<![CDATA[Lab Best Practice - Archives]]> https://health.ucdavis.edu/lab-best-practice en-US hourly 1 https://wordpress.org/?v=4.9.3 <![CDATA[Reconstituted Whole Blood Transfusion Exchange in Neonatal Hyperbilirubinemia]]> https://health.ucdavis.edu/blog/lab-best-practice/reconstituted-whole-blood-transfusion-exchange-in-neonatal-hyperbilirubinemia/2021/09 Wed, 15 Sep 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/reconstituted-whole-blood-transfusion-exchange-in-neonatal-hyperbilirubinemia/2021/09 Alison Chan, D.O., Grace Fortes Monis, M.D., Ph.D.

The blood bank occasionally will receive requests for a transfusion exchange in a neonate with hyperbilirubinemia. Neonates with hyperbilirubinemia may necessitate hospital admissions with approximately 5-10% of all neonates needing medical intervention. Neonatal hyperbilirubinemia can be due to unconjugated or conjugated bilirubin, with the former being more important for early detection and treatment to prevent serious complications such as bilirubin-induced encephalopathy. Unconjugated bilirubin is insoluble in the body and is tightly bound to serum albumin. There is a small fraction of unbound unconjugated bilirubin that can diffuse into tissues such as the brain leading to toxicity. Conjugated bilirubin is nontoxic as it is water soluble and can be easily excreted from the body. Common causes of unconjugated hyperbilirubinemia include hemolytic disease of the newborn, G6PD deficiency, resorption of a cephalohematoma, breast-feeding jaundice, breast-milk jaundice, Gilbert syndrome, and many more. Phototherapy is commonly used to convert unconjugated bilirubin into the water soluble and excretable form and can reduce the need for exchange transfusions. In more severe cases and urgent cases of hyperbilirubinemia an exchange transfusion using reconstituted whole blood can be considered.

Reconstituted whole blood, also known as reconstituted red blood cells, refer to the combination of red blood cells and plasma to achieve a specific volume of a targeted hematocrit. The literature suggests that the use of reconstituted whole blood for the exchange transfusion is immunologically safer and better than using whole blood. The benefits of an exchange transfusion include removal of serum unconjugated (indirect) bilirubin, possible circulating mother antibodies, and any antibody-coated neonate red cells from the circulation. Other benefits of using reconstituted whole blood include providing compatible red cells to correct the associated anemia and albumin which can bind additional bilirubin to further decrease the serum levels of indirect bilirubin. Reconstituted whole blood can also be used in exchange transfusions of neonates with severe anemia.

At UC Davis Medical Center, the decision to perform an exchange transfusion is determined by the clinical team. Although there is no cutoff at which the total bilirubin levels must reach to consider an exchange transfusion, the clinical team considers factors such as risk factors for development of severe hyperbilirubinemia, how quickly they need to remove the bilirubin, whether the infant is symptomatic with moderate or advanced clinical signs of bilirubin-induced neurologic dysfunction, and whether intensive phototherapy has failed, when making their decision. Tools that can be used to help determine the appropriate medical intervention for treating unconjugated bilirubinemia include the BIND score and the newborn hyperbilirubinemia assessment calculator. The BIND score determines the presence and severity of acute bilirubin encephalopathy in infants with no other identifiable etiologies for neurologic dysfunction. The newborn hyperbilirubinemia assessment calculator uses the total bilirubin level and presence of additional risk factors to stratify the infant into different groups and their recommended course of treatment.

Once the request for a reconstituted whole blood exchange transfusion is made, the transfusion medicine team is notified and at which they work with the clinical team to coordinate timing of the procedure. As part of the work flow, a type and screen and direct antiglobulin test need to be done on the neonate’s and mother’s blood to help select the appropriate red cell unit for reconstitution. It is important to have the mother’s type and screen so that a red cell unit lacking the antigens to any possible antibodies that the mother may have passively transferre

Key Points

  • The clinical team makes the ultimate decision to perform a reconstituted whole blood exchange transfusion.
  • The entire workflow can take up to 3 hours to prepare the reconstituted whole blood unit.
  • The reconstituted unit will expire 24 hours from the date and time that the unit of RBCs is washed.

References

  • Epomedicine. Neonatal Jaundice (NNJ) : Approach [Internet]. Epomedicine; 2015 Sep 2 [cited 2021 Aug 18].
  • Gharehbaghi MM, Hosseinpour SS. Exchange transfusion in neonatal hyperbilirubinaemia: a comparison between citrated whole blood and reconstituted blood. Singapore Med J. 2010;51(8):641-644.
  • Kakkar B, Agrawal S, Chowdhry M, Muthukumaravel PJ, Makroo RN, Thakur UK. Exchange transfusion in neonatal hyperbilirubinemia: A single Centre experience from Northern India. Transfus Apher Sci. 2019;58(6):102655. doi:10.1016/j.transci.2019.09.008
  • Olusanya BO, Imam ZO, Emokpae AA, Iskander IF. Revisiting the Criteria for Exchange Transfusion for Severe Neonatal Hyperbilirubinemia in Resource-Limited Settings. Neonatology. 2016;109(2):97-104. doi:10.1159/000441324
  • Sharma D, Rai S, Iyengar S, Jain B, Sao S, Gaur A, Sapra R. Efficacy of Whole Blood Reconstituted (WBR) in Exchange Transfusion (ET) in Hemolytic Disease of New Born (HDN) —A Study of 110 Cases. Open Journal of Blood Diseases, Vol. 3 No. 1, 2013, pp. 15-20. doi: 10.4236/ojbd.2013.31004.
  • These ND. Chapter 18 Liver and gall bladder. In: Kumar V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Philadelphia, PA: Elsevier; 2015: 852-852.
  • Wong RJ, Bhutani VK. Unconjugated hyperbilirubinemia in term and late preterm infants: management. Uptodate. Updated 12/9/2019. Accessed 9/2/2021.
  • Wong RJ, Bhutani VK. Unconjugated hyperbilirubinemia in the newborn: interventions. Uptodate. Updated 8/21/2020. Accessed 9/2/2021.
]]> 0 <![CDATA[Comparison of SARS-CoV-2 Specimen Types: Nasal Swabs, Nasopharyngeal Swabs and Beyond]]> https://health.ucdavis.edu/blog/lab-best-practice/comparison-of-sars-cov-2-specimen-types-nasal-swabs-nasopharyngeal-swabs-and-beyond/2021/09 Thu, 09 Sep 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/comparison-of-sars-cov-2-specimen-types-nasal-swabs-nasopharyngeal-swabs-and-beyond/2021/09 Nam Tran, Ph.D.; Samer Albahra, M.D.

INTRODUCTION

The nasopharyngeal (NP) swab serves as the primary specimen type for respiratory molecular pathogen detection.1 During the novel coronavirus infectious disease (COVID) pandemic of 2019, the NP swab became the centerpiece for detecting the severe acute respiratory syndrome – coronavirus – 2 (SARS-CoV-2). Unfortunately, NP swab collection can be extremely uncomfortable for patients. Combined with swab shortages observed early in the pandemic, many facilities pursued alternate specimen types such as saliva.2 Now, over a year later, as supply chains improve, the use of oropharyngeal (OP), mid-turbinate (MT) and anterior nares (AN) swab are now commonly used for COVID-19 testing.

LABORATORY BEST PRACTICE

The Infectious Disease Society of America (IDSA) recommends the use of NP swab, MT swab, AN swab, saliva or a combined AN/OP swab rather than an OP swab alone for SARS-CoV-2 RNA testing in symptomatic individuals suspected of having COVID-19.1 This recommendation comes with several caveats. Studies show that NP, MT, and AN swabs are comparable. Other studies suggest MT and AN may lose some sensitivity compared to NP specimens, with AN having a relative sensitivity ranging from 82 to 88%.3 AN achieves the highest concordance with NP when viral load is >1,000 RNA copies/mL4. Saliva specimens also exhibits good performance; however, this complex specimen type may create more variability due to inconsistent production of saliva by patients, and the potential for variable viscosity due to hydration status or other factors.5 Oropharyngeal swab, despite being more tolerated by patients, are the least desirable since data suggests this specimen type exhibits a higher false negative rate.1 To this end, UC Davis Health has approved only NP and AN swabs for SARS-CoV-2 polymerase chain reaction (PCR) testing at this time. The AN swab option provides a less invasive alternative for collection, with NP swab still being recommended. Saliva and MT swab samples are not approved at our institution.

PRE-ANALYTIC FACTORS IMPACTING COVID-19 TEST PERFORMANCE

In addition to the specimen type, it is worth discussing pre-analytic factors influencing SARS-CoV-2 testing performance.6 Specifically, the majority of factors impacting SARS-CoV-2 detection occurs before the test itself. Factors such as:

  • Specimen collection quality: How well a healthcare professional collects any swab sample influences the amount of SARS-CoV-2 for testing. Even with a perfectly sensitive and specific PCR test, the lack of SARS-CoV-2 RNA on a swab results in a negative result regardless of a patient’s COVID-19 status.
  • Patient viral load at the time of collection: The SARS-CoV-2 virus also has a part to play in test performance. Viral load may vary over time as the infection progresses and perhaps favor one compartment in the body over another.4,7 Symptom and vaccination status may also influence the viral load. Early COVID-19 studies suggest viral load from the NP region may vary back and forth from a high to low viral load state over the course of an intensive care unit stay. Serial swabbing of patients may also impact detection. Collection intervals of <24 hours may result in false negative results on subsequent specimens due to the virus having insufficient time to repopulate the collection site. Figure 1 shows internal data at UC Davis Health evaluating the SARS-CoV-2 viral RNA load between paired NP versus AN swabs. There is a statistically significant reduction in mean viral load for AN swabs compared to NP specimens.
Figure 1. Comparison of Viral Load for NP versus AN Swabs via Quantitative Digital Droplet RT-PCR
Figure 1. Comparison of Viral Load for NP versus AN Swabs via Quantitative Digital Droplet RT-PCR.
Viral Load of paired NP (blue) versus AN (yellow) swab samples based on targeting of two regions (N1 and N2) within the SARS-CoV-2 nucleocapsid gene. The test is validated for investigational use only. The limit of detection of this assay has been determined to be 600 copies/mL based on the FDA reference panel.
  • Specimen matrix: Sample viscosity impacts the performance of common SARS-CoV-2 tests.5,6 For example, many PCR tests require pipetting steps which may be automated via robotics or performed manually. High viscosity could prevent accurate pipetting and compromise test performance. Viscosity issues are often observed in saliva and lower respiratory tract samples. Additives may be used to reduce viscosity but at the price of further diluting a sample and reducing the viral load for testing.
  • Presence of interfering or diluting substances: Patients may use nasal medications or other compounds which may directly interfere with SARS-CoV-2 assays.6 These compounds may also potentially dilute the sample to reduce viral load below an assay’s detection limit.
  • Specimen transportation conditions: Samples should be transported correct media which contains compounds to limit degradation of viral nucleic acids and inhibit growth of contaminating bacteria.4,6 Transport on ice is also recommended to further minimize specimen degradation. Delays in transportation may, again, impact specimen quality prior to testing.

SUMMARY

At UC Davis Health, NP and AN swab samples are acceptable specimen types for SARS-CoV-2 RNA testing by PCR across all platforms. The AN swab is less invasive for patients but may exhibit lower sensitivity compared to the NP swab. Many other factors influence SARS-CoV-2 test sensitivity including sample quality, presence of interfering substances, and delayed/inappropriate transport conditions. It is important for healthcare providers to be aware of these factors and weigh the pros and cons between NP versus AN swabs.

REFERENCES

  1. IDSA Guidelines on the Diagnosis of COVID-19: Molecular Diagnostic Testing (Published 12/23/2020): Accessed on August 24, 2021.
  2. National Public Radio website: Accessed on August 24, 2021.
  3. Zhou Y, O’Leary TJ. Relative sensitivity of anterior nares and nasopharyngeal swabs for initial detection of SARS-CoV-2 in ambulatory patients: Rapid review and meta-analysis. PLOS One 2021.
  4. Callahan C, Lee RA, Lee GR, et al. Nasal swab performance by collection timing, procedure, and method of transport for patients with SAR-CoV-2. J Clin Microbiol 2021;59:e00569-21.
  5. Landry ML, Criscuolo J, Peaper DR. Challenges in the use of saliva for detection of SARS-CoV-2 RNA in symptomatic outpatients. J Clin Virol 2020;130:104567.
  6. Vandenberg O, Martiny D, Rochas O, et al. Considerations for diagnostic COVID-19 tests. Nat Rev Microbiol 2021;19:171-183.
  7. Lim AY, Cheong HK, Oh YJ, et al. Modeling the early temporal dynamics of viral load in respiratory tract specimens of COVID-19 patients in Incheon, the Republic of Korea. Int J Infect Dis 2021;108:428-434.
]]> 0 <![CDATA[Role of Quantitative Serology Testing for COVID-19]]> https://health.ucdavis.edu/blog/lab-best-practice/role-of-quantitative-serology-testing-for-covid-19/2021/08 Mon, 23 Aug 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/role-of-quantitative-serology-testing-for-covid-19/2021/08 Ryan Thomas, MD; Samer Albahra, MD; Scott Bainbridge, CLS; Nam K Tran, PhD

Introduction

The current pandemic has led to considerable interest in COVID-19 related laboratory testing. The widespread distribution of the various vaccines, the emerging genetic variants of SARS-CoV-2 circulating around the world, and their impact on testing efficacy, accuracy, and interpretation are all prominent topics. Multiple assays using various methodologies have been developed and implemented in the clinical diagnostic laboratory setting. At UC Davis Health we primarily utilize molecular methods, namely reverse transcriptase (RT) polymerase chain reaction (PCR) assays, for detection of the virus but point of care testing as well as serologic testing is available [1]. Serologic assays—particularly quantitative serologic tests—are an increasingly significant topic in the assessment of vaccination response and risk of breakthrough infections. Until recently, the serologic tests available at our institution were qualitative assays [1]. The goal of this blog article is to discuss a semi-quantitative serology test recently implemented at UC Davis Health. Information presented below is current at the time of posting this article.

Viral Structural Proteins

SARS-CoV-2 has four structural proteins, nucleocapsid (N), spike (S), membrane (M) and envelope (E) (figure 1) [1,2,3,4]. The S protein consists of two subunits, S1 which contains the Receptor Binding Domain (RBD, the target of neutralizing antibodies) and S2. The nucleocapsid and spike proteins are considered the main immunogens and are widely used in the development of serologic tests or immunoassays [2,3,4].

SARS-CoV-2 Structure and Molecular Targets
Figure 1. SARS-CoV-2 Structure and Molecular Targets. Common SARS-CoV-2 virus antigenic targets include spike, envelope, and nucleocapsid proteins [1].

Serologic Testing

Serology testing measures the host antibody response in the form of immunoglobulins (Ig) such as IgM, IgA, or IgG following infection and/or vaccination. IgM production begins as early as three days post-infection with IgG appearing as early as seven days post-infection. By day 14, the majority of individuals should have a detectable antibody response. False negative serology results may occur if testing is performed too soon after exposure where antibody production is non-detectable (“window period”) [1].

Anti-nucleocapsid serology tests can be used in settings where prior infection with SARS-CoV-2 is suspected [2,3,4]. The nucleocapsid protein is relatively conserved across the various coronaviruses (including non-SARS-CoV-2), making the anti-nucleocapsid serology assays less specific (and prone to false positive results) than their anti-spike assay counterparts. The anti-spike serology assays detect antibodies developed after prior infection but can also detect antibodies developed after vaccination [2,3,4]. However, with the emergence of genetic variants of SARS-CoV-2, concern has arisen over the ability of these assays to detect antibodies to viral variants.

The previous UC Davis serology assay was the LIAISON® SARS-CoV-2 S1/S2 IgG assay, which detected IgG antibodies using a recombinant-expressed spike protein that encompassed both the S1 and S2 domains of the spike protein [1,5]. The S1 subunit is unique in that it also contains the RBD, therefore antibodies against this region are most likely to serve as effective neutralizing antibodies. Studies suggest the LIAISON® SARS-CoV-2 S1/S2 IgG assay correlated well with neutralizing antibody testing [6]. This was a qualitative assay developed for detection of antibodies to SARS-CoV-2 in human serum and plasma. It was not developed with the intention of quantifying the antibodies present [5].

The LIAISON® SARS-CoV-2 TrimericS IgG assay is the 2nd generation of Diasorin’s immunoassay, developed for the semi-quantitative determination of IgG antibodies to the SARS-CoV-2 spike protein [7,8]. The assay uses the complete recombinant-expressed trimeric spike protein. The use of these larger antigen, as opposed to shorter fragments such as those in the previous assay or the commonly used RBD region, provides a larger number of epitopes to capture antibodies, making the assay more sensitive [7,8]. This is useful in the setting of spread of new viral variants, where there was concern for markedly reduced antibody detection in the setting of the variant’s mutations. The assay exhibits a sensitivity of 99.7% and specificity of 99% [8].

The assay is intended as an aid in the diagnosis of COVID-19, but because of its semi-quantitative nature it also has theoretical utility in the study of the immune status of infected and/or vaccinated patients by providing a quantification of IgG antibodies against SARS-CoV-2. In the setting of vaccinated individuals, quantitative serologic testing could offer insight as to who is more susceptible to breakthrough infection after vaccination—an uncommon but important occurrence [9]. These benefits are theoretical however, and the clinical applicability of a semiquantitative result is currently unknown [7].

Results of the LIAISON SARS-CoV-2 TrimericS IgG assay will be reported as a quantitative result in arbitrary units per milliliter (AU/mL) along with a Positive or Negative result. The cutoff for a positive result in our lab is 13 AU/mL, as compared to the cutoff of 15 AU/mL on the S1/S2 IgG assay. The limitations of this assay are similar to those of other immunoassays. Positive SARS-CoV-2 serology results alone are not diagnostic, and as alluded to previously they do not definitively indicate that a patient has had prior SARS-CoV-2 infection [1]. Additionally, not all antibodies produced against a virus, such as SARS-CoV-2, confer immunity [1].

References

  1. Tran, N., Cohen, S., Waldman, S., & May, L. (2021). Review of COVID-19 Testing Methods. Retrieved 22 August 2021.
  2. Brochot, E., Demey, B., Touzé, A., Belouzard, S., Dubuisson, J., & Schmit, J. et al. (2020). Anti-spike, Anti-nucleocapsid and Neutralizing Antibodies in SARS-CoV-2 Inpatients and Asymptomatic Individuals. Frontiers In Microbiology, 11. doi: 10.3389/fmicb.2020.584251
  3. MacDonald, A. (2021). COVID-19 Antibody Testing: S vs. N Protein. Retrieved 22 August 2021.
  4. Rikhtegaran Tehrani, Z., Saadat, S., Saleh, E., Ouyang, X., Constantine, N., & DeVico, A. et al. (2020). Performance of nucleocapsid and spike-based SARS-CoV-2 serologic assays. PLOS ONE, 15(11), e0237828. doi: 10.1371/journal.pone.0237828
  5. FACT SHEET FOR HEALTHCARE PROVIDERS: DiaSorin Inc. LIAISON® SARS CoV-2 S1/S2 IgG assay. (2021). Retrieved 23 August 2021.
  6. Muecksch F, Wise H, Batchelor B, et al. Longitudinal serological analysis and neutralizing antibody levels in coronavirus disease 2019 convalescent patients. J Infect Dis 2021;233:389-398
  7. FACT SHEET FOR HEALTHCARE PROVIDERS: DiaSorin Inc. LIAISON SARS-CoV-2 TrimericS IgG. (2021). Retrieved 23 August 2021.
  8. Bonelli, F., Blocki, F., Bunnell, T., Chu, E., De La O, A., & Grenache, D. et al. (2021). Evaluation of the automated LIAISON® SARS-CoV-2 TrimericS IgG assay for the detection of circulating antibodies. Clinical Chemistry And Laboratory Medicine (CCLM), 59(8), 1463-1467. doi: 10.1515/cclm-2021-0023.
  9. Bergwerk, M., Gonen, T., Lustig, Y., Amit, S., Lipsitch, M., & Cohen, C. et al. (2021). Covid-19 Breakthrough Infections in Vaccinated Health Care Workers. New England Journal Of Medicine. doi: 10.1056/nejmoa2109072
]]> 0 <![CDATA[An Overview of Prosthetic Joint Infection (PJI) Definition and Diagnosis]]> https://health.ucdavis.edu/blog/lab-best-practice/an-overview-of-prosthetic-joint-infection-pji-definition-and-diagnosis/2021/07 Tue, 13 Jul 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/an-overview-of-prosthetic-joint-infection-pji-definition-and-diagnosis/2021/07 Jasper Zheng, M.D. and Anna Maria Romanelli, Ph.D.

The numbers of primary total hip and knee arthroplasties have been increasing over time, with 332,000 total hip and 719,000 total knee arthroplasties performed in 2010 in the United States. It is estimated that 3,481,000 primary total knee and 572,000 primary total hip arthroplasties will be performed annually in the US by 2030. With the increase in prosthetic joint implantations, serious complications of prosthetic joint infections (PJI) of the hip and knee is also on the rise.

The diagnosis of prosthetic joint infections (PJI) poses a significant challenge since real evidence-based guidelines to aid clinicians in choosing the most accurate diagnostic strategy are lacking. Despite a significant amount of basic and clinical research in this field, many questions pertaining to the definition of infection as well as diagnosis and management of these infections remains unanswered. Clinical practice guidelines for the diagnosis and management of PJI have been proposed by a number of organizations, including the 2021 European Bone and Joint Infection Society (EBJIS) criteria, the 2018 International Consensus Meeting (ICM) criteria, the 2013 ICM criteria, the 2013 Infectious Disease Society of America guidelines (IDSA), and the 2011 Infection Society (MSIS) criteria. These guidelines stress the importance of using a multi-disciplinary approach to aid in the diagnosis of PJI, requiring supporting evidence from clinical examination, laboratory results, microbiological culture identification, histological interpretation, and intraoperative findings. These guidelines are meant to serve as an educational tool designed to assist practitioners in providing appropriate care for patients. It is anticipated that consideration of these guidelines may help reduce morbidity, mortality and the costs associated with PJI.

In this Lab Best Practice Blog, we will review the criteria used to establish infection and explore the process of diagnosing prosthetic joint infection.

Risk Factors of Prosthetic Joint Infection

Patient-related risk factors for prosthetic joint infection include prior revision arthroplasty or prior same site prosthetic joint infection, tobacco use, obesity, rheumatoid arthritis, malignancy, immunosuppression and diabetes mellitus.

Postoperative risk factors include wound healing complications (such as superficial infection, hematoma, delayed wound healing, wound necrosis, or dehiscence), atrial fibrillation, myocardial infarction, urinary tract infection, prolonged hospital stay, and at any time postoperatively, Staphylococcus aureus bacteremia. It is important to consider all of these potential factors when assessing for risks of postoperative PJI.

Categorization of Prosthetic Joint Infection

The classification scheme useful for identification of PJI is simply based on the time to infection, classified as early, delayed, or late onset. Early onset PJI occurs less than three months after the last surgery. These infections are most commonly initiated at the time of operation, through intraoperative contamination, and are usually caused by relatively virulent microorganisms. Delayed onset PJI occurs after 3 months but before 12 or 24 months. Different authors have used different time points to differentiate between delayed and late onset PJIs. However, regardless of the cutoff used, the common theme is that these infections are also typically acquired at the time of surgery but are caused by less virulent microorganisms such that the overt presentation of infections does not occur within the first 3 months. Late onset PJI, usually occurs 12-24 months after surgery and is likely due to a hematogenous infection but may also be due to extremely indolent infection initiated at the time of surgery.

Pathogens involved in Prosthetic joint infection

Timing of infection can serve as a clue to ]]> 0 <![CDATA[Hepatitis B Serologic Testing Methods]]> https://health.ucdavis.edu/blog/lab-best-practice/hepatitis-b-serologic-testing-methods/2021/06 Thu, 10 Jun 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/hepatitis-b-serologic-testing-methods/2021/06 Luke Dang, M.D., Scott Bainbridge, C.L.S., Nam Tran, Ph.D.

Introduction

Hepatitis B virus (HBV) was first discovered in 1965. Briefly, HBV is a DNA virus from the Hepadnaviridae family which is spread via contaminated body fluids(1). After exposure, the virus enters hepatocytes and integrates its circular, partially double-stranded DNA genome (3.2 kb in size) to the host cell nucleus as a covalently closed circular DNA (cccDNA) intermediate, which acts as a stable nuclear template for viral replication(2). This mechanism enables the virus to chronically infect the host and reactivate at a later date (secondary to immunocompromise as in the case of transplantation, chemotherapy, immunosuppression or infection (as in the case of HIV)). Reverse transcription of the cccDNA then results in assembly and exocytosis of new viral particles. Hepatitis B exhibits geographic variation and is classified into 9 genotypes (A-I), with some differences in disease manifestations, although this remains an area of ongoing research(3).

Clinical Manifestations and Epidemiology

Hepatitis B virus infection exhibits a range of manifestations in the acute and chronic time frames(4). Acute infection can be sub-clinical or clinical in severity and can cause both icteric (~30%) or anicteric hepatitis (~70%). In a small subset of patients (~0.5%), acute infection can lead to fulminant hepatic failure secondary to immunologic lysis of the virus-infected hepatocytes. For chronically infected patients, long term exposure to viral antigens can lead to immune-mediated liver injury as well as direct viral cytotoxicity. This ongoing viral insult eventually can lead to chronic hepatitis, cirrhosis, or hepatocellular carcinoma, although other patients may remain asymptomatic carriers. The incubation period ranges from 30-120 days and is detectable within 30-60 days.

Although preventable by immunization(5), hepatitis B remains a significant worldwide cause of morbidity and mortality. The World Health Organization estimated that in 2015, 257 million individuals had chronic hepatitis B and 887,000 individuals died as a result of hepatitis B (secondary to cirrhosis and hepatocellular carcinoma)(6). In endemic areas, hepatitis B is transmitted both vertically (from mother to child at birth) and horizontally (via to infected body fluids, e.g. sexual contact or needlestick exposure). Chronic infection is more common in those infected at lower ages compared to infected adults. A subset of individuals with hepatitis B are coinfected with HIV or hepatitis D as well. Hepatitis D is a subviral agent which is dependent upon the presence of HBV for replication(7). Infection can occur coincident to HBV infection (coinfection) or subsequent to chronic or latent HBV infection (superinfection). Coinfection of hepatitis D with HBV is regarded as the most severe chronic hepatitis, although it can also be avoided via hepatitis B vaccination.

Diagnostic Serologic Testing for Hepatitis B

Since infected individuals may not display any symptoms upon infection and hepatitis B cannot be specifically identified from other viral etiologies clinically, confirmation of hepatitis B infection relies upon laboratory testing (Figure 1). Additionally, although treatment of acute infection is primarily supportive, chronically infected patients with immune active phase disease(8) or with other indications(9) may be treated with anti-viral medications. Given the severity of long-term complications of this entity (cirrhosis, HCC), appropriate identification of infected patients is a critical clinical task.

Figure 1. Hepatitis B Virus Structure Simplified 0 <![CDATA[Plasma Exchange with Albumin Replacement for Alzheimer’s Disease]]> https://health.ucdavis.edu/blog/lab-best-practice/plasma-exchange-with-albumin-replacement-for-alzheimers-disease/2021/05 Wed, 26 May 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/plasma-exchange-with-albumin-replacement-for-alzheimers-disease/2021/05 Miao Tian, MD PhD, Grace Fortes Monis, MD PhD

The guidelines on the use of therapeutic Apheresis in clinical practice published in JCP 2019 has showed strong consideration for the addition of a new fact sheet on Alzheimer’s disease (AD) based on the preliminary data on the use of therapeutic plasma exchange (PE) in treating AD. However, due to limited published evidence for this therapeutic effect at that time, the disease was not selected for inclusion in the guideline (1).

A recent study published by Boada et al in October 2020 (2) provides strong evidence of the therapeutic effects of PE in AD. This 14-month, randomized, controlled, phase 2b/3 clinical trial examined the effects of plasma exchange with albumin replacement in patients with mild to moderate AD. A total of 347 patients were randomized into three PE treatment groups with different doses of albumin and intravenous immunoglobulin replacement, and into one placebo (sham PE) group. The patients received 6-week period of weekly conventional PE followed by 12-month period of monthly low volume PE. A total of 4709 apheresis procedures were performed over 41 sites in Spain and the United States. The authors discovered that PE treated patients with moderate AD performed significantly better than placebo for co-primary endpoints (the Alzheimer’s Disease Cooperative Study–Activities of Daily Living (ADCS-ADL) and Alzheimer’s Disease Assessment Scale–Cognitive Subscale (ADAS-Cog)) and for global assessment endpoints (the clinical dementia rating sum of boxes (CDR-sb) and Alzheimer’s Disease Cooperative Study-Clinical Global Impression of Change (ADCS-CGIC)). The study indicates that PE with albumin replacement could slow cognitive and functional decline in AD patients.

Alzheimer’s disease is the most common neurodegenerative disease that affects memory, thinking and behavior (3). The extracellular aggregates of amyloid β peptide (Aβ) and intracellular neurofibrillary tangles of phosphorylated tau protein deposits are hallmarks of AD pathology. However, the initial biological trigger of the disease process is unknown (4). No pharmacological treatments available so far for AD have yet been shown to stop or slow the disease progress, although some neurotransmission modulators such as AChEI and NMDAR antagonist may temporarily improve symptoms (5,6). The preclinical research studies have shown that human albumin is able to bind Aβ (7) and the Aβ levels of cerebrospinal fluid may be modified by sequestration of albumin bound-Aβ in the peripheral blood which alters the balance to induce CSF Aβ to pass to plasma in animal models (8-11). These basic research studies proposed that Aβ could be a valid therapeutic target. Indeed, the initial pilot study of PE in AD patients showed that 7 patients undergoing 6 PE in 3 weeks and 1 year of follow-up had more stable cognitive status scores than expected (12). In a phase 2 clinical trial, the safety, tolerability and preliminary efficacy of PE with 5% albumin in 42 mild to moderate AD patients were evaluated (13). This 21-week, double blind and controlled study showed that patients treated with PE had improvement in memory and language functions, which persisted after PE was discontinued. The above mentioned larger and more rigorous phase 2b/3 clinical trial (2) further confirmed the therapeutic effects of PE in AD patients.

Plasma exchange is often used to treat hematologic, immunological and metabolic disorders (1). These recent studies support that PE could also be applied as a new therapeutic approach for AD.

References

  1. Padmanabhan A, Connelly-Smith L, Aqui N, et al. Guidelines on the Use of Therapeutic Apheresis in Clinical Practice – Evidence-Based Approach from the Writing Committee of the American Society for Apheresis: The Eighth Special Issue. J Clin Apher. 2019 Jun;34(3):171-354. 0 <![CDATA[Fine Needle Aspiration (FNA)]]> https://health.ucdavis.edu/blog/lab-best-practice/fine-needle-aspiration-fna/2021/05 Fri, 14 May 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/fine-needle-aspiration-fna/2021/05 Abebe Teklu, MD, Alaa Afify MD

    FNA is a simple, safe, and Cost-effective procedure for patient with a mass. FNA is a biopsy procedure and should be considered in the same light as a surgical biopsy. It is a diagnostic tool and can effectively triage patients for further investigation, surgery, or other therapeutic options (1).

    Indications of FNA: (4)

    • Any mass: palpable or visualized by an imaging method can be sampled, but there should be a reasonable expectation of obtaining useful information from the procedure.

    Contraindication of FNA: (4)

    • Clinically insignificant small lymph nodes, vague induration, or asymmetries
    • Lung FNA: advanced emphysema, severe pulmonary hypertension, marked hypoxemia, mechanical ventilatory assistance.
    • Abdominal FNA: Rare; Bile peritonitis, peritonitis, pancreatitis, hemorrhage, infection needle tract implantation of malignancy
    • Post-FNA tissue infarction which may interfere with subsequent histologic interpretation.

    Complications (2; 3; 5)

    • Bleeding, small hematoma
    • Pneumothorax, very rare (transthoracic FNA, aspiration of breast or supraclavicular/ axillary region
    • Transthoracic FNA using larger needles (180gauge or larger): Rarely, deaths have been reported due to pulmonary hemorrhage or tension-pneumothorax in emphysematous patients.

    FNA includes the following events:

    • Collection of pertinent clinical data
    • Sampling: Using 22-gauge or smaller needles
    • Specimen preparation and staining
    • Interpretation
    • Communication and reporting

    Pre-FNA Requirements:

    • Informed consent and patient education
    • Clinical information: Patient’s name, identification number, sex, age, tumor location and size, physical and imaging characteristics of the lesion, presenting symptoms and duration

    Who can perform FNA?

    • Superficial lesion: Pathologist, clinicians, or radiologist
    • Deep seated lesion: Radiologist, Pulmonologist Gastroenterologist, Radiologists

    Specimen preparation and staining: (1)

    • Wet-fixed (Using 95% ethanol) and air-dried smears
    • Romanowsky or modified Wright-Giemsa stain (air-dried smears)
    • Papanicolaou or hematoxylin-eosin stain (wet-fixed smears).
    • Large tissue fragments should be picked up gently with a pipette or needle and placed directly in formalin for cell block preparation.

    Ancillary Studies:

    • Immunohistochemical stains on cell block
    • Microbiological culture
    • Electron microscopy
    • Flow cytometry
    • Cytogenetics and molecular studies

    Interpretation:

    • Involves assessment of cell morphology, Cell-to-cell interaction, tissue fragment architecture and extracellular matrix

    Diagnostic Categories: (1, 6)

    • Inadequate/unsatisfactory: acellularity/hypocellularity, poor fixation, poor preparation (crush artifact), poor staining, excessive blood obscuring cellular details, excessive necrosis or debris
    • Benign: No evidence of malignancy
    • Atypical Cells present: Atypical in appearance and malignancy is an unlikely possibility
    • Suspicious for malignancy: Definite diagnosis of malignancy can not be rendered because the malignant cells are too few in number or there are some features of malignancy but lack overtly malignant cells
    • Malignant

    References

    1. Cytology : Diagnostic Principle and Clinical Correlates: Fifth edition Edmund S. Cibas, MD; Barabara S. Ducatman, MD.
    2. Smith EH. The hazards of fine-needle aspiration biopsy. Ultrasound Med Biol 1984;10:629–634.
    3. Davies JD, Webb AJ. Segmental lymph node infarction af]]> 0 <![CDATA[Race and eGFR: Addressing Health Disparities in Chronic Kidney Disease]]> https://health.ucdavis.edu/blog/lab-best-practice/race-and-egfr-addressing-health-disparities-in-chronic-kidney-disease/2021/04 Thu, 29 Apr 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/race-and-egfr-addressing-health-disparities-in-chronic-kidney-disease/2021/04 Alexander Ladenheim, M.D., Nam Tran, Ph.D., Baback Roshanravan, M.D., M.S., M.S.P.H., Brian Young, M.D.

      Race and Health Disparities in Chronic Kidney Disease

      Starting on May 4, 2021, UC Davis Health will calculate estimated glomerular filtration (eGFR) rate without a parameter for race. African Americans are at high risk for chronic kidney disease (CKD). The US Renal Data System (USRDS) 2018 Annual Report showed that black Americans in older age groups experienced higher rates of CKD compared to white Americans of similar age.1 This finding is also shared by the Jackson Heart Study, an epidemiologic study of black Americans in Mississippi, with the rate of CKD in this population close to 20%.2 Black Americans have a marked increased risk for end-stage renal disease (ESRD), with an age-sex-standardized incidence ratio of 2.9 compared to white Americans in 2016.3 Also troubling, black patients with ESRD exhibit a lower rate of kidney transplant compared to persons of other races.4 It is in this context that we need to reevaluate measures of kidney function that are estimated differently based on self-reported African American race.

      Calculation of eGFR

      The use of eGFR is an important clinical tool in the diagnosis of chronic kidney disease (CKD). Its primary role is to facilitate a simpler interpretation of biomarkers of renal disease, primarily creatinine and cystatin C. Currently, 89% of labs surveyed by the College of American Pathologists (CAP) report eGFR alongside serum creatinine.

      Several equations to calculate eGFR have been developed, with each iteration theoretically becoming more sensitive and specific for detecting CKD. The Modification of Diet in Renal Disease (MDRD) Study equation and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation are the most widely used equations for estimating GFR in adults. In each, regression models using serum creatinine and available demographic data helped to develop eGFR equations that correlated with measured GFR,5 with adjustments made for age, sex, and race (Figure 1).

      Variables in eGFR Calculation
      Figure 1. Variables in eGFR Calculation: The figure illustrates factors that influence eGFR calculations. Increasing serum/plasma creatinine, increasing age, and female sex decrease eGFR, while African American race increases eGFR calculations.


      The MDRD equation (Figure 2), published in 1999, is still the most commonly used method to calculate eGFR, according to the 2018 CAP chemistry survey. One issue with the study is that the study population was predominately white and male (40% female, 80% white, 12% black).6

      The CKD-EPI equation (Figure 2) was introduced 10 years later and is presently used at UC Davis Health for eGFR. Benefits of the CKD-EPI equation included having a larger and more diverse study cohort for the training and internal validation groups. However, races other than black or white represented a very small proportion of the study (6%), and the external validation group was less diverse (10% black, 87% white).7

      Comparison of the MDRD and CKD-EPI Equations
      Figure 2. Comparison of the MDRD and CKD-EPI Equations: This comparison of the MDRD and CKD-EPI equations shows the relative weights of each of the parameters mentioned above. In the MDRD equation, African American race increases eGFR by approximately 21%; in the CKD-EPI equation, it increases eGFR by approximately 16%. 0 <![CDATA[Acid Fast Bacilli (AFB) Smear and Culture Testing]]> https://health.ucdavis.edu/blog/lab-best-practice/acid-fast-bacilli-afb-smear-and-culture-testing/2021/03 Tue, 16 Mar 2021 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/acid-fast-bacilli-afb-smear-and-culture-testing/2021/03 Jacob Donnelly, M.D., Pathology Resident, Jasper Zheng, M.D., Pathology Resident
      Anna Romanelli, Ph.D., Medical Director of Clinical Microbiology Laboratory

      Introduction:

      It is a foundational principle for any laboratory test procedure that the value of the test heavily relies on using specimens that have been properly collected, labelled, handled or stored prior to and during the testing process. Microbiological tests are not as standardized as some other lab tests; the way in which a sample is processed and the results are interpreted depends heavily on the information provided with the specimen. Erroneous results as a result of specimen mis-management can affect patient care and outcomes, as well as hospital infection control, patients’ length of stay in the hospital, costs and laboratory efficiency. The initial collection of samples for microbiology testing is critical, since errors that occur at this stage cannot be corrected at a later time, and since mistakes require collection of new specimens.

      The focus of this lab best practices blog is to review the appropriate specimen collection and processing for Acid Fast Bacilli (AFB) smear and culture testing. Both AFB smear and culture tests are performed at the Sacramento County Public Health Department.

      AFB smear microscopy refers to the quick and inexpensive microscopic examination of clinical specimens using a fluorochrome stain. One must take note that smear microscopy is unable to differentiate among different species of Mycobacteria, and it is also unable to detect the viability of bacilli. Furthermore, few other organisms can be stained even when using staining methods specific to mycobacteria, such as Nocardia spp.

      The AFB culture refers to the process of inoculating a clinical specimen onto culture media; including Becton-Dickinson Mycobacteria Growth Indicator Tubes (B-D MGIT broth) and a Lowenstein-Jensen (L-J) media slant. It is then incubated at 37°C for up to six weeks. Lastly, it is examined for growth or no growth.

      Methodology:

      • AFB smear - fluorochrome staining
      • AFB culture - B-D MGIT system and conventional culture media.

      Specimen Collection

      Specimen Source(s):

      • UCD Health Systems
      • Hospital Based Clinics / HBC
      • Physician Clinics Network / PCN
      • Others

      Materials:

      • Parafilm
      • Sterile containers
      • Small and large plastic biohazard bags
      • Biohazard stickers

      Acceptable Specimen Type(s):

      • Sputum
      • Bronchial Washing
      • Pleural Fluid

      Sputum:

      • Specimen/Sputum collection best practice for initial diagnosis of tuberculosis:
        - Early morning, deep cough specimen, three (3) consecutive days collection.
        - A minimum of eight (8) hours must be allocated between specimens if sputum is collected within the same 24-hour period,
        - Minimum acceptable specimen volume: two (2) mL
        - Refrigerate until transported

      Bronchial Washing:

      • Preferred minimum acceptable volume: two (2) mL
      • Refrigerate until transported

      Pleural Fluid:

      • Preferred minimum volume: two (2) mL
      • Refrigerate until transported
      Figure1_BlueFluid
      Figure 1: *Blue fluid represented in the photo is used as an example of 2-3 ml or the amount of fluid needed from the patient.

      Unacceptable Conditions:

      1. Specimens that are not labeled with the patient name or identifier.
      2. Clinical specimen collected within eight hours of previous specimen.
      3. Clinical specimen received greater]]> 0 <![CDATA[The Importance of Trephine Biopsy and Aspirate in Bone Marrow Analysis]]> https://health.ucdavis.edu/blog/lab-best-practice/the-importance-of-trephine-biopsy-and-aspirate-in-bone-marrow-analysis/2021/02 Tue, 16 Feb 2021 00:00:00 -0800 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/the-importance-of-trephine-biopsy-and-aspirate-in-bone-marrow-analysis/2021/02 Gopal Patel, Zarir Karanjawala, Denis M. Dwyre

        The morphologic analysis of bone marrow specimen is an essential part of comprehensive evaluation in the diagnosis of hematologic and certain non-hematologic disorders. Some of the most common indications for bone marrow examination include a) unexplained cytopenias, cytosis, or abnormal blood cell morphology, suggestive of bone marrow pathology. b) diagnosis, staging, and therapeutic follow-up of hematologic malignancies, c) investigation of suspicious metastatic disease or bony lesions on imaging study, d) unexplained organomegaly, e) fever of unknown origin with suspicion for microbial infection or occult hematologic malignancy, and f) investigation of iron stores, lipid/glycogen storage disorders, or nutritional deficiencies (1). In this lab best practice blog, we will review the complementary roles of peripheral blood smear and bone marrow core biopsy and aspirate in the diagnosis of various hematologic malignancies.

        1) Peripheral Blood Smear

        Although patients with hematologic diseases may present with various sign and symptoms, an abnormal complete blood count (CBC) during routine medical visits is a common indication that prompts further investigation and eventual bone marrow evaluation. Therefore, examination of CBC data and peripheral blood smears are the first diagnostic step in defining a hematologic disease. Peripheral blood often provides clues to the underlying bone marrow pathology (Fig. 1). For example, when myelodysplastic syndrome (a bone marrow failure disorder) is suspected, a well stained peripheral blood smear is very important for the evaluation of cytopenias. Dysplastic neutrophils (hypolobated nuclei, hypogranular cytoplasm, etc.) and platelet size and granularity are best evaluated in peripheral blood smears. In cases of suspected myelofibrosis, tear-drop cells and/or leukoerythroblastic changes are often seen in peripheral blood smears. Some suspected hematopoietic malignancies are evaluated based on routine peripheral blood data in asymptomatic patients and bone marrow work-up is performed to determine the extent and/or progression of disease. For example, chronic lymphocytic leukemia is defined by the presence of monoclonal B-cell population, with characteristic morphology and immunophenotype, in peripheral circulation. WHO 2016 recommends morphologic evaluation and manual 200 leukocyte differential counts in peripheral blood smear as part of complete bone marrow evaluation (2). According to a recent study, a peripheral blood smear review performed for the assessment of abnormal cell morphology, hematolymphoid neoplasm, leukocytosis, circulating blasts, and parasitic infection has significant added clinical value (3).

        Peripheral blood smear
        Fig. 1: Peripheral blood smear: This peripheral blood smear shows anemia (increased central pallor in RBCs), leukocytosis with monocytosis and circulating blasts, which prompted bone marrow evaluation.

        2) Bone marrow evaluation

        A bone marrow evaluation is called for when available clinical and laboratory results cannot adequately explain the hematological abnormalities. Generally, a thorough bone marrow evaluation may include trephine bone marrow core biopsy, touch imprints of a bone marrow core biopsy, aspirate smears, spicule crush, and aspirate clot preparation (4). In addition, a bone marrow investigation invariantly includes some of the ancillary studies, such as special stains, cytochemistry, immunohistochemical studies, immunophenotypic analysis (flow cytometry), cytogenetics, molecular genetics, and other specialized investigations.

        The International Council for Standardization in Hematology (ICSH) has prepared a set of guidelines based on pref]]> 0 <![CDATA[The SARS-CoV-2 Variant and its Impact on Diagnostic Testing]]> https://health.ucdavis.edu/blog/lab-best-practice/the-sars-cov-2-variant-and-its-impact-on-diagnostic-testing/2021/01 Fri, 08 Jan 2021 00:00:00 -0800 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/the-sars-cov-2-variant-and-its-impact-on-diagnostic-testing/2021/01 Nam K. Tran, PhD1; Chris Miller, PhD2; Sarah Waldman, MD3
        1Department of Pathology and Laboratory Medicine; 2Center for Immunology and Infectious Diseases; 3Division of Infectious Diseases, Department of Internal Medicine

        Introduction

        The new SARS-CoV-2 mutant (VOC 202012/01) has become the dominant variant in the United Kingdom (UK)1 with the first known case in the United States identified on December 29, 2020.2 Over sixty percent of COVID-19 infections in the UK are now attributed to the new variant.1 The new variant is defined by 23 mutations, 13 of which are non-synonymous point mutations. In addition, there are 4 deletions and 6 synonymous point mutations.3,4 The non-synonymous mutations include a series of spike protein mutations, including a mutation in the receptor binding domain (RBD). Other notable mutations include a stop codon in ORF8. There are 6 synonymous mutations with 5 in ORF1ab (C913T, C5986T, C14676T, C15279T, C16176T), and one in the M gene (T26801C).

        Viral mutations are not unexpected, this is an unusually large number of mutations in a single cluster. Mutations that enhance the virus’s capacity to spread among people provide a new variant with an advantage over the ancestral strain and enable the new variant to become dominant is its capacity to spread. In the case of SARS-CoV-2 VOC 202012/01, it is believed this RBD mutation increases the affinity of the S protein for the ACE-2 receptor on human cells which in turn enhances the transmissibility between people as any virus that a naïve person encounters is more likely to bind to its receptor.3,4 To date, there is no evidence that VOC 202012/01 increases COVID-19 severity.

        However, the ability of some molecular diagnostic assays to detect the VOC may be affected by these mutations.5 Commercially available SARS-CoV-2 molecular assays often target the ORF region, as well as genes encoding for envelope protein (E), S and/or nucleoprotein (N). Therefore, the mutations in the new SARS VOC could theoretically impact the accuracy of assays that target ORF8 and S. However, as all commercial assays target two or more viral genes, the loss of ORF8 or S signal should not significantly affect assay performance. At present, three commercial assays target a combination of ORF8 or S with other targets.5,6 Assays targeting ORF8 and S are NOT used at UC Davis Health.

        Laboratory Best Practice

        The new SARS-CoV-2 variant is detectable by the tests currently offered at UC Davis Health. Figure 1 identifies gene targets for the four UC Davis Health assays, all of which have received emergency use authorization (EUA) by United States Food and Drug Administration (FDA) and is summarized here7-10:

        • Our high throughput platform (Roche cobas 6800) targets the ORF1ab region and E gene,
        • Our rapid point-of-care platform similarly targets the ORF1ab region, but also detects the N gene.
        • Our medium throughput and “urgent” testing platforms used at UC Davis Health are based on the original Centers for Disease Control and Prevention (CDC) assay targeting two regions within the N gene (N1 and N2).

        The selection of ORF1ab and N targets is due to their highly conserved nature (i.e., less likely to mutate) and their unique sequence specific for SARS-CoV-2. In contrast, the use of the E gene serves as a pan-Sarbecovirus marker.7 It must be noted that N and E mutations do exist but have not increased in prevalence since these variants appear to be no more infectious than non-mutants.

        Molecular Targets UCDavisHealth SARS CoV-2 R]]></content:encoded> <slash:comments>0</slash:comments> </item> <item> <title><![CDATA[Red blood cell antigen phenotyping and genotyping]]></title> <link>https://health.ucdavis.edu/blog/lab-best-practice/red-blood-cell-antigen-phenotyping-and-genotyping/2020/12</link> <pubDate>Wed, 02 Dec 2020 00:00:00 -0800</pubDate> <dc:creator>Laboratory Best Practice blog</dc:creator> <category/> <guid isPermaLink=https://health.ucdavis.edu/blog/lab-best-practice/red-blood-cell-antigen-phenotyping-and-genotyping/2020/12 Anupam Mitra, MBBS, MD Pathology Resident, PGY2
        Sarah Barnhard, MD Medical Director of Transfusion Services

        The main purpose of testing prior to transfusion is to provide the most compatible blood to the patient in order to minimize the risk of hemolytic transfusion reactions. The type and screen are the first two tests required as pre-transfusion testing. As the name suggests, these are two tests: “type”- to detect the ABO and Rh type of the patient’s red blood cells and “screen” – to detect the presence of antibody(ies) against RBC antigen(s). Antibody/antigen complex formation is thermal range dependent. Antibodies against RBC antigens are optimally reactive at either warm (at or above body temperature) or cold (below body temperature) thermal amplitudes. Warm antibodies are usually acquired and of IgG type. They react at or above 37C. Cold antibodies are usually naturally occurring and of IgM type. They react below 37C.1 

        1. What is RBC phenotyping?

        The phenotype of RBCs (RBC phenotyping) refers to determining the type of antigens present on the RBC. The ABO/Rh type in the ‘type and screen’ is performed on all patients requiring transfusions. However, an extended antigen phenotype may also be performed. This determines the antigen expression other than the A, B or D antigens. Red blood cell antigen extended phenotyping is almost always performed as a reflex test. That is, extended phenotyping usually supplements routine pre-transfusion testing in patients with clinically relevant alloantibody(ies) or in patients who are at risk for making clinically relevant alloantibody(ies). Four versions of RBC extended phenotyping panels are performed in the transfusion services laboratory - ‘cold phenotype’; ‘full warm phenotype’; ‘complete phenotype’ and ‘limited phenotype’.

        As the name suggests, the ‘cold phenotype’ is a panel that determines the expression of all antigens with common corresponding cold-reacting antibodies (M, N, P, Lea and Leb). The ‘full warm phenotype’ is a panel that determines the expression of all the antigens with common clinically significant corresponding antibodies that are warm reacting (K, E, e, C, c, Fya, Fyb, Jka, Jkb, S and s). The ‘complete phenotype’ is a panel that determines the expression of all antigens with common corresponding antibodies, either warm reacting (K, E, e, C, c, Fya, Fyb, Jka, Jkb, S and s) or cold reacting (M, N, P, Lea and Leb). In some cases, a ‘limited phenotype’ panel is performed to detect one or a few specific antigens. RBC phenotyping is always performed from a pre-transfusion specimen to avoid interference from transfused red blood cells.2-3

        1.1 Indication to perform a red blood cell antigen full warm phenotype
        A full warm phenotype may be done in several different settings.2-3

        1.1.1 To prevent RBC antibody formation- patients who are receiving chronic transfusions are exposed to multiple foreign RBC antigens repeatedly over a long period of time, which increases the possibility of developing new alloantibodies. Thus, performing a full warm phenotype prior to transfusions allows the transfusion services laboratory to provide fully or partially phenotype-matched units for these patients to prevent development of alloantibody(ies). The clinical indication for a full warm RBC phenotype prior to transfusions are as follows-

        1. Newly diagnosed sickle cell disease
        2. Patients with sickle cell disease who have not previously had a full warm phenotype performed
        3. Other hemoglobinopathies that are transfusion dependent

        1.1.2 To prevent additional RBC antibody formation- pa]]> 0 <![CDATA[CAP Inspections and the importance of informatics…]]> https://health.ucdavis.edu/blog/lab-best-practice/cap-inspections-and-the-importance-of-informatics/2020/11 Mon, 30 Nov 2020 00:00:00 -0800 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/cap-inspections-and-the-importance-of-informatics/2020/11

        The College of American Pathology (CAP), systematically reviews and accredits laboratories across the United States to ensure these laboratories meet certain safety and patient care criteria. The CAP facilitates this important role through the Laboratory Accreditation Program, which accredits each lab with powers granted from the Centers for Medicare and Medicaid Services (CMS)1. These site visits are unique in that they are performed by other practicing laboratory professionals and occur every 2 years to asses compliance. Checklist are made available to each lab before the site visit is performed so laboratory personal can compile, organize, and review the required information. Compiling this data often forces laboratory admins to spend sufficient time ensuring this data is collated to the needs of CAP and that the data is accurate. Having expertise in informatics can facilitate this and alleviates many of the time-consuming tasks required for this documentation. Thus, reinforcing informatics critical role in all pathology departments.

        One such checklist item that became time consuming, both for our admin staff, as well as our LIS analytics team, was the American Society of Clinical Oncology (ASCO) / CAP estrogen and progesterone receptor testing in breast cancer guidelines2. These guidelines allow laboratories the ability to establish standard operating procedure to ensure the validity of low positive or negative interpretations with these biomarkers. At our institution, the Beaker LIS environment went live mid-way through our CAP visit cycle, making this process more complicated. Thankfully we had developed an in-house pathology search engine PathSearch (Michael Erickson), which combines our old LIS data, with new Beaker data.

        PathSearch allows pathologists to search for cases by keywords and extract the data into a spreadsheet which can be used for further analysis. For the CAP estrogen and progesterone receptor reporting we built a small program to further breakdown the results into a suitable table. The receptor data is not stored as a discrete field but rather in either the final diagnosis or separately in an addendum. In order to search for receptor status only once per case, all case text across all addendums were merged into a single text field and then queried using regular expressions to identify receptor status. The result is noted as a new column for each case/row and tabulated at the end to produce the final data needed.

        For this specific task the CAP required reporting on total breast carcinoma or DCIS for a given period, the age range and mean age of the patients, the number of cases per tumor grade, the receptor status (ER, PR and HER2) case counts by age (pre- or post-menopausal), and the receptor status (ER, PR and HER2) case counts by tumor grade.

        By empowering our physicians with the ability to query for data we have shortened the time needed to generate a report and reduced the amount of people needed for such a project. Of course, there are limitations to keep in mind. For example, the regular expressions are a form of advanced text search and depends on an understanding of all the written possibilities of the data being queried. Since free text search is often difficult, due to complexity of natural language as well as difference in writing styles amongst physicians, having discrete data points for individuals findings, such as receptor status for a case, can be hard coded, allowing for simplified data analysis. As a result, this may not be as comprehensive and may require further verification. Ultimately having a specialist in informatics within the laboratory allows the department to take control of their data and use it seamlessly with their regulatory and business needs. However, anticipating which records will be needed for regulatory parameters is not always defined ahead of time as biomarkers and prognostic status evo]]> 0 <![CDATA[Rapid Combined Flu A/B and SARS-CoV-2 RNA Polymerase Chain Reaction Testing for Emergency and Ambulatory Care Settings]]> https://health.ucdavis.edu/blog/lab-best-practice/rapid-combined-flu-ab-and-sars-cov-2-rna-polymerase-chain-reaction-testing-for-emergency-and-ambulatory-care-settings/2020/11 Tue, 03 Nov 2020 00:00:00 -0800 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/rapid-combined-flu-ab-and-sars-cov-2-rna-polymerase-chain-reaction-testing-for-emergency-and-ambulatory-care-settings/2020/11

        Nam K. Tran, Ph.D.1; Larissa May, M.D.2; Allen Hall, M.D.3; Tom Bullen, M.D.3;
        Sarah Waldman, M.D.4; John Rodrigo, M.D.1; Julia Loegering, B.S.1; Shelley Gillott, C.L.S.1;
        Stacy Yee, CLS1; Taylor Howard, MD1

        1Dept. of Pathology and Laboratory Medicine; 2Dept. of Emergency Medicine; 3UC Davis Health Community Physicians; and 4Division of Infectious Diseases, Dept. of Internal Medicine

        Introduction

        The primary means for diagnosing SARS-CoV-2 infection is by reverse transcriptase (RT) real-time polymerase chain reaction (PCR). Tests used early in the novel coronavirus (COVID-19) pandemic were based on the Centers for Disease Control and Prevention (CDC) assay targeting several regions within the SARS-CoV-2 nucleoprotein (N) gene.1 High throughput tests, including those deployed at UC Davis Health (cobas® 6800, Roche Diagnostics, Indianapolis, IN) targeted open reading frame (ORF) 1ab and the envelope protein (E) genes (Figure 1).2 Unfortunately, these early RT-PCR tests used “batched” testing schemes (e.g., needing to test multiple samples at one time) to optimize reagent use, and required operation by licensed high complexity laboratory personnel, and exhibiting testing turnaround times that were not compatible with “STAT” (<1 hour) needs.

        SARS-CoV-2 Diagnostic Targets
        Figure 1. SARS-CoV-2 Diagnostic Targets: The figure illustrates common antigen and molecular targets for SARS-CoV-2 diagnostic testing. Genes targeted by Roche assays versus CDC assays are for illustrative purposes only and do not represent exact locations.

        Point-of-care (POC) SARS-CoV-2 molecular testing solutions emerged March 27, 2020. One POC platform relied on isothermal nucleic acid amplification techniques. However, these early POC tests exhibited less than ideal clinical sensitivity due to their isothermal detection methods.3 To this end, for most of the COVID-19 pandemic, the world lacked a viable high sensitivity rapid POC RT-PCR solution to accelerate decision making in at risk populations and facilitate patient isolation, and contact tracing.

        On November 3, 2020, UC Davis Health deployed the first commercially available high sensitivity rapid POC PCR platform (cobas® Liat, Roche Diagnostics, Indianapolis, IN) for detecting SARS-CoV-2 and also differentiating between influenza A and B viruses. The test will be rolled out in the Emergency Department and select clinics within the health system. Testing can be performed by Clinical Laboratory Improvement Amendment (CLIA) waived users including physicians and nurses. The primary specimen type will be nasopharyngeal (NP) swabs at this time. For SARS-CoV-2 detection, this assay targets regions in ORF1ab and N genes (Figure 1)4 which exhibits clinical sensitivity and specificity of >99.9% with 100% agreement with to our high through / high sensitivity cobas 6800 laboratory-based PCR method. Internal validation at UC Davis Health has found the cobas Liat sensitivity to remain comparable to the cobas 6800 even with low viral load samples. This rapid POC PCR platform has been used since early 2018 for influenza A/B, respiratory syncytial virus (RSV), and Group A Streptococcus testing and will now add SARS-CoV-2 testing capability to the emergency department and ambulatory care settings. Implementation of this rapid POC PCR platform continues the Department of Pathology and]]> 0 <![CDATA[GI biopsies with lymphoid aggregates: What does this mean?]]> https://health.ucdavis.edu/blog/lab-best-practice/gi-biopsies-with-lymphoid-aggregates-what-does-this-mean/2020/10 Thu, 15 Oct 2020 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/gi-biopsies-with-lymphoid-aggregates-what-does-this-mean/2020/10 Ananya Datta Mitra, M.D.
        Hooman H. Rashidi, M.D.
        Karen Matsukuma, M.D., Ph.D.

        Background

        Because the gastrointestinal (GI) tract is a site of continuous challenge by foreign antigens, it contains a well-developed immune system component. The upper aerodigestive tract and small and large intestines have endogenous lymphoid tissue, including the Waldeyer ring in the oropharynx, Peyer patches in the terminal ileum, and mucosal lymphoid aggregates in the appendix. In contrast, the esophagus and stomach are not associated with a significant amount of organized lymphoid tissue under normal conditions but can acquire lymphoid aggregates under constant antigenic stimulation, such as in the setting of gastric Helicobacter pylori infection.

        The most common specimens encountered by GI pathologists in daily practice are endoscopic biopsies of the upper and lower GI tract. A subset of these will contain lymphoid tissue, which may be mentioned in the pathology report. To aid in understanding what is meant when lymphoid tissue is described in the pathology report, we provide the following definitions:

        Lymphoid tissue: A general term to describe a collection of B-cells, T-cells, and support cells. Lymphoid tissue is normally concentrated along the mucosal surfaces of the body (tonsils, Peyer patches) and can also be acquired at sites of chronic antigenic stimulation. The primary lymphoid tissues are bone marrow and thymus (sites of lymphocyte development); the normal secondary lymphoid tissues include mucosa-associated lymphoid tissue (MALT, mentioned above) and the lymph node, which both serve similar functions. In contrast to mucosa-associated lymphoid tissue which is non-encapsulated, a lymph node is a specialized type of lymphoid tissue that is in continuity with the lymphatic system and enclosed within a fibrous capsule.

        Other terms used to describe specific types of lymphoid tissue are:

        • Lymphoid aggregate/infiltrate: A collection of B cells, T cells, and supporting cells, present within the stroma of various organs. The term can be used to describe endogenous lymphoid tissue or acquired lymphoid tissue.
        • Lymphoid follicle: Similar to a lymphoid aggregate (sometimes used interchangeably) but typically refers to a more discrete collection of B cells, T cells, and supporting cells. There are two types of lymphoid follicle:
        • Primary follicles are lymphoid follicles that do not yet contain a germinal center (described below). They are precursors to secondary follicles and are composed predominantly of small naïve B cells and inconspicuous supporting cells. Because primary follicles appear monotonous, they can occasionally raise concern for lymphoma.
        • Secondary follicles contain germinal centers. The presence of a germinal center indicates activation of adaptive (antigen-specific) immunity and typically is a feature of a reactive rather than a neoplastic process.
        • Germinal center: The site where antigen-presenting cells interact with naïve B-cells to initiate an antigen-specific immune response. It is the slightly paler circular area within the secondary lymphoid follicle (Figure 1) and is composed predominantly of B cells, with rare T-cells and scattered support cells. Of note, the darker area surrounding the germinal center (the mantle zone) consists predominantly of B-cells and can be thought of as residual primary follicle surrounding the germinal center.
        Lymphoid follicle
        Figure 1. Lymphoid follicle with germinal center (i.e., secondary follicle).

        Terms fr]]> 0 <![CDATA[Introduction to Therapeutic Drug Monitoring and the Clinical Laboratory's Role]]> https://health.ucdavis.edu/blog/lab-best-practice/introduction-to-therapeutic-drug-monitoring-and-the-clinical-laboratorys-role/2020/09 Wed, 23 Sep 2020 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/introduction-to-therapeutic-drug-monitoring-and-the-clinical-laboratorys-role/2020/09 Raymond Gong, M.D., Pathology Resident
        Nam K. Tran, Ph.D., HCLD (ABB), FAACC, Professor of Clinical Pathology

        Introduction:

        Therapeutic drug monitoring (TDM) is the practice of measuring drug concentrations in order to tailor dosages and maintain therapeutic levels in a patient’s bloodstream [1-2]. The goal of TDM is to improve clinical outcomes by improving efficacy, limiting toxicity, and subsequently reduce the overall cost of drug therapy. It is an interdisciplinary process that includes clinicians, pharmacists, and laboratory professionals and combines knowledge of pharmacokinetics, pharmacodynamics, the patient's clinical setting (including various preanalytical factors such as dosage, dosing interval, patient characteristics, sample type, and timing of sample collection), and analytical factors within the clinical laboratory. TDM is the standard of care for monitoring therapy with many drugs and is particularly valuable for drugs that are used for an extended period of time, show pharmacokinetic variability, and/or known to a have a narrow therapeutic index.

        While the benefits of TDM are clear, a review of the literature shows that monitoring practices may not always be ideal. Based on earlier studies of TDM programs for digoxin and phenytoin, it had been reported that as many as 70% to 80% of TDM assays performed on inpatients were inappropriate [3]. This was attributed to over-monitoring as well as under-monitoring with providers failing to detect trends in TDM assay results. A more recent study focusing on TDM of vancomycin in pediatric inpatients revealed that up to 94% of TDM assays were inappropriate in that particular institution, owing to mistiming of sample collection and lack of clinical indication [4]. Other studies, including international research, that are based on provider survey data indicate that while TDM is accessible, there has been concern over a perceived lack of clinical value, unawareness on how to use TDM, and wide variation in TDM practice [5-8]. These studies highlight the importance of knowledge of TDM guidelines and appropriate interpretation of results, which are needed to ensure effective TDM utilization, avoid excessive healthcare costs, and prevent adverse drug events.

        Laboratory Best Practice:

        At UC Davis Health, assays for use in TDM are offered for a variety of drug analytes including antiepileptic drugs, immunosuppressive drugs, and antimicrobials. Given appropriate clinical indication and collaboration with the Clinical Laboratory, UC Davis Health clinicians are able to order drug concentration testing for select medications. Placing orders in Epic Hyperspace provides a dialog box such as that shown in the image below (annotated with a red arrow):

        As indicated by the red arrow, a reference link to the Laboratory Test Directory is available to providers for further information about laboratory tests.

        Using peak gentamicin concentration as an example, accessing the link will take providers to a corresponding webpage within the Laboratory Test Directory, as shown in the following image which only partially displays the information on test specifications that is available.

        As seen in the preceding image, recommendations for sample collection timing are given for drugs in which TDM has an established role. This information is intended to guide providers on appropriate sample collection, demonstrating one of the ways the Laboratory Test Directory acts as a resource for providers and an exa]]> 0 <![CDATA[Challenges and Implications of False Negative COVID-19 Testing]]> https://health.ucdavis.edu/blog/lab-best-practice/challenges-and-implications-of-false-negative-covid-19-testing/2020/08 Mon, 17 Aug 2020 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/challenges-and-implications-of-false-negative-covid-19-testing/2020/08 Ying Liu, M.D., Pathology Resident
        Anna Romanelli, Ph.D., Medical Director of Clinical Microbiology Laboratory

        As communities across the U.S. have struggled to cope with the effects of the COVID-19 pandemic, many have focused on the lack of widespread testing as a major roadblock to reopening the country and go back to some sort of normalcy. However, as progress has been made on this front, concern has shifted to testing accuracy, predominantly with antibody testing, which is designed to identify prior infection. The goal of this blog article is to review the challenges and implications associated with false negative COVID-19 testing.

        Challenges with COVID-19 Diagnostic Testing:

        In general, antibody tests identify prior infection, while molecular diagnostic testing identifies current active infection. There are two main issues that are worth understanding for COVID-19 diagnostic testing. The first issue relates to who gets tested. At the start of the pandemic in the U.S, availability of testing kits was limited, which placed restrictions on who got tested. During this time, to get a coronavirus diagnostic test, a person was asked to meet certain criteria, such as having symptoms of COVID-19, the disease caused by the coronavirus, or having had close contact with a person confirmed to be infected. In turn, asymptomatic people who are thought to be the major “spreaders” of COVID-19 were not being tested due to the paucity of testing kits. Fast forward to three months later, testing is being performed on a wider scale to include both symptomatic and asymptomatic individuals. Implementing this broader strategy of testing demonstrated a much higher incidence of COVID-19 than previously reported. In order to accurately interpret this uptick in COVID-19 positive cases, we need to understand how well the current diagnostic tests rule out COVID-19 infection. This leads us to our second issue which is the testing itself.

        There is already growing concern that the diagnostic test for COVID-19 is not reliable. Current testing has the potential to come back positive in some people who are not infected with SARS-CoV-2 and negative in people who are in fact infected. A false positive result could mistakenly label a person infected, which has implications causing unnecessary isolation mandates and needless contact tracing. A false negative result, on the other hand, is significantly more consequential, because an infected person who may be asymptomatic will not be required to quarantine and has the potential to infect others. When people have false negative results, they tend to go back to their normal routine or “life before COVID-19”. Social distancing is not being practiced, masks are “optional” in their daily lives and they put themselves in high-risk environments. With imperfect tests, a negative result means only that a person is less likely to be infected. Important testing parameters to keep in mind for a negative test are pre-test probability and the test's sensitivity and specificity. The greater the sensitivity, the less likely it will miss real cases. The greater the specificity, the more likely uninfected individuals will be correctly deemed negative. The current SARS-CoV-2 diagnostic PCR assay performed at UC Davis has a sensitivity of greater than 97.5% and specificity of 100%. It can detect several orders of magnitude above the limit of detection using a typical nasopharyngeal swab specimen.

        SARS-CoV-2 is an enveloped positive sense RNA virus and current molecular tests are designed to target one or more common regions, include the envelope (E), nucleocapsid (N), spike protein (S), and the open reading frame (ORF) region. Assays used by the Center for Disease Control and Prevention (CDC) and others target multiple regions within a gene (1,2). In a meta-analysis review, the authors performed a systematic review and critical appraisal of literature]]> 0 <![CDATA[Specimen Collection Practices for Microbiologic Culture]]> https://health.ucdavis.edu/blog/lab-best-practice/specimen-collection-practices-for-microbiologic-culture/2020/07 Wed, 15 Jul 2020 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/specimen-collection-practices-for-microbiologic-culture/2020/07

        Alexander Ladenheim, M.D., Pathology Resident
        Melanie Rilloraza, C.L.S. (ASCP), Microbiology Supervisor
        Nam K. Tran, Ph.D., Director of Clinical Chemistry
        Anna Romanelli, Ph.D., Medical Director of Clinical Microbiology Laboratory

        Topics

        1. Introduction
        2. Expired Collection Products
        3. Anaerobic Cultures
        4. Lukens Traps for Mucus/Aspirate Specimen Collection
        5. Surveillance Cultures (MRSA and C. difficile)
        6. Other Cultures Requiring Special Media
        7. References

        Introduction

        The culturing of microorganisms remains the mainstay of laboratory testing for infectious disease, even in an age of rapid and more cost-effective molecular testing. For cultures to be reliable, special attention needs to be paid to the collection, transport, and processing of these specimens. The goal of microbiologic culture is the preservation of viable clinically relevant organisms, specifically ones which are likely to be pathogenic. A negative culture result is less helpful for ruling out disease since there is always possibility that organisms were rendered nonviable by incorrect collection or handling. Similarly, positive cultures can be confounded by the presence of contaminant microorganisms and rendered extremely hard to interpret.

        Specimen collection is a team effort and involves the clinical team, couriers, and laboratory personnel, each of whom can have a drastic influence on the quality of the final result. The clinical team in many ways sets the parameters of testing by forming a differential diagnosis and choosing many of the basic conditions: the method of collection, an appropriate site, and sampling. The laboratory, in turn, makes a commitment to providing education and resources for appropriate collection and to promptly and correctly process received specimens. Furthermore, if a sample is compromised or otherwise unlikely to provide useful diagnostic information, the lab has a responsibility to reach out to the clinical team to educate and coordinate the collection of an optimal sample. This is perhaps just as important as rapidly and accurately reporting results but easily overlooked in the hustle and bustle of clinical care.

        What follows is a discussion of several common problems in specimen collection:

        The “sniff test”: Expired collection products

        Expired media cannot be relied upon for specimen collection. It is not uncommon for expired blood culture vials and swab kits to accumulate in the myriad supply closets of the hospital, but these should be identified and replaced. The unit manager, charge nurse, or other person responsible for inventory management can obtain replacements with a call to UCD Supply Chain Distribution or Microbiology.

        The use of expired media is unacceptable both from a laboratory accreditation perspective (by the requirements of the College of American Pathologists) and from a patient care perspective; it leads to the risk of both false negative and false positive results. Collection media is a specially designed, pH balanced, sterile blend of food for microorganisms and myriad other components1 which can include:

        • Reducing agents to promote the growth of anaerobic bacteria (inactivated by oxygen)
        • Selective agents to promote the growth of particular microorganisms
        • Resins and charcoal to neutralize antibiotics and promote growth

        Depending on the specific type of collection media, some of these components are more labile than others, which can result in a shorter shelf-life. Some specialized collection media (such as thioglycolate broth) are so labile that indicator dyes are added to show when a vial of medium is no longer usable.2 In addition, the majority of collection media are not subject to quality control testing by users (i.e., the laboratory) and are considered exempt ]]> 0 <![CDATA[Review of COVID-19 Testing Methods]]> https://health.ucdavis.edu/blog/lab-best-practice/review-of-covid-19-testing-methods/2020/06 Tue, 16 Jun 2020 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/review-of-covid-19-testing-methods/2020/06 Nam Tran, Ph.D., HCLD (ABB), FAACC
        Stuart Cohen, M.D.
        Sarah Waldman, M.D.
        Larissa May, M.D.


        Introduction

        The current pandemic has created significant interest in COVID-19 related laboratory testing. Given the rapidly evolving testing landscape, it has become challenging to keep up with the numerous assays available on the market. Some assays have been marketed with questionable performance in recent weeks, while reagent shortages have forced many hospital laboratories to adopt multiple testing modalities to keep up with demand – creating additional educational challenges when describing COVID-19 testing.1,2 The goal of this blog article is to clarify the current state of testing for COVID-19 and provide recommendations for best practices. Information presented below is current at the time of posting this article.


        SARS-CoV-2 Diagnostic Testing

        As of May 28, 2020, molecular and antigen testing are the only techniques capable of detecting the SARS-CoV-2 virus. Briefly, SARS-CoV-2 is an enveloped positive sense RNA virus. Molecular approaches are designed to target one or more genes (Figure 1).3 Common target genes include the envelope (E), nucleocapsid (N), spike protein (S), and the open reading frame (ORF) region. Some assays may target multiple regions within a gene, such as the assay used by the Center for Disease Control and Prevention (CDC). Molecular methods are primarily based on reverse transcriptase (RT) polymerase chain reaction (PCR). Other methods do exist but are less common. Early data (late 2019) suggested RT-PCR SARS-CoV-2 assays having a false negative rate of about 30%.4 Presently, the primary UC Davis assay has a sensitivity of greater than 97.5%, specificity of 100%, and can detect << 50 copies/mL of virus. On average, a typical nasopharyngeal swab sample from a symptomatic patient carries about 106 copies/mL of virus – several orders of magnitude above the detection limit modern PCR assays.5 False negatives are largely attributed to variable viral shedding of COVID-19 and/or bad specimen collection technique or processing. Specimen type also influences sensitivity, with nasopharyngeal swabs, nasal and mid-turbinate still being preferred for testing of symptomatic individuals.6 At UC Davis NP swabs are accepted with other specimen types currently under validation. Testing from lower respiratory tract specimens, such as bronchoalveolar fluid, are presently performed through Sacramento County Public Health Lab.

        Figure 1. SARS-CoV-2 Structure and Molecular Targets. Common SARS-CoV-2 virus antigenic targets include spike, envelope, and nucleocapsid proteins. Spike proteins are considered more specific and are responsible for binding to ACE2 receptors and facilitate invasion into host cells. Molecular assays target the genes for these proteins.

        Point-of-care testing is also available, however current methods relying on isothermal methods exhibit poor sensitivity for SARS-CoV-2.7 Recent reports indicate these methods have up to 15% false negatives, and therefore, not considered appropriate for screening asymptomatic individuals. Any negative result would require confirmation by RT-PCR.

        More recently, SARS-CoV-2 viral antigen testing has become available and could also be used at the point of care. Instead of detecting the viral RNA, antigen testing employs immunoassay techniques for detecting specific viral proteins.8 The benefits associated with antigen testing is speed (~15 minutes) and cost-effectiveness. Unfortunately, viral antigen kinetics following infection remains unknown. It is believed that SARS-CoV-2 antigens can be found shortly after RNA is detectable. Manufacturer data suggests a sensitivity of about 80% and specificity of 100% based on remnant nasopharyngeal swab viral transport media samples (n = 143) as well as prospectively collected samples (n = 48). More data are needed before widespread adoption of SARS-CoV-2 antigen testing can be made. To this end, molecular approaches remain the primary means for detecting SARS-CoV-2.

        Figure 2. Conceptual Drawing of the Antibody Response Following SARS-CoV-2 Infection. This figure is intended for educational use only and approximates the antibody response to SARS-CoV-2 infection. Following SARS-CoV-2 infection, viral RNA and an


        Serology Testing

        Serology testing measures the host antibody response following infection.9 SARS-CoV-2 serology testing is also based on immunoassay techniques and measures immunoglobulins (Ig) such as IgM, IgA, or IgG. Both IgA and IgM appear early and are less specific, while IgG is produced later and is more specific.10 IgG antibody levels may correlate with potential immunity (Figure 2). Data suggests IgM production begins as early as three days post-infection with IgG appearing as early as seven days post-infection. By day 14, the majority of individuals should have a detectable antibody response. Some, but not all, serology assays experience false positives due to previous exposure to non-SARS-CoV-2 human coronaviruses. Additionally, false negative serology results may occur if testing is performed too soon after exposure where antibody production is non-detectable (“window period”). To this end, it's important to note that positive SARS-CoV-2 serology results alone are not diagnostic, nor does it definitively indicate that a patient has had prior SARS-CoV-2 infection.1 It is also important to understand that not all antibodies produced against a virus, such as SARS-CoV-2, confer immunity. At present, no serology assay to date can differentiate between non-neutralizing and neutralizing antibodies, and it is not known how long we maintain immunity (if any) following SARS-CoV-2 infection.

        The current UC Davis serology assay targets IgG antibodies against the S1/S2 domains of the spike protein and exhibits a sensitivity of 97.6% and specificity of 99.3%. Serology assay specificity is critical to success for COVID-19 due to the low disease prevalence impacting the positive predictive value of the test.1 Exclusion of IgM and IgA testing is due to the lack of specificity as well as more recent data showing IgG often rapidly rising in parallel to IgM following SARS-CoV-2 infection.10 Total immunoglobin tests (IgA, IgM, and IgG) have also emerged claiming the ability to detect high affinity antibodies to enhance sensitivity, while maintaining acceptable specificity.12 More studies are needed to compare the performance of all these assays under real world conditions. Regardless, it is recommended that COVID-19 serology1: (a) results should be interpreted in the context of the expected predictive values, positive and negative, (b) testing used to support the diagnosis of COVID-19 in patients presenting within 9 to 14 days of symptom onset in conjunction with molecular testing, (c) testing used to support observational epidemiological studies evaluating the prevalence of disease, or (d) testing used to help establish a diagnosis when patients present with late complications of COVID-19 illness, such as multisystem inflammatory syndrome in children. SARS-CoV-2 serology SHOULD NOT be used alone as a COVID-19 diagnostic test without paired molecular diagnostics, to determine immune status post-COVID-19, guide personal protective equipment use, or returning to the workplace decisions. Commercially available “rapid” serology tests (lateral flow assays) are not recommended for any setting due to significant false negative and false positive rates.


        Other Chemistry Tests

        In addition to the testing approaches above, routine biochemical testing can aid the diagnosis and management of COVID-19.13 Procalcitonin (PCT) is presently used as a biomarker of bacterial infection and for antimicrobial stewardship. During a viral infection, interferon gamma production inhibits PCT release, thus COVID-19 patients should present with normal PCT levels. Although true for the majority of patients, some recent studies suggest PCT values may be elevated in individuals with severe COVID-19. This observation may be due to the presence of bacterial co-infection, and/or tissue injury-mediated release of PCT due to COVID-19 related tissue damage. Interleukin-6 (IL-6) is another biochemical marker under discussion for COVID-19. Non-surviving COVID-19 patients presented with significantly higher IL-6 compared to survivors. Unfortunately, IL-6 is presently available as a referral laboratory test and should only be used if the result would prompt any management change. Lastly, coagulation testing may help identify patients at risk for COVID-19 related coagulopathy. D-dimer values are significantly higher in patients with severe COVID-19.


        References

        1. Centers for Disease Control and Prevention COVID-19 Serology Guidelines: Accessed on May 28, 2020
        2. Infectious Disease Society of America Primer: May 4, 2020.
        3. Wu F, et al. A new coronavirus associated with human respiratory disease in China. Nature 2020;579:265-269.
        4. Wang W, et al. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA 2020;323:1843-1844.
        5. Wolfel R, et al. Virologic assessment of hospitalized patients with COVID-19. Nature 2020;581;465-469.
        6. Infectious Disease Society of America Guidelines for the Treatment and Management of Patients with COVID-19 (Updated May 6, 2020): Accessed on May 28, 2020.
        7. United States Food and Drug Administration Press Release (May 14, 2020): Accessed on May 28, 2020
        8. Quidel Sophia 2 SARS Antigen FIA product insert: Accessed on May 28, 2020.
        9. American Society for Microbiology Communications (May 19, 2020): Accessed on May 28, 2020.
        10. Long QX, et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nature Medicine 2020.
        11. Diasorin LIAISON SARS-CoV-2 S1/S2 IgG product insert (EN – 200/007-798, 03 – 2020-5).
        12. Roche Diagnostics SARS-CoV-2 serology assay: Accessed on May 28, 2020.
        13. International Federation for Clinical Chemistry (IFCC) Guide on COVID-19 (May 25, 2020): Accessed on May 28, 2020.
        ]]> 0 <![CDATA[Pathology Residency in the Era of Shelter-in-Place]]> https://health.ucdavis.edu/blog/lab-best-practice/pathology-residency-in-the-era-of-shelter-in-place/2020/06 Mon, 15 Jun 2020 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/pathology-residency-in-the-era-of-shelter-in-place/2020/06 Samer Albahra, M.D., Clinical Informatics Fellow
        John Paul Graff, D.O., Assistant Professor, Hematology and Informatics

        Introduction

        COVID-19 and the resulting pandemic has forced everyone to rethink when person-to-person interactions are required. Thus, reducing physical interaction such as handshakes, socially distancing, and in many instances meeting remotely. These remote meetings have leveraged often existing platforms, but raising their importance to dependent technologies.1,2

        Because of the telehealth bill (Coronavirus Preparedness and Response Supplemental Appropriations Act) and subsequent changes to remote sign out, nearly all pathology meetings (educational or otherwise) have moved to the digital space. In our institution, we have not relied on a single software solution to facilitate the video conferencing, but instead use three major vendors: Microsoft Teams4, Cisco Webex5, and Zoom6. In this post, we outline some of the benefits and consequences of each solution.

        Microsoft Teams comes with Office 365 subscription and as a result is typically already available at most institutions. Microsoft's solution is not only a video conferencing tool but also supports text-based communication using channels, direct messages and group messaging. All of the mediums support additional based communication such as using a white board, starting a video call, or using integrations such as polling. Additionally, a wiki is provided to share more static text as well as a files section to upload references for residents. Teams also integrates the Outlook calendar making joining and scheduling meetings straight forward. Teams is limited in the number of video callers that can be active at any given time, which appears to be limited to 20 users.

        Cisco Webex is the work horse of most institutions conference extension solution, meaning it allowed remote guests to attend an otherwise in-person meeting. As a result, your institution might already have this licensed and available. Of course, Webex support's video conferencing as well with up to 200 attendees on a single call. Once in a call, Webex also supports a chat room as well as a rich polling feature. However, the chat's do not persist once the call ends for the attendee. Overall, Webex is a session-based solution contrast to Teams which offers continuity between meetings akin to working in an office. Webex is a bit more difficult to use for video conferencing where there is not a single presenter.

        Zoom is the current sweetheart to the video conferencing world and for one key reason: ease of use. This used to be true in its initial incarnation which allowed you to join a call by just clicking a link. However, this lack of authorization and security lead to random attendees from the Internet joining each other's call for “fun”. This has resulted in a complete lock down of Zoom, requiring you not only to sign in but most now require some sort of password for the conference session. Furthermore, the native applications (iOS, macOS, and Android) have been discovered to use insecure practices so it's highly recommended to only use the web client. Zoom does support up to 100 callers with video and is similar to Webex in that the interaction is session based.

        Table 1. Highlights the Pros/Cons of the three major video conferencing solutions.
        Figure 1. Screenshot of Microsoft Teams demonstrating the channel capability.
        https://health.ucdavis.edu/blog/lab-best-practice/vitreous-fluid-for-primary-intraocular-lymphoma/2020/05 Sara Kwong, M.D., Cytopathology Fellow
        Alaa Afify, M.D., Professor and Director of Cytology

        Introduction:

        Primary intraocular lymphomas (PIOLs) are a subset of primary central nervous system lymphoma arise from retina, vitreous, or optic nerve-with or without central nervous system (CNS) involvement.1 Most PIOLs is usually an aggressive diffuse large B-cell lymphoma although a small subset of the PIOLs can be T cell origin.2 The diagnosis of PIOL is often challenging because the clinical symptoms overlap with other pathologic conditions such as viral induced uveitis or autoimmune uveitis. Patients are initially treated with steroids or antiviral medication with no clinical improvement. The presence of malignant cells or tissue is required for the diagnosis of PIOLs. In addition, immunohistochemical stains, flow cytometric analysis, cytokine analysis, and immunoglobulin heavy chain gene rearrangements can provide invaluable information to support the diagnosis.3 Approximately 60-80% of PIOL will have CNS involvement and the accurate diagnosis is imperative for patients to receive timely treatment, which may lead to an improved mortality rate.4

        Lab Best Practice:

        Specimen may be obtained via fine needle aspiration of the vitreous fluid or pars plana vitrectomy. The retrieved specimen must be delivered to laboratory in a timely manner to minimize cellular degeneration and to maximize the diagnostic yields.5 Even with the maximum effort, the specimen could have insufficient material to evaluate due to hypocellularity of the sample. Therefore, it may require several biopsies to reach an unequivocal diagnosis.6 It is important to note the prior treatment history, especially for patients who has received steroid because steroids have cytolytic effect on malignant lymphoma cells and could lower the diagnostic yield.7

        Microscopically, PIOL show large lymphocytes (2-4 times the size of a normal lymphocyte) with scant cytoplasm, increased nuclear to cytoplasmic ratio, and coarse, immature chromatin with prominent nucleoli.8 These malignant cells can be identified using hematoxylin-eosin stain or Papanicolau stain, however, Giemsa or Diff-Quik stain is better to reveal the cytologic details of the cells.2 Cytology specimen has shown to have variable sensitivity in detecting intraocular malignancy that ranges from 31 to 66.7%,9 and one study reported a sensitivity of 83.3% of detecting PIOL.10 In addition, immunohistochemical stains can be performed, if there is a sufficient number of tumor cells, which often shows positivity immunohistochemical activity for B-cell markers, such as CD20 and CD22.

        Flow cytometry is a very helpful technique to diagnose B-cell lymphomas and to discriminate infections and uveitis,11 however, it has shown limited utility in diagnosing PIOL due to the low cellularity nature of the specimen, which often yields limited or nondiagnostic findings.8

        Cytokine analysis may also provide helpful information. B-cell lymphoma cells secretes high level of interleukin (IL)-10 and it is significantly increased in the vitreous fluid from patients with PIOL.12 On the contrary, in inflammatory conditions, such as uveitis, high level of IL-6 is produced. It has shown that the ratio of IL-10 to IL-6 greater than 1 is suggestive of malignancy. One study shows that IL-10 to IL-6 ratio of greater than 1 has sensitivity of 74.3% and a specificity of 75.0% in diagnosing PIOL.13

        Polymerase chain reaction (PCR) is another powerful technique to detect the monoclonal gene rearrangement of immunoglobin heavy chain (IgH), and the presence of which is highly supportive of lymphoma cells. One study showed that 100% (50 out of 50 cases) tested PIOL cases had monoclonal IgH gene rear]]> 0 <![CDATA[Staining Methods in Frozen Section: Best Lab Practices]]> https://health.ucdavis.edu/blog/lab-best-practice/staining-methods-in-frozen-section-best-lab-practices/2020/03 Sun, 15 Mar 2020 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/staining-methods-in-frozen-section-best-lab-practices/2020/03 Alejandro S. Mendoza, M.D., Surgical Pathology/GI Fellow
        John Bishop, M.D., Health Sciences Clinical Professor, Vice Chair for Clinical Services, Department of Pathology and Laboratory Medicine, UC Davis

        Introduction

        Tissue staining during a frozen section is one of the most important steps in obtaining an accurate pathologic diagnosis. Visualization and discernment of microscopic details can become difficult if staining is done improperly. Here, we will review the two (H&E, Toluidine blue) common staining methods used during a frozen section, compare their advantages and limitations, and outline best lab practices in achieving optimal staining result.

        H&E

        H&E is the most commonly used of all the various staining methods available in frozen section. H&E is simple to perform, inexpensive and reliable. The two main dye components are hematoxylin and eosin.

        Hematoxylin is a natural dye derived from the Haematoxylon campechianum logwood tree, a tree native to Campeche’s Mexican state. It is a basic dye that stains acidic cell components such as nucleic acids, glycosaminoglycans, and acid glycoproteins, into a blue-purple hue. 1,2,3 Eosin is an acidic dye and serves as an excellent counterstain to hematoxylin that targets the cytoplasm of cells, specifically mitochondria, secretory granules, and collagen.3 It gives differing shades of red and pink to the cytoplasm of different types of cells and different types of connective tissues.4

        H&E can be performed in two different methods: progressive or regressive. In the progressive method, the tissue is initially stained with a hematoxylin solution that contains an excess of aluminum salts or acid, which increases nuclei affinity.5 Washing with water follows before a counterstain is applied. Variation in color is due to the selective affinity of tissue components to hematoxylin. With the regressive method, overstaining the tissue section with a neutral hematoxylin solution is the initial step. An acid alcohol is then used to remove excess stain, followed by an alkaline solution to achieve a neutralized tissue section. Stain intensity is controlled by visual examination with a microscope. The progressive method yields more reproducible results, but the regressive method offers a sharper degree of differentiation.6,7

        Advantages: H&E yields excellent morphology, providing excellent contrast between cellular components.5 H&E provides better visualization of individual cell necrosis, keratinization, and keratin pearls, which are frequent histologic findings in well­differentiated squamous cell carcinoma. H&E offers stability of stained tissue lasting years without fading. H&E stain is also compatible when combined with various oxidants, mordants, and differentiating agents.8 H&E is reliable and simple to perform.

        Limitations: One of the main limitations of H&E is the length of the staining procedure. It can take from 3 to 5 minutes to prepare one slide, depending on practice protocol. Due to the number of stains used, performing H&E during a frozen section can sometimes become challenging especially when multiple parts or sections are processed at once. H&E also poorly stains elastic material, reticular fibers, basement membranes, and lipids. It only provides limited insight into the specific biochemical nature of the tissue, specifically only the acidic and basic components.9

        Toluidine Blue

        Tolonium chloride, aka Toluidine blue (TB), is a cationic (basic) thiazine metachromatic dye which has a high affinity for acidic tissue components and turns nucleic acid blue and polysaccharides purple.10 When it binds to glycosaminoglycans, it give off a reddish purple staining called me]]> 0 <![CDATA[Concerns Regarding Biotin Interference: Two Years Later]]> https://health.ucdavis.edu/blog/lab-best-practice/concerns-regarding-biotin-interference-two-years-later/2019/10 Tue, 15 Oct 2019 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/concerns-regarding-biotin-interference-two-years-later/2019/10 Miao Tian, M.D., Pathology Resident
        Nam Tran, Ph.D., Director of Clinical Chemistry

        Introduction: Biotin interference for immunoassay-based laboratory tests has been recognized for decades (1-3). In recent years, the emergence of high dose biotin supplementation and unproven regimens for treating multiple sclerosis has brought biotin interference back into the spotlight. In November 2017, The United States Food and Drug Administration (FDA) issued a safety warning stating that biotin can interfere with laboratory tests (4). This warning was made based on a case in which a patient taking high dose biotin died after a potentially falsely low troponin lab result. However, it must be noted that biotin interference was not confirmed and troponin measurements following the event were made post-mortem. Nonetheless, this FDA warning prompted significant concern by patients and healthcare providers alike. Now two years later, is biotin interference still a problem?

        In a previous blog article, we discussed biotin is an essential co-factor for several carboxylases responsible for gluconeogenesis and metabolism of fatty acids and certain amino acids (5). High concentrations of biotin in patient samples can compete the biotinylated reagents for binding to streptavidin thus interfere the reaction of the immunoassays. Reference intervals for serum biotin concentrations are 0.05-0.83 ng/mL (6). The biotin interference threshold, where the assay exhibits >10% change in results, for some assays have been reported to be 20 ng/ml (7)

        Katzman BM et al at studied the prevalence of biotin supplement usage and plasma biotin concentrations among 1944 outpatients presenting to the emergency department (2). They found that serum concentration of biotin was 10 ng/mL (the lowest known interference threshold) or higher in 7.4% of outpatients presenting to the ED, indicating that the biotin supplement use is not rare among the patient population at the Mayo Clinic in Rochester, MN. However, there could be a sampling bias where patients taking biotin may more tend to attend the survey than those who were not taking biotin, so the prevalence measured here could be higher than actual. It is worth noting that the prevalence of biotin interference may also be geographic dependent, as another study in Australia showing less than 1% of patients presenting to ED had over 10 ng/ml serum concentration (8).

        Laboratory Best Practice: For laboratory best practice, the most effective strategy to mitigate the biotin interference would be an early intervention. First and foremost, public education and patient awareness is the primary line of defense. Patients who need laboratory testing should be notified to refrain from taking supplements and allow several hours or days to “wash out” any residual biotin. One of the pharmacokinetic studies of biotin shows that for patients taking biotin doses of up to 5 mg twice a day. or 10 mg once a day, serum biotin levels could reach <30 ng/ml at 8-hour post-ingestion; while for those taking biotin at >10 mg daily or patient has kidney insufficiency, longer period of delaying sample collection is needed (9). Patients also need disclose their biotin intake to the clinicians and the detailed medication history should be documented to the electronic medical record.

        Another strategy to avoid the biotin interference is to utilize assays that are not susceptible to biotin interference. However, this may not be feasible for many institutions, and assays that are not affected by biotin may be impacted by other immunoassay interferences such as heterophilic antibodies and fibrin. With that said, upcoming assays are incorporating various countermeasures against free biotin without significantly altering assay performance.

        It is worth noting that biotin deficiency is very rare, there is no official recommende]]> 0 <![CDATA[Optimal Utilization of Thromboelastrography (TEG)]]> https://health.ucdavis.edu/blog/lab-best-practice/optimal-utilization-of-thromboelastrography-teg/2019/09 Sun, 15 Sep 2019 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/optimal-utilization-of-thromboelastrography-teg/2019/09 Ananya Datta Mitra, M.D.
        Denis M. Dwyre, M.D.

        Thromboelastography (TEG) was first described by Dr. Hartert in Germany almost 60 years ago. However, it came to clinical practice almost 25 years following its discovery [1]. This test measures the viscoelastic changes associated with the entire coagulation process and provides a global assessment of the hemostatic function. It was initially used in 1980s for monitoring coagulation in orthotopic liver transplantation. Over the following decades, technological advances have resulted in significant improvements in the test leading to its current use as a point-of-care device, however, keeping the basic principles unchanged. There are multiple applications of TEG in clinical care including but not limited to surgical and trauma patients, liver transplantation, acute and chronic liver disease, optimal blood utilization, coagulopathies (like hemophilia), cardiac bypass, sepsis, pregnancy and postpartum hemorrhage, neonatal care, veterinary medicine and drug monitoring [2]. Although TEG has been used widely to guide hemostatic therapies, and some consider it to be superior to conventional laboratory assays, many clinicians and authors claim that these tests are not completely validated. In this blog, we are going to review TEG as a viscoelastic global tool regulating hemostatic therapies and its optimal utilization in current clinical practice.

        General principles of TEG

        In traditional kaolin activated TEG, whole blood at 37 degrees centigrade is placed into a sample cup in which a pin is suspended by a torsion wire. The cup then rotates either clockwise or anticlockwise. As the blood clots, platelets and fibrin forming in the cup adhere to the pin which is measured as torque via a torsion wire. Results are graphically displayed in real-time. TEG with platelet mapping is a modality to measure platelet function, especially in patients taking antiplatelet medications. It consists of three components: arachidonic acid (AA), which is sensitive to aspirin, adenosine diphosphate (ADP), which is sensitive to clopidogrel, and an activator substituting for thrombin, used for comparison to calculate percent inhibition. For TEG platelet mapping, results show underlying hemostasis but also shows receptor-specific platelet function and inhibition. However, TEG has not been shown to consistently predict total bleeding risk.

        Current indications of TEG [3]

        1. Express the function of and identify dysfunction in the patient's hemostasis system
        2. Reduce the use of unnecessary blood products and reduce thrombotic complications
        3. Distinguish between anatomical (surgical) and coagulopathic bleeding
        4. Differentiate primary from secondary fibrinolysis, including the consumptive phase
        5. Provide a personalized platelet function and inhibition assessment for patients known to be on anti-platelet medications

        General limitations of TEG

        In this modern era of personalized healthcare, an ideal test on blood coagulation still does not exist. Although TEG has been in clinical use and has convincingly demonstrated its usefulness to help improve outcomes in cardiac surgery, Cochrane database systematic review of 9 RCTs with a total of 776 participants, found a decreased amount of bleeding when TEG were utilized but without a decrease in morbidity or mortality [4].

        Blood coagulation is a complex process and involves the interactions between the tissue factor and the endothelium with other components like blood flow, vessel size, and local vessel wall biology that determine the quantity and functional activity of the membrane-bound pro- and anticoagulation factors, which cannot be quantified in vitro. The main principle behind TEG is measuring blood coagulation in vitro, with or without an additional activator, rather than flow within an endothelialized vasculature. Thus, the TEG tracing is not refle]]> 0 <![CDATA[The Pre-Operative Type and Screen: Why Timing is Everything!]]> https://health.ucdavis.edu/blog/lab-best-practice/the-pre-operative-type-and-screen-why-timing-is-everything/2018/09 Sat, 15 Sep 2018 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/the-pre-operative-type-and-screen-why-timing-is-everything/2018/09 Sarah Barnhard M.D., Associate Medical Director of Transfusion Services
        Brandon Thomas C.L.S., M.T.(ASCP), Transfusion Services Supervisor
        David Unold M.D., Transfusion Services Faculty
        Grace Monis M.D., Ph.D., Medical Director of Therapeutic Apheresis
        Hanne Jensen M.D., Medical Director of Transfusion Services

        Background

        The clinical laboratory is one of the most highly regulated services in the hospital, and transfusion services is one of the most highly regulated areas in the lab! The FDA's Center for Biologics Evaluation and Research (CBER) regulates biological products for human use under applicable federal laws1. The AABB sets standards for transfusion medicine2 and these are incorporated as state law in California3. Other organizations such as the College of American Pathology also set standards for our laboratory's accreditation4. All this oversight ensures our laboratory's processes and protocols produce accurate results.

        Pre-transfusion testing requirements are delineated by all regulatory, accreditation, and standards organizations. Pre-transfusion testing is incredibly important, because for each transfusion given within UC-Davis Health (over 20,000 RBC units in 2017!) this testing ensures the donor blood is compatible with the recipient.

        The “type and screen” (T&S) is the first step in determining donor/recipient compatibility. Even though it is one order and one blood draw, it is 3 different tests. The T&S determines the ABO blood type of the patient, determines the Rh blood type of the patient (specifically, whether the D antigen in the Rh blood group is present or not), and screens the patient for any non-ABO antibodies that may have developed against donor red blood cells.

        The blood supplier tests the ABO blood type, the Rh blood type, and the non-ABO antibody screen for all blood donors too5. UCDMC's transfusion services laboratory even confirms blood donor testing!

        Does all of this fulfill the regulatory requirements for pre-transfusion testing? Partially. The type and screen must also be drawn at the right time. AABB's standards state6:

        5.14 Pre-transfusion tests for allogeneic transfusion shall include ABO group and Rh type. In addition, for whole blood, red blood cells and granulocyte components, pretransfusion testing for unexpected antibodies to red cell antigens shall be performed.

        5.14.3.1 When clinically significant antibodies are detected additional testing shall be performed.

        5.14.3.2 A sample shall be obtained from the patient within 3 days of the scheduled transfusion in the following situations.

        1) If the patient has been transfused in the preceding 3 months with blood or a blood component containing allogeneic red cells.

        2) If the patient has been pregnant within the preceding 3 months.

        3) If the history is uncertain or unavailable.

        Lab Best Practice

        So how do we translate these regulatory requirements into best practices for patients being scheduled for elective surgery?

        The patient should be evaluated for their risk of needing blood transfusions. The maximum surgical blood order schedule (MSBOS) is used to determine general risk based on the procedure7. Patients with a >5% probability of transfusion based on procedure type are considered of sufficient risk to prepare for a possible transfusion8. Pre-op clinical evaluation is prudent to assess for co-morbidities that impact this risk stratification as well.

        For patients undergoing applicable elective procedures, a type and screen should be drawn within 3]]> 0 <![CDATA[Cytochrome P450 Genetic Testing: Should we be testing everyone?]]> https://health.ucdavis.edu/blog/lab-best-practice/cytochrome-p450-genetic-testing-should-we-be-testing-everyone/2017/12 Fri, 15 Dec 2017 00:00:00 -0800 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/cytochrome-p450-genetic-testing-should-we-be-testing-everyone/2017/12 Nam K. Tran, Ph.D., M.S., F.A.C.B., Director of Clinical Chemistry and POCT
        Kristin Grimsrud, D.V.M., Ph.D., Assistant Professor, Dept. of Pathology and Laboratory Medicine
        Jeffrey Gregg, M.D., Senior Director of Clinical Pathology and Director of Molecular Diagnostics
        Jennifer Jeffries, C.L.S., Send Outs Supervisor

        Introduction

        Cytochrome P450s (CYP) are a superfamily of heme containing enzymes that play a role in drug metabolism, hormone synthesis/breakdown, and other metabolic pathways.1 The role of CYP in drug metabolism has gained renewed interest for precision medicine and pharmacogenetic testing to provide means to tailor drug therapy. Different medications can be metabolized by various subfamilies of CYP to facilitate bioactivation and alter pharmacokinetic properties. For example CYP3A4 is responsible for metabolizing over 60% of prescribe medications, while CYP2D6 is known to have variants that may present as rapid or slow metabolizers. Patients that have “fast” phenotypes can rapidly metabolize medications and result in sub-therapeutic drug levels. Alternately, slow metabolizers may increase drug half-life and lead to toxicity. Early studies involving the breast cancer drug, tamoxifen, suggested patients with CYP2D6 slow metabolizer genotype may be at risk for treatment failures.2 Since then, the role of CYPs for other medications, including opioids has gained interest—renewing demand for CYP genetic screening programs.3

        Lab Best Practice

        As the field of precision medicine and pharmacogenetics grows, we see an increased use of CYP testing. However, unregulated CYP screening of patients is currently not recommended. First, the majority CYP genetic analyses are performed at referral laboratories—making these tests both expensive and requiring extended turnaround times (i.e., 2-5 days). Next, Centers for Medicare and Medicaid Services (CMS) may not reimburse for CYP testing (e.g., CYP2C9 for warfarin therapy)4, therefore, the list of referral laboratories performing CYP screening remains limited. Also, despite early evidence of CYP2D6 polymorphisms impacting tamoxifen therapy, later studies suggest CYP2D6 genetic variants are only part of the picture and the role CYP testing remains controversial. 5-7 Lastly, not all CYP assays are created the same. The majority of CYP analyses are considered “Laboratory Developed Tests”, which have been known to differ between facilities. In other words, not all tests are created equal.

        At UC Davis Medical Center (UCDMC), there is a growing demand for CYP testing. Cases of CYP genetic testing typically involve patients refractive to pharmacotherapy. However, many of these requests are not backed by objective laboratory data to suggest the presence of CYP variants. In one case, CYP testing was requested for a suspected slow metabolizer, however upon drug level analysis by the laboratory, it was found the patient had no drug levels at all. Obviously, it is not possible to be a slow metabolizer or to determine CYP phenotype if the patient has no detectable drug levels. A more common scenario is a request for the “CYP panel” that looks at multiple enzymes and their variants since a patient is reportedly refractive to one medication. Not all CYPs play a role for a single drug, and some drugs are metabolized by more than one CYP subfamily versus another. To this end, the UCDMC Clinical Laboratory will only approve CYP testing if at least one of the following conditions are met:

        • In addition to clinical presentation, objective laboratory data is available such as parent drug and/or metabolite level testing showing abnormal concentrations to suggest the presence of a CYP variant.
        • There is a scientific basis]]> 0 <![CDATA[Molecular Pathogen Detection at the Point of Care: Next Generation Flu Testing!]]> https://health.ucdavis.edu/blog/lab-best-practice/molecular-pathogen-detection-at-the-point-of-care-next-generation-flu-testing/2017/11 Thu, 16 Nov 2017 00:00:00 -0800 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/molecular-pathogen-detection-at-the-point-of-care-next-generation-flu-testing/2017/11 Nam K. Tran, Ph.D., M.S., F.A.C.B., Director of Clinical Chemistry and POCT
          Larissa May, M.D., M.S.P.H., M.S.H.S., Professor and Director of Emergency Department Antimicrobial Stewardship
          Shelley Gillot, CLS Specialist (POCT)
          Stacy Yee, CLS Specialist (POCT)

          Introduction

          For years, antigen-based rapid influenza diagnostic tests (RIDT) have been used to identify patients with influenza at the point of care. These RIDTs employ antibodies against proteins found on the influenza virus to generate results in less than 30 minutes.1 Despite the convenience of RIDTs, concerns have been raised by the Centers for Disease Control and Prevention (CDC) about the clinical sensitivity of these assays. False negative rates are common especially when influenza rates are high. Novel strains of influenza virus, such as the H1N1 swine flu, have been shown to be undetectable by RIDTs.1,2 Due to these concerns, the United States Food and Drug Administration (FDA) reclassified RIDTs from being a “Class I” device to a “Class II” device—requiring additional safeguards to improve overall RIDT performance.3 These new regulations will go into effect January 12, 2018 and have forced many manufacturers to discontinue production of RIDTs. Hospitals may continue to use RIDTs until their existing stockpiles are depleted, however, an RIDT alternative will necessary for the next influenza season.

          Conceptual Workflow for Molecular Pathogen Detection using Polymerase Chain Reaction

          Figure 1. Conceptual Workflow for Molecular Pathogen Detection using Polymerase Chain Reaction: The figure illustrates a sample containing pathogens. Nucleic acids represented as DNA are cycled through denaturation by heat (?) (i.e. separate the standards of DNA), amplification by PCR via DNA polymerase (DNA pol), and cooled to reform doubled standard DNA steps. In the end, the nucleic acids are amplified many times and identified (ID).

          Laboratory Best Practice

          Molecular respiratory pathogen detection technologies have been available for nearly a decade. Many of these molecular assays employ polymerase chain reaction (PCR) to detect pathogen nucleic acids (Figure 1). However, these highly sensitive assays were labor intensive and could only be operated in the clinical laboratory environment. Interestingly, in January 2015, the first point-of-care (POC) molecular influenza A/B test was approved by the FDA—spawning a new generation of easy-to-use rapid molecular testing at the bedside.4 Rather than using antibodies against proteins found on the influenza virus (i.e., RIDTs), molecular assays target the virus' genetic make-up. In the case of influenza virus, molecular assays amplify the organism's ribonucleic acid (RNA) for detection. Using the same nasopharyngeal sample type, these molecular POC systems detect and differentiate influenza A/B in as little as 15 minutes. These POC molecular assays are not limited to influenza testing and may be employed to detect respiratory syncytial virus (RSV) Group A Streptococcus, and also Clostridium difficile. Beginning January 2018, UC Davis Medical Center will begin deployment of a PCR-based POC influenza and RSV test for the Emergency Department and our Primary Care Network.5 We discuss laboratory best practices utilizing these innovative technologies to ensure optimal patient care and test utilization:

          Molecular Influenza Testing in Clinic Settings: Molecular influenza testing is not required for every patient that presents with signs and symptoms of influenz]]> 0 <![CDATA[Anti-Factor Xa for Monitoring of Unfractionated Heparin Therapy]]> https://health.ucdavis.edu/blog/lab-best-practice/anti-factor-xa-for-monitoring-of-unfractionated-heparin-therapy/2017/10 Mon, 16 Oct 2017 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/anti-factor-xa-for-monitoring-of-unfractionated-heparin-therapy/2017/10

          Andy D Jones, M.D., Pathology Resident
          Sarah Barnhard, M.D., Assistant Professor and Associate Director, Transfusion Services
          Grace Monis, M.D., Ph.D., Assistant Professor, Transfusion Services

          Background
          Recent developments in the field of coagulation testing have shown that the test previously used to monitor therapeutic dosing of unfractionated heparin (UFH) in hospitalized patients has serious drawbacks. Developments in laboratory testing, reduced cost of reagents, and comparative studies have suggested superiority in monitoring UFH with Antifactor Xa levels as compared to the more traditional activated partial thromboplastin time (aPTT).

          Table 1: Critical values and interference values for Antifactor Xa assay at UC Davis Medical Center.
          Table 1

          Mechanism of action of UFH
          Heparin is a naturally-occurring glycosaminoglycan polymer that has a physiologic anti-coagulant function.1 It exists as polymers of varying sizes (20,000 – 50,000 kDa) naturally, and as manufactured unfractionated heparin. (Fractionation produces concentrations of heparin molecules of similar sizes, as in low molecular weight heparin.2)

          As an indirect thrombin inhibitor, the eccentric pentasaccharide sequence of the heparin molecule binds to anti-thrombin, causing a conformational change. This change increases the activity of antithrombin (ATIII), which has effects on both activated thrombin (Factor IIa) and Factor Xa. The net result is an inhibition of the coagulation cascade (leading to anti-coagulation).1

          While heparin is a naturally occurring molecule, unfractionated heparin is most commonly manufactured from the mucosal tissues of slaughtered pigs and cows (gut and lung mucosa, respectively). It has a short half-life (~ 2 hours), is low cost, has non-renal elimination, and is readily reversible in the inpatient setting.3

          Historical measurement of UFH activity
          Traditional laboratory measurement of heparin activity was accomplished via the Activated Partial Thromboplastin Time (aPTT) test. The test involves obtaining a patient’s plasma (in a negatively-charged centrifuged sodium-citrate tube). The citrate reversibly chelates the available calcium, leading to inhibition of activation of the coagulation proteins (i.e. ensuring the sample doesn't irreversibly clot before arriving at the lab). Excess calcium is then added to the tube, and the clotting time measured.1 This provides an estimate of the functional performance of the intrinsic pathway. Typical estimates of appropriate anticoagulation (depending on patient populations) range from 1.5 – 2.5 fold increase in clotting time over the patient’s baseline.2

          Problems with aPTT in UFH monitoring
          While the aPTT assay has long been used in monitoring patient response to UFH, laboratorians have noticed an increasing array of complications that render the aPTT an unfavorable choice in this scenario.

          Pre-analytic variables that reduce the efficacy of aPTT include diurnal variation coagulation factors, which can result in a spuriously low aPTT (morning sampling) or high aPTT (evening sampling); variations in the concentration of citrate in the collection tube, which can result in a spuriously high aPTT; and underfilled sample tubes, which can result in a spuriously high aPTT.3

          Analytic variables have also been shown to have a significant impact on aPTT monitoring of UFH therapy, including the reagents used (including variations from lot to lot) and the instrument used. Whil]]> 0 <![CDATA[Best Practices for Cardiac Troponin Testing: Part I]]> https://health.ucdavis.edu/blog/lab-best-practice/best-practices-for-cardiac-troponin-testing-part-i/2017/07 Fri, 28 Jul 2017 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/best-practices-for-cardiac-troponin-testing-part-i/2017/07

          Nam K. Tran, Ph.D., M.S., F.A.C.B., Associate Professor and Director of Clinical Chemistry
          Ezra Amsterdam, M.D., Distinguished Professor, Cardiovascular Medicine
          Stacey Howell, M.D., Resident Physician, Internal Medicine
          Bryn Mumma, M.D., MAS, Assistant Professor, Emergency Medicine
          Javier E. López, M.D., M.A.S., Assistant Professor, Cardiovascular Medicine

          Background

          Cardiac troponin (cTn) is the preferred biomarker that aids in the diagnosis of acute myocardial infarction (MI) as defined by the 3rd Universal Definitions of MI.1 This month's blog (Part 1 of 3) will review the clinical use of the current cTn assay and discuss the transition to new units of measurement. Future blogs will focus on a next generation of cTn assays.

          Recommendations from the 3rd Universal Definition of MI

          Acute MI should be diagnosed when there is clinical evidence of myocardial ischemia and injury that culminates in necrosis.1 Myocardial ischemia is determined by one of the following: (a) clinical symptoms, (b) new or presumed new significant ST-segment-T-wave changes or new left bundle branch block, (c) development of pathological Q waves on electrocardiogram, (d) imaging evidence of new loss of viable myocardium or new regional wall motion abnormality, or identification of an intracoronary thrombus by angiography or autopsy.

          Cardiac troponin is the most specific blood marker of myocardial injury. To diagnose a MI, a rise and/or fall of cTN level above the 99th percentile of the upper reference limit (URL) needs to be associated with at least one of the above clinical findings of ischemia. At the 99th percentile level the assay should exhibit an imprecision of ≤10% coefficient of variation (CV). This 99th percentile level is often defined from a normal population provided by the assay manufacturer and verified locally by hospital laboratories upon implementation. At the 99th percentile mark, 99% of subjects have a level below the cutoff. Assay imprecision helps determine if cTn changes are due to myocardial injury or due to analytical and biological variability. A CV ≤10% at this 99th percentile is considered sufficient to detect “real” changes in cTN. Assays with CV >10% make determination of change more difficult and should not be used.

          Recommended cTn assay units

          The 3rd Universal Definition of MI establishes that cTn results be presented in nanograms per liter (ng/L) to result in whole numbers. For example, a current cTn level of 0.04 ng/mL would be the same as 40 ng/L when the units are changed. This change will reduce misinterpretations when future next-generation assays intending to improve sensitivity are made available.

          Laboratory Best Practice at UC Davis Medical Center

          Our clinical laboratory currently employs the 99th percentile cTn cutoff where assay imprecision is ≤10% (0.04 ng/mL). Starting on September, cTn units will change to ng/L as recommended by our multidisciplinary team including Cardiovascular Medicine, Emergency Medicine, and Pathology and Laboratory Medicine. You are reading this blog as a part of an educational program to members of our clinical community prior to this change. In future blogs, we will discuss a next-generation high-sensitivity cTn assay, and its anticipated implementation in 2018 at UC Davis Medical Center.

          References

          1. Thygesen K, Alpert JS, Jaffe AS, et al.; Writing Group on behalf of the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction. Third universal definition of myocardial infarction. Glob Heart. 2012 Dec;7(4):275-95.
          0 <![CDATA[Biotin Interference in Clinical Immunoassays: The Dose Makes the Interference]]> https://health.ucdavis.edu/blog/lab-best-practice/biotin-interference-in-clinical-immunoassays-the-dose-makes-the-interference/2017/06 Thu, 15 Jun 2017 00:00:00 -0700 Laboratory Best Practice blog https://health.ucdavis.edu/blog/lab-best-practice/biotin-interference-in-clinical-immunoassays-the-dose-makes-the-interference/2017/06 Guofeng “George” Gao, M.D., Resident Pathologist
          Nam Tran, Ph.D., M.S., F.A.C.B., Director of Clinical Chemistry and POCT

          Background

          Biotin, also known as Vitamin B7, is a co-factor in fatty acid metabolism, amino acid degradation, and gluconeogenesis. The recommended daily intake (RDI) for biotin is extremely low—about 30 µg/day.1 Given the low RDI, biotin deficiency is rare. In recent years, there has been a surge in biotin supplementation for various reasons including as a beauty product. Biotin is now found in multivitamin preparations, as well as cosmetic products. Interestingly, there is discussion that high dose biotin (100-300 mg/day) may have a role in treating certain diseases such as Multiple Sclerosis (MS).2

          The increased use of biotin by patients creates a potential problem with modern laboratory immunoassays.1 Biotin is used to conjugate antibodies for routine immunoassay tests including thyroid stimulating hormone (TSH), cardiac troponins, carcinoembryonic antigen (CEA), among many others. In vitro diagnostic (IVD) manufacturers leverage biotin's affinity to streptavidin to conjugate large molecules such as antibodies for their assays. There have been reports of biotin interference causing misdiagnosis of thyroid disorders.3 These case studies generate great concern not only for endocrinology patients, but also those with suspected myocardial infarction being tested with a biotinylated cardiac troponin immunoassay.

          Lab Best Practice

          Biotinylated immunoassays are common, with biotin being one of many interfering substances that can impact laboratory testing. First and foremost, IVD manufacturers have evaluated the performance of their assays against biotin at concentrations at or near 56 ng/mL.4 At 56 ng/mL, we would not expect any biotin interference if patients meet and do not exceed the RDI of 30 µg/day. Furthermore, the rapid clearance of biotin (half-life =2 hours) works to our favor whereby even at 5 and 10 mg/day concentrations, the levels should not reach levels to interfere with modern biotinylated immunoassays. Concerns may arise in the emergency care setting for ruling out myocardial infarction. However, again, the rapid clearance of biotin works in the assay's favor. Studies have shown that most patients presenting to the emergency department had experienced chest pain hours before admission5—providing time for any biotin to clear the body. Additionally, serial cardiac troponin testing current requires at least two measurements to be made over a few hours. Therefore it is unlikely that biotin would impact cardiac troponin testing. At UCDMC, we conducted a pilot interference study evaluating the impact of biotin therapy on TSH testing. We compared the performance of a biotinylated assay (cobas, Roche Diagnostics) against an assay that does not use biotin conjugation (i1000, Abbott Laboratories). There was no statistical or clinical difference between the assays among patients taking between 2.5 to 10 mg/day of biotin (Figure 1). To this end, the lab best practice for managing patients with biotin therapy is to: (a) educate patients that they should not take biotin before having blood drawn for laboratory testing, and (b) clinicians should be aware of biotin interference, but the concentration of biotin (not just the presence of the molecule) makes the interference.

          Figure 1. The figure illustrates no statistical or clinical significance in performance between the cobas and i1000 platforms among patients taking 2.5 to 10 mg/day of biotin.