duties seem simple enough, but the fact that the laboratory transacts business with approximately 14,000 submitters throughout the state of Texas complicates the task.
                                             
The following information illustrates and quantifies the majority of the work that the group does:

  1. More than 800,000 NBS specimens are processed annually. Each of those kits are processed by Container Prep
  2. The Check-In group accessions almost 6,000 specimens daily. Ninety percent of those specimens are received in containers that the group ships, cleans, recycles, and processes.
  3. Container Prep receives almost 100 orders daily and receives an average of 50 calls daily
  4. At least 80-90 cold boxes are cleaned and shipped back daily.
  5. Over 1,000 cans are received daily for the TB, Refugee and the TX Health Steps programs.

Container Prep also manages requests for any type of special shipment. To note one, they send more than 200 time-sensitive media annually to support Influenza Surveillance efforts. This type of media may also be requested on an “as needed” basis. Also, in the event of an outbreak, such as in the case of mumps and food-borne diseases, the group prepares to send out necessary supplies for submitters.

In addition, Container Prep has the added responsibility of picking up biohazard waste for all of the different floors at the DSHS Laboratory in Austin. Recently, the group volunteered to lead in the calibration and return of more than 200 centrifuge units. This is a Clinical Laboratory Improvement Amendment (CLIA) regulation that has to be completed every two years.

With full staff, turn-around time for filling orders is typically five days from receipt of the order. Ideally, a fully staffed group consists of eight members. However, due to transitions, turnovers and organization changes, the group is now down to five members. Despite that constraint, the Container Prep team has managed to maintain a two or three day turn-around time for orders.

Container Prep provides the utmost in customer service and education to the laboratory’s stakeholders. Dawni Allen has been the manager of this department for almost a year; the one thing that she takes the most pride in about her team is that every member goes the extra mile to help submitters. She says calls have been routed to the department that may not be related to containers at all, yet her staff investigates the nature of the call and provides guidance on what to do and who to contact. “My staff care. They help customers in every way they can, going to great lengths if they need to.”

One of the biggest challenges for Container Prep so far was the 2009 H1N1 Response. Although this was before her tenure as the group’s manager, Allen recounts how the staff gave a lot of their time and effort so that they could meet the needs of submitters. The group was a vital component of the response because there was a dramatic increase in specimen submission to the DSHS Laboratory, thus requiring extra hours to complete orders for submitters in a timely manner.

Bob Russin has been in Container Prep for approximately 13 years. He recalls one of his most memorable experiences was when the department assisted in the Anthrax response almost 10 years ago. He says that the group gained recognition when it was asked to store almost 300 items that tested negative for Anthrax. “This was a golden opportunity for us to prove that we can work beyond our scope and that we can do more than just clean cans and ship out boxes”, Russin explains. For this endeavor, Container Prep also created a database to register all the items stored and maintained the chain of custody when returning items to law enforcement authorities.

“Working at Container Prep is just different every day,” Allen says. She adds that the group performs work that may be tedious and unrewarding at times, but it is critical to supporting the testing capacity of the Laboratory Services Section. The sentiment is also echoed by Russin as he explains, “In this department, you get a sense of accomplishment at the end of each day. That is where I get my satisfaction from.”

*Dawni Allen recently accepted a new position as a Program Specialist for the Emergency Preparedness Branch. While transitioning to her new role, she continues to provide guidance and training to Container Prep until her position is filled.

by Jan Adversario

 

Student Programs in the Laboratory

The Laboratory works in many ways to further its mission to improve health and well-being in Texas by promoting health, preventing disease and injury, and protecting from and responding to health threats. One of the ways it fulfills this mission is by educating and training the next generation of public health professionals. At the DSHS Laboratory in Austin there are several programs that give students the opportunity to gain experience in the public health setting.

The Laboratory works specifically with three local universities: The University of Texas (UT), Texas State University, and Austin Community College. There are currently three students working in the Laboratory through the UT intern program. This structured program allows students to conduct goal-oriented, one-semester research projects in epidemiology, laboratory science, or field biology under the mentorship of state and local public health personnel.

Three UT students are currently working in the Laboratory under the guidance of seasoned mentors. Andrew Cheng is working with Aaron Benfield, PhD, in the Molecular Laboratory, on a project to subtype Salmonella Enteritidis isolates that the Laboratory received in 2010, which are potentially connected with the egg Salmonella outbreak. Storey Zimmerman is working with Cathy Snider, in the Parasitology Laboratory, on “Data Analysis on Fecal Ova and Parasite Results for Refugees Entering Texas.” Zimmerman is analyzing data from the years 2005–2006 and examining specimen results by a number of demographic factors, including age, gender, and country of origin. Sandy Pingali is working with Rachel Lee, PhD, in the DNA Diagnosis Laboratory, on the optimization of DNA extraction from dried blood spots. Dr. Lee’s intern from last semester, Lauren Rego, went on to UT’s honors program and is still working on the newborn screening pilot study for Severe Combined Immunodeficiency Disorders.

Texas State University sends their Clinical Laboratory Science students for a one week observation of the Laboratory. They visit the Newborn Screening and Microbiology departments. During their time at the Laboratory, the students spend at least a half a day, upwards to a full day, with each team in these departments.

The Bio Technology Department of Austin Community College sends certificate program students to the Specimen Acquisition and Media Preparation areas for first-hand experience in a laboratory. Students come for one month. While in check-in, they participate in learning Texas Health Steps Check-in procedures as well as working in Container Preparation. The students are taught to make agar, dispense it, take pH, weigh reagents, and label in Media Preparation. Employees, Denise Alamo, in Chemistry Check-in; Hong Trinh, in Media Preparation; and Jennifer Gonzales, in Virology, are all graduates from this program.

In addition to supporting three area schools, there are two other programs where students visiting the Laboratory may not come from local universities. The Laboratory works in conjunction with the Austin State Hospital’s internship program. This program is a five-year degree plan for Clinical Laboratory Science majors. After completing four years of education in the university setting, interns spend their fifth year primarily at the Austin State Hospital. As part of the program, they are sent to the Laboratory at various times throughout the year. During their visit, students may stay between half a day and a week in a particular testing laboratory or section of the Laboratory. The students visit the Laboratory’s Microbiology and Newborn Screening branches.

APHL fellow working in the CT Laboratory

The Association of Public Laboratories (APHL) also sponsors a fellowship program. APHL member laboratories, including the DSHS Laboratory, host a fellow for one to two years. The member laboratories submit the proposed project for the fellow, and the laboratories are only allowed one fellow per laboratory. The purpose of the program is twofold:

it allows scientists to gain experience in a public health laboratory and it accomplishes the Centers for Disease Control and Prevention’s (CDC) strategic goal of providing strong local, state, and federal public health organizations support in surveillance, prevention, and control programs. Currently, Alona Umali, PhD, is working in the Chemical Threat (CT) Laboratory on developing a method for the determination of cyanide metabolites by LC-MS/MS. This project is under the mentorship of David Klein, PhD. The laboratory was fortunate to acquire current employees and former APHL Fellows Linda Su in the CT Laboratory and Mark Fountain, PhD, in Newborn Screening, through the fellowship program.

By taking part in these five educational programs, the Laboratory is a partner in contribution to the education and development of future members of the public health workforce.

by Cindy Crouch


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.


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THE LABORATORIAN
April 2011, Volume Two, Issue Four
(Publication #E14-13156)
Published by DSHS Laboratory Services Section
PO Box 149347, MC 1947
Austin, TX 78714

512 458 7318
888 963 7111, ext 7318 Toll Free
email The Laboratorian

 

LABORATORY DIRECTOR (ACTING)
Sherry Clay
512 458 7318
email Sherry

NEWSLETTER EDITOR
Jimi Ripley-Black
512 458 7318, ext 6505
email Jimi

Last updated July 06, 2012