Casualties of COVID-19: Measuring the Length, Width, and Depth of a Pandemic’s Impact

An editorial in Nature on August 12, 2020 entitled, “How to stop COVID-19 fuelling a resurgence of AIDS, malaria, and tuberculosis” provided four suggested solutions specifically for these diseases in the wake of Sars-CoV-2. For reference, here are the four approaches suggested as written in the editorial (#4 in detail):

  1. Hospitals and health authorities in affected cities and regions must recognize that AIDS, malaria and TB are surging again.
  2. Researchers must continue to refine their models using more real-world data.
  3. There is a need for public-information campaigns
  4. These campaigns cannot on their own keep surgeries and wards open, or equipment functioning. The resurgence of infectious diseases has created a greater demand for tests, treatments and research. All of these need more funding. 

Do those strike you as odd? The entire economy of nations along with the focus of their healthcare has been derailed and distracted by COVID-19 and the solution for these diseases is to recognize them, improve models, inform the public and seek more funding. You are either completely in tuned with the author in seeing that more funding is needed or you are a bit miffed that, in the wake of all that is happening, THESE guys want more money?

The US is a major contributor to the Global Fund for HIV, TB, and Malaria (the largest funder of these activities) and the total pledges to date for the GF approach $69 billion dollars with the US providing $54 billion (92%). From 2008 to 2016, the US contribution increased almost every year from $840 million to $1.65 billion annually until 2015 when it was frozen at $1.35 billion until 2019. In 2020, prior to the COVID-19 pandemic (i.e., during calendar year 2019 when the fiscal year 2020 budget was being planned), the amount from the US dropped to $958 million (2010 levels), representing a 30% drop in funding. So, to recap: The Global Fund started the year down by nearly 30% of what had been available, COVID-19 derailed all activities and drained the fiscal resources of patients and nations, and now, the progress that has been made on these diseases has been set back bay possibly a decade. The situation couldn’t be more desperate and, YES, the program needs a massive increase in funding. But, to be very clear, that massive increase pre-dated COVID-19 and represents something more distressing underneath.

I was fortunate to give the Michelle Rablais lecture at the ASCP Annual Meeting in Phoenix in 2019 where I carefully laid out the costs of controlling JUST malaria (not to mention TB and HIV) and demonstrated for the audience that as the number of cases get smaller and smaller (because your measures are so successful), the cost of finding the remaining cases goes up. As we successfully approach elimination or eradication of a disease, the final push requires at least the same but often more funding to make it across the finish line. This is not an opinion but is based on an enormous amount of data from other diseases as well as from the world’s experience with the first malaria eradication campaign. For HIV, we can’t eliminate it or eradicate it but we have converted it to a chronic disease and, therefore, infrastructure and funding to support patients ongoing is needed and by any form of math has to increase as the population lives longer and more people are added to the disease pool (although those numbers had been greatly reducing). Tuberculosis in its simplest form is a disease of poverty related to lack of access to drugs and healthcare, cramped living conditions, etc. When a pandemic derails the economy and causes the poor to become even more poor, tuberculosis is going to surge.

To the authors of this editorial I offer a gracious thank you and note with a heavy heart that the estimate of $28.5 billion additional dollars being needed to make up the ground lost by COVID-19 does also include the ground they had already lost by defunding principles trending over the last 4 years for global health.

But at least the countries that struggle with these diseases only have HIV, TB, and malaria to worry about, right? Wrong. In almost every low- to middle-income country where HIV, TB, and malaria are or have been major health challenges, hypertension, diabetes, cancer, cardiovascular disease, stroke, and mental health are equivalent or worse health problems than compared with high income countries. Do not be dissuaded by sheer numbers and always consider the outcomes, pre-COVID-19. For cancer, mortality in the US averages around 35% while in Africa it is closer to 80%. In full COVID-19 response mode, cancer programs—fledgling, underfunded, and disorganized—became non-existent and are only now (nearly 6 months after closing) starting to re-open and find their way back to where they were—fledgling, underfunded, and disorganized! Diabetics cannot go 6 months without insulin, hypertensive patients cannot have unregulated blood pressure, etc. While in the safety of a high-income country, makeshift systems, telehealth, contactless visits, etc. were brought on board to keep some semblance of a healthcare system in place, cancer patients were delayed in receiving diagnoses and treatment due to rationing of time and elimination of “elective” procedures.

As the data continues to be tallied and as models continue to be developed to understand just how much we have lost from our failed response to COVID-19 as a world and certainly as a nation, please do not slough off the staggering “additional” deaths that are going to be reported because of patients who didn’t have access to their regular health system. Every person from November 2019 until the end of this pandemic whose death occurred because their regular supply lines were disrupted, their planned treatments were cancelled, their medical supplies were not available, or their access to life-saving interventions were delayed is just as much a casualty from COVID-19 as a directly infected patient who succumbs to the disease. Our recent experience as a nation with the disasters in Puerto Rico around both the confusing death tolls from the hurricanes as well as the total death toll from the fiscal challenges of their medical system (prior to COVID-19) should serve as valuable lessons. Let us not come out of the other side of this pandemic with a similar disregard for the value of every human life or without an understanding of how our individual and collective mistakes as a nation have lead directly to these effects.

milner-small


-Dan Milner, MD, MSc, spent 10 years at Harvard where he taught pathology, microbiology, and infectious disease. He began working in Africa in 1997 as a medical student and has built an international reputation as an expert in cerebral malaria. In his current role as Chief Medical officer of ASCP, he leads all PEPFAR activities as well as the Partners for Cancer Diagnosis and Treatment in Africa Initiative.

Massive COVID-19 Testing: 30 Million Tests/Week

Population COVID-19 testing

Population-wide testing to identify symptomatic and asymptomatic infections could be a powerful tool to control Coronvirus Disease 2019 (COVID-19) spread, but current global testing capacity does not permit widespread testing of asymptomatic individuals. These tests are still limited to individuals who are symptomatic with limited availability to those with recent exposure to an infected person.

Because of the high prevalence of asymptomatic COVID-19 infections, proposals from the Rockefeller Foundation for disease mitigation include widespread and frequent testing of the US population. In the United States, diagnostic testing for SARS-CoV-2, the causative virus of COVID-19 is currently >2 million per week. Estimates for US testing needs for population wide surveillance range from 30 to 300 million per week. In order to scale testing by an order of magnitude, novel technologies and rethinking current testing paradigms are needed. The NIH has initiated a rapid funding program to develop SARS-CoV-2 testing, and these new technologies may play a part. However, we can broadly conceptualize key problems to address in population-wide testing in the US. The first is high-sensitivity testing which identifies active infection and can be performed with massive throughput. The second is the logistics of gathering hundreds of thousands of samples to each testing laboratory each day.

Next Generation Solutions to COVID testing

Emerging technologies using targeted next-generation sequencing have been suggested as a potential solution to population-wide testing. The key features include 1) extraction free amplification 2) an easily collected specimen such as saliva, 3) nucleotide barcodes to enable sample pooling, and 4) a limited number of targets (to allow deeper sequencing, i.e. higher sensitivity). Illumina is selling a whole genome test for SARS-CoV-2, but this limits sequencing to 3,000 tests/ run. Another recent approval for a private testing lab uses only one target, and may allow it to increase to 100,000 tests/ day. And a recent protocol for LAMP-Seq in pre-print outlines how this could work in a scheme below. An attractive aspect of this approach is decentralized specimen processing.

Whereas Bill Gates has supported a portfolio approach to vaccines placing multiple bets on different processes in parallel, a similar approach should be applied to multiplexed sequencing methods. Two sequencing runs can be performed on a single instrument in a single day, which can process several thousand samples. However, sequencing is not the only step in sequencing; library preparation and specimen handling take significant amounts of time too.

Laboratory Logistics

This technology would represent an exponential expansion in analytic testing capacity, but clinical labs will require a similar escalation in logistic capacity. The largest clinical laboratories in the world process less than 100,000 samples per day. Clinical laboratories have a long history of automation with the first robotic specimen track systems developed in the 1980s. Engineering and clinical lab expertise should thus partner to innovate on methods to handle high volumes. This level of investment for an issue that is likely to fade in 2 years, is not attractive to most private health systems, so public investment from multiple states in regional reference labs is needed.

It is still hard to conceive the necessary scale up in sample processing can be achieve within the time frame needed, so I would also propose a de-centralized sample processing approach. This would include self-collection of saliva (a safe, effective sample type with similar sensitivity as nasopharyngeal swabs), drop-off sites, and processing at places like Pharmacies (>90% of Americans live within 5 miles of a pharmacy and they could be authorized to administer tests- just as they administer vaccines). This would introduce pre-analytic problems, but if the goal is frequent and high rates of testing, then we will have to accept certain losses in sensitivity (which currently is arguably better than it needs to be). Interestingly, pre-analytic concerns with saliva have not led to sample instability or degradation of RNA causing false negatives, as described in my last post. However, other factors could affect saliva quality: smoking, age, and genetic factors of water: protein ratio affecting viscosity.

Testing solutions should be considered in the context of the planned testing network. The specimen type should be easy for the patient to provide, processed with existing laboratory equipment and resulted electronically. For example, current COVID-19 testing is based on sample collections requiring a healthcare worker encased in personal protective equipment (PPE) utilizing a swab device. Testing needs to progress to a simpler solution such as saliva which can be collected by the patient in the absence of a swab or PPE. Preliminary studies have demonstrated that saliva is sample type comparable to nasopharyngeal swab. The ideal saliva sample would be collected into an existing collection tube type (e.g. red-top tubes) which are already compatible with existing laboratory automation. In aggregate, a person could spit into a tube at-home, have the tube sent to a laboratory, and in the laboratory the tube would be directly placed onto an automated robotic track system. 

Laboratory professionals need to provide a comprehensive plan for regional and national laboratory networks which can scale to provide overwhelming force to COVID-19 testing. No other profession or governmental organization understands testing as much as we do. Our understanding of managing samples from collection to result should be applied to the pandemic at hand. Until now most laboratorians in the US have focused on the immediate needs of providing testing for symptomatic patients and healthcare workers.

Vision for automated COVID-19 testing

One could envision an automated line of testing that moves samples through processing to allow multiplexing and combinations of samples to allow large numbers of patients to be tested at once (see below). This is feasible in some specialized centers, but would require investments in automation, bioinformatics, and interfaces for a seamless process (figure below). If testing mostly asymptomatic patients, it may also be possible to do this on pooled samples. The number of samples to pool would depend on the likelihood to having a positive result (this would require sequencing all individuals in a pool).

This represents a synthesis of ideas in decentralized specimen collection, laboratory automation and massive testing throughput with Next-Generation Sequencing, but unfortunately this is not yet a reality.

References

  1. Jonathan L. Schmid-Burgk et al. LAMP-Seq: Population-Sclae COVID-19 Diagnostics Using Combinatorial Barcoding. bioRxiv 2020.04.06.025635.
  2. The Rockefeller Foundation. National Covid-19 Testing Action Plan Pragmatic steps to reopen our workplaces and our communities. 2020.
  3. Cahill TJ, Cravatt B, Goldman LR, Iwasaki A, Kemp RS, Lin MZ et al. Scientists to Stop COVID-19.  OR Rob Copeland, Wall Street Journal (2020) The Secret Group of Scientists and Billionaires Pushing a Manhattan Project for Covid-19. April 27
  4. https://www.illumina.com/products/by-type/ivd-products/covidseq.html

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

The Story of the Mott Cell, COVID-19 and the Cute Little Mouse

I have worked in hematology for many years, and there are certain things that never fail to excite technologists. Working in New Hampshire, it was always exciting to sickle cells or malaria, something common to some, but not common in our patient population. I now work in Baltimore, and see sickle cells nearly every day, and we come across malaria not too infrequently, but we still share good examples and save them for training. When we see something different or unusual, we always share the finding. Cells may need to be sent to the pathologists for a pathology review, and we always check back to see the pathologist’s identification and comments. Medical Technologists by nature are a curious bunch, and we always want to see ‘cool’ things. I wrote a blog two years ago about the only patient I have ever seen with Trypanosoma (Hematology Case Study: The Race to Save a 48 Year Old Man from a Rare Disease). Last month I wrote about Blue-green cytoplasmic inclusions (COVID-19 Patients with “Green Crystals of …” STOP! Please Don’t Call Them That). So, when I saw something else ‘cool’ and different on a peripheral smear, and then saw it AGAIN, on another patient, and saw other techs here in the US and in other countries were also mentioning these, because it’s my nature, I got curious.

When I write these blogs, I often feel a little bit like the mouse in the children’s story “If You Give a Mouse a Cookie”, by Laura Joffe Numeroff. It’s about an adorable little mouse who asks for a cookie, and then decides he needs a glass of milk to go with it, and then he needs a straw, and it goes on and on, in a circle, back to the beginning. Maybe it’s that the mouse is a little ADD, but I like to believe that he’s just creative and curious. I start with an idea, and often go off on many tangents before a blog is finished and comes back to where I started.. When I started writing this, it was because I saw an interesting cell, and I started exploring, and found that others had seen them, too. Then I started looking through my textbooks for references and information, and searched for recent research or studies, and then I wanted to find out more… just like that mouse.

There are some things that we learn about in school and we may see on CAP surveys, but no matter where you work, they are still rarely seen, so are a novelty. Mott cells are one of these things. I have a collection of Hematology texts from grad school and years of teaching Hematology. Several of these don’t even mention Mott cells, but, when they do, it’s barely a sentence in a discussion of plasma cells. I happen to have a very old copy of Abbott Laboratories “The Morphology of Human Blood Cells” . The one with the red cover, from 1975. The term Mott cell does not appear in this manual, but they do show pictures and describe “Plasma cells with globular bodies (Grape, Berry or Morula cells)”, and describe these globules as “Russell bodies”.1 So some of us who have been working in the field for many years may remember Russell bodies and Morula cells, or Grape cells, even if the term Mott cell is not familiar. Regardless of what we or textbooks call them, they tend to trigger a memory because the images are so unique.

So, again, I’m a bit like that mouse and getting distracted with the background. Why am I writing this blog? In recent months I have seen cells identified as plasmacytoid lymphocytes and Mott cells in several hospitalized patients. I have heard reports of these cells in other facilities as well. So, like a good medical technologist, I got curious about Mott cells. What are they, and what is their significance? And why are we seeing more of these now?

Mott Cells are named after surgeon F.W. Mott. In the 1890’s, William Russell first observed these cells with grape like globular inclusions, but did not recognize what the inclusions were or their significance. Russell examined the cytoplasmic globular inclusions and assumed that these cells were fungi. Ten years later, Mott described cells he called morular cells. He recognized that these cells were plasma cells and the inclusions were indicative of chronic inflammation. Thus, today we refer to these cells as Mott cells, morular cells or grape cells, and the inclusions as Russell bodies.2

Hematology texts describe Mott cells as morphologic variations of plasma cells packed with globules called Russell bodies. We know that plasma cells produce immunoglobulin. When the plasma cells produce excessive amounts of immunoglobulin, and there is defective immunoglobulin secretion, it accumulates in the endoplasmic reticulum and golgi complex of the cells, forming Russell bodies. Russell bodies are eosinophilic, but in the staining process the globulin may dissolve and they therefore appear to be clear vacuoles in the cell under the microscope. Thus, a plasma cell with cytoplasm packed with these Ig inclusions is called a Mott cell.

Mott recognized that these atypical plasma cells were present in inflammation. Plasma cells are not typically seen on peripheral blood smears and constitute less than 4% of the cells in a normal bone marrow. Yet, on occasion, we can see plasma cells, including Mott cells, on peripheral blood smears in both malignant and non-malignant conditions. Mott cells are associated with stress conditions occurring in a number of conditions including chronic inflammation, autoimmune diseases, lymphomas, multiple myeloma, and Wiskott–Aldrich syndrome.3

So, why are we seeing an increased mention of Mott cells now? We seem to be seeing these on patients testing positive for SARS-CoV-2. I have seen cells on patients at my facility that resemble Mott cells. I belong to a Hematology Interest group and over the past few months I have seen several people post pictures of Mott cells, cells with Russell bodies, and plasmacytoid lymphocytes identified on peripheral blood smears of COVID-19 patients. Other techs chimed in with comments that they have also seen these cells recently. I have even seen a comment propose that these cells are indicative of COVID-19 infection.

SARS-CoV-2 definitely causes inflammatory processes and stress conditions in the body, so it makes sense that we may see these cells in COVID-19 positive patients.

Figure 1 shows a Mott cell on an image from Parkland Medical Center Laboratory, Derry, NH. A Mott cell was identified by pathologist in a male patient who tested negative for COVID-19 at the time the sample was drawn, and subsequently tested positive. Mariana Garza, a Medical Technologist working at Las Palmas Medical Center in El Paso, TX shared a case of a 59 year old diabetic male, diagnosed with COVID-19. The patient’s WBC was 31 x 103/μL. Two Mott cells were identified by pathologist on his differential. So, the curious little mouse in me researched some more.

Image 1. Mott cell. Photo courtesy Parkland Medical Center, Laboratory, Derry, NH.

Several published research papers have studied morphologic changes in peripheral blood cells in COVID-19 patients. As we now know, SARS-CoV-2 affects many organs including the hematopoietic and immune systems. A study in Germany showed that COVID-19 patients exhibited abnormalities in all cell lines; white blood cells, red blood cells and platelets. Increased WBC counts were seen in 41% of samples in their study. Differentials performed on study patients showed lymphocytopenia in 83%, and monocytopenia in 88%. Red blood cell morphology changes were noted. Platelet counts ranged from thrombocytopenia to thrombocytosis, but giant platelets were noted across the board.4

Mott cells are indicative of chronic inflammation and may have significance in association with COVID-1. In the above mentioned study, aberrant lymphocytes were noted in 81% of patients who were SARS-CoV-2 positive, and observable in 86% of the same patients after they tested negative. The paper shows plasmacytoid lymphocytes and Mott cells amongst these aberrant lymphocytes. Moreover, morphologic changes in neutrophils, such as a left shift and pseudo‐Pelger‐Huët anomaly, decreased after virus elimination but changes in lymphocytes, indicators of chronic infection, remained.4

Another study also reported reactive or plasmacytoid lymphocytes and Mott cells observed in peripheral blood.4,5 Researchers at Northwick Park Hospital, UK, presented a case study of a 59 year old male with COVID-19 with a normal WBC and thrombocytosis. His differential revealed lymphocytopenia. His differential also showed lymphoplasmacytoid lymphocytes and Mott cells. In their conclusions it is stated that “In our experience, the lymphocyte features illustrated above are common in blood films of patients presenting to hospital with clinically significant Covid‐19. The observation of plasmacytoid lymphocytes supports a provisional clinical diagnosis of this condition.”5

Can these variant plasma cells, along with other commonly seen morphological changes, be used as part of a diagnostic algorithm for SARS-Cov-2 infection? As we see more COVID-19 patients there will be more, larger studies done and more Mott cells identified. Some disorders, such as Epstein Barr Virus and Dengue Fever are characterized by distinct viral changes in cells. However, since Mott cells can be seen in many conditions, these alone could not be considered diagnostic, but the indications are that these cells, along with the entire differential and morphological patterns, could prove to be a straightforward and easy to perform supplementary diagnostic tool. More, larger studies need to be done. It was concluded in the German study, that this pattern of morphologic changes in cells could be further investigated and validated with a larger blinded study, and that this information could lead to the development of a morphologic COVID‐19 scoring system.4 In the meantime, keep an eye out for Mott cells. These should not be ignored and should be in some way noted because they may be of future diagnostic use. That’s all or now, folks! Something to dig deeper into in another blog! The mouse strikes again!

Many thanks to Nikki O’Donnell, MLT, Parkland Medical Center, Derry, NH and Mariana Garza, MT, Las Palmas Medical Center in El Paso, TX for sharing their case studies and photos.

Becky Socha MS, MLS(ASCP)CMBB

References

  1. Diggs, LAW, Sturm, D, Bell,A. The Morphology of Human Blood Cells, Third edition. Abbott Laboratories. 1975.
  2. ManasaRavath CJ, Noopur Kulkarni, et al. Mott cells- at a glance. International Journal of Contemporary Mudeical Research 2017;4(1):43-44.
  3. Bavle RM. Bizzare plasma cell – mott cell. J Oral Maxillofac Pathol. 2013;17(1):2-3.doi: 10.4103/0973-029X.110682.
  4. Luke, F, Orso, E, et al. Coronavirus disease 2019 induces multi‐lineage, morphologic changes in peripheral blood cells:eJHaem. 2020;1–8.
  5. Foldes D, Hinton R, Arami S, Bain BJ. Plasmacytoid lymphocytes in SARS-CoV-2 infection (Covid-19). Am J Hematol. 2020;1–2. https://doi.org/10.1002/ajh.
  6. Numeroff, Laura. If You Give a Mouse a Cookie, 1985.

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

False Negatives in COVID-19 Testing

I left for vacation at the beginning of June thinking “once I get back, all of this COVID stuff will be quieted down.” …Well that wasn’t quite the case and testing for novel Coronavirus has continued to be very important. In fact, this last weekend I was tested by occupational health. It came back negative, but I’m am very enthusiastic to get alternative specimen types validated; those Nasopharyngeal swabs are quite…uncomfortable. Luckily, my test was processed at our institution which gets results back in 24-48 hours. However, with the resurgence around the country, turnaround times are backing up to 7-8 days. One solution has been the widely used IDNOW point of care platform. However, there has been significant concern over false negatives produced by this platform. One reason the sensitivity is different is because this platform performs isothermal amplification of nucleic acid. This method amplifies RNA at a stable temperature instead of cycling the temperature as in real-time PCR.

Colleagues at my institution reflexed any negative IDNOW samples to the m2000 Real-Time PCR assay for SARS-CoV-2 for one month. Within that time, over 500 samples were tested and the IDNOW was found to have missed 21% of positive cases (prevalence rate of 5%)2. One the positive side, it had a 98% negative predictive value, which helped rule out COVID19 infection. However, as prevalence rates are increasing, a high negative predictive value isn’t as important as sensitivity.

One study drew much attention when it claimed the IDNOW had a sensitivity of 52% in a New York City academic institution (Basu)4. However, this seems to be an outlier compared to other studies of this platform: one large multi-center study found positive percent agreement (equivalent of sensitivity when a gold standard test hasn’t been established) of 74%1. The highest PPA of 88%3 for the IDNOW was found in a study that indicated it can be completed in 17 minutes, whereas another quick instrument (but not point of care instrument: Xpert Xpress, 45min) had a PPA of 98%2.

Myself and other colleagues looked more closely at the clinical characteristics of false negative test results on the IDNOW. Overall, we found 82% PPA, and 8 patients with false negative tests. Interestingly, a majority of these patients were tested over 2 weeks after their initial onset of symptoms. The virus is known to be at its highest levels at the beginning of symptom onset. So the test may not be limited, but it should be used in the correct clinical context (< 2weeks from symptom onset). After that time, other RT-PCR based tests are more appropriate.

As clinical laboratorians, we often hear: “the right test for the right patient at the right time.” Now with so many platforms available for use in different contexts, we should help guide clinicians to Choose Wisely.

References

  1. Harrington A et al. Comparison of Abbott ID Now and Abbott m2000 methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs from symptomatic patients. JCM 2020. PMID: PMID: 32327448
  2. McDonald et al. Diagnostic Performance of a Rapid Point of Care Test for SARS-CoV-2 in an Urban ED Setting. Academ. Emerg. Med. 2020. PMID: 32492760
  3. Zhen W et al. Clinical Evaluation of Three Sample-To-Answer Platforms for the Detection of SARS-CoV-2. JCM 2020. PMID: 32332061
  4. Basu A et al. Performance of the rapid Nucleic Acid Amplification by Abbott ID NOW COVID-19 in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a New York City academic institution. BioRxiv 2020.

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

COVID-19 Patients with “Green Crystals of …” STOP! Please Don’t Call Them That

Blue-green cytoplasmic inclusions in neutrophils and monocytes are a novelty in hematology. It is rare to see these inclusions on peripheral smears, and when we do, there is excitement, but sadness too, because, when noted, they usually indicate a poor prognosis and impending death. Thus, we have heard them called “green crystals of death” or “death crystals.” I know I would not want to read a family member’s medical chart and see reference to “death crystals.” It’s an insensitive term, and one the medical community is trying to discourage. And, in fact, though it typically does indicate a poor prognosis, not all cases lead to death. In published reports, it has been shown that short term mortality in patients with these crystals is about 60%.1

These rare inclusions are refractile and irregular in shape, and are found in neutrophils, and occasionally in monocytes. Color seems to be subjective here. They call them green when inclusions in photos or cells I am looking at look very blue to me. The color perceived may depend on the type of stain (Giemsa, Wright or Wright-Giemsa) used and how fancy we get in color names and descriptions. Or, maybe I’m just color blind! Some people (like my husband) are “lumpers” and call anything blue-green, blue, or green, but don’t recognize subtleties of colors. Thus, I guess to make everyone happy, or to compromise, the blue-green description may fit them best.

Image 1. Blue-green inclusions seen in neutrophils. Photos courtesy of Alana D. Swanson. UMMC

These blue-green inclusions were originally reported in patients with hepatic injury and failure. Laboratory results include elevations in AST, ALT and LDH. More recently, there have been cases with no evidence of hepatic injury. Researchers are now finding that these crystals can occur in patients with tissue injury other than liver, and in patients with multiorgan failure. In patients with no liver injury, what is a common factor is that LDH is elevated, indicating tissue injury. Additionally, along with these crystals, lactic acid levels can be used as a predictor of survival. Higher levels of lactic acidosis at the time crystals are noted is a negative predictor of survival.2

In trying to determine the clinical significance of these crystals, they have been subject to a number of different stains to determine their content. The association with hepatic failure led researchers to hypothesize that the crystals were a bile product in circulation. Since then, the crystals have been found to be negative in bile stains. When stained with other stains, Oil Red O showed positive in neutrophils, indicating high lipid content. The inclusions did not stain positive with iron stain or myeloperoxidase. Acid fast stains showed the inclusions to be acid fast positive.3 These crystals also show an interesting similarity to sea-blue histiocytes, which further associates them with tissue injury. After analysis, it is now thought that these crystals contain lipofuscin-like deposits representing lysosomal degradation products, and may be present in multiple types of tissue injury.2

With the current pandemic, I have seen reports of these crystals in COVID-19 patients. I have heard of fellow technologists seeing these, and a recent paper described the first reported cases in patients with COVID-19. These recent incidences may lead to new information about exactly what clinical significance they hold. About one third of COVID-19 patients have elevated ALT and AST, though it is not yet clear whether the liver dysfunction is directly caused by the virus, due to sepsis, or other complications of patient comorbidities. Many COVID-19 patients have mild disease, yet some develop severe pneumonia, respiratory complications, and multiorgan failure. Mortality is increased in these severely affected patients. To better understand and manage treatment for COVID-19, physicians seek to identify biological indicators associated with adverse outcomes.1

In a New York City study, Cantu and colleagues reported on six COVID-19 patients who presented with blue-green crystals in neutrophils and/or monocytes. All six patients had an initial lymphocytopenia, and significantly elevated AST, ALT, LDH and lactic acid at the time the crystals were noted. All of the patients had comorbidities, yet only two of the six presented with acute liver disease. Interestingly, in the six cases reported on in the study, only one had blue-green inclusions reported from the original manual differential. The others were found retrospectively when correlating the cases with patients known to have elevated ALT and AST. All patients died within 20 days of initial diagnosis.1

The consensus of several papers in the last few years is that these crystals are being underreported. As seen in the above study, the crystals were originally seen in just one of the six patients. A look back revealed the other cases. With an increase in COVID-19 cases in our facilities, these blue-green crystal inclusions may be a novelty that is wearing off. We may see a rise in their presence, and need to be able to recognize and report them. This information is important to report if clinicians are to use these crystal inclusions along with acute transaminase and lactic acid elevations to predict poor patient outcomes.

Clinicians, hematologists, and laboratory technologists should be educated and have a high level of awareness of these inclusions. The University of Rochester conducted a study a few years ago that noted that, because these crystals are rare, techs may not be on the lookout for them. Once techs see them, they seem to be on the alert and more are reported. The hospital instituted an “increased awareness” campaign, which resulted in an increase in detection. This revealed cases that were not related to liver injury, including patients with metastatic cancer and sepsis. However, an important correlating factor was that all of the patients had mild to severe elevations in liver enzymes. With more awareness, we are starting to see them in patients without hepatic injury, but with other inflammation and tissue injury.4

Image 2. Blue- green crystal inclusions seen in a patient diagnosed with sepsis and multiorgan failure. Photo courtesy of Karen Cable, YRMC.

Let’s raise our level of awareness of these maybe-not-so-rare crystal inclusions. And, please be sure to call them by their preferred name, blue-green neutrophil inclusions! Let’s not talk about death crystals or crystals of death.

Many thanks to my colleague Alana D. Swanson, MLS(ASCP)CM , University of Maryland Medical Center and Karen Cable, Hematology Section Lead, Yavapai Regional Medical Center, Arizona, for the photos used in this blog. 

References

  1. Cantu, M, Towne, W, Emmons, F et al. Clinical Significance of blue-green neutrophil and monocyte cytoplasmic inclusions in SARS-CoV-2 positive critically ill patients. Br J Haematol. May 26, 2020.
  2. Hodgkins, SR, Jones, J. A Case of Blue-Green neutrophil inclusions. ASCLS Today. 2019;32:431.
  3. Hodgson, T.O., Ruskova, A., Shugg, C.J., McCallum, V.J. and Morison, I.M. Green neutrophil and monocyte inclusions – time to acknowledge and report. Br J Haematol, 2015;170: 229-235.
  4. Patel,N, Hoffman,CM, Goldman,BJ et al. Green Inclusions in Neutrophils and Monocytes are an Indicator of Acute Liver Injury and High Mortality. Acta Haematol. 2017;138:85-90

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

A Resident’s Perspective of SARS-CoV-2 Testing Using the Double Diamond Model of Design Process

During the 2019-2020 residency interview season, I “courted” – no better way to describe those interactions over lunch–several potential co-residents, who were eager to know why I came to University of Chicago (NorthShore) for my residency. My answers and those of my fellow residents would help the candidates determine how high they should rank our program, so I enthusiastically recalled things I liked when I interviewed at NorthShore about a year earlier. I had also recently completed my first microbiology rotation in residency and I had enjoyed seeing all of those factors work synergistically to improve patient health outcomes through improved testing. So passionately, I shared how I fell in love with the physical structure of the department which has almost all the labs and offices one floor, the automation of the labs-especially the core and microbiology labs, the capability and regular expansion of its molecular laboratory, the people and of course, “the feel” about NorthShore.

With these experiences, I looked forward to my second microbiology in March 2020, where I would learn more about the diagnostics of various microorganisms–E. coli: Gram negative short stubby/broad shouldered rods vs. Pseudomonas aeruginosa, Gram negative long slender rods, etc. (Un)fortunately, March came, but the novel coronavirus (SARS-CoV-2) had other plans for my learning. Cases of Coronavirus disease 19 (COVID-19), caused SARS-CoV-2[1] were increasing rapidly in the US, so laboratories, including ours had rapidly implement testing. Rather than have morning rounds and other educational activities where the differential diagnoses of several clinically relevant microorganisms were discussed, we had virtual and in-person meetings discussing what to do about one virus. These continued and by the middle of March, we had become the only non-government lab in Illinois and second in the Midwest that had developed a clinical PCR test for SARS-CoV-2. I was excited to be part of that success, but more so, about learning how we achieved that as a team.

Our approach could be summarized using the Double diamond or 4D model of design process which consists of four phases: Discover, Define, Develop and Deliver (Figure 1).

Figure 1. Double diamond or 4D model of design process which consists of four phases: Discover, Define, Develop and Deliver. Plan Do Study Act (PDSA) is an iterative model of quality improvement embedded in the 4D design process.
  1. In the discover phase, a phase of divergent thought [2] and exploration, we identified from events in China and other parts of the world as well as some other states in the US that the community we care for could potentially be affected by the COVID-19 outbreak.
  2. The next phase- define- is a convergent phase where the problem to be solved, as well as the resources available and resources needed to solve it are delineated [2]. As we transitioned from the discovery to define phases-and recalling the 2009 H1N1 influenza outbreak about 10 years ago- it became evident that an epidemic of a relatively fatal respiratory virus which we knew very little about was heading our way. As clinical laboratory professionals, our objective was to help identify members of the community who had been infected through testing so appropriate steps could be taken to sequester and care for them. Among our available resources was our molecular laboratory, but like most laboratories outside the Centers for Disease Control and Prevention, CDC we lacked the reagents, primers and authorization to run the test.
  3. Develop is the next phase in the process and this is a divergent phase where the team explores and refines potential solution to the issues and selects one[2]. This is often followed by the convergent deliver phase where one of the solutions from the develop phase is implemented. Feedbacks which are used for projects are also received during this phase[2]. But, the outbreak continued to evolve rapidly [3] with briskly increasing positivity rates[4] and some of the solutions we considered would require some time to be implemented and/or have long turnaround times. For instance, since we had a roust molecular laboratory, one option was to develop our assays and test in-house, while another was to send the samples to outside labs where they could be run. Running the tests in-house would have a shorter turnaround time and would be more efficient, which is extremely important considering the severity of COVID-19.
  4. Deliver is the last phase of the process.  We decided to develop a SARS-CoV-2 RT-PCR test at our institution, but we also knew we needed to put logistics and protocols in-place to deliver our solution.  For example, COVID-19 presents with flu-like symptoms but flu is common between December and March[5-7] so it would be impractical to expect to test all patients with flu-like symptoms – at least with the limited resources we had. In any case, it was clear that we would not have an ideal amount of time or information to develop and implement the perfect solution. As such, the revolving and fluid nature of the develop and deliver phases of our response is best depicted using the Plan Do Study Act (PDSA), an iterative model of quality improvement. As shown in Fig. 1, we developed and validated our assay, as well as developed an initial protocol for screening patients and logistics for patient-centered delivery in the “Do” step. Importantly, we also reviewed the effectiveness of these operations, and made necessary changes corresponding in the “Study” and “Act” steps respectively.

The prompt decision to implement in-house COVID-19 testing at NorthShore has proven to be the right one. To date we have tested 75,000 specimens and nearly 20,000 tests have been positive. Success which was possible because of the factors which made me come to NorthShore, amongst others. The LEAN, bright and capacious design of the department limits the innate barriers of hierarchical organizational structure; encouraging seamless horizontal and vertical intradepartmental consultation and collaboration as COVID-19 led us into uncharted territory. Also, having a molecular lab that regularly expands its capability made the decision to test in-house relatively easy. In addition, having an automated microbiology lab made it easier for staff to be flexible and deal with the various demands of testing for a new bug in a pandemic. And of course, the people at NorthShore who are ready to volunteer, take up new roles or change shifts to accommodate the demands of a rapidly evolving pandemic, stay in constant communications and provide feedback, and who make everything else at NorthShore work!

References

  1. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200130-sitrep-10-ncov.pdf?sfvrsn=d0b2e480_2
  2. Council, Design. “Eleven lessons: Managing design in eleven global companies-desk research report.” Design Council (2007).
  3. https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/summary.html
  4. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200324-sitrep-64-covid-19.pdf?sfvrsn=723b221e_2
  5. https://www.who.int/news-room/q-a-detail/q-a-coronaviruses#:~:text=symptoms
  6. https://www.cdc.gov/flu/symptoms/symptoms.htm
  7. https://www.cdc.gov/flu/about/season/flu-season.htm
  8. Christoff, Patricia. “Running PDSA cycles.” Current problems in pediatric and adolescent health care 48.8 (2018): 198-201.

Adesola Akinyemi, M.D., MPH, is a first year anatomic and clinical pathology resident at University of Chicago (NorthShore). He is interested in most areas of pathology including surgical pathology, cytopathology and neuropathology -and is enjoying it all. He is also passionate about health outcomes improvement through systems thinking and design, and other aspects of healthcare management. Twitter: @AkinyemiDesola

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

COVID-19 Testing Explained

By this point I believe we are all tired of reading and talking about COVID. However based on reading comments on social media, it’s quite clear that there are a lot of misconceptions about COVID testing. For starters COVID-19 is the disease caused by the SARS-CoV-2 virus. So all of the tests we are using to assist in the diagnosis of COVID-19 are really looking for signs that the person was infected with SARS-CoV-2. There are also 3 main categories of tests for SARS-CoV-2 based on the target of the assay: RNA, antigen, and antibody.

Diagnosis of COVID-19 should be based on clinical symptoms, risk of exposure, test results and timeline. The diagnostic tests based on detection of SARS-CoV-2 RNA are the most commonly used and reliable for diagnosis of COVID-19.1 All of these assays are based on amplifying the viral RNA to detect the presence of the RNA. Most assays use some form of PCR to amplify the virus, however because the virus is RNA-based it has to be converted to cDNA with reverse transcriptase PCR before amplification and detection. TMA or transcription-mediated amplification is another chemistry that can be used to amplify the RNA to a detectable level. Both PCR and TMA based assays are very sensitive at detecting the virus especially within the first week after symptoms develop.1,2 Due to the RNA-based nature of the SARS-CoV-2 genome, the mutation rate is anticipated to be high. Most of the RNA-based assays have adopted a strategy to target 2 different areas of the viral genome to prevent missing the presence of the virus due to a mutation in the primer binding site.

A SARS-CoV-2 antigen test received EUA in early May. The test is designed with immunofluorescence-based lateral flow. This type of test is designed to detect SARS-CoV-2 proteins present on the outside of the virus. In general, this class of test is cheaper and faster than RNA-based testing however it is less sensitive (80% clinical sensitivity).3 The clinical specificity of antigen assays is shown to be 100%,3 therefore a positive result is reliable. These tests can be used for screening; however patients with negative results may still need to proceed to testing by an RNA-based method. Antigen based tests is typically more sensitive during the same timeframe when PCR testing is more sensitive, ie earlier in the course of disease.

SARS-CoV-2 antibody tests are the last class of tests. Seroconversion appears to occur within 7-14 days of symptom onset2 or 15-20 days post exposure to the virus.4 There are many different tests to choose from to determine if the patient has previously been exposed to SARS-CoV-2. The assays range from lateral flow cassettes to high throughput chemiluminescent based assays. Some of the SARS-CoV-2 antibody assays detect IgG, IgM, IgA or some combination of the 3 including total antibody without differentiating between the three. The latest studies have shown that some patients develop IgM first, some with IgG, and others had both IgG and IgM develop at the same time.5 Therefore differentiating IgG from IgM is not providing a timeline for acute infection as we have seen in response to other viruses. Although sensitivity and specificity vary widely between manufacturers total antibody detection appears to be more sensitive than IgG or IgM detection alone.4 The FDA recently pulled numerous assays off of the market due to poor performance.

It is important to note that even with the most sensitive and specific antibody test, these tests cannot determine if a patient has protective immunity. Unfortunately we don’t know enough about immunity with regards to COVID yet. Early studies are promising, showing that some level of antibody will likely provide protection from future exposure. We don’t know if there is a threshold of antibody that needs to be present before a patient is immune, will the immunity only decrease the severity and not prevent reinfection, and how long the antibodies are maintained after exposure. These will be important questions to answer before the clinical utility of antibody testing can be realized. Right now the test is useful to determine is a patient was previously exposed to SARS-CoV-2 and is helpful to address epidemiological questions with regards to prevalence of COVID-19 in the community. The antibody test should not be used for diagnosis of current infection due to the delay to seroconvert after exposure.

References

  1. Sethuraman, N., Jeremiah, S. S., & Ryo, A. (2020). Interpreting Diagnostic Tests for SARS-CoV-2. JAMA. doi:10.1001/jama.2020.8259
  2. Wolfel, R., Corman, V. M., Guggemos, W., Seilmaier, M., Zange, S., Muller, M. A., . . . Wendtner, C. (2020). Virological assessment of hospitalized patients with COVID-2019. Nature, 581(7809), 465-469. doi:10.1038/s41586-020-2196-x
  3. Quidel Sofia®2 SARS Antigen FIA. https://www.quidel.com/sites/default/files/product/documents/EF1438900EN00_0.pdf 5/29/2020.
  4. Lou, B., Li, T. D., Zheng, S. F., Su, Y. Y., Li, Z. Y., Liu, W., . . . Chen, Y. (2020). Serology characteristics of SARS-CoV-2 infection since exposure and post symptom onset. Eur Respir J. doi:10.1183/13993003.00763-2020
  5. Long, Q. X., Liu, B. Z., Deng, H. J., Wu, G. C., Deng, K., Chen, Y. K., . . . Huang, A. L. (2020). Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med. doi:10.1038/s41591-020-0897-1

-Tabetha Sundin, PhD, HCLD (ABB), MB (ASCP)CM,  has over 10 years of laboratory experience in clinical molecular diagnostics including oncology, genetics, and infectious diseases. She is the Scientific Director of Molecular Diagnostics and Serology at Sentara Healthcare. Dr. Sundin holds appointments as Adjunct Associate Professor at Old Dominion University and Assistant Professor at Eastern Virginia Medical School and is involved with numerous efforts to support the molecular diagnostics field. 

Review: Blood Supplies During the COVID-19 Pandemic

When I first thought of writing a blog on blood supplies amid the COVID-19 pandemic, it was early March. Fast forward a couple months and a lot of things have changed. So, where were we, and where are we now?

January 6, 2020

At this time, most people in the US were not even aware of the novel coronavirus. (unless you were taking my Introduction to Human Disease course and were searching online for media articles about infectious disease!)

I first became aware of this ‘mystery’ virus in early January, when I was teaching an online Winter session course called Introduction to Human Disease. I developed this course a number of years ago as a STEM course for non-science majors. The intent of the course is to familiarize students with diseases and disease terminology that they will use in their everyday lives. The course gives students a chance to learn basic medical concepts that will enable them to become their own (or their family’s) medical advocate. In addition, the course covers many diseases that are ‘in the news’ and allows students to gain some knowledge and insight into the myths and facts surrounding these diseases. Topics covered include general mechanisms of disease, including inflammation, infectious disease, immunity, heredity, and cancer. Emphasis is placed on emerging and pandemic …. so, when this disease emerged, we were right there to take note!

I asked the students to find an article in the media on an infectious disease, and to summarize and answer questions about the article and the mechanism of the disease. Three students chose different articles about this yet unnamed mystery illness affecting people in Wuhan, China. We had active discussion board conversations about this emerging severe respiratory disease and pneumonia, that at the time had infected around 40 people, with no reported deaths, and no human to human transmission. In my comments, I compared this novel virus to seasonal influenza, H1N1, SARS and MERS and tried to reassure students that this would hopefully follow the same path as H1N1 or SARS and MERS.

Feb 21, 2020

The first confirmed case in the United States was on Jan 21 in Washington state. (CDC)1 On Jan 31, the Health and Human Services Secretary declared Coronavirus a Public Health Emergency in the US. (HHS.gov)2 We began hearing news of restrictions on flights from China, passengers affected on Princess Cruise ships and outbreaks at a long term care facility in Washington State. 

As a Medical laboratory Scientist, I became concerned with this virus early on, and started watching statistics. I was concerned not only for the health of my family, friends and coworkers, but also for the health or our laboratories and our blood supply.

The first journal articles I read about COVID-19 and blood safety were published in Transfusion Medicine Reviews on Feb 21, 2020. In the very early days of this novel coronavirus, researchers in China reviewed publications about SARS and MERS to help give us a better understanding of SARS-CoV-2, the virus that causes our current pandemic of COVID-19. When discussing blood safety, one of the first things to consider is if the virus is transmittable via blood transfusions. If the virus is transmittable, we also must consider if there is an asymptomatic time when there is virus in the blood. One review stated that SARS, MERS and SARS-CoV-2 can all be found in the serum or plasma, but, at the time of this review, it was still uncertain if SARS-CoV-2 could be transmitted from those with pre-symptomatic or asymptomatic infections.3

March 18, 2020

On March 18, Blood Transfusion published an article written by a group at several Blood Centers in a few provinces in China. This article discussed efforts to minimize the impact of blood shortages due to COVID-19. It was noted that the rising pandemic had had a profound impact on the number of blood donations, and on blood safety. Because it was now recognized that there is a long incubation period and a significant number of asymptomatic cases, this posed a huge challenge in recruiting blood donors. In China, strictly restricted mobility led to a decrease in donations across the country. Donors were recruited through various methods, including the use of social media. Social distancing during blood donations and thorough cleaning and disinfecting of donor areas were enforced. Screening procedures were enhanced to include temporary isolation of blood products for 14 days after collection and delaying release for clinical use. At the same time, donors were followed up until the expiration of the products. If a donor was found to test positive for COVID-19 after donation, the blood products were recalled. These new protocols in place were helping to insure adequate donations and the safety of blood products. ne interesting note is that this article referred to the epidemic as “effectively controlled” and that “normal medical services had been resumed”.4

Meanwhile, in the US, American Red Cross was pleading for blood donors. On March 17 it was reported that 2,700 mobile blood drives had been cancelled at a loss of 86,000 units of blood potentially collected. On March 21, 4 days later, that number had risen to more than 5,000 blood drives canceled at a loss of 170,000 units. As more schools, workplaces, churches and college campuses closed down in response to the pandemic, those institutions had to cancel their blood drives. Social distancing guidelines and shelter in place orders resulted in fewer people donating blood. In addition, an FDA mandate from February, that people who had traveled to areas with COVID-19 outbreaks should wait at least 28 days before donating blood, most likely contributed to the shortage. Dr. Justin Kreuter, from the Mayo Clinic Blood Donor Center, stated that the blood shortage was not due to more COVID-19 patients needing blood products. Rather, “it’s a lack of donations coming in.”5

April 1, 2020

procedures that the Chinese had instated. Mobile blood drives were shut down, but collection centers remained open. TV commercials, radio ads, You Tube videos and social media called for blood donors, assuring them that this was essential and that donating blood was safe. Donations were arranged through appointments only, and potential donors contacted and verbally screened for symptoms and risk factors before appearing to donate. On arrival at the centers, temperatures were taken and travel and symptoms questions were asked before a donor was allowed to enter the center. The use of masks and social distancing, along with extra cleaning and donor chair decontamination between donors were all implemented.

In an effort to open up the pool of potential donors, the FDA reviewed current studies and epidemiological data and concluded that certain donor eligibility criteria could be modified without compromising the safety of the blood supply. On April 2, 2020 the FDA approved several important changes in donor qualifications. These revisions included the following:

  • For male donors deferred for having sex with another male: the recommended deferral period changed from 12 months to 3 months.
  • For female donors deferred for having sex with a man who had sex with another man: the recommended deferral period changed from 12 months to 3 months
  • The deferral period for recent tattoos and piercing was changed from 12 months to 3 months
  • For people who have traveled to malaria-endemic areas, the recommended deferral period was changed from 12 months to 3 months. In addition, the guidance notes that deferral can be waived for these donors, provided the blood components are pathogen-reduced using an FDA-approved pathogen reduction device.
  • For donors who spent time in European countries or on military bases in Europe who were previously deferred due to potential risk of transmission of Creutzfeldt-Jakob Disease or Variant Creutzfeldt-Jakob Disease, the FDA has eliminated the deferrals and these individuals may now qualify to donate.6

Despite loosening requirements, advertising, and calls from the blood centers for additional donors, the shortages remained. To address the decline in blood product availability, it became essential to review the principles of patient blood management (PBM). PBM is defined as “the timely application of evidence-based medical and surgical concepts designed to maintain hemoglobin concentration, optimize hemostasis and minimize blood loss in an effort to improve patient outcome.”7 Firstly, elective procedures were put on hold, thus freeing up units for the most needy patients. Despite this, many blood banks still had their standing orders decreased. In many cases, Blood bank Medical Directors approved changes in transfusion triggers. At the hospital where I work, the transfusion trigger was changed from a hemoglobin of 8g/dL to 7 g/dL. New changes of SOP were approved to issue to all patients, except females of child bearing age, Rh positive units instead of more scarce Rh negative units. We also have a large NICU unit and baby units were not available from ARC, so we were using the newest units available, when necessary for these patients.

By April 8, 15,000 blood drives had been cancelled across the US, at a potential loss of almost 500,000 donated units. One technologist reported in an online Blood bank professionals group, that “Our supplier downgraded us in terms of standard inventory (about 40%), but our transfusion numbers have dropped at least as much.”8 With the decrease in usage and the careful patient blood management, blood needs were met.

May 12, 2020

AABB began sending out a weekly COVID impact survey for hospital transfusion services survey in late March. Many questions on the survey, and the resulting charts and graphs, are related to COVID convalescent plasma practices and procedures (details in my next blog!), but one important graph produced by this survey shows the increase in inventory wastage due to changes related to COVID-19. These changes due to COVID-19 can be a decrease in patients and elective surgeries or changes in transfusion protocols. In early April, in the first few weeks of the survey, 25%-28% of hospitals responding reported an increase in inventory wastage. This corresponds to when donors started coming back to donate, and usage dropped. This percent of hospitals reporting wastage increased each week until the week of May 4-7 when 54% of hospitals reported inventory wastage. This may be due to several factors. The units collected at the end of March and early April, have reached their 42 day expirations. Donors came out initially in response to the call for blood, but now, these units have expired, and it has not yet been 56 days when these donors can donate again. Usage also decreased during this time. COVID patients have not generally had heavy use of red cells, in particular, and doctors have been very conservative in usage with all patients. For the week of May 11-14, as more hospitals are planning to resume elective surgeries, and for the first time in the 8 weeks, fewer hospitals (52.0%) reported an increase in wastage due to changes related to the pandemic. Of the 100 respondents, 59% reported they are resuming “some” elective surgeries before mid-May and 28.0% are doing so after mid-May.9

What does this mean for the future of our blood supply during this pandemic? On May 12, a group of Blood bank professionals, when asked in an informal online survey, had had varying answers. These were likely dependent on location, both geographic and city vs. rural, and size of the hospital. One comment was that “We have gone from huge shortages to throwing away massive units not being used. Hospital is empty.” Another tech said “We were way overstocked a week ago, now we’re dipping way below average.” Technologists in Florida, Oregon and Pennsylvania reported low inventory. Techs in Ohio and Maryland reported their inventory to be very healthy. But these reports could easily vary between areas of the individual state, and even different hospitals in the same city. Another technologist commented “We had a mass of donors when this all started and now all those units are expiring!” The shortage of donors will likely continue, but may relax a bit with some states beginning to lift restrictions. We likely won’t see a huge drove of donors, all at once, which is actually good because it will spread out expiration dates. But, though things may be opening up, it is unlikely that we will see blood drives at schools, workplaces and churches for some time, and this is a huge source of our countries blood supply.

We have seen a big swing in both inventories and usage. After elective and with surgeries have been put on hold for months, we may see an increase over the typical number of elective surgeries, which will mean we will see an increase in blood usage, and with a lack of donors, inventories may drop again.

As far as blood safety, we know now that SARS-CoV-2 did not follow the path of SARS and MERS. We know that it can definitely be transmitted from person to person, and can be transmitted by people who are asymptomatic. But, we also know that, in general, respiratory viruses are not known to be transmitted by blood transfusion. So, from what we know at this time, it is likely not necessary to routinely screen blood products for SARS-CoV-2, and not necessary to isolate blood products after collection and delay release of the products. It is recommended that blood centers encourage self-deferral for donors who have traveled to a COVID-19 affected area or been in contact with an infected person in the past 14 days and to screen donors carefully for fever and respiratory symptoms. With these practices in place, we can ensure an adequate and safe blood supply. We will continue to see swings in volumes, but with careful patient blood management, we will ride these waves and come out on top. Thanks to all our wonderful Blood Bank Technologists who are helping manage our country’s blood supplies!

References

  1. First Travel-related Case of 2019 Novel Coronavirus Detected in United States – CDC, January 21, 2020
  2. https://www.hhs.gov/about/news/2020/01/31/secretary-azar-declares-public-health-emergency-us-2019-novel-coronavirus.html
  3. L. Chang, Y. Yan, L. Wang Coronavirus disease 2019: coronaviruses and blood safety. Transfus Med Rev (2020)
  4. Xiaohong, et al. Blood transfusion during the COVID-19 outbreak, Blood Transfusion (2020)
  5. https://newsnetwork.mayoclinic.org/discussion/critical-blood-shortages-because-of-covid-19/
  6. https://www.fda.gov/media/92490/download
  7. http://sabm.org
  8. Facebook Blood Bank professionals page, May 12, 2020
  9. http://www.aabb.org/advocacy/regulatorygovernment/Documents/AABB-COVID-19-Impact-Survey-Snapshot.pdf

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Laboratory Safety and COVID-19: References You Need to Know

Three months ago, life in the laboratories in these United States carried on as usual, and no one could probably have predicted where we stand today. The COVID-19 pandemic has changed the way laboratorians work everywhere. Some staff have had hours cut because of decreased workloads, other labs worked around the clock to bring new testing on board, and others dealt with staffing shortages due to illness. It has been a wild ride, and through it all, a great many safety issues have arisen. Common lab practices are now viewed through a new lens- is it acceptable to bring hematology slides for review into a clean pathologist’s office? Can we wear surgical masks worn in the lab into the break room? There are many good questions, but some of the answers can be found using references offered from reliable sources. Not everything you read online can be believed, but here are some references that may be necessary and that provide important information.

The pandemic has created a world-wide shortage of PPE, and some have wondered what can be done as resources diminish. The CDC has some good information about calculating how long PPE can be used and how long it can last. There are good guidelines about re-use and extended use of PPE.

PPE Burn Rate Calculator:

https://www.cdc.gov/coronavirus/2019-ncov/hcp/ppe-strategy/burn-calculator.html

Strategies to Optimize the Supply of PPE and Equipment:

https://www.cdc.gov/coronavirus/2019-ncov/hcp/ppe-strategy/index.html

There are specific references regarding respirators and how they should be used.

Respiratory Protection During Outbreaks: Respirators versus Surgical Masks

Understanding the Use of Imported Non-NIOSH-Approved Respirators

Proper N95 Respirator Use for Respiratory Protection Preparedness

Some laboratory disinfectants have become more difficult to purchase. The gold standard for disinfection remains a 10% bleach solution, but there are many other options that can be used as well.

Disinfectants for Use Against SARS-CoV-2 (EPA List N):

https://www.epa.gov/pesticide-registration/list-n-disinfectants-use-against-sars-cov-2

EPA’s Registered Antimicrobial Products Effective Against Human HIV-1 and Hepatitis B Virus:

https://www.epa.gov/pesticide-registration/list-d-epas-registered-antimicrobial-products-effective-against-human-hiv-1

The CDC also offers laboratories a set of COVID-19 guidelines for performing testing, biosafety issues, waste management, and protection against aerosols. These guidelines are thorough, and they can be very helpful should safety challenges arise.

Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19):

https://www.cdc.gov/coronavirus/2019-nCoV/lab/lab-biosafety-guidelines.html

Many of these references are updated regularly, so be sure you go to go to the source when making safety policy about COVID-19 tasks.

Laboratorians are now literally on the front lines during this novel coronavirus pandemic. While many public and commercial services have been scaled back, restaurants are closing, and many people are staying or working at home, lab staff are doing their level best to keep coming to work despite the extremely unusual circumstances and hardships.

I am here to serve as well. If you have questions about how to safely navigate this national (and global) emergency while working in the lab, ask me (info@danthelabsafetyman.com). I will do my best to provide any lab safety resources you may need. Make sure the decisions you make during these days are safe, sound, and based on the most recent resources available to you.

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.

Extraction-free and Saliva COVID-19 Testing

Much has changed quickly with SARS-CoV-2 virus (COVID-19) testing. Several commercial options are now available. Labs have less problems getting control material (positive samples are no longer in short supply). And labs that opted to bring on testing are now running multiple versions of COVID-19 molecular tests with a combination of high speed platforms or high throughput. Rapid cartridge tests are used for clearing people from the ED/ removing contact isolation on inpatients while the high throughput assays are used for routine screening.

However, several bottlenecks still exist. There are shortages of nucleic acid extraction kits, collection swabs and viral transport media. Fortunately, some recent studies have demonstrated preliminary evidence for using alternative sample types, collection methods, and storage conditions.

One of the first tenets of molecular diagnostics is isolation and purification of nucleic acid. Therefore, it was surprising to see a report on an extraction-free COVID-19 protocol from Vermont (Bruce EA et al.). This study initially analyzed two patient samples and showed drops in sensitivity of ~4Ct cycles. While this would not be suitable for low level detection, many viral samples have high levels of virus that still would permit detection. The team went on to test this method on 150 positive specimens from the University of Washington and found 92% sensitivity with 35% sensitivity at the low viral load range (Ct value> 30). This was improved with a brief heat inactivation step (Table 1). This was similarly seen in a study from Denmark, where brief heat inactivation of extraction-free methods (Direct) had 97% specificity in 87 specimens (Table 2).

Table 1. Vermont study comparing sensitivity of direct RT-PCR (no extraction step) with the validated results of 150 specimens coming from the University of Washington.
Table 2. Denmark study found extraction-free protocols (Direct) were comparable to extracted RNA (MagNA Pure extraction method) detection in 87 specimens.

Some similar studies out of Chile also showed extraction-free protocols on a larger number of specimens, and they reported a loss in sensitivity varying from 1-7 Ct cycles depending on the primers used.

Figure 1. P1 and P2 are patient 1 and 2. NSS indicates a nasal swab sample where RNA was extracted. RNA indicates a sample with no RNA extraction.

As this novel Coronavirus has an RNA-based genome, RNA is the target of molecular tests. As RNA is susceptible to degradation, there have been concerns over sample storage. Should it be refrigerated? Frozen? How do multiple freeze-thaw cycles impact specimen stability? Are there viable alternatives to viral transport media? One preliminary study explored these questions very nicely. They took X multiple sample types (NP, BAL, saline storage media) and stored them at 20C, 4C, -20, and -70 for multiple days up to 1 week and then analyzed the level of virus detected. In each case, the loss in sensitivity was minimal (<2 Ct cycles from day 0 to day 7) at room temperature with comparable results at lower temperatures (Table 3).

Table 3. Stability of SARS-CoV-2 RNA detected by the Quest EUA rRT-PCR. VCM- viral culture media; UTM-R Copan’s transport medium; M4-microtest media; BAL- bronchoalveolar lavage.

Lastly, alternative sample types such as saliva will help break the bottleneck in swabs and viral transport media. I was surprised to hear about this being a suitable alternative. Having worked with saliva for DNA analysis, I know it can be contaminated, of variable quantity, includes digestive enzymes and is viscous (slimy). These are not characteristics a lab would look for in a specimen type being used for high-throughput testing where several sample failures could occur. But these researchers from Yale showed measurable levels of SARS-CoV-2 that facilitated even higher sensitivity than nasopharyngeal swabs (Wylie AL et al).

Figure 2. SARS-CoV-2 titers are higher in the saliva than nasopharyngeal swabs from hospital inpatients. (a) All positive nasopharyngeal swabs (n = 46) and saliva samples (n = 39) were compared by a Mann-Whitney test (p < 0.05). Bars represent the median and 95% CI. Our assay detection limits for SARS-CoV-2 using the US CDC “N1” assay is at cycle threshold 38, which corresponds to 5,610 virus copies/mL of sample (shown as dotted line and grey area). (b) Patient matched samples (n = 38), represented by the connecting lines, were compared by a Wilcoxon test (p < 0.05). (c) Patient matched samples (n = 38) are also represented on a scatter plot.

With a much-needed increase in testing for this country, optimizations need to be implemented to improve efficiency. These steps alone will not be enough, but if we can have extraction-free testing of saliva collected at home, this would provide a substantial benefit to bringing easy testing to everyone.

UPDATE: Since this was written, the first FDA EUA was authorized for an at-home saliva collection kit for use at the Rutger’s clinical genomics lab (https://www.fda.gov/media/137773/download).

References

Please note: many of these references were on pre-print servers and have not been peer-reviewed.

  1. Bruce EA, Huang ML, Perchetti GA, et al. DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP. 2020. https://www.biorxiv.org/content/10.1101/2020.03.20.001008v2.full#T2
  2. Wyllie AL, Fournier J, Casanovas-Massana A, Campbell M et al. Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. medRxiv 2020. https://www.medrxiv.org/content/10.1101/2020.04.16.20067835v1#disqus_thread
  3. Fomsgaard AS, Rosentierne MW. An alternative workflow for molecular detection of SARS-CoV-2 – escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020. https://www.medrxiv.org/content/10.1101/2020.03.27.20044495v1.full.pdf
  4. Rogers AA, Baumann RE, Borillo GA, et al. Evaluation of Transport Media and Specimen Transport Conditions for the Detection of SARS-CoV-2 2 Using Real Time Reverse Transcription PCR. JCM 2020.
  5. Beltran-Pavez C, Marquez CL, Munoz G et al. SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction. bioRxiv 2020. https://www.biorxiv.org/content/10.1101/2020.03.28.013508v1.full.pdf

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.