FDA Guidance to Develop Novel Molecular Diagnostic Tests for SARS-CoV-2

A laboratory advisory from the CDC:

The Food and Drug Administration (FDA) issued new guidance on February 29, 2020, for laboratories to be able to develop novel coronavirus (COVID-19) molecular diagnostics tests and begin use prior to obtaining Emergency Use Authorization (EUA). This permits laboratories that are CLIA certified and meet requirements to perform high complexity testing to start offering severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular diagnostic testing after validation is completed as outlined in the guidance. Laboratories should submit an EUA request to the FDA within fifteen business days after validation. FDA will be hosting a webinar to provide more information on March 2, 2020, at 3 pm ET.

Clinical laboratories should contact their state health departments for guidance if they have a suspected COVID-19 case specimen. Clinical laboratories should NOT attempt viral isolation from specimens collected from COVID-19 persons under investigation (PUIs). For interim guidelines for collecting, handling, and testing clinical specimens from PUIs for COVID-19, please see the CDC Coronavirus Disease 2019 (COVID-19) website.

The Forensic Pathologist as Patient Advocate

Patient advocates are simply people who care about patients as fellow human beings enough to act on that care. Forensic pathology fascinates many people, but hardly anyone realizes how strongly forensic pathologists advocate for patients.

Forensic pathologists have the responsibility of identifying human remains and determining the cause and manner of death for individuals that die suddenly and unexpectedly. Most often, we accomplish this mission by performing an autopsy. Death makes many people uncomfortable, and we’re accustomed to grim jokes about their work when meeting someone. Typically these comments carry an undertone that because decedents cannot talk, we don’t need interpersonal skills. Not only is this untrue, comments such as these provide an educational opportunity.

It is true that our patients have already died, but the relatives of our patients are very much alive. Those relatives have needs that we work to provide and questions we strive to answer. The most common question relatives have is “Why did my loved one die?” which is precisely what the pathologist is working to determine. We regularly talk with relatives of decedents that we’ve examined. We can tell family members why death occurred, including any implications that the death has for remaining members of the family. We can also help families begin to work through the social and bureaucratic requirements that death brings for those still living, such as the need to make arrangements for the disposition of the body and the need for a death certificate. (For example, after a person dies, that person’s financial accounts are frozen until a death certificate becomes available to unlock the accounts.)

Forensic pathologists work to develop a good relationship with the decedent’s relatives. Because anger and bargaining are part of grieving, conversations with relatives sometimes begin as though the relative and the pathologist are adversaries, but with time and compassion, the relationship usually transforms into a more appropriate professional relationship. A particularly important aspect of family interactions is listening to a grieving relative, because listening with care helps someone who is grieving. Attempting to build a good relationship with the decedent’s relatives does not mean that the pathologist is a blind advocate for the family. We won’t change the cause of death so that the family can reap more financial benefit from the death, for example. Lies will not help someone pass through the process of grieving in a healthy way – truth, time, and patient, loving care are the necessary therapeutic measures.

In the case of homicides, forensic pathologists advocate for the decedent by calling the death what it is and then testifying to the medical facts of that death when a suspect is tried in court. The pathologist testifies to the medical aspects of what caused death without trying to ensure that the suspect is either convicted or acquitted. Trying to sway the jury’s verdict is the work of attorneys; presenting the medical facts of why and how the decedent died is the work of the pathologist.

Forensic pathologists advocate for public health by providing an accurate cause of death. Death certificate data provide an essential component for assessing public health, and those data are an important determinant for allocation of medical research funds and for interventions to improve public health.

Like other pathologists, forensic pathologists typically do their work quietly in the background, advocating for their unique patients in their own special way. People give little thought to professional interactions with a forensic pathologist until forced to do so; in that difficult time we try to serve as best we can.

-Gregory G. Davis, MD, FASCP graduated from Vanderbilt Medical School and trained in pathology at Vanderbilt University Medical Center, Nashville, TN, followed by a fellowship in forensic pathology at the San Diego County Medical Examiner Office in San Diego, CA. Dr. Davis then joined the faculty at the University of Alabama at Birmingham, where he currently serves as a Professor and as Director of the Forensic Division of the Department of Pathology. Dr. Davis also serves as Chief Coroner/Medical Examiner for Jefferson County, Alabama, the county in which Birmingham is located. Dr. Davis has earned a Master of Science in Public Health from the UAB School of Public Health. His research interest is the application of epidemiology to the study and practice of forensic pathology, especially drug abuse. He has published 74 peer-reviewed manuscripts, including serving as lead author on the 2013 opioid position paper of the National Association of Medical Examiners. He is currently working as chair of a panel revising and updating the NAME opioid position paper for expected publication in 2020. He serves on the editorial boards of the Journal of Forensic Sciences and Forensic Science, Medicine, and Pathology. Dr. Davis is a Fellow At-Large Director on the Board of Directors of the American Society for Clinical Pathology.

Respiratory Protection in the Days of the Novel Coronavirus

In the peak of the flu season we might see many people wearing masks in physician offices and hospitals. In the news today, as the 2019 Novel Coronavirus (SARS-CoV-2) continues to spread, we see many images of people wearing different types of respirators, some are N95 respirators and others are surgical masks. Not all experts agree on the efficacy of these pieces of personal protective equipment in the face of viruses, but if you’re going to use them, it is important to know how, when and why.

OSHA’s Respiratory Protection standard (1910.134) provides information about requirements for staff who may potentially be exposed to airborne pathogens. These requirements include specific instructions for choosing the proper respirator, for providing fit-testing, and for user training. The College of American Pathologists (CAP) also expects labs to determine the risk of airborne pathogen exposure for each employee, and they require labs to have a plan which outlines engineering and work practice controls that reduce exposure potential.

The purpose of a respirator is to protect the employee from contaminated or oxygen-deficient air. Therefore, two classes of respirators are common; air-purifying respirators which use filters to remove contaminants from the air you breathe, and atmosphere-supplying respirators which provide clean air from an uncontaminated source. These types of respirators can also be classified further as tight-fitting or loose-fitting.  Tight-fitting respirators need a tight seal between the respirator and the face and/or neck of the user in order to work properly. For now, let’s focus on the air-purifying respirators which are in high demand these days as a potential 2019-nCoV pandemic looms.

In the laboratory, N95 respirators are probably the most commonly-used respirators, often used for protection against tuberculosis and other airborne pathogens. These respirators filter out 95% of airborne pathogens that are 0.3 microns or larger. While the exact size of the 2019-nCoV is not yet known, most coronaviruses are slightly larger than 0.1 microns. Does that mean a N95 respirator (recommended by the CDC) will not offer protection from the coronavirus? Not necessarily.

According to biosafety expert Sean Kaufman (www.saferbehaviors.com), the filter in the N95 respirator works three ways- through interception, impaction, and diffusion. Interception collects larger particles which are blocked by mask fibers, and impaction collects larger particles which have too much inertia to be moved around the filter fibers. Diffusion occurs as smaller particles are bombarded with larger air molecules and are pushed against filter fibers. Most of the bacteria or virus particles are removed from the airstream making the respirator quite useful and protective (HEPA filters on a Biological Safety Cabinet work in much the same way).

Employees who may need to wear a tight-fitting respirator as part of their job are required to have fit-testing every year. This is required by OSHA, and contracted employees (such as pathologists) should be fit-tested as well. Employees who may need such respirators would be those who work in microbiology labs, cytology techs who participate in patient procedures, and others. Labs should perform a risk assessment for each job category to determine the type and level of potential harmful airborne exposure.

Procedure masks, such as those handed out when people suspect they have the flu, are not technically considered respirators. Often, the person who is sick will wear these masks in order to prevent the spread of droplets when coughing or sneezing. They can protect others in the area, but they do not protect the user from harmful airborne pathogens or vapors.

Can these surgical masks be useful for the healthy public when a coronavirus is present? Sean Kaufman says “yes. If you wear a surgical mask in a potentially contaminated environment (on a commuter bus, for example),” Kaufman says, “it can keep you from touching your nose or mouth- two major routes of entry for viruses. Behaviorally speaking, these masks do offer some protection.”

Knowing when and why you use a respirator is vital, but knowing how to use it is important as well. Tight-fitting respirators should never be used without fit-testing to make sure the correct size is being used. Otherwise, the protection offered will be limited. Make sure your staff is properly trained and protected to work in environments where the air is not safe to breathe, and teach others about the usefulness of respirators when the flu and other viruses are lurking!

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

EUA for 2019-nCoV Test

On February 4th, the FDA announced an Emergency Use Authorization for the CDC’s 2019 Novel Coronavirus real-time RT-PCR Diagnostic Panel. Here’s the press release:

Audience: Clinical Laboratory Professionals

Subject: Laboratory Update: Information about Emergency Use Authorization for  2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel

Level: Laboratory Update

This message is to ensure that clinical laboratories are aware that CDC has developed a new laboratory test kit called the CDC’s 2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, for use in testing patient respiratory specimens for 2019-nCoV. On February 4, 2020, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) to enable emergency use of the test kit in the United States. All EUA documents are available on the FDA website.

The test kit will be available for ordering today from the International Reagent Resource (IRR). Formerly, U.S. diagnostic testing for 2019-nCoV was only being conducted at CDC; however, the FDA EUA and distribution of the tests will allow 2019-nCoV testing to take place at laboratories designated by CDC.  This includes U.S. state and local public health laboratories and Department of Defense (DoD) laboratories.

Clinical laboratories should NOT attempt viral isolation from specimens collected from 2019-nCoV persons under investigation (PUIs). For interim guidelines for collecting, handling, and testing clinical specimens from PUIs for 2019-nCoV, please see the CDC 2019 Novel Coronavirus website

The FDA website lists current EUA assays, and also includes a link to terminated EUA assays. Each pathogen-specific EUA includes the device-specific Letter of Authorization, fact sheets, and manufacturer instructions/package inserts. These documents are updated when amendments are made (e.g., additional specimen types, extraction methods, procedural clarifications), so check the website routinely to ensure your laboratory staff members have the most up-to-date information.

Additional Resources

If you have any questions, please contact LOCS@cdc.gov.

Patient Interaction

Medical school councilors have good intentions in mind when they steer medical students who realize that direct patient care isn’t their strong suit into pathology. But I am different kind of pathologist – the one who sees (or talks to) patients every day. I am a member of unique subspecialty – Transfusion Medicine – which is the most patient-centric subspecialty of all pathology subspecialties. And, contrary to the popular wisdom, I like seeing patients.

Don’t get me wrong though, my heart and soul still live in the lab, deeply rooted in understanding test performance, troubleshooting and quality control. But direct patient care helps to put all the work I have done in the lab into a perspective.

One program that became especially dear to my heart is our chronic RBC exchange program for the kids and adults with sickle cell anemia who have high risk of developing serious complications from the disease, such as stroke, acute chest syndrome, and severe iron overload. As an apheresis physician I see these patients quite frequently due to the nature of the program – chronic RBC exchanges every 4 to 6 weeks. This also means that I quickly had to learn quite a lot not only about managing the exchanges, but also about patients’ success and failures, spend time explaining to parents the benefits of the program and engaging them to maintain compliance with rigorous schedule. The work is not immediately rewarding. All the adjustments I do to the plan of care show changes in lab values in a month or two at best. But it is not entirely about numbers. Another aspect that makes this program special is when you notice that the kids you treat are doing better at school, have less ED visits and overall live a more fulfilling life.

Sometimes the patient interaction is not as direct as in the case of the sickle cell RBC exchange program. For example, being part of the obstetric team that cares for the patient with severe hemolytic disease of fetus and newborn is also extremely rewarding. And the more challenging clinical question is the more rewarding it is in the end. Just this summer we had a patient who developed an antibody to very high frequency antigen that is present in 99.7% of the population and finding the right donor for intrauterine transfusion involved quite a few people in at least 3 cities.  When all the pages, phone calls, emails, and personal conversations between me and residents, obstetricians, anesthesiologists, pediatricians, and blood suppliers result in a positive outcome for mom and baby – I feel elated. And who wouldn’t?! That is why I enjoy what I do!

-Aleh Bobr MD is currently the medical director of blood bank and tissue services at University of Nebraska Medical Center in Omaha, NE. He did his residency in Anatomic and Clinical pathology and Fellowship in Transfusion Medicine at Mayo Clinic Rochester, MN. Prior to that he did his post-doctoral research fellowship in Immunology with focus on dendritic cell biology at University of Minnesota and Yale University. He received his medical degree from Vitebsk State Medical University in Vitebsk, Belarus. Current interests include application of apheresis, platelet refractoriness.

Prozone Effect in the Fecal Immunochemical Test (FIT)

Case History

An 80 year old female had a history of chronic iron deficiency anemia with unknown cause and comorbidities included hypothyroidism, congestive heart failure (CHF), severe aortic stenosis and COPD. The patient presented at the ED with initial presentation with increasing shortness of breast, NYHA class 3-4. She was admitted to the hospital for further treatment for CHF, as well hyperventilation, sleep apnea and COPD. Her serum iron and iron saturation were tested and results were 2 umol/L (reference range for iron: 10-29 umol/L) and 7% (reference range: 14-51%), respectively. Part of her investigations included a qualitative fecal test to screen for gastrointestinal bleeding. The immunochemical fecal occult blood test was performed using a CLIA waived Hema Screen SpecificTM POCT test (Immunostics, Inc, USA) in the hospital lab. Hema Screen Specific test is a qualitative, sandwich dye conjugated immunoassay that uses a combination of monoclonal and polyclonal antibodies to detect the globin component of hemoglobin in the fecal samples. The manufacture recommended using Hema Screen Specific test in routine physical examines, hospital monitoring of bleeding in patients and for screening for colorectal cancer or gastrointestinal bleeding for any source (statement from the product package insert).

 The specimen submitted to the lab was markedly red (Image 1), yet Hema screen test returned a negative result. Since this device is designed to detect occult blood in fecal samples, a prozone effect was suspected, as the stool appeared to contain overt hemorrhage. The specimen was reanalyzed with serial dilutions by a factor of 5, 10, and at 100 × dilution. The FIT result became clearly positive for blood (Image 2). The patient received a colonoscopy, which revealed internal hemorrhoids, severe diverticulosis in the left colon, as well as multiple angiodysplastic lesions. One such lesion was in the ascending colon and was actively bleeding at the time of colonoscopy. The others, which were not bleeding, were distributed in the proximal ascending colon, hepatic flexure, and proximal transverse colon. All angiodysplastic lesions were treated with argon plasma coagulation.

Image 1. Fecal specimen demonstrating overt hemorrhage.
Image 2. Fecal immunochemical test performed on the patient sample submitted. Serial dilutions of fecal specimen were performed. At the dilution factor of 1:100, the result showed positive. Saline was used to dilute the fecal sample.

Moreover, we have tested the device with another bloody fecal sample during the initial evaluation. When an appropriate dilution factor was used, the prozone effect begins to lose its interference as show in Image 3.

Image 3. Fecal immunochemical test (FIT), showing control (C) and test (T) lines, applied to serial dilutions of fecal specimen (B ─ undiluted, C ─ 10 ×, D ─ 100 ×, E ─ 1 000 ×, F ─ 10 000 ×, G ─ 100 000 ×). At 10 × dilution, the prozone effect begins to lose its interference, and at 100 × the test is clearly positive. At dilutions higher than 1 000 ×, the concentration of blood becomes too low to return a positive result. (Image 3 provided by Dr. Andrew Lyon, PhD, DABCC, FCACB, Lab Medicine & Pathology, Saskatchewan Health Authority)

Discussion

The prozone effect (or Hook effect) has long been appreciated as a source of interference in immunoassays.1 It typically occurs in sandwich assays, of which the FIT test is an example.2 When the concentration of the analyte is excessively high, it oversaturates the capture and detection antibodies in favor of forming single antibody:analyte complexes, rather than sandwiches. This results in a false negative result where the assay is unable to detect the analyte. The solution to the prozone effect is serial dilution to lower the concentration of the analyte.

The FIT test is designed to detect microscopic amounts of blood, hence its function in screening for fecal occult blood. A number of hospital labs use this test in an acute care setting to screening bleeding in patients. However, its capacity is oversaturated in specimens containing overt hemorrhage, as in our patient. In these cases it is nevertheless important to prove that the red color of the specimen is truly due to blood, as bright red stool can be caused by a wide range of dietary factors. Some examples are red food coloring, beets, cranberries, and tomato juice.3 If these possibilities are not ruled out, the patient may become subject to the risks of unnecessary endoscopy. Serial dilution of the specimen is extremely useful in this type of situation.

References

  1. Dasgupta A, Wahed A. Clinical Chemistry, Immunology and Laboratory Quality Control: A Comprehensive Review for Board Preparation, Certification and Clinical Practice. Amsterdam: Elsevier; 2014. 2.11.
  2. Allison JE, Fraser CG, Halloran SP, Young GP. Population Screening for Colorectal Cancer Means Getting FIT: The Past, Present, and Future of Colorectal Cancer Screening Using the Fecal Immunochemical Test for Hemoglobin (FIT). Gut and Liver. 2014 Mar;8(2):117-30. https://doi.org/10.5009/gnl.2014.8.2.117
  3. Picco MF. Stool color: When to worry [Internet]. Mayo Clinic; 2019 Feb 19 [cited 2019 Feb 23]. Available from: https://www.mayoclinic.org/stool-color/expert-answers/faq-20058080

-Hao Li, MD is a currently a first year anatomical pathological resident at Western University, London ON, Canada. Prior to be a pathology resident, he was a neurosurgery resident at the University of Saskatchewan, Saskatoon SK, Canada. When he was at the University of Saskatchewan, he spent his third year primarily in neuropathology, with also some general anatomical pathology and clinical pathology. Through these experiences, he has come to realize that his passion and calling lay more in pathology than in surgery. He has successfully transferred into pathology, and started a new residency in anatomical pathology in July 2019. Having a background in the clinical neurosciences, he hopes to eventually pursue a fellowship in neuropathology, and possess the skill set to practice both anatomical pathology and neuropathology.