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 transferred to her baby is prepared. The red blood cell unit will be a type O unit that is leukocyte reduced, CMV negative, sickle cell trait negative, irradiated, washed, and crossmatch compatible. The red blood cells are washed and concentrated to achieve a high hematocrit before combining it with the plasma. The washed red cells are sent to hematology where the hematocrit is checked. Type AB, CMV negative, irradiated plasma is thawed out for the reconstitution. The final reconstituted whole blood product has a typical target hematocrit of 40-50%. The entire workflow can take up to 3 hours to prepare the reconstituted whole blood unit, but sometimes can be faster. A key consideration for this request is the timing since in some cases, the neonates are being transferred in from an outside hospital. Additionally, once the whole blood is reconstituted, it only has a shelf life of 24 hours from when the RBC unit was washed. In the case the reconstituted whole blood unit is no longer needed, the product is wasted since whole blood is not transfused to general patients. Thus, the certainty of completing the exchange transfusion must be determined by the clinical team. It is recommended that the total bilirubin should be checked to help make the decision. If the bilirubin is down trending, a reconstituted whole blood unit may not be necessary.

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.

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.

• 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.

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].

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]. Because semiquantitative SARS-CoV-2 antibody assays are not standardized, and the performance characteristics of each semiquantitative SARS-CoV-2 antibody test is uniquely established, results from different semiquantitative SARS-COV-2 antibody assays are not comparable [1,5,6]. As stated in previous best practice blogs articles, 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, to guide personal protective equipment use, or for return-to-workplace decisions [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

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 the pathogen identity. It is important to keep in mind that some pathogens present a significantly higher risk than others.

1. Early onset (less than three months after surgery):
   • Staphylococcus aureus
   • Anaerobes
   • Polymicrobial infection
2. Delayed onset (three to twelve months after surgery):
   • Coagulase-negative staphylococci
   • Cutibacterium (Propionibacterium) species
   • Enterococci spp.
3. Late onset (greater than twelve months after surgery)
   • Staphylococcus aureus
   • Gram-negative bacilli
   • Beta-hemolytic streptococci

Pathogenesis

The pathogenesis of prosthetic joint infections by pathogens is dependent on the formation of biofilm. Biofilms are complex communities of microorganisms embedded in an extracellular matrix that forms on surfaces. Pathogens will adhere to orthopedic hardware, and proliferate with elaboration of exopolysaccharides known as glycocalyx, which after a certain period, will coalesce into biofilm. This biofilm microenvironment serves as a barrier that renders host defenses and antimicrobials less effective. Complicating matters further, pathogens hidden deep in the biofilm have characteristic indolent low metabolic rate which also prevent accurate culture identification. Together, S. epidermis, S. aureus and Pseudomonas aeruginosa make up almost 75% of the biofilms found in medical devices. S. aureus and Staphylococcus epidermis are the most common biofilm-forming bacteria.

Diagnosis of prosthetic Joint Infection

As mentioned previously, there are a number of criteria published, but given its simplicity and wide use, the Musculoskeletal Infection Society (MSIS) 2011 diagnostic criteria appear to be favored by the orthopedic specialty to help establish PJI.

Preoperative Evaluation of Suspected Prosthetic Joint Infection:

1. Clinical manifestation
   • Patient with joint prosthesis and symptoms of infection
     • Joint pain, warmth, erythema, induration, incision site edema, sinus tract, persistent wound drainage, wound dehiscence, joint effusion, and/or fever

Then

2. Plain radiograph
   • should be performed in all patients with a suspected prosthetic joint infection
   • Radiographic findings suggesting possibility of PJI:
     • abnormal lucency larger than 2 mm in width at the bone-cement interface
     • changes in the position of prosthetic components
     • cement fractures
     • periosteal reaction
     • motion of components on stress views
   • leukocyte scans, positron emission tomography (PET) scans, computed tomography (CT) scans, magnetic resonance imaging (MRI) scans, or bone scans are not useful for routine diagnostic evaluation in most cases of suspected PJI
3. Sedimentation rate or C-reactive protein (CRP) tests
   • When suspected prosthetic joint infection is not clinically apparent, Sedimentation rate or C-reactive protein (CRP) tests should be performed.
   • Timing dependent
     • early-onset PJI
       • ESR is not useful
       • CRP is often >100 mg/L
     • delayed and late onset PJI
       • ESR is often >30 mm/hour
       • CRP is often >10 mg/L
     • if both ESR and CRP are negative, the likelihood of PJI is low.
     • PJI may be present in the setting of normal ESR and CRP
       • Possible late PJI
       • infection due to pathogens of low virulence
       • prior antibiotic use
       • immunosuppression

Warning:

• Following joint replacement surgery, the CRP may require two to three weeks to return to normal preoperative values, and the ESR may require up to a year to return to normal preoperative values.
  Interpret with caution in the setting of coexistent chronic inflammatory disease, which can also elevate serum inflammatory markers.

Followed by

4. Arthrocentesis:
   • Arthrocentesis is performed for all patients with suspected acute prosthetic joint infection.
     • Unless the diagnosis is clinically evident, surgery is planned, and antimicrobials can be safely withheld prior to surgery.
     • May not be necessary if surgery is planned and the result is not expected to alter management.
   • Arthrocentesis is advised in patients with a chronic painful prosthesis with unexplained elevated sedimentation rate or CRP level.
   • If the patient is medically stable, withhold antimicrobial therapy for at least 2 weeks prior to collection of synovial fluid for culture will increase the likelihood of recovering an organism.
   • Synovial fluid may be sent for culture in a sterile tube (ideally a red-top tube with no additives and a tube with an anticoagulant such as ethylenediaminetetraacetic acid to guard against clotting) or in blood culture bottles. If blood culture bottles are used, synovial fluid also should also be sent in a sterile container for Gram stain. Use of blood culture bottles may increase the likelihood of recovering nonpathogenic skin contaminants; in such cases, culture results should be interpreted in the context of the Gram stain result.
5. Interpretation of synovial fluid analysis
   • Time of onset dependent
     • early-onset PJI : synovial fluid cell count is often >10,000 cells/microL (>90 percent neutrophils)
     • delayed- and late-onset PJI : the synovial fluid cell count is often >3000 cells/microL (80 percent neutrophils)
   • The most accurate cell count threshold for knee PJI:
     • 1630 cells/microL (sensitivity and specificity 84 and 82 percent, respectively)
     • with 60 percent neutrophils (sensitivity and specificity 80 and 77 percent, respectively) [73].
   • The most accurate cell count threshold for hip PJI:
     • 2582 cells/microL (sensitivity and specificity 80 and 85 percent, respectively)
     • with 66 percent neutrophils (sensitivity and specificity 82 percent).
   • Use of saline irrigation to obtain synovial fluid may confound the cell count.

Figure 1. Summary of PJI evaluation.

Intraoperative evaluation

1. Histopathological Assessment and Interpretation During Frozen Section
   • Presence of polymorphonuclear cells is indicative of an acute inflammatory reaction supporting the diagnosis of PJI

Diagnosis requires a threshold of at least five PMNs per HPF (sensitivity >80%, specificity >90 percent) in periprosthetic tissue.

Evaluation of Suspected Prosthetic Joint Infection Culture

1. When to send culture:
   • In patients with a sinus tract, drainage (collected by aspiration) should be sent for culture; swabs from the sinus tract should not be sent given discordance with deep cultures.
   • In patients with fever or other systemic manifestations of infection, blood cultures (two sets) should be obtained.
2. Cultures of a superficial wound or sinus tract are often positive because of microbial colonization from the surrounding skin and should therefore be avoided.
3. Culture of prosthesis
   • If the prosthesis is removed, the implant or its components can be cultured in enrichment broth. However, the risk of contamination during specimen processing is high.
   • Cultures of periprosthetic tissue provide the most reliable means of detecting a pathogen
     • - sensitivity of these cultures ranges from 65 to 94 percent
4. At least three to six intraoperative tissue specimens should be sampled for culture.
5. Diagnosis of PJI established from culture
   • may be established in the setting of two or more periprosthetic cultures with phenotypically identical organisms (a combination of preoperative synovial fluid aspiration culture and intraoperative tissue culture or ≥2 intraoperative tissue cultures)
   • Growth of a virulent microorganism (eg, S. aureus) in a single specimen of a tissue biopsy or synovial fluid may also represent PJI.
6. Cultures are negative for 7-39% of patients with suspected prosthetic joint infection.
   • Cultures are more likely to be positive for early-onset prosthetic joint infection.
   • Cultures are more likely to be negative if insufficient tissue was sent or if only swabs were collected.
   • Culture yields are more likely to be diminished and negative if antibiotics were administered prior to culture collection.
   • Negative cultures can be attributed to slow growing and difficult to detect variants of staphylococci, or fastidious pathogens that includes Coxiella Burnetii, Brucellosis, Bartonellosis, Abiotrophia defectiva, Granulicatella adiacens, Mycoplasma, Mycobacteria, and Fungi
   • Cultures may be negative due to prolonged transport time to the microbiology laboratory.
   • Swab cultures have a low sensitivity and should be avoided.
7. To detect cases of low-grade infection, antimicrobial therapy should be discontinued at least two weeks before tissue specimens are obtained.
8. If revision surgery is planned, perioperative prophylaxis should not be administered until after tissue specimens have been collected for culture.

Summary

The diagnosis of prosthetic joint infection involves first establishing whether the joint is infected and then, if it is, defining the involved microorganism(s). The goal of treatment is to cure infection, prevent recurrence, and achieve a pain-free, functional joint. This can best be achieved by a multidisciplinary team, including an orthopedic surgeon, clinical microbiologist, and infectious diseases specialist. Antimicrobial agents alone, without surgical intervention, ultimately usually fail. The quality of surgical debridement is critical. Prosthetic joint infection (PJI) is a serious complication of prosthetic joint implantation. The epidemiology, microbiology, clinical manifestations, and diagnosis described here all serve as an important piece of the puzzle required to provide effective prevention, management, and treatment of prosthetic joint infection.

Resources:

1. Zimmerli W, Trampuz A, Ochsner PE. Prosthetic-joint infections. N Engl J Med. 2004;351(16):1645-1654. doi:10.1056/NEJMra040181
2. Parvizi J, Tan TL, Goswami K, et al. The 2018 Definition of Periprosthetic Hip and Knee Infection: An Evidence-Based and Validated Criteria. J Arthroplasty. 2018;33(5):1309-1314.e2. doi:10.1016/j.arth.2018.02.078
3. Osmon DR, Berbari EF, Berendt AR, et al. Diagnosis and management of prosthetic joint infection: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2013;56(1):e1-e25. doi:10.1093/cid/cis803
4. Tomas X, Bori G, Garcia S, et al. Accuracy of CT-guided joint aspiration in patients with suspected infection status post-total hip arthroplasty. Skeletal Radiol. 2011;40(1):57-64. doi:10.1007/s00256-010-0940-2
5. Berbari, E. et al. Prosthetic joint infection: Epidemiology, microbiology, clinical manifestations, and diagnosis. In: UpToDate, Post TW (Ed), UpToDate, Waltham, MA. (Accessed on 07/05/2021.)
6. Douglas R. Osmon, Elie F. Berbari, Anthony R. Berendt, Daniel Lew, Werner Zimmerli, James M. Steckelberg, Nalini Rao, Arlen Hanssen, Walter R. Wilson, Diagnosis and Management of Prosthetic Joint Infection: Clinical Practice Guidelines by the Infectious Diseases Society of America, Clinical Infectious Diseases, Volume 56, Issue 1, 1 January 2013, Pages e1–e25, https://doi.org/10.1093/cid/cis803
7. Jämsen E, Huhtala H, Puolakka T, Moilanen, Risk factors for infection after knee arthroplasty. A register-based analysis of 43,149 cases. T J Bone Joint Surg Am. 2009 Jan; 91(1):38-47.
8. Peersman G, Laskin R, Davis J, Peterson M., Infection in total knee replacement: a retrospective review of 6489 total knee replacements. Clin Orthop Relat Res. 2001 Nov; (392):15-23.

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.

Hepatitis B screening should be performed on patients with signs and symptoms suggestive of acute or chronic hepatitis as well as asymptomatic individuals with a history of or high risk of exposure (e.g., HIV or hepatitis C infected, multiple sexual partners, history of intravenous drug use, end-stage renal disease, household contacts of known hepatitis B infected individuals, chronic liver disease, incarcerated, parents who were born in geographic areas with hepatitis B prevalence >8%) or those at high risk of complications (individuals born in countries with a high hepatitis B prevalence, pregnant women, immunocompromised patients, donors for blood or tissue products, infants born to infected mothers)⁽¹⁰⁾.

Serologic testing for multiple viral antigens and the corresponding antibodies allows for identification of both acute and chronic infections. In the setting of acute infection, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B DNA (HBV DNA) are present, and IgM anti-HBc (antibody against the hepatitis B core antigen) appears shortly thereafter. The presence of IgM anti-HBc and HBsAg, without anti-HBsAg is diagnostic of acute infection (see Table 1 for summary of interpretation of serologic results). Recovery after acute infection is marked by the reduction of serum HBV DNA and seroconversion to anti-HBe and anti-HBs (accompanied by disappearance of HBeAg and HBsAg, although rarely HBsAg and anti-HBs may coexist (1.2% in a study by Lee et al)⁽¹¹⁾. Class switching of the IgM anti-HBc to IgG anti-HBc also occurs after initial infection. Therefore, individuals with a prior history of natural infection will have IgG anti-HBc as well as anti-HBs. Conversely, the presence of only anti-HBs without anti-HBc is indicative of a vaccinated individual. Individuals with a chronic infection may have persistent HBeAg, with continued circulation of HBsAg (and HBV DNA), with IgG but not IgM anti-HBc, without anti-HBs. Detection of HBsAg for longer than six months after acute infection is diagnostic of chronic infection. Although quantitation of serum HBV DNA may be used clinically to assess ongoing HBV replication, serum HBV DNA may be negative in inactive chronic HBV. Since recovery from acute infection is accompanied by disappearance of HBV DNA, PCR testing for quantitation of serum HBV DNA occurs more frequently in the assessment of chronic infection rather than the acute setting or during initial screening.

Table 1. Interpretation of Hepatitis B Serological Results. Adapted from UpToDate. Hepatitis B virus: Screening and diagnosis.; Terrault NA, Lok ASF, McMahon BJ, et al. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology 2018; 67:1560.

Hepatitis B e Antigen and Chronic Hepatitis B Infection

The hepatitis B e antigen (HBeAg) is a marker of replication and infectivity. In the setting of acute infection, conversion from HBeAg to anti-HBe typically occurs before the transition from HBsAg to anti-HBs. However, in the setting of chronic hepatitis B infection, seroconversion may not be observed. Chronic HBV infection is divided into multiple phases (see Figure 1 from Terrault et al)^(12-13). In the immune-tolerant phase, HBeAg is positive, HBV DNA levels are high, alanine transaminase (ALT) and aspartate transaminase (AST) levels are normal to minimally elevated, and there is minimal hepatic inflammation or fibrosis. The immune-active phase is characterized by lower HBV DNA levels, with intermittently or constant ALT/AST levels, and chronic, moderate to severe hepatic inflammation (with or without fibrosis). The inactive phase has negative HBeAg, positive anti-HBe, low HBV DNA, normal AST/ALT, and absence of hepatic inflammation (variation in degree of fibrosis). The immune-active phase can be further divided by HBeAg status, and management recommendations have been made in the past based on this basis (Terrault et al, see Figure 2 below).

Occult HBV Infection and Reactivation in the Transplantation Setting

Isolated anti-HBc without HBsAg or anti-HBs can occur during acute infection when anti-HBc is primarily IgM, when anti-HBs has waned (years after recovery from natural infection), in the setting of HBsAg mutations which evade testing or with low titers of HBsAg which are below testing threshold cutoffs. Testing for anti-HBc can also help detect patients with occult infection, that is, patients with detectable HBV DNA by PCR but who are HBsAg seronegative. These patients may have low serum HBV DNA levels, however transplantation of livers from these individuals may result in infection upon transplantation⁽¹⁴⁾. Additionally, patients with occult hepatic infection may experience reinfection upon receipt of immunosuppressive regimens as in the case of bone marrow or stem cell transplant⁽¹⁵⁾. Therefore, diligent hepatitis B screening of patients during transplantation evaluation is appropriate. Patients positive for HBsAg and HBeAg are at higher risk for reactivation in the transplant setting^(16-17).

Testing Platform at UC Davis Health

The UC Davis Health Hepatitis B e antigen and antibody test are now performed on the Diasorin Liason XL instrument on serum or plasma samples – providing faster turnaround times and exhibiting excellent clinical performance.

References

1. Castaneda D, Gonzalez AJ, Alomari M, Tandon K, Zervos XB. From hepatitis A to E: A critical review of viral hepatitis. World J Gastroenterol. 2021 Apr 28;27(16):1691-1715.
2. Rybicka M, Bielawski KP. Recent Advances in Understanding, Diagnosing, and Treating Hepatitis B Virus Infection. Microorganisms. 2020; 8.
3. Revill PA, Tu T, Netter HJ, Yuen LKW, Locarnini SA, Littlejohn M. The evolution and clinical impact of hepatitis B virus genome diversity. Nat Rev Gastroenterol Hepatol. 2020;17:618–634.
4. UpToDate. Hepatitis B virus: Clinical manifestations and natural history. Accessed on May 18, 2021.
5. Centers for Disease Control and Prevention. Hepatitis B. Accessed on May 18, 2021.
6. World Health Organization Hepatitis B Factsheet. Accessed on May 18, 2021.
7. Centers for Disease Control and Prevention. Hepatitis D. Accessed on May 18, 2021.
8. European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017;67:370–398.
9. Jeng WJ, Lok AS. Should Treatment Indications for Chronic Hepatitis B Be Expanded? Clin Gastroenterol Hepatol. 2020.
10. UpToDate. Hepatitis B virus: Screening and diagnosis. Accessed on May 18, 2021.
11. Lee WM, King WC, Schwarz KB, Rule J, Lok ASF; HBRN Investigators. Prevalence and clinical features of patients with concurrent HBsAg and anti-HBs: Evaluation of the hepatitis B research network cohort. J Viral Hepat. 2020 Sep;27(9):922-931.
12. Croagh CM, Lubel JS. Natural history of chronic hepatitis B: phases in a complex relationship. World J Gastroenterol. 2014;20:10395–10404.
13. Terrault NA, Lok ASF, McMahon BJ, et al. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology 2018; 67:1560.
14. Dickson RC, Everhart JE, Lake JR, Wei Y, Seaberg EC, Wiesner RH, Zetterman RK, Pruett TL, Ishitani MB, Hoofnagle JH. Transmission of hepatitis B by transplantation of livers from donors positive for antibody to hepatitis B core antigen. The National Institute of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database. Gastroenterology. 1997 Nov;113(5):1668-74.
15. UpToDate. Hepatitis B virus reactivation associated with immunosuppressive therapy. Accessed on May 18, 2021.
16. Dhédin N, Douvin C, Kuentz M, Saint Marc MF, Reman O, Rieux C, Bernaudin F, Norol F, Cordonnier C, Bobin D, Metreau JM, Vernant JP. Reverse seroconversion of hepatitis B after allogeneic bone marrow transplantation: a retrospective study of 37 patients with pretransplant anti-HBs and anti-HBc. Transplantation. 1998 Sep 15;66(5):616-9.
17. Lau GK, Leung YH, Fong DY, Au WY, Kwong YL, Lie A, Hou JL, Wen YM, Nanj A, Liang R. High hepatitis B virus (HBV) DNA viral load as the most important risk factor for HBV reactivation in patients positive for HBV surface antigen undergoing autologous hematopoietic cell transplantation. Blood. 2002 Apr 1;99(7):2324-30.

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.
2. Boada M, López OL, Olazarán J, et al. A randomized, controlled clinical trial of plasma exchange with albumin replacement for Alzheimer's disease: Primary results of the AMBAR Study. Alzheimers Dement. 2020 Oct;16(10):1412-1425.
3. Ferri CP, Prince M, Brayne C, et al. Global prevalence of dementia: a Delphi consensus study. Lancet. 2005 Dec 17;366(9503):2112-7
4. Montine TJ, Phelps CH, Beach TG. National Institute on Aging-Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease: a practical approach. Acta Neuropathol (Berl). 2012;123:1-11
5. Kemp PM, Holmes C, Hoffmann S, et al. A randomised placebo controlled study to assess the effects of cholinergic treatment on muscarinic receptors in Alzheimer’s disease. J Neurol Neurosurg Psychiatry. 2003;74:1567-1570.
6. Tariot PN, Farlow MR, Grossberg GT, et al. Memantine treatment in patients with moderate to severe Alzheimer disease already receiving donepezil. JAMA. 2004;291:317.
7. Costa M, Ortiz AM, Jorquera JI. Therapeutic albumin binding to remove amyloid-𝛽. J Alzheimers Dis. 2012;29:159-170.
8. Roberts KF, Elbert DL, Kasten TP, et al. Amyloid-beta efflux from the central nervous system into the plasma. Ann Neurol. 2014;76:837- 844
9. DeMattos RB, Bales KR, Cummins DJ, et al. Peripheral anti-A antibody alters CNS and plasma A clearance and decreases brain A burden in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A. 2001;98:8850-5
10. DeMattos RB, Bales KR, Cummins DJ, et al. Brain to plasma amyloid-beta efflux: a measure of brain amyloid burden in a mouse model of Alzheimer’s disease. Science. 2002;295:2264-7
11. Marques MA, Kulstad JJ, Savard CE, et al. Peripheral amyloid-beta levels regulate amyloid-beta clearance from the central nervous system. J Alzheimers Dis. 2009;16:325-329.
12. Boada M, Ortiz P, Anaya F, et al. Amyloid-targeted therapeutics in Alzheimer’s disease: use of human albumin in plasma exchange as a novel approach for A𝛽 mobilization. Drug News Perspect. 2009;22:325- 339
13. Boada M, Anaya F, Ortiz P, et al. Efficacy and safety of plasma exchange with 5% albumin to modify cerebrospinal fluid and plasma amyloid-𝛽 concentrations and cognition outcomes in Alzheimer’s disease patients: a multicenter, randomized, controlled clinical trial. J Alzheimers Dis. 2017;56:129-143

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 after fine needle aspiration. J Clin Pathol 1982;35:855–857.
4. Frable WJ. Fine needle aspiration biopsy. A review. Hum Pathol 1983;14:9–28.
5. Sinner WN. Complications of percutaneous transthoracic needle aspiration biopsy. Acta Radiol Diagn 1976;17:813–828
6. Diagnostic Accuracy Studies of Fine-Needle Aspiration Show Wide Variation in Reporting of Study Population Characteristics: Implications for External Validity. Robert L. Schmidt, MD, PhD, MBA, Krishna K. Narra, MD, MS, Benjamin L. Witt, MD, Rachel E. Factor, MD, MHS Journal: Archives of Pathology & Laboratory Medicine (2014) 138 (1): 88–97

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).

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

Refuting the Race Parameter in the Calculation of eGFR

Several studies suggest that the equations used to calculate eGFR may overestimate GFR in black Americans, potentially leading to underdiagnosis of CKD. Data from the USRDS annual report⁸ and several recent studies^9-10 show that black Americans have a higher prevalence of risk factors for CKD, such as hypertension, diabetes, and abnormal adjunct laboratory markers for renal disease despite a lower prevalence of CKD (eGFR < 60 mL/min) in this population as currently calculated. This implies that the use of eGFR alone as a screening tool may not be sensitive enough to find CKD in this population.

Race is a Social Construct with Limited Utility in Addressing Biologic Variability

A number of studies have examined the transferability of the eGFR equations to non-American black populations, such as populations in Democratic Republic of Congo, Ghana, and Ivory Coast. As noted in Omuse et al., the issue is not settled, and various studies have assessed the utility of the MDRD and CKD-EPI equations, with and without the race coefficient, for use in these populations with competing results.¹¹ This in itself highlights the artificial nature of race as a social rather than biological construct and its limited utility as applied to estimating GFR. In fact, the race coefficient in the MDRD and CKD-EPI equations is presumably correcting for some other poorly characterized factor which raised the baseline serum creatinine levels in the persons in the training population for these two equations.

Although in clinical nephrology eGFR is not used in isolation to diagnosis CKD, Eneanya et al. succinctly points out the potential consequences of imprecision in eGFR in black persons, which include the way that drugs are administered or withheld based on eGFR, how quickly patients are referred to nephrology for the management of CKD, clinical trial eligibility, and access to renal transplant waitlists.

The authors also point out several flaws in the clinical categorization of race, including the nature of race as a social construct that does not correlate well to underlying genotypic or metabolic differences among persons. Race parameters in the calculation of eGFR do not account for persons who identify as multiracial or decline to answer clinicians’ questions about race. Further, clinicians may improperly assign race based on arbitrary phenotypic factors such as skin tone or hair.¹² On the laboratory side, the problems of uncertainty in this arbitrary categorization are acknowledged in the way eGFR is currently reported, with values with and without the race coefficient simultaneously reported, leaving it for the clinicians to decide how to interpret eGFR in their patient’s case.¹³

Equations based on the measurement of serum levels of cystatin C have been developed and are being analyzed in various populations; for the purpose of comparability, there now exists a reference standard for cystatin C.¹⁴ The CKD-EPIcys equation mentioned earlier was developed in 2012. One advantage of cystatin C as a biomarker is that its levels are not dependent on an individual’s muscle mass or diet, and studies have borne out that race and body surface area need not be factored into equations utilizing cystatin C to calculate eGFR.¹⁵ There remain concerns about the accuracy of cystatin C, particularly in the setting of comorbid heart and liver disease which are both common in patients with CKD.¹⁶

Review of UC Davis Health Data

As part of the UCDH Working Group to address the question of using race in the calculation of eGFR, the Department of Pathology studied the feasibility of changing or eliminating the reporting of race from eGFR reporting. Approximately 1 month of serum creatinine values and corresponding eGFR data was obtained from Beaker, along with each patient’s age and sex, from 373 patients over age 18 who provided 497 specimens (range: 1-10 specimens per patient). Our findings showed, as expected, a higher prevalence of CKD as defined by eGFR < 60 mL/min/1.73m² utilizing the eGFR equation without a race parameter (NAA equation) compared to the equation with the parameter for race (AA equation). Patients’ eGFRs were classified into subgroups corresponding to the major breakpoints in the staging of CKD (>90, 60-90, 30-60, and <30 mL/min/1.73m²). Our data showed that a large number of patients in higher risk groups would either be reassigned from stage 3 to stage 4 CKD, or reassigned from CKD-negative to CKD-positive, simply by removing the race parameter from the calculation of their eGFR.

The tests were compared in terms of positive and negative agreement¹⁷ to evaluate whether utilizing the NAA equation alone would be adequately sensitive (no missed cases of CKD) without adding too many new cases, which could either be cases of CKD that were previously missed or false positives. There was perfect positive agreement utilizing the NAA equation, analogous to 100% sensitivity. Negative agreement was 91.2% for males and 93.4% for females, meaning that overall only a relatively small number of patients were reassigned to CKD positive status. These cases, as noted above, could represent cases of CKD that would be missed if the AA equation was utilized to calculate eGFR. However, they could also represent false positives.

To determine what the impact of utilizing the NAA equation exclusively on this population might be, a chart review was performed on the patients who were CKD positive by the NAA equation but CKD negative by the AA equation. Risk factors for CKD, such as hypertension and diabetes were common among these patients. Other risk factors included inflammatory arthritis and other autoimmune diseases, renal donors, and renal transplant recipients. To summarize, many of the patients in this group with borderline eGFRs have significant risk factors for CKD and at the least warrant close follow-up.

A chart review was not performed on patients with higher eGFRs (> 60 mL/min). It is worth noting that the largest number of patients reassigned to a lower risk group by the AA equation were those with eGFRs between 60-90 mL/min as calculated by the NAA equation. It is unclear what the clinical consequences would be of reassigning these patients to the higher risk group by calculation with the NAA equation. Importantly and as noted earlier, eGFR is not diagnostic of CKD in isolation for patients with eGFRs > 60 mL/min. In clinical practice, nephrologists take a comprehensive look at the clinical picture, adjunct laboratory values, and trends in renal biomarkers/eGFR to determine the diagnosis of CKD.

Action Plan

The UCDH Working Group, with an eye to national standards being developed by a national working group addressing this same problem (National Kidney Foundation/American Society of Nephrology Task Force on Reassessing the Inclusion of Race in Diagnosing Kidney Disease), will change the reporting of calculated eGFR. A single eGFR calculated without the race parameter will be reported as a discrete value in the chart. Using this calculation is more sensitive for the detection of CKD. This supports the goal of narrowing some of the disparities in CKD, including the higher prevalence of CKD in African Americans compared to white patients, the higher rate of progression to ESRD, and transplant eligibility.

Additionally, in a comment, without perpetuating racial phenotypes, there is acknowledgement that eGFR based on creatinine may have person-to-person variability. A recommendation for alternate measures of GFR, such as cystatin C or measurement of kidney clearance, is given for situations in which more precision is desired. Acknowledging variability in creatinine-based calculations of eGFR will give the clinician the flexibility for shared decision making in initiating therapy in these patients, recommendation for transplant waiting lists, and ensuring clinical trial eligibility based on the patient’s overall clinical status instead of a single number.

Clinical Impact of these Changes

The change to a single eGFR value calculated by CKD-EPI without a parameter for race is intended to increase the sensitivity of the eGFR calculation for chronic kidney disease. From a practical standpoint, more patients will appear to have lower eGFRs than prior to this change by approximately 16%. For African-American patients, this may cause anxiety or concern over what an apparent new abnormal lab value. Patients should be reassured that although eGFR is important and useful in interpreting creatinine, the diagnosis of chronic kidney disease does not hinge on any single calculated eGFR, and that the entire clinical context, including medical history, medication history, and adjunct laboratory markers of renal disease are crucial to the diagnosis.

However, there are a number of clinical contexts in which this small change in eGFR can be of crucial importance. Patients being considered for renal transplant will be more likely to reach transplant threshold and in a shorter time when the race parameter is not used,¹⁸ as guidelines advise an eGFR threshold of <20 mL/min before moving to transplantation. On the other hand, the Kidney Donor Risk Index (KDRI) uses creatinine to predict the risk of graft failure in deceased donor renal allografts, and it also uses race independently in its calculation. African-American race has a hazard ratio of 1.196 in the KDRI calculation (approximately 20% increased risk of graft failure). However, a recent study suggested that race is, as in the case of eGFR, likely a ‘convenient’ substitute for some other poorly characterized negative prognostic factor. Julian et al. convincingly suggest that polymorphisms in apolipoprotein L1 (APOL1) may in part account for poorer outcomes in some allografts from African-American donors.¹⁹

Chronic renal disease frequently co-occurs with cardiac disease, and eGFR is a prognostic indicator and clinical predictor in patients with heart failure.²⁰ Furthermore, measures of eGFR may influence the dosing and choice of medical therapy, as angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and mineralocorticoid receptor antagonists (MRAs) can cause increases in serum creatinine.²¹ However, patients are at risk of undertreatment if these medications are withheld due to small decreases in eGFR.²²

In the case of acute kidney injury (AKI), it is worth considering that part of the definition of AKI involves a significant change in serum creatinine from the patient’s baseline. In the acute phase, estimates of GFR are not particularly useful as GFR does not decline in a linear fashion with the rise seen in serum creatinine. However, where eGFR has utility is establishing where the patient with AKI stood in terms of baseline renal function (prior to AKI), as the presence of CKD can have an increased risk of subsequent AKI and vice versa.²³ With this reporting change, in African-American patients, some individuals with borderline eGFRs may now be classified as having CKD by the score without race parameter and this may influence management of AKI in the ED or inpatient setting.

Acknowledgements

We are grateful to Dr. Lydia Howell and all of the UCDH Working Group participants for their advice and coordination in developing the action plan and helping to make these changes possible.

References

1. United States Renal Data System*. “Chapter 2: Identification and Care of Patients with CKD.” In 2018 USRDS annual data report: Epidemiology of kidney disease in the United States, vol. 1. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases; 2018.
2. Flessner M, Wyatt S, Akylbekova E et al. “Prevalence and Awareness of CKD Among African Americans: The Jackson Heart Study.” American Journal of Kidney Diseases 2009;53(2): 238-247.
3. United States Renal Data System*. “Chapter 1:Incidence, Prevalence, Patient Characteristics, and Treatment Modalities.” In 2018 USRDS annual data report: Epidemiology of kidney disease in the United States, vol. 2. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases; 2018.
4. United States Renal Data System*. “Chapter 6: Transplantation.” In 2018 USRDS annual data report: Epidemiology of kidney disease in the United States, vol. 2. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases; 2018.
5. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDKD). “Estimating Glomerular Filtration Rate.” (n.d.). Retrieved from https://www.niddk.nih.gov/health-information/professionals/clinical-tools-patient-management/kidney-disease/laboratory-evaluation/glomerular-filtration-rate/estimating. Accessed July 15, 2020.
6. Levey A, Bosch J, Lewis J et al. “A More Accurate Method To Estimate Glomerular Filtration Rate from Serum Creatinine: A New Prediction Equation.” Ann Intern Med 1999;130(6):461-70.
7. Levey A, Stevens L, Schmid C et al. “A New Equation to Estimate Glomerular Filtration Rate.” Ann Intern Med 2009;150(9): 604-612.
8. United States Renal Data System*. “Chapter 1: CKD in the General Population.” In 2018 USRDS annual data report: Epidemiology of kidney disease in the United States, vol. 1. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases; 2018.
9. Peralta C, Lin F, Shlipak M et al. “Race differences in prevalence of chronic kidney disease among young adults using creatinine-based glomerular filtration rate-estimating equations.” Nephrology Dialysis Transplantation 2010;25(12): 3934-3939.
10. Anker N, Scherzer R, Peralta C et al. “Racial disparities in creatinine-based kidney function estimates among HIV-infected adults.” Ethnicity and Disease 2016;26(2): 213-220.
11. Omuse G, Maina D, Mwangi J et al. “Comparison of equations for estimating glomerular filtration rate in screening for chronic kidney disease in asymptomatic black Africans: A cross sectional study.” BMC Nephrology 2017;18(1): 369.
12. Eneanya N, Yang W, Reese, P. “Reconsidering the Consequences of Using Race to Estimate Kidney Function.” JAMA 2019;322(2): 113-114.
13. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDKD). “Reporting Glomerular Filtration Rate.” (n.d.). Retrieved from https://www.niddk.nih.gov/health-information/professionals/clinical-tools-patient-management/kidney-disease/laboratory-evaluation/glomerular-filtration-rate/reporting. Accessed July 15, 2020.
14. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDKD). “Update on Cystatin C.” (n.d.). Retrieved from https://www.niddk.nih.gov/health-information/professionals/clinical-tools-patient-management/kidney-disease/laboratory-evaluation/glomerular-filtration-rate/update-cystatin-c. Accessed July 15, 2020.
15. Levey A, Inker L, & Coresh J. “GFR estimation: From physiology to public health.” American Journal of Kidney Diseases 2014;63(5): 820-834.
16. Levey A, Coresh J, Tighiouart H et al. “Measured and estimated glomerular filtration rate: current status and future directions.” Nature Reviews Nephrology 2020;16(1): 51-64.
17. Food and Drug Administration (FDA). “Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests - Guidance for Industry and FDA Staff.” (March 2007). Retrieved from https://www.fda.gov/regulatory-information/search-fda-guidance-documents/statistical-guidance-reporting-results-studies-evaluating-diagnostic-tests-guidance-industry-and-fda. Accessed July 15, 2020.
18. Zelnick LR, Leca N, Young B, et al. Association of the Estimated Glomerular Filtration Rate With vs Without a Coefficient for Race With Time to Eligibility for Kidney Transplant. JAMA Netw Open. 2021;4(1):e2034004. doi:10.1001/jamanetworkopen.2020.34004. PMID 33443583.
19. Julian BA, Gaston RS, Brown WM, et al. Effect of Replacing Race With Apolipoprotein L1 Genotype in Calculation of Kidney Donor Risk Index. Am J Transplant. 2017;17(6):1540-1548. doi:10.1111/ajt.14113. PMID 27862962.
20. Viswanathan G, Gilbert S. The cardiorenal syndrome: making the connection. Int J Nephrol. 2010;2011:283137. Published 2010 Oct 4. doi:10.4061/2011/283137. PMID 21151533.
21. Scully P, Goldsmith D. The management of end-stage heart failure and reducing the risk of cardiorenal syndrome. Clin Med (Lond). 2013;13(6):610-613. doi:10.7861/clinmedicine.13-6-610. PMID 24298112.
22. Chahal RS, Chukwu CA, Kalra PR, Kalra PA. Heart failure and acute renal dysfunction in the cardiorenal syndrome. Clin Med (Lond). 2020;20(2):146-150. doi:10.7861/clinmed.2019-0422. PMID 32188648.
23. Makris K, Spanou L. Acute Kidney Injury: Diagnostic Approaches and Controversies. Clin Biochem Rev. 2016;37(4):153-175. PMID 28167845.

*The data reported here have been supplied by the United States Renal Data System (USRDS). The interpretation and reporting of these data are the responsibility of the author(s) and in no way should be seen as an official policy or interpretation of the U.S. government

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

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 than three (3) days from collection.
4. Specimen leaking on arrival.
5. Insufficient specimen volume (less than 2 mL).
6. Interfering Substances

Result Report Timing

1. AFB smears are reported within 24 hours of receipt
2. AFB cultures are reported:
   (a) when growth occurs and identification is made
   or
   (b) at 7th weeks when no growth occurs

Possible Results:

AFB Smear:

Number of AFB observed at 100x magnification:
1+ (Rare) = 1-9 / 100 fields
2+ (Few) = 1-9 / 10 fields
3+ (Moderate) = 1-9 / field
4+ (Many) = greater than 1-9 / field
AFB Not Found
Smear Not Performed

AFB Culture:

• Identification of Mycobacteria species
• No growth of Mycobacteria after 6 weeks
• Unsatisfactory

Patient instructions:

Collection of Sputum/Phlegm/Saliva Sample

Please carefully follow the steps below.

1. Gargle with water immediately prior to obtaining a sputum specimen to reduce the number of oral bacteria. Do not use a mouthwash or any other gargle.
2. Open the lid of the container.
3. Try coughing as strong as you can while holding the tissue over your nose and mouth.
4. When you are ready to spit, do it into the cup/vial, spitting any phlegm with saliva. If you are not able to produce any phlegm while coughing, spit saliva into the cup/vial.
5. Press the rim of the specimen container under the lower lip to catch all the expectorated or coughed sputum.
6. Cough deeply and expectorate sputum (not saliva) into the cup.
7. Close the lid securely and notify your caregiver that your specimen is ready for transport to the laboratory.

NOTE: To have enough sample, it is permitted to spit multiple times into the container. Spitting 2-3 times into the container should provide enough material for testing.

Close the cap by following instructions in the next step to secure your cup/vial.

Please note: If lid comes off and there is leakage, we will destroy the sample for the safety of our lab technicians and your sample will NOT be processed.

Cup Instructions

1. If possible, rinse outside of sterile container to remove any excess sputum/ saliva. Allow time for drying.
2. Secure sample container lid / make sure it is sealed with strip of Parafilm
3. Place individual specimen in corresponding individual small / large biohazard bag
4. Include the copy of order slip with each specimen (order slips are to be placed in the biohazard order slip pocket)

5. After sample is secure, wash hands thoroughly

Resources:

1. Grant, Lindsay R, et al. “Procedures for Collection of Induced Sputum Specimens from Children.” Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America, Oxford University Press, Apr. 2012.
2. “How to Collect and Ship COVID-19 Samples.” MicroGen Diagnostics, 28 Apr. 2020.
3. “Acid-Fast Bacilli (AFB) Smear and Culture.” Acid-Fast Bacilli (AFB) Smear and Culture | State Public Health Laboratory | Health &amp; Senior Services.
4. “Header.” ISDH: Sputum Collection.

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).

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 preferred best practices that is widely utilized and accepted for the collection and reporting of bone marrow specimen (1). Together the aspirate and trephine biopsy provide a complementary and comprehensive evaluation of the bone marrow. The final bone marrow report requires the integration of peripheral blood findings, bone marrow core and aspirate findings, evaluated together with the results of supplementary tests such as cytogenetics, immunophenotyping, and molecular genetics studies. Therefore, most authorities advocate obtaining both the aspirate and trephine biopsy, and clinicians must attempt to collect both for an optimal bone marrow evaluation.

The sequence of obtaining marrow specimen is controversial. Collection of core biopsy first releases thromboplastic elements that can cause clotting and compromise the quality of aspiration. Procurement of aspirate first cause aspiration artifacts in core biopsy (hypocellularity, sinusoidal hemorrhage, etc.). Nonetheless, the sequence is unimportant if both specimens are obtained as part of the same procedure. Many different techniques and biopsy equipment exist that have greatly facilitated bone marrow sampling with minimal artifacts and distortions (5).

2a) Bone marrow trephine biopsy

Trephination is the oldest surgical procedure for which archaeological evidence exists (6). Its use for pathologic bone marrow sampling was first described in 1903 by G. Pianese (7). Current guidelines from WHO recommends collection of ≥ 1.5 cm long bone marrow core with ≥ 10 partially preserved intertrabecular areas.

Bone marrow core biopsy can be a painful process for patients. Additionally, biopsy processing takes a longer time and multiple steps because it requires additional processing, namely decalcification. However, it allows in situ morphologic and immunophenotypic analysis that are important for accurate assessment of cellularity, architecture, infiltrative processes, and spatial relationships between hematopoietic elements (Fig. 2). Unexplained pancytopenia and leucoerythroblastic-like blood smear are indications for core bone marrow biopsy because they are likely to demonstrate marrow fibrosis (reticulin and collagen). In cases where adequate aspiration material is not obtained (i.e. fibrosis or cell packing), an additional core biopsy, after disaggregation, may provide additional cellular material for flow cytometry, cytogenetics, and molecular analysis. Core biopsy is mandatory in the cases with dry tap to determine the nature of pathologic process (7). Core biopsy is superior for evaluation of microorganisms, aplastic anemia, different phases of myeloproliferative neoplasms, plasma cell myeloma, and in tuberculosis and sarcoid granulomas (8, 9). In hematolymphoid malignancies the formation and localization of lymphoid aggregates can be demonstrated on core biopsy. Classic Hodgkin lymphoma can be evaluated on a core biopsy. Core biopsy is more useful than an aspirate in assessing amyloid deposition and for staging metastatic malignancies to the bone. Core biopsy is essential in initial staging of many pediatric non-hematopoietic malignancies, such as rhabdomyosarcoma, neuroblastoma, PNETs, and Ewing sarcoma. Many adult non-hematopoietic malignancies, such as sarcoma of the bone, also require bone marrow biopsy for staging.

2b) Bone marrow aspirate

Bone marrow aspiration is a reliable and rapid method of evaluating bone marrow pathology (10). It is especially preferred in clinically urgent cases because aspirate slides are easy to make and usually can be stained with readily available stains without need for elaborate processing. The advantage of bone marrow aspirate lies in its ability to allow cytomorphologic analysis and cell enumeration (Fig. 3). Bone marrow aspirate is important in the evaluation of acute leukemias and MDS, where an accurate enumeration of the blasts is essential for diagnosis. Aspirate is also important in diseases with patchy marrow involvement (i.e. plasma cell dyscrasias). However, with the advancement in cytogenetics and molecular techniques, and the ability to incorporate such data for diagnostic and prognostic purposes, the bone marrow aspirate has become even more important. Hematolymphoid malignancies can evolve and change both morphologically and immunophenotypically as diseases progress due to accumulation of additional mutation over time. Therefore, molecular analysis is the best tool to follow up with disease progression in these patients. Unfortunately, trephine bone marrow biopsy material cannot be used for molecular study since decalcification process degrades DNA and RNA. However, aspirate and its derivatives (i.e. clot preparation) can be used for molecular studies. In WHO 2016 classification of tumors of hematopoietic and lymphoid tissues, many leukemias and lymphomas are classified based upon molecular studies. For example, the diagnosis of acute promyelocytic leukemia is based upon the molecular findings of PML-RARA fusion gene generated by the t(15; 17) chromosomal translocation. Therefore, bone marrow aspirate is important and must be collected along with trephine biopsy.

Conclusion

Bone marrow evaluation is a complex, but essential multimodal process for the diagnosis of hematologic malignancies and many non-neoplastic disorders. Every attempt should be made to collect and submit both the trephine core biopsy and bone marrow aspirate along with peripheral blood sample since they together provide the most comprehensive and complementary information in the diagnostic work-up of hematolymphoid malignancies.

References

1. Lee, S.-H. et. al. (2008) ICSH guidelines for the standardization of bone marrow specimens and reports. Int. Jnl. Lab. Hem. 30, 349-364.
2. Swerdlow, S. H. et. al. (Eds): WHO classification of tumors of haematopoietic and lymphoid tissues (Revised 4th ed.). IARC: Lyon 2017.
3. Beckman, A. K. et. al. (2020) Clinician-ordered peripheral blood smears have low reimbursement and variable clinical value: a three-institution study, with suggestions for operational efficiency. Diagnostic Pathology. 15, 112-201.
4. Bain, B. J. (2001) Bone marrow aspiration. J. Clin. Pathol. 54, 657-663.
5. Islam, A. (2007) Bone marrow aspiration before bone marrow core biopsy using the same bone marrow biopsy needle: a good or bad practice? J. Clin. Pathol. 60, 212-215.
6. Parapia, L. A. (2007) Trepanning or trephines: a history of bone marrow biopsy. Brit. J. Haematology. 139, 14-19.
7. Riley, R. S. et. al. (2004) A pathologist’s perspective on bone marrow aspiration and biopsy: I. Performing a bone marrow examination. J. Clin. Lab. Analysis. 18, 70-90.
8. Kaur, M. et. al. (2014) Diagnostic value of bone marrow aspiration and biopsy in routine hematology practice. J. Clin. Diag. Res. 8, 13-16.
9. Bain, B. J. (2001) Bone marrow trephine biopsy. J. Clin. Pathol. 54, 737-742.
10. Afkhami, A. et. al. Peripheral blood smears, bone marrow aspirate trephine and clot biopsies: methods and protocols, page 257-270, in Day, C. E. (Eds.): Histopathology methods and protocols. Humana Press: New York 2014.

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 here^7-10:

• 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.

Situations may arise where only one out of two targets are detected. Detection of only one gene does not necessarily indicate the presence of a new variant and may be the result of low viral load. For example, with the high throughput assay at UC Davis Health, an ORF1ab positive, but E-gene negative result is still considered positive for SARS-CoV-2 RNA.⁷ It has been observed that ORF1ab positive/E-gene negative result combinations occur most frequently when patients are convalescing and present with very high cycle threshold (Ct) values suggestive of low viral loads. This phenomenon is due differing ratios of ORF1ab, and E genes produced during SARS-CoV-2 replication. Studies suggest ORF1ab and N sub-genomic material exist in higher quantities than E, thus, as patients recover, the E-gene RNA is the first to become undetectable. The same logic applies if encountering assays outside of UC Davis Health that relies on detecting ORF8 or S with other genetic targets (e.g., ORF1ab and/or N), thus not detecting ORF8 or S gene while detecting other targets does not necessarily indicate the patient is infected by the new SARS-CoV-2 variant. It must be noted that clinical laboratories do not report which genes are detected nor provide Ct-values for interpretation. The FDA has not approved this application at this time.

Presently, the only way to definitively identify the new SARS-CoV-2 variant is by sequencing. UC Davis is part of the SARS-CoV-2 Sequencing for Public Health Emergency Response, Epidemiology and Surveillance (SPHERES) program under the CDC, a national genomic consortium to monitor changes in the virus during the pandemic.¹¹ Additionally, we have collaborated with University of California San Francisco and the Chan-Zuckerberg BioHub since March 2020 to sequence samples to monitor for changes locally. Suspected variants from clinical samples are sent to the California Department of Public Health Viral and Rickettsial Disease Laboratory (VDRL) in Richmond for sequencing.

References

1. Center for Disease Control and Prevention, Interim: Implications of the Emerging SARS-CoV-2 Variant VOC 202012/01, Accessed on December 30, 2020.
2. Washington Post article, Accessed on December 30, 2020.
3. Public Health England –Investigation of novel SARS-CoV-2 variant: Variant of Concern 202012/01 Technical Brief 1, Accessed on December 30, 2020.
4. Public Health England –Investigation of novel SARS-CoV-2 variant: Variant of Concern 202012/01 Technical Brief 2, Accessed on December 30, 2020.
5. ThermoFisher Scientific website, Accessed on December 30, 2020.
6. United States Food and Drug Administration (FDA) Emergency Use Authorization website, Accessed on December 30, 2020.
7. Roche Molecular Systems cobas 6800/8800 SARS-CoV-2 assay information for use, Accessed on December 30, 2020.
8. Roche Molecular Systems cobas Liat SARS-CoV-2 & Flu A/B assay information for use, Accessed on December 30, 2020.
9. GenMark ePlex SARS-CoV-2 assay information for use, Accessed on December 30, 2020.
10. Beckton Dickenson BioGX SARS-CoV-2 assay information for use, Accessed on December 30, 2020.
11. Centers for Disease Control and Prevention SPHERES Program website, Accessed on December 30, 2020.

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- patients with RBC alloantibody(ies) are at increased risk of developing other alloantibodies, especially if they are exposed to more immunogenic antigens. Performing RBC antigen phenotyping after identifying alloantibodies is critical to provide best matched transfusions and prevent additional antibodies from forming.  This is performed either by a full warm phenotype or limited phenotype based on the reflex testing pathway.

1.1.3 To prepare for medications that interfere with all testing. In patients receiving anti-CD47 (such as Hu5F9), the medication is known to interfere with the red blood cell antibody screen. A full warm phenotype is performed prior to drug administration to allow phenotype matched transfusions during the period the medication is administered when new antibodies cannot be detected and thus allow safe transfusions.

1.2 Indication to perform a red blood cell antigen cold phenotype
Phenotyping the patient’s red blood cell antigens corresponding with common antibodies that are cold-reactive is typically performed when the patient has made a cold-reacting antibody. Common scenarios include anti-M (a naturally occurring antibody common in children) or anti-Lewis (a naturally occurring antibody common in pregnancy).

1.3 Indication to perform a red blood cell antigen complete phenotype
A complete phenotype is performed when the patient has multiple antibodies that are both cold-reacting and warm-reacting. This determines the patient’s antigen profile for K, E, e, C, c, Fya, Fyb, Jka, Jkb, S, s, M, N, Lea and Leb antigens.

1.4 Indication of performing a limited antigen phenotype
A limited or partial phenotype determines one or several specific RBC antigens instead of a full or complete phenotype. It is done in the following situations-

1.4.1 To help investigate the specificity of antibodies (patients develop antibodies against the antigens they lack). If an antibody panel identifies one or several alloantibodies of unclear specificity, performing a limited antigen phenotype may help determine the specificity of the antibody(ies).

1.4.2 In any patient that has made an antibody, the laboratory performs phenotyping for the corresponding antigen as well as any antigens which are more immunogenic.

1.4.3 To evaluate risk of hemolytic disease of the newborn. Paternal or cord blood RBC antigen typing determines the antigen expression that corresponds to the maternal antibody.

1.4.4 Rarely performed after a transfusion to determine if the RBC recovery is as expected. Determining the patient’s own RBC phenotype in the post-transfusion circulating RBCs and compared it to pre-transfusion antigen typing can aid in determining the RBC recovery.

1.5 Sample collection
Blood is collected in EDTA tube (lavender top). EDTA chelates calcium and thus acts as an anticoagulant. The plasma in the sample is used for antibody testing while the red blood cells are used for antigen phenotyping.

1.6 Assay time and technique
Usually, the phenotype panels listed require testing time of 1.5-2 hrs. Determining the phenotype of some antigens requires an extended incubation time while others are determined rapidly. The assays are performed using known monoclonal or polyclonal antisera incubated with the patient’s red blood cells. Coombs reagent is required when using monoclonal IgG based antisera to induce agglutination and determine if the antigen is present.

1.7 What are the clinical implications?
In patients who require extended antigen phenotype matching, the antigen profile of any donor units that are transfused match the NEGATIVE antigens in the patient’s antigen profile in addition to any antibodies. If the patient is not chronically transfused and one or several antibodies are detected for the first time, transfusion services will perform extended antigen typing for highly immunogenic antigens and provide packed red blood cells from a donor who lacks the pertinent antigen(s). This will avoid a hemolytic transfusion reaction and formation of new antibodies. If the patient’s care will require lifelong chronic transfusions, a full warm RBC phenotype is performed prior to all transfusions. At times, the negative antigen profile is difficult to match and transfusion services pathologists need to risk stratify the antigens by immunogenicity to find the best available match. By performing this reflex testing and obtaining donor red blood cell units that are extended phenotype matched, we prevent hemolysis from current antibodies and prevent new antibodies from forming.

1.8 Limitations to RBC Phenotyping
Red blood cell antigen phenotyping cannot be performed in certain situations:

1.8.1 The Coombs test is positive (DAT+). In autoimmune hemolytic anemia (AIHA), the patient’s red blood cells are coated in IgG or IgM, an autoimmune phenomenon. The presence of the IgG/IgM causes false positive phenotype testing, and thus the antigen profile cannot be reliably determined.

1.8.2 The patient has been recently transfused. The RBC antigen phenotype is performed on a sample of the patient’s own RBCs. In the setting of recent transfusion, circulating RBCs consist of a mixture of the patient’s RBCs and the donor’s RBCs. Thus, phenotyping will not represent the patient’s native RBCs. The clinical history of transfusion is very important in this setting.

1.8.3 RBCs are coated with drugs and cause interference (anti-CD47). If the patient’s RBCs are coated with medications, then the antigen profile cannot be reliably determined.             

2. What is RBC genotyping?

As the name suggests, red blood cell antigen expression may also be determined using genetic testing. Current testing allows the patient’s genomic DNA to be isolated and red cell antigen genotyping performed to predict the red cell antigen phenotype for select antigens. This is a send-out test, currently performed at Versiti laboratories. There are two panels available - Red cell genotyping panel (44 antigens) and a STAT panel (24 antigens).⁴

2.1 Indication of doing RBC antigen genotype - 

1. Patient’s red blood cell antigens cannot be reliable phenotyped due to recent history of receiving blood transfusion
2. Prior to significantly interfering drugs (such as anti-CD47)
3. To evaluate for genetic changes such as partial antigens that may explain alloantibodies but positive phenotyping
4. In rare cases when uncommon antibodies form and antigen profiling beyond the common antigens mentioned is required. 

2.2 Sample collection
EDTA (lavender top) blood- 5 ml

2.3 Methodology and turn-around time
For the 44-panel assay, 72 PCR-hybridization probes are used in 36 polymerase chain reactions to identify the alleles associated with 44 blood group antigens. For the STAT Panel, 32 PCR-hybridization probes are used in 16 polymerase chain reactions to identify the alleles associated with 24 blood group antigens.4 The 44-antigen panel turn-around time is 2-5 days, whereas the STAT (24-antigen panel) panel turn-around time is 24-48 hours.

2.4 Limitations
The RBC genotype is performed using specific probes against RBC antigen alleles. Thus, any mutations outside of the targeted region will not be detected. Novel mutations leading to altered or partial antigen expression and null phenotypes may not be detected. Results from hematopoietic stem cell transplant recipients may not match the genotype obtained from other tissues.⁴

References:

1. Harmening, Denise. Modern Blood Banking and Transfusion Practices 5th Edition. Philadelphia PA, FA Davis Company, 2005.
2. Fung MK, Grossman BJ, Hillyer CD, Westhoff CM Editors. AABB Technical Manual 18th Edition. Bethesda MD, AABB Press, 2014.
3. Reid ME, Lomas-Francis C, Olsson ML. The Blood Group Antigen Facts Book 3rd Edition. London UK, Elsevier. 2012.
4. Red Blood Cell Genotyping Panels. Versiti Laboratories, Blood Center of Wisconsin. Information online at: https://www.versiti.org/Custom/Files/Versiti/68/682cf64b-4508-4714-ae29-7f1a414b7a1e.pdf 

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.

Terms frequently used in GI pathology reports

Reactive lymphoid aggregate: A lymphoid aggregate that demonstrates a germinal center. The presence of a germinal center is typically indicative of a reactive rather than a neoplastic process (but should be taken in the context of the entire case).

Prominent lymphoid aggregate: The term “prominent” is a descriptor often used to describe a lymphoid aggregate in GI mucosa that is larger than expected (thus possibly accounting for the endoscopic impression of a polyp). Unless otherwise stated, when composed of primary or secondary follicles, there are no features worrisome for lymphoma.

Atypical lymphoid aggregate: A lymphoid aggregate that lacks the typical morphologic and immunophenotypic features of a reactive lymphoid aggregate but is not diagnostic of lymphoma.

How does a pathologist distinguish a reactive lymphoid aggregate from lymphoma?

Some histologic features can help to distinguish reactive lymphoid aggregates from lymphoma (Table 1, Figure 2). However, it is essential to keep in mind that distinction between a reactive and a neoplastic lymphoid process is often not possible based on morphology alone. Moreover, certain lymphomas (e.g., extranodal MALT lymphoma) may develop in association with reactive lymphoid infiltrates, especially in the stomach. Thus, immunohistochemistry, gene rearrangement studies, and other ancillary studies are frequently necessary for further evaluation.

Table 1. Histologic features that help distinguish reactive lymphoid aggregates from lymphoma.

Histologic featureReactive lymphoid aggregateLymphomaSizeUsually smallUsually LargerArchitecture of the GI mucosaPreserved mucosal architecture with lymphoid follicles sitting above the muscularis mucosaeDisrupted mucosal architecture with lymphoid cells pushing aside or destroying glands (or crypts) and/or disrupting the muscularis mucosaeGerminal centersPresentTypically, absentCytologyPolymorphous: Mixture of large and small cells, including plasma cells; minimal cytologic atypiaMonomorphous: Cells typically are similar in size and shape; may show nuclear membrane irregularitiesLymphoepithelial lesions(lymphocytes invading and destroying glands or crypts)AbsentCan be present

What does a pathologist do when encountering a lymphoid aggregate in the GI mucosa?

The following is the algorithm commonly followed here at UC Davis for evaluation of a lymphoid aggregate found in the GI tract (Figure 3):

IHC: Immunohistochemistry

FISH: Fluorescence in situ hybridization

ISH: In situ hybridization (RNA)

Monoclonal B-cell/T-cell proliferation: An abnormal collection of B or T-cells demonstrating clonality but not always diagnostic of a specific lymphoma.

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 example of how the Clinical Laboratory facilitates TDM. As alluded to earlier, communication of available guidelines helps to ensure proper TDM utilization.

Continuing to use gentamicin, an aminoglycoside antibiotic, as an example, the following is a brief discussion to illustrate potential factors that come into play in TDM and their clinical significance.

Aminoglycosides are a class of antibiotics primarily used against aerobic gram-negative bacteria and act by binding to the 30S ribosomal subunit of bacterial mRNA, inhibiting protein synthesis as a result [1]. In addition to gentamicin, aminoglycosides include tobramycin and amikacin. Aminoglycosides are generally prescribed by intramuscular or intravenous injection due to their poor bioavailability through the oral route that is attributable to poor penetration of tissues. The distribution of aminoglycosides in the body is mainly restricted to the extracellular fluid compartment with the exception of bile, renal cortex, and endolymph of the inner ear, with the latter two sites being significant in the discussion of aminoglycoside toxicity [9]. Notable toxic effects include acute renal tubular necrosis and delayed-onset vestibular and cochlear sensory cell destruction [1].

The vast majority of an administered dose of an aminoglycoside is renally eliminated, primarily by glomerular filtration [1, 10]. As clearance is highly dependent on renal function, underdevelopment (such as in neonates and infants) or impairment of glomerular filtration causes accumulation of these drugs and potential nephrotoxicity and/or ototoxicity. Monitoring of drug concentrations and subsequent dose adjustments are important in the management of patients receiving aminoglycosides, especially in those with compromised renal function.

While approaches to parenteral aminoglycoside dosing differ and depend on the individual patient, two general dosing strategies are commonly used in adults: multiple-daily dosing and extended-interval (or once-daily) dosing [11]. Based on the recognized pharmacodynamic and pharmacokinetic parameters of aminoglycosides, recommendations for target concentrations (peak, trough, and random) and timing of sample collection for concentration monitoring have been established and are available for both multiple-daily dosing and extended-interval dosing strategies. Such recommendations for TDM of aminoglycosides are utilized at UC Davis and are provided in the Laboratory Test Directory [12].

Besides the measurement of drug concentrations, the Clinical Laboratory also aids in TDM through other available tests that may be useful in assessing manifestations of toxicity. For example, with aminoglycosides and other renally eliminated drugs, it is known that renal function affects the rate of drug clearance and thus affects the dosing approach and risk of toxicity. Standard tools to readily evaluate renal function include measurement of serum creatine concentration, creatine clearance, and estimated glomerular filtration rate, which, in the case of aminoglycoside therapy, are routinely monitored at baseline and over the duration of therapy [11].

Conclusion:

Given their pattern of distribution within the body, dependence on renal elimination, and potential for toxicity, aminoglycosides serve as a common practical example of TDM. Many other drugs are available for concentration monitoring at UC Davis Health and many of the principles demonstrated in the example of aminoglycosides still apply. It is worth noting that the Clinical Laboratory contributes to TDM through other testing methods (e.g., renal function testing, etc.) in addition to concentration monitoring.

While the Clinical Laboratory plays an important role in TDM, it is necessary to reiterate that it works with many other members of the healthcare system. An understanding of the patient's clinical setting and pharmacologic properties of the drug are crucial to the interpretation and use of drug concentration monitoring. This also entails recognition of the various factors that may affect interpretation. Knowledge of factors such as dosage, dosing interval, patient characteristics, sample type, and the timing of sample collection is key to utilizing TDM effectively.

References:

1. Burtis CA, Bruns DE. Tietz Fundamentals of Clinical Chemistry and Molecular Diagnostics. Saunders; 2014.
2. Birkett DJ. Therapeutic drug monitoring. Aust Prescr. 1997;20:9-11.
3. Bates DW. Improving the use of therapeutic drug monitoring. Ther Drug Monit. 1998;20(5):550-5.
4. Suryadevara M, Steidl KE, Probst LA, Shaw J. Inappropriate vancomycin therapeutic drug monitoring in hospitalized pediatric patients increases pediatric trauma and hospital costs. J Pediatr Pharmacol Ther. 2012;17(2):159-65.
5. Sorrento TA, Bonanza KC, Salisbury DW. Pharmaceutical services in a capitated geriatric care program. Am J Health Syst Pharm. 1996;53(23):2848-52.
6. Wallerstedt SM, Lindh JD. Prevalence of Therapeutic Drug Monitoring for Antidepressants and Antipsychotics in Stockholm, Sweden: A Longitudinal Analysis. Ther Drug Monit. 2015;37(4):461-5.
7. Leung D, Ensom MHH, Carr R. Survey of Therapeutic Drug Monitoring Practices in Pediatric Health Care Programs across Canada. Can J Hosp Pharm. 2019;72(2):126-132.
8. Choi R, Woo HI, Park HD, Lee SY. A nationwide utilization survey of therapeutic drug monitoring for five antibiotics in South Korea. Infect Drug Resist. 2019;12:2163-2173.
9. Craig WA. Optimizing aminoglycoside use. Crit Care Clin. 2011;27(1):107-21.
10. Mathews A, Bailie GR. Clinical pharmacokinetics, toxicity and cost effectiveness analysis of aminoglycosides and aminoglycoside dosing services. J Clin Pharm Ther. 1987;12(5):273-91.
11. Gonzalez LS, Spencer JP. Aminoglycosides: a practical review. Am Fam Physician. 1998;58(8):1811-20.
12. UCDH Pharmacy and Therapeutics Committee. UCDMC Adult Aminoglycoside Dosing and Monitoring Guidelines. UC Davis Health. April 2018. https://intranet.ucdmc.ucdavis.edu/pharmacy/clinical/Guidelines/AminoglycosideDosingMonitoring.pdf

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, they screened all the publications from major database, and selected five published studies involving 957 patients in China (either confirmed cases or cases under suspicion of Covid-19). The false negative rates of RT-PCR SARS-CoV-2 assays were calculated using a multilevel mixed-effect logistic regression model and resulted in a range from 2% to 29%, which also indicated up to 29% of patients could have an initial RT-PCR false-negative result (3). However, because of the heterogeneity of sensitivity among the five studies, the certainty of the evidence was low. Even though the evidence is limited, the concern of false negative RT-PCR results is not negligible.

It is also important to be aware that detection of viral RNA does not necessarily indicate the virus is transmissible. The duration of virus shedding dropped after 20 days post-onset of symptoms. The genetic material of dead viral particles can remain within the epithelial cells and can be detected. This was supported by a recent study, viral shedding from sputum has been shown to extend beyond symptom duration (4,5). Although we have learned a lot about this virus in a short period of time, these are still fundamental questions we have to answer in order to make intelligent decisions on how to approach testing (6).

Another challenge is the need for establishing reference standards for measuring the sensitivity of SARS-CoV-2 test in asymptomatic patients as it is crucial to identify people who are infected, so they are isolated to limit their interactions with people who are at higher risk. Unfortunately, there is no data available to measure test sensitivity in asymptomatic patients. It was believed that viral loads in asymptomatic patient may be different than patients with symptoms. However, that is also debatable, as one other study analyzing a small number of patients also reported viral load of asymptomatic patients was as high as that of symptomatic patients (7).

Antibody Test

Antibody testing can identify prior infection by using a surface protein or an array of virus peptides to capture antibodies specific to the virus in patients' blood. It has been shown in a recent study analyzing 134 samples, using colloidal gold-based immunochromatographic strip method targeting the viral antibody, the sensitivity of the assay is up to 93%, 8-14 days after onset of symptoms (8). There are limitations regarding the utilization of antibody-based testing. A better understanding of the importance of the “testing window” or timeline of antibody testing and what the test result means are important to overcome the limitations. The timeline of development of new antibody can be delayed because validation of a new test using the antibodies from the blood of infected people always takes time. Clinically, immunoglobulins (Ig) such as IgM, IgA, or IgG are used for immunoassays. IgG is more specific than IgA and IgM, and the levels of IgG may correlate with immunity, but it typically appears 7 days post infection. IgM may appear as early as 3 days post infection, but IgM and IgA are less specific. It is believed by day 14 post infection, although virus-targeting antibody is detectable, this lag period indicates this test cannot be used for early detection of infection within the first week or so. The fact that this test is most accurate 2 weeks after onset of symptoms makes it unrealistic to be used for the diagnosis of this highly infectious disease (9,10). We have to be aware that if testing is performed during this “window period” (too soon after exposure where antibody production is non-detectable), false negative serology results may occur. Ongoing research is trying to determine if virus-targeting antibodies provide some immunity to SARS-CoV-2, how long the antibodies last and whether past infection will protect from future infection.

A recent study screened 9,530 specimens using Diazyme SARS-CoV-2 IgG serology assay, and all positive results (N=164) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the levels of neutralizing antibodies and the magnitude of positive SARS-CoV-2 serology was correlated. The authors demonstrated the three widely available clinical serology assays positively correlated with SARS-CoV-2 neutralization activity, and confirmed that using a two-platform approach for COVID-19 serology positive individuals greatly improved positive predictive value for neutralization (11). Of note, the UC Davis serology assay actually has similar platforms as those used in the current study.

Conclusion

In summary, performing a single COVID-19 test could lead to false negative results and misdiagnosis. Clinicians should not trust unexpected negative results in a patient with typical symptoms and known exposure (assuming false negative) if pretest probability is high. Therefore, a combined diagnostic workflow integrated with both molecular detection (detects current active infection) and antibody testing (prior infection/immunity) should be implemented to provide a high quality and cost-effective diagnostic solution. This approach should also include this diagnostic workflow at different time points throughout the course of disease.

References

1. Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579:265-269.
2. UC Davis Labroatary Best Practise Blog (updated on July 15, 2020) https://blog.ucdmc.ucdavis.edu/labbestpractice/index.php/2020/06/16/review-of-covid-19-testing-methods/ Accessed on Aug 15, 2020.
3. Arevalo-Rodriguez I, Buitrago-Garcia D, Simancas-Racines D, et al. False-negative results of initial RT-PCR assays for COVID-19: a systematic review. medRxiv. April 21, 2020.
4. Rawlins EL, Hogan BL. Ciliated epithelial cell lifespan in the mouse trachea and lung. Am J Physiol Lung Cell Mol Physiol. 2008;295(1):L231-L234.
5. Wölfel R, Corman VM, Guggemos W, et al. Virological assessment of hospitalized patients with COVID-2019. 2020;581(7809):465-469.
6. Woloshin S, Patel N, Kesselheim A. False negative tests for SARS-CoV-2 infection-challenges and implications. New Engl. J. Med. 2020;383:e38.
7. Lee S, Kim T, Lee E, et al. Clinical course and molecular viral shedding among asymptomatic and symptomatic patients with SARS-CoV-2 infection in a community treatment center in the republic of Korea. JAMA Intern Med.Published online August 06, 2020.
8. Pan Y, Li X, Yang G, et al. Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19 patients. medRxiv. Jan 1, 2020.
9. Hu E. COVID-19 Testing: Challenges, Limitations and Suggestions for improvement. Preprints. 2020,2020040155.
10. Zhang W, Du RH, Li B, et al. Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes, Emerging Microbes & Infections. 2020;9(1):386-389.
11. Suhandynata RT, Hoffman MA, Huang D, et al. Commercial Serology Assays Predict Neutralization Activity Against SARS-CoV-2. July 10, 2020.

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.

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.

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.

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.

Discussion

Overall, these platforms have allowed work to continue and current demand and utilization is high. Having knowledgeable staff assist and have time dedicated to accelerating adoption of these platforms for all department members is a key factor to success. In addition to hospital VPN and EMR access which should be established and facilitated at the institutional level, with remote desktop protocols as needed to allow seamless workflow remotely. Educational meetings and conferences have performed well through all platforms, with usual hiccups such as inadequate hardware (webcam/microphone/etc.)³, personal home internet speeds, cumbersome security, and version control.

The platforms excel at sharing live microscope images (with practice) and can simulate real time sign-out. We employ two methods, resident driven and attending driven scope sessions. In resident driven sessions, the resident/fellows present the case virtually, during initial review of cases, while the attending comments. During sign-out the attending can share the screen of the prepared report, and make edits changes in real-time, along with final review of the case(s). This workflow also applies to clinical pathology practice by facilitating sharing of flow cytometry histograms, SPEP/IFE, and other clinical test.

It's also worth noting there are other contenders to consider. Almost every person owns a Google account and therefore can use Google Hangouts/Meets.⁸ This solution allows you to quickly create a meeting and share links with attendees, however calls are limited to 150 users and 10 video users (paid accounts can have 25 video users). Overall, Google provides dependable video conferencing, but it may not be the best quality. Similar to Microsoft Teams, Slack offers a software solution meant to facilitate remote work with a remote office space.⁷ Slack calling is extremely easy to use and has a feature that stands out from the rest: annotation on the presenting user's display. This feature could be immensely helpful for slide sessions allowing trainees to highlight regions of interest quickly. Also, worth a mention, FaceTime allows group calling now and is a quick alternative for small groups (32 user limit) but of course requires an iOS or macOS operating system. Messages on macOS does also offer a screen sharing option which includes the ability to request remote control and two-way audio.

Finally, HIPAA enforcement has been changed during the pandemic, but will likely return after the crisis. With that said using platforms that have already been validated by your institution for security and have existing business agreements should be explored first. Sufficient to say, there are several technologies that can facilitate remote pathology practice and leveraging the existing platforms at your institution should be prioritized as all the platforms share similar functionality.

References

1. Marshall CL, Petersen NJ, Naik AD, et al. "Implementation of a regional virtual tumor board: a prospective study evaluating feasibility and provider acceptance". Telemed J E Health. 2014;20(8):705?711. doi:10.1089/tmj.2013.0320
2. El Saghir NS, Keating NL, Carlson RW, Khoury KE, Fallowfield L. "Tumor boards: optimizing the structure and improving efficiency of multidisciplinary management of patients with cancer worldwide". Am Soc Clin Oncol Educ Book. 2014;e461?e466. doi:10.14694/EdBook_AM.2014.34.e461
3. Graff JP, Wu ML. "The Nokia Lumia 1020 smartphone as a 41-megapixel photomicroscope". Histopathology. 2014;64(7):1044?1045. doi:10.1111/his.12355
4. Microsoft
5. Cisco
6. Zoom
7. Gofine M, Clark S. "Integration of Slack, a cloud-based team collaboration application, into research coordination". J Innov Health Inform. 2017;24(2):936. Published 2017 Jun 30. doi:10.14236/jhi.v24i2.936
8. Chan T, Joshi N, Lin M, Mehta N. "Using Google Hangouts on Air for Medical Education: A Disruptive Way to Leverage and Facilitate Remote Communication and Collaboration". J Grad Med Educ. 2015;7(2):171?173. doi:10.4300/JGME-D-14-00545.1

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 rearrangement.¹⁴ This test requires a minimum of 15 atypical lymphoid cells.¹⁵

Recently, the detection of a mutation in myeloid differentiation factor 88 (MYD88) gene (usually the canonical L265P mutation) has shown to provide diagnostic value. One study showed that this mutation was detected in 69% of primary vitreoretinal lymphoma, the most common form of intraocular lymphoma, with or without CNS involvement.¹⁶ This test utilizes the minimum amount of genomic DNA (10-20ng) for pyrosequencing.¹⁷

Conclusion:

In summary, the diagnosis of PIOL can be extremely challenging, especially if the specimen yields low cellularity. Although a repeat sampling can be performed and may yield more cellular specimen for a definitive diagnosis, it is not always clinically feasible. On the other hand, several ancillary studies can be performed to improve the diagnostic specificity; however, these results must be interpreted in combination with clinical course, radiologic and cytologic evaluations.

References:

1. Swerdlow SH, Campo E, Harris NL, Jaffe ES, and PileriS A, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4^th Lyon, 2017.
2. Faia LJ and Chan CC. Primary Intraocular Lymphoma. Arch Pathol Lab Med. 2009;133(8):1228-1232.
3. Sen HN, Bodaghi B, Hoang PL, and Nussenblatt Primary intraocular lymphoma: diagnosis and differential diagnosis. Ocul Immunol Inflamm. 2009;17(3):133-141. PMID: 19585354.
4. Gonzales JA and Chan CC. Biopsy techniques and yields in diagnosing primary intraocular lymphoma. Int Ophthalmol. 2007;27(4):241-250.
5. Karma A, von Willebrand EO, Tommila PV, PaetauAE, Oskala PS, Immonen Primary Intraocular lymphoma: improving the diagnostic procedure. Ophthalamology 2007;114(7):1372-1377.
6. Tang, LJ, Gu CL, and Zhang P. Intraocular lymphoma. Int J Ophthalmol, 2017;10(8):1301-1307.
7. Chan CC and Wallace DJ. Intraocular lymphoma: update on diagnosis and management. Cancer Control. 2004;11(5):285-295.
8. Farkas T, HarbourJW, and Davila RM. Cytologic diagnosis of intraocular lymphoma in vitreous aspirates. Acta Cytol. 2004;48(4):487-491.
9. Hwang CS, Yeh Steven, and Bergstrom CS. Diagnostic vitrectomy for primary intraocular lymphoma: when, why, how? Int Ophthalmol 2014;54(2):155-171.
10. Margolis R. Diagnostic vitrectomy for the diagnosis and management of posterior uveitis of unknown etiology. CurrOpin Ophthalmol. 2008;19:218-224.
11. Davis JL, Miller DM, and Ruiz P. Diagnostic testing of vitrectomy specimens. Am J Ophthalmol. 2005;140:822-829.
12. Cassoux N, Giron A, Bodaghi B, Tran THC, Baudet S, et al. IL-10 measurement in aqueous humor for screening patients with suspicion of primary intraocular lymphoma. Invest Ophthalmol Vis Sci. 2007;48(7):3253-3259.
13. Wolf LA, Reed GF, Buggage RR, Nussenblatt RB, and Chan CC. Vitreous cytokine levels. Ophthalmology. 2003;110(8):1671-1672.
14. Chan CC. Molecular pathology of primary intraocular lymphoma. Trans Am Ophthalmol2003;101:275-292.
15. Wang Y, Shen D, Wang VM, Sen HN, Chan CC. Molecular biomarkers for the diagnosis of primary vitreoretinal lymphoma. Int J Mol Sci. 2011;12(9):5684-5697.
16. Fend F, Ferreri AJ and Coupland SE. How we diagnose and treat vitreoretinal lymphoma. Br J Haematol. 2016;173(5):680-692.
17. Cummings PJ, Ahmed R, Durocher JA, Jessen A, Vardi T, et al. Pyrosequencing for microbial identification and characterization. J Vis Exp. 2013;(78).

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 metachromasia.¹¹ This property is particularly useful in Mohs surgery for basal cell carcinoma (BCC) because the contrast of BCC’s blue aggregates with the reddish purple metachromasia of glycosaminoglycans in the surrounding stroma helps to delineate very small nests or a few clusters of tumor cells from adnexal structures.

Advantages: Toluidine blue staining is faster and simpler than H&E and requires no counterstain.¹¹ TB is a reliable stain and requires only one primary staining agent as opposed to H&E. Minimizing the staining time reduces patient waiting, facilitates patient flow, and frees up staff to perform other tasks.¹² The metachromasia in BCCs and microcystic adnexal carcinoma(MAC) can be helpful in detecting small numbers of infiltrating tumor cells that otherwise might be missed.^13,14 Mast cells and mucopolysaccharides in the stroma surrounding BCC may be highlighted in a magenta hue, prompting a diligent search for residual tumor. Mast cells have bright purple metachromatic granules on TB and are abundant in the stroma of BCCs. Abundant mast cells may be indicative of residual tumor.⁸

Limitations: Nuclear detail is inferior compared to H&E.¹¹ A lot of pathologists/Mohs surgeons are not trained with TB and therefore may find it difficult to use TB at first. It may take a while for them to become familiar with the staining pattern of TB.⁸ As with H&E, elastic material, reticular fibers, nerve fibers, and fat are difficult to identify.

Best Lab Practices

Whatever is the preferred staining method for frozen section, the following general best lab practices helps to ensure an optimal staining result.

• Wear appropriate personal protective equipment to avoid injury and cutaneous absorption.
• Optimal staining is achieved with 5-6 µm thick sections.
• Do not allow frozen sections to air-dry at any time during the staining procedure for better preservation of tissue morphology.
• Drain slides after each step to avoid injury and cutaneous absorption.
• Change staining solutions on a regular basis according to acceptable laboratory protocol. Consideration must be given to number of slides stained as well as to time in use.
• Store chemicals in a cool, dry, well-ventilated area, and flammables in an approved cabinet or room.
• Ensure adequate ventilation and regularly check fume contaminant hoods that can minimize the amount of inhaled chemical vapor.
• Quality control should be performed daily before proceeding with patient specimens.

Common Problems with Rapid H&E Staining

1. Weak Hematoxylin Staining:
   Due to autolysis or poor fixation, over-decalcification, inadequate staining time, excessive de-staining, weak hematoxylin due to carryover, contaminants, thin sections, and inadequate removal of alcohol or insufficient pre-rinsing with water prior to staining with hematoxylin
2. Excessive Hematoxylin Staining:
   Due to drying of tissue, excessive staining times, too weak or inadequate de-staining time, thick section.
3. Weak Eosin Staining:
   Due to too high Eosin pH, contaminant in the alcohol rinse, deteriorating eosin due to excessive carryover, thin sections, inadequate staining time, over-differentiation by subsequent 95% alcohol.
4. Excessive Eosin Staining:
   Due to stronger dye solution (can be due to excessive evaporation), use of isopropyl alcohol as the rinsing agent, thick sections, excessive staining times
5. Water Beads or White Haze under the Cover­slip:
   Due to incomplete dehydration of the section, formed by mixture of water and the clearing agent.
6. Eosin Bleeding from the Tissue Section:
   Due to inadequate clearing of alcohol, which will remain within the tissue section and causes the bleeding.

Sample Rapid H&E Staining Method

1. 95% ethyl alcohol (10 dips)
2. Formalin 10%, Phosphate Buffered (10 dips)
3. Distilled water (10 dips)
4. Hematoxylin Stain, Harris Modified (30 seconds)
5. Distilled water (10 dips)
6. Distilled water (10 dips)
7. 95% ethyl alcohol (10 dips)
8. Eosin Y (15 seconds)
9. 95% ethyl alcohol (10 dips)
10. 95% ethyl alcohol (10 dips)
11. 100% ethyl alcohol (10 dips)
12. 100% ethyl alcohol (10 dips)
13. Clearing agent (10 dips)
14. Clearing agent (10 dips)
15. Coverslip with compatible mounting medium

Common Problems with Toluidine Blue

1. Weak Toluidine Blue Staining:
   Due to autolysis or poor fixation, over-decalcification, inadequate staining time, excessive de-staining/rinsing with water, weak solution due to water carryover, contaminants, thin sections
2. Excessive Toluidine Blue Staining:
   Due to drying of tissue, excessive staining times, inadequate de-staining/rinsing with water, thick sections
3. Water Beads or White Haze under the Coverslip:
   Due to incomplete dehydration of the section, formed by mixture of water and the clearing agent

Sample Toluidine Blue Staining Method

1. Alcoholic formalin (10 dips)
2. Tap water (10 dips)
3. Tap water (10 dips)
4. 1% T-blue (40 seconds)
5. Tap water (10 dips)
6. 70% ethanol (10 dips)
7. 95% ethanol (10 dips)
8. 100% ethanol (10 dips)
9. 100% ethanol (10 dips)
10. Clearing agent (10 dips)
11. Clearing agent (10 dips)
12. Coverslip with compatible mounting medium

Conclusion

Preference for staining method in frozen section is usually based on individual training or personal experience. The advantages and limitations of each technique should be considered, whether the preference is H&E or TB. It is important to recognize that staining characteristics, vary with technique, brand of the solution/stain, and experience. Firm understanding of the principles of the H&E or TB staining methods is crucial in troubleshooting for problems. Tissue staining should be reproducible and reliable. Excellent visualization and discrimination of histologic detail of tissue sections are crucial in a rapid and accurate pathologic diagnosis during a frozen section. Lastly, other stains such as Oil red O for fat, Hematoxylin and periodic acid Schiff (PAS) to highlight glycogen and mucoproteins, and some immunohistochemical stains are also available during a frozen section and should be considered if available.

References

1. Bancroft JD, Gamble M, editors. Theory and Practice of Histological Techniques. 6th ed. New York: Churchill Livingstone;2008.
2. Bancroft JD, Stevens A. Histopathological Stains and Their Diagnostic Uses. New York: Churchill Livingstone; 1975.
3. Junqueira LC, Carneiro J. Basic Histology. 11th ed. New York: McGraw Hill, Inc.; 2005.
4. Ross MH, Wojciech P. Histology: A Text and Atlas. 5th ed. Baltimore: Lippincott Williams & Wilkins; 2006.
5. Larson K, Ho HH, Anumolu PL, Chen TM. Hematoxylin and eosin tissue stain in Mohs micrographic surgery: a review. Dermatol Surg. 2011 Aug;37(8):1089-99. doi: 10.1111/j.1524-4725.2011.02051.x. Epub 2011 Jun 2. Review. PubMed PMID: 21635628.
6. Preece A. A Manual for Histologic Techniques. 3rd ed. Boston: Little, Brown, and Company; 1972.
7. Luna L, editor. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York: McGraw-Hill, Inc.; 1968.
8. Humphreys TR, Nemeth A, McCrevey S, Baer SC, Goldberg LH. A pilot study comparing toluidine blue and hematoxylin and eosin staining of basal cell and squamous cell carcinoma during Mohs surgery. Dermatol Surg. 1996 Aug;22(8):693-7. PubMed PMID: 8780761.
9. Wittekind D. Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain. Biotech Histochem 2003;78:261-70.
10. Sridharan, G; Shankar, AA (2012). Toluidine Blue: A review of its chemistry and clinical utility. J Oral Maxillofac Pathol. 16: 251–5. doi:10.4103/0973-029X.99081.
11. Aslam A, Aasi SZ. Frozen-Section Tissue Processing in Mohs Surgery. Dermatol Surg. 2019 Dec;45 Suppl 2:S57-S69. doi: 10.1097/DSS.0000000000002260. PubMed PMID: 31764292.
12. Todd MM, Lee JW, Marks VJ. Rapid toluidine blue stain for Mohs’ micrographic surgery. Dermatol Surg. 2005 Feb;31(2):244-5. PubMed PMID: 15762224.
13. Tehrani H, May K, Morris A, Motley R. Does the dual use of toluidine blue and hematoxylin and eosin staining improve basal cell carcinoma detection by Mohs surgery trainees? Dermatol Surg. 2013 Jul;39(7):995-1000. doi: 10.1111/dsu.12180. Epub 2013 Mar 6. PubMed PMID: 23465179.
14. Wang SQ, Goldberg LH, Nemeth A. The merits of adding toluidine blue-stained slides in Mohs surgery in the treatment of a microcystic adnexal carcinoma. J Am Acad Dermatol. 2007 Jun;56(6):1067-9. PubMed PMID: 17504725.
15. Carson, Freida L., and Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press, 2015. 120-121.

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 recommended daily intake for biotin in the US. Standard multivitamin in the market contains 30 – 60 µg which is a suggested biotin intake for adults. Doses under 2,500 µg/day make up the majority of biotin sales, and such dosages have no risk for immunoassay interference. Biotin sales with more than 2,500 µg doses actually have declined over the past three years which may be attributed to public education about biotin interference (10). At very rare situations where the patients have certain conditions like multiple sclerosis, higher doses (e.g. more than 5,000 µg) may be prescribed by physician. Such a high dose has a potential to lead to test interference. However, these high doses are under physician's direct care and should be recorded in patient's electronic medical records. Taken together, biotin interference for immunoassay-based laboratory tests is very rare. With public education and more and more patients' awareness, the biotin interference eventually will not be a problem in the future.

References

1. Samarasinghe S, Meah F, Singh V, Basit A, Emanuele N, Emanuele MA, Mazhari A, Holmes EW. Biotin interference with routine clinical immunoassays: understand the causes and mitigate the risks. Endocr Pract. 2017 23(8):989-998
2. Katzman BM, Lueke AJ, Donato LJ, Jaffe AS, Baumann NA. Prevalence of biotin supplement usage in outpatients and plasma biotin concentrations in patients presenting to the emergency department. Clin Biochem. 2018 60:11-16
3. Bowen R, Benavides R, Colón-Franco JM, Katzman BM, Muthukumar A, Sadrzadeh H, Straseski J, Klause U, Tran N. Best practices in mitigating the risk of biotin interference with laboratory testing. Clin Biochem. 2019 Aug 29. In press
4. FDA Safety Communication. The FDA Warns that Biotin May Interfere with Lab Tests. 2017 Nov 28. Available from: https://www.fda.gov/medical-devices/safety-communications/fda-warns-biotin-may-interfere-lab-tests-fda-safety-communication
5. Mcmahon RJ. Biotin in Metabolism and Molecular Biology. Annu Rev Nutr 2002 22:221–239.
6. LabCorp, LabCorp Vitamin B7 (Biotin) test catalog entry [Internet], Available from: https://www.labcorp.com/test-menu/36691/vitamin-bsub7-sub.
7. Roche Diagnostics. Elecsys T3 package insert, 2017-10, V 2.0 English.
8. Trambas CM, Liu KC, Luu H, Louey W, Lynch C, Yen T, Sikaris KA. Further assessment of the prevalence of biotin supplementation and its impact on risk. Clin Biochem. 2019 65:64-65.
9. Grimsey P, Frey N, Bendig G, Zitzler J, Lorenz O. Population pharmacokinetics of exogenous biotin and the relationship between biotin serum levels and in vitro immunoassay interference. Int J Pharmacokinet 2017 2:247–256
10. Roche Diagnostics 2018. Available from: https://biotinfacts.roche.com/understan

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 reflective of the role of endothelium to coagulation. Inherently the test is a poor predictor of platelet adhesion and von Willebrand’s disease related bleeding diathesis.

Moreover, an abnormal TEG in a patient lacking clinically relevant bleeding does not require transfusion of blood components. A single test or patient-related factor seldom guides the decision to transfuse blood components or initiate/correct antithrombotic therapy. Studies have also shown that preoperative TEG data are poor predictors of postoperative bleeding. Furthermore, the TEG results do not correlate the effects of hypothermia during surgeries, especially in cardiac cases as TEG is performed at 37°C [5,6].

TEG has a low sensitivity and specificity, which significantly varies in different populations. Patients taking anticoagulants and antiplatelet agents are a major concern in the trauma setting. Recently in a cohort study, it was noted that TEG was normal despite high international normalized ratio (INR) values in a large percentage of patients receiving warfarin. This might be related to the use of kaolin as a part of TEG procedure. Kaolin activates the intrinsic coagulation cascade, which cannot effectively detect alterations in the extrinsic coagulation cascade caused by warfarin [7]. This is a good example of how TEG may miss a potentially clinically significant coagulopathy. Hence, INR is still the gold standard of monitoring warfarin therapy. Several important blood tests also cannot be currently replaced by TEG, such as P2Y12 platelet function assay to guide clopidogrel therapy, D-Dimer to exclude venous thrombo-embolism (VTE) in low-risk outpatients, and advanced thrombophilia diagnostic tests.

Coagulopathies in massive trauma is related to the activation of the fibrinolytic system and is a major cause of increased mortality from traumatic hemorrhage [8]. In such settings, TEG is useful in detecting the fibrinolysis process. However, there are limitations in using TEG over conventional plasma biomarkers of fibrinolytic activation, e.g. plasmin-α2-antiplasmin (PAP) complex. Studies have shown that using PAP as a biomarker for fibrinolysis has detected fibrinolytic activation in over 80% of severely injured patients [9], whereas TEG detected hyperfibrinolysis in only 5–18% of the cases. This occurred due the that fact that TEG detects fibrinolysis only when the tPA (tissue plasminogen activator) levels are five times the normal. Moreover, rapid inhibition of tPA by plasminogen activator inhibitor (PAI)-1 may result in false negative rates of detection of fibrinolysis by TEG [9,10]. Thus, antifibrinolytic therapies with tranexamic acid (TXA) cannot be optimized in these patients based on TEG data [11]. Also, there is conflicting evidence for TEG usefulness in trauma patients. A recent Cochrane database systematic review found inadequate data to compare the accuracy of TEG versus PT/INR in the diagnosis of trauma-induced coagulopathy.[12] The review concluded that these tests are still in the phase of clinical research. Moreover, recent studies have shown that the use of platelet mapping with TEG appears to be limited by its nonspecific findings of platelet receptor inhibition in determining anti-platelet therapy in certain patient populations [13].

Moreover, sample collection and processing need to be standardized in TEG in order to reduce inter-observer variabilities between laboratories. There is a continuous requirement for adequate maintenance, quality control, and supervision of personnel performing the test when testing occurs away from the controlled environment of the laboratory, as when used as a point of care device. The instrument requires multiple daily calibrations which should be performed by trained personnel and under standardized techniques. Sometimes this can be more expensive, as well as more time consuming, than conventional coagulation testing in the lab. Lastly, excessive TEG testing impacts laboratory workflow by competing with other time sensitive coagulation assays and intraoperative TEG. Each TEG run generally takes 30 minutes to an hour to complete and only a few cases can run simultaneously, unlike conventional lab coagulation testing. Therefore, optimization of TEG use is an important concern in providing appropriate patient laboratory testing.

Lab Best Practices

TEG has been used in different clinical settings with different algorithms at UC Davis Health (UCDH). However, the rationale behind ordering platelet mapping with TEG has not been evaluated and needs to be evaluated in order to improve patient care and appropriate laboratory utilization. Optimizing resources with a goal of improving patient care begins with determining the justification for testing with TEG, with or without platelet mapping, in different UCDH clinical settings.

Although TEG is recommended by NICE (National Institute for Health and Care Excellence) guidelines to help detect, manage, and monitor hemostasis in cardiac surgery patients (NICE guidelines, 2014), current clinical guidelines do not strongly recommend TEG for use in additional settings due to the lack of high-quality evidence. Recently updated guidelines of the European Society of Anesthesiology recommended viscoelastic hemostatic assays (TEG) to guide the management of perioperative bleeding and severe peripartum hemorrhage even though with the low level of evidence [14].

Based on our preliminary evaluations on TEG ordering practices, the Laboratory has designed the following best practice algorithm to follow when ordering kaolin TEG and/or TEG with platelet mapping. The goal of this algorithm is to optimize TEG utilization for patients needing emergent coagulation/TEG testing and support on-label use of the platform.

Future directions

Evaluation of TEG test ordering practices at UCDH need to be completed, and evaluation of adherence to guidelines analyzed. Adjustments to the algorithm will be made based upon the evaluation. Additionally, in the area of coagulation research, future studies may determine and refine the indications for TEG and evaluate if TEG can measure the effect of antiplatelet therapy, detect hyporesponsiveness, and predict the risk of bleeding or thromboembolic complications. The potential of TEG to improve the quality of antithrombotic therapy is a promising avenue for experimental and clinical research. The novel concept of personalized health care can be applied to the laboratory, including both anticoagulation and antiplatelet therapy monitoring.

Acknowledgements:

Dr. Nam Tran and Leslie Freeman.

References

1. Hartert H. [Not Available]. Klinische Wochenschrift 1948;26:577-583.
2. Othman M, Kaur H. Thromboelastography (TEG). Methods in molecular biology 2017;1646:533-543.
3. TEG® Hemostasis Analyzer, Model 5000, package insert
4. Wikkelsø A, Wetterslev J, Møller AM, Afshari A. Thromboelastography (TEG) or thromboelastometry (ROTEM) to monitor haemostatic treatment versus usual care in adults or children with bleeding. Cochrane Database Syst Rev. 2016 Aug 22;(8):CD007871.
5. Enriquez LJ, Shore-Lesserson L. Point-of-care coagulation testing and transfusion algorithms. British journal of anaesthesia 2009;103 Suppl 1:i14-22.
6. Rhee AJ, Kahn RA. Laboratory point-of-care monitoring in the operating room. Current opinion in anaesthesiology 2010;23:741-748.
7. Dunham CM, Rabel C, Hileman BM, et al. TEG(R) and RapidTEG(R) are unreliable for detecting warfarin-coagulopathy: a prospective cohort study. Thrombosis journal 2014;12:4.
8. Gall LS, Davenport RA. Fibrinolysis and antifibrinolytic treatment in the trauma patient. Current opinion in anaesthesiology 2018;31:227-233.
9. Raza I, Davenport R, Rourke C, et al. The incidence and magnitude of fibrinolytic activation in trauma patients. Journal of thrombosis and haemostasis : JTH 2013;11:307-314.
10. Leebeek FW, Rijken DC. The Fibrinolytic Status in Liver Diseases. Seminars in thrombosis and hemostasis 2015;41:474-480.
11. Cole E, Davenport R, Willett K, et al. Tranexamic acid use in severely injured civilian patients and the effects on outcomes: a prospective cohort study. Annals of surgery 2015;261:390-394.
12. Nakayama Y, Nakajima Y, Tanaka KA, Sessler DI, Maeda S, Iida J, Ogawa S, Mizobe T. Thromboelastometry-guided intraoperative haemostatic management reduces bleeding and red cell transfusion after paediatric cardiac surgery. Br J Anaesth. 2015 Jan;114(1):91-102.
13. Lam H, Katyal N, Parker C, Natteru P, Nattanamai P, Newey CR, Kraus CK. Thromboelastography With Platelet Mapping is Not an Effective Measure of Platelet Inhibition in Patients With Spontaneous Intracerebral Hemorrhage on Antiplatelet Therapy. Cureus. 2018 Apr 22;10(4):e2515.
14. Roullet S, Pillot J, Freyburger G, Biais M, Quinart A, Rault A, Revel P, Sztark F. Rotation thromboelastometry detects thrombocytopenia and hypofibrinogenaemia during orthotopic liver transplantation. Br J Anaesth. 2010 Apr;104(4):422-8.

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 30 days of the scheduled procedure. This initial T&S allows transfusion services to fulfill AABB standard 5.14.3.1 – When clinically significant antibodies are detected, additional testing shall be performed. Epidemiologic studies reveal that 1-3% of the general patient population will have a non-ABO red blood cell antibody⁹. After a positive antibody screen is detected, the additional testing (antibody identification and finding/crossmatching the compatible blood) takes several hours or sometimes several days to complete. It is impractical to expect this testing to be done emergently the day of surgery. Massive transfusion 'universal blood' is also not guaranteed safe to provide when the antibody screen is positive since it may be incompatible.

Since it is logistically challenging to confirm history in most cases, UC Davis Medical Center's hospital transfusion policy requires a type and screen to be drawn from every patient within 3 days of their scheduled transfusion whether they have a history of transfusion/pregnancy or not¹⁰. A second T&S should therefore be drawn within 3 days of surgery. Any products ordered for surgery will be crossmatched using this fresh sample.

Conclusion

Appropriate pre-op planning ensures blood availability for the date/time of surgery and reduces the risk of a canceled procedure. Following this lab best practice helps to certify the right blood gets to the right patient at the right time. It reduces the risk of adverse events or canceled procedures and improves system efficiency. It's a win/win for everyone, most importantly for our patients.

References:

1. Information online at https://www.fda.gov/BiologicsBloodVaccines/default.htm. Accessed 9/11/18.
2. Information online at http://www.aabb.org/tm/Pages/default.aspx. Accessed 9/11/18.
3. Information online at https://www.cdph.ca.gov/Programs/OSPHLD/LFS/Pages/BloodBanksandBiologics.aspx. Accessed 9/11/18.
4. Information online at http://www.cap.org/web/home/lab/accreditation/laboratory-accreditation-program. Accessed 9/11/18.
5. Information online at https://www.redcrossblood.org/biomedical-services/blood-diagnostic-testing/blood-testing.html. Accessed 9/11/18.
6. Standards for Blood Banks and Transfusion Services 31^st Effective April 1, 2018. AABB Press.
7. On the intranet at http://intranet.ucdmc.ucdavis.edu/pacu/Transfusion%20Blood%20Bank/MSBOS.pdf. Accessed 9/11/18.
8. Frank, SM et al. Reducing Unnecessary Preoperative Blood Orders and Costs by Implementing an Updated Institution-Specific MSBOS and a Remote Electronic Blood Release System. Anesthesiology. 2014 Sep; 121(3): 501–509.
9. Zhengtong P and Arpad S. Prevention of surgical delays by pre-admission type and screen in patients with scheduled surgical procedures: improved efficiency. Blood Transfus. 2015 Apr; 13(2): 310-312.
10. On the intranet at http://intranet.ucdmc.ucdavis.edu/policies/patient_care_standards/line_management_therapy/xiii-12.shtml. Accessed 9/11/18.

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 for targeting a CYP or set of CYPs for a specific drug. Broad CYP genetic analysis requires prior approval and review by the laboratory test utilization staff.

Other considerations include patients with normal CYP genotypes presenting with abnormal drug levels. These abnormal drug levels may be caused by polypharmacy where drugs compete for common CYP pathways. Some medications and even foods can also act as CYP inducers and inhibitors. Lastly, CYPs are not the only enzymes that metabolize drugs. Glucuronidation, glutathione, and sulfation pathways are common and not routinely tested. Further studies are needed to determine the clinical value of CYP analysis and proper test utilization.

References

1. Zanger UUM, Schwab M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharm Ther 2013;138:103-141.
2. Goetz MP, Kamal A, Ames MM. Tamoxifen pharmacogenomics: the role of CYP2D6 as a predictor of drug response. Clin Pharmacol Ther 2008;83:160-166.
3. Medscape Article: https://www.medscape.com/viewarticle/771480, Accessed on December 12, 2017.
4. Centers for Medicare and Medicaid Services (CMS) announcement: https://www.cms.gov/Outreach-and-Education/Medicare-Learning-NetworkMLN/MLN MattersArticles/downloads/MM6715.pdf, Accessed on December 12, 2017.
5. Schroth W, Goetz MP, Hamann U, et al. Association between CYP2D6 polymorphisms and outcomes among women with early stage breast cancer treated with tamoxifen. JAMA. 2009;302:1429-36.
6. Regan MM, Leyland-Jones B, Bouzyk M et al.; Breast International Group (BIG) 1-98 Collaborative Group. CYP2D6 genotype and tamoxifen response in postmenopausal women with endocrine-responsive breast cancer: the breast international group 1-98 trial. J Natl Cancer Inst. 2012;104:441-51.
7. Rae JM, Drury S, Hayes DF, et al. CYP2D6 and UGT2B7 genotype and risk of recurrence in tamoxifen-treated breast cancer patients. J Natl Cancer Inst. 2012;104:452-60.

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.

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 influenza to make antiviral treatment decisions.⁶ Once influenza activity has been identified in the community, a clinical diagnosis of influenza can be made for outpatients with signs/symptoms consistent with suspected influenza. This workflow is very effective during periods of peak influenza activity in the community. In some cases, indiscriminate molecular influenza testing may unneccessarily increase costs and providing little to no clinical value.

Molecular Influenza Testing in Pregnant Women: The American College of Obstetrics and Gynecology (ACOG) and Society for Fetal Medicine recommends empiric treatment with antiviral therapy following CDC guidelines for pregnant women presenting with influenza-like illness.⁷ Molecular influenza testing is not required in this population. Molecular influenza testing could be wasteful or lead to treatment delays.

Molecular Influenza Testing in Children: The influenza infection rate is higher among infants and children, and consequently, influenza-related complications are also higher in this high-risk population. To this end, children may benefit from rapid molecular influenza testing. However, caution is advised since other respiratory diseases, such as pertussis (“whooping Cough”) may be present. It is highly recommended that at-risk children should be evaluated for other pathogens using molecular respiratory viral/bacterial panels available at UC Davis Medical Center. In the Sacramento area, pertussis infection rates have increased seven-fold from 2009 to 2010, therefore, it is recommended to include molecular testing for this pathogen in at risk children.⁸

Environmental Contamination: The high sensitivity of molecular assays is a “double-edged sword”. It is true that these assays outperform many RIDTs, however, there is an increased risk for false positive results.⁹ Unclean operating environments, poor specimen collection techniques and hand hygiene may lead to false positives. It is recommended that molecular POC operators rigorously maintain environmental cleanliness and follow manufacturer instructions for sample collection and testing.

References

1. World Health Organization website: http://apps.who.int/iris/bitstream/10665/44304/1/ 9789241599283_eng.pdf, Accessed on November 1, 2017.
2. Center for Disease Control and Prevention website: https://www.cdc.gov/h1n1flu/ guidance/rapid_testing.htm, Accessed on November 1, 2017.
3. United States Food and Drug Administration website: https://www.fda.gov /downloads/AdvisoryCommittees/CommitteesMeetingMaterials/MedicalDevices/MedicalDevicesAdvisoryCommittee/MicrobiologyDevicesPanel/UCM517283.pdf, Accessed on November 1, 2017.
4. United States Food and Drug Administration website: https://www.fda.gov/ NewsEvents/Newsroom/PressAnnouncements/ucm429127.htm, Accessed on November 1, 2017.
5. Binnicker MJ, Epsy MJ, Irish CL, et al. Direct detection of influenza A and B virus in less than 20 minutes using a commercially available rapid PCR assay. J Clin Microbiol 2015;53:2353-2354.
6. Center for Disease Control and Prevention website: https://www.cdc.gov/flu/professionals/ diagnosis/molecular-assays.htm, Accessed on November 2, 2017.
7. American Association for Obstetrics and Gynecology Immunization Website: http://www.immunizationforwomen.org/, Accessed on November 2, 2017.
8. Sacramento County Department of Public Health website: http://www.dhhs.saccounty.net /PUB/Pages/AZ-Health-Info/Pertussis-Whooping-Cough-Update.aspx, Accessed on November 10, 2017.
9. John A, and Price CP. Existing and emerging technologies for point-of-care testing. Clin Biochem Rev 2014;35:155-167.

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. Mean (SD) bias was 0.13 µIU/mL (P=0.094).

References

1. Chun KY. Biotin interference in diagnostics. http://clinchem.aaccjnls.org/content/63/2/619, Accessed on June 1, 2017
2. Sedal F, Papeix C, Bellanger A, et al. High doses of biotin in chronic progressive multiple schlerosis: a pilot study. Mult Scler Relat Disord 2015;159.
3. Kummer S. Biotin treatment mimicking Grave's disease. N Engl J Med 2016;375:704
4. Roche Diagnostics Product TSH Assay Product Insert, Accessed on June 1, 2017
5. DeVon HA, Hogan N, Ochs AL, et al. Time to treatment for acute coronary syndromes: the cost of indecision. J Cardiovasc Nurs 2011;25:106.