the process of antigen presentation

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Antigen Processing and Presentation

• Primary and Secondary Immune Responses
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Original Author(s): Antonia Round Last updated: 17th July 2023 Revisions: 9

• 1 Antigen Presentation
• 2.1 MHC Class I Molecules
• 2.2 MCH Class II Molecules
• 3.1 T Cell Receptors
• 3.2 Co-Receptors
• 4 Clinical Relevance – Autoimmune disease

T cells can only recognise antigens when they are displayed on cell surfaces. This is carried out by  Antigen-presenting cells (APCs) , the most important of which are dendritic cells, B cells, and macrophages. APCs can digest proteins they encounter and display peptide fragments from them on their surfaces for other immune cells to recognise.

This process of antigen presentation allows T cells to “see” what proteins are present in the body and to form an adaptive immune response against them. In this article, we shall discuss antigen processing, presentation, and recognition by T cells.

Antigen Presentation

Antigens are delivered to the surface of APCs by Major Histocompatibility Complex (MHC) molecules. Different MHC molecules can bind different peptides. The MHC is highly polygenic and polymorphic which equips us to recognise a vast array of different antigens we might encounter. There are different classes of MHC, which have different functions:

• MHC class I  molecules are found on all nucleated cells (not just professional APCs) and typically present intracellular antigens such as viruses.
• MHC class II molecules are only found on APCs and typically present extracellular antigens such as bacteria.

This is logical because should a virus be inside a cell of any type, the immune system needs to be able to respond to it. This also explains why pathogens inside human red blood cells (which are non-nucleated) can be difficult for the immune system to find, such as in malaria.

Whilst this is the general rule, in cross-presentation extracellular antigens can be presented by MHC class I, and in autophagy intracellular antigens can be presented by MHC class II.

Antigen Processing

Before an antigen can be presented, it must first be processed . Processing transforms proteins into antigenic peptides.

MHC Class I Molecules

Intracellular peptides for MHC class I presentation are made by proteases and the proteasome in the cytosol, then transported into the endoplasmic reticulum via TAP (Transporter associated with Antigen Processing) to be further processed.

They are then assembled together with MHC I molecules and travel to the cell surface ready for presentation.

Fig 1 – Diagram demonstrating the production of peptides for MHC class I presentation

MCH Class II Molecules

The route of processing for exogenous antigens for MHC class II presentation begins with endocytosis of the antigen. Once inside the cell, they are encased within endosomes that acidify and activate proteases, to degrade the antigen.

MHC class II molecules are transported into endocytic vesicles where they bind peptide antigen and then travel to the cell surface.

Fig 2 – Diagram showing processing of antigens for MHC Class II presentation by a dendritic cell

The antigen presented on MHCs is recognised by T cells using a T cell receptor (TCR) . These are  antigen-specific .

T Cell Receptors

Each T cell has thousands of TCRs , each with a unique specificity that collectively allows our immune system to recognise a wide array of antigens.

This diversity in TCRs is achieved through a process called V(D)J recombination during development in the thymus. TCR chains have a variable region where gene segments are randomly rearranged, using the proteins RAG1 and RAG2 to initiate cleavage and non-homologous end joining to rejoin the chains.

The diversity of the TCRs can be further increased by inserting or deleting nucleotides at the junctions of gene segments; together forming the potential to create up to 10 15 unique TCRs.

TCRs are specific not only for a particular antigen but also for a specific MHC molecule. T cells will only recognise an antigen if a specific antigen with a specific MHC molecule is present: this phenomenon is called  MHC restriction .

Co-Receptors

As well as the TCR, another T cell molecule is required for antigen recognition and is known as a co-receptor. These are either a CD4 or CD8 molecule:

• CD4 is present on T helper cells and only binds to antigen-MHC II complexes.
• CD8 is present on cytotoxic T cells and only binds to antigen-MHC I complexes.

This, therefore, leads to very different effects. Antigens presented with MHC II will activate T helper cells and antigens presented with MHC I activate cytotoxic T cells. Cytotoxic T cells will kill the cells that they recognise, whereas T helper cells have a broader range of effects on the presenting cell such as activation to produce antibodies (in the case of B cells) or activation of macrophages to kill their intracellular pathogens.

Clinical Relevance – Autoimmune disease

It is important to note that APCs may deliver foreign antigens or self-antigens. In the case of autoimmune diseases, self-antigens are presented to T cells, which then initiates an immune response against our own tissues.

For example, in Graves’ disease , TSHR (thyroid stimulating hormone receptor) acts as a self-antigen and is presented to T cells. This then activates B cells to produce autoantibodies against TSHRs in the thyroid. This results in the activation of TSHRs leading to hyperthyroidism and a possible goitre.

[start-clinical]

Clinical Relevance - Autoimmune disease

[end-clinical]

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Antigen processing and presentation: Cytosolic and Endocytic pathway

August 3, 2020 Gaurab Karki Immunology 0

Antigen processing and Antigen presentation

• Antigen processing is a metabolic process that digests the proteins into peptides which can be displayed on the cell membrane together with a class-I or class-II MHC molecules and recognized by T-cells.
• Antigen presentation is the process by which certain cell in the body especially antigen presenting cells (APCs) express processed antigen on their cell surface along with MHC molecules in the form recognizable to T cell.
• If antigen is presented along with class-I MHC molecule, it is recognized by CD8 + Tc-cell and if presented along with class-II MHC molecule, it is recognized by CD4 + TH cells.

On the basis of types of antigen to be processed and presented, antigen processing and presenting pathway are of two types:

Cytosolic pathway of antigen processing and presentation

• Cytosolic pathway processed and presented the endogenous antigens i.e. those generated within cell eg. Viral infected cells, tumor cells and intracellular pathogens ( M . tuberculosis, Histoplasma capsulatum).
• The processed antigen is presented on the cell membrane with MHC-class I molecule which is recognized by CD8 + Tc-cell for degradation.

Steps involved in cytosolic pathways are:

• Proteolytic degradation of Ag (protein) into peptides
• Transportation of peptides from cytosol to RER
• Assembly of peptides with class I MHC molecules

i. Proteolytic degradation of proteins into peptides:

• Intracellular proteineous antigen are larger in size to be bound to MHC molecule.
• So, it is degraded into short peptides of about 8-10 amino acids.
• These proteins are degraded by cytosolic proteolytic system present in cell called proteasome.
• The large (20S) proteasome is composed of 14 sub-units arranged in barrel-like structure of symmetrical rings.
• Some, but not all the sub-units have protease activity.
• Proteins enter the proteasome through narrow channel at each end.
• Many proteins targeted for proteolysis have a small protein called ubiquitin attached to them.
• Ubiquitin attached to them ubiquitin-protein complex consisting of 20S proteasome and 19S regulatory component added to it.
• The resulting 26S proteasome cleaves peptide bonds which is ATP-dependent process.
• Degradation of ubiquitin protein complex is thought to occur within the central hollow of the proteasome to release peptides.

ii. Transportation of peptides from cytosol to Rough Endoplasmic Reticulum (RER):

• Peptides generated in cytosol by proteasome are transported by TAP (transporter associated with antigen processing) into RER (Rough endoplasmic reticulum) by a process which require hydrolysis of ATP.
• TAP is membrane spanning heterodimer consisting of two proteins, TAP1 and TAP2.
• TAP has affinity for peptides having 8-16 amino acids.
• The optimal peptide length required by class-I MHC for binding is nine, which is achieved by trimming the peptides with the help of amino-peptidase present in RER. Eg. ERAP.
• In addition to it, TAP favor peptides with hydrophobic or basic carboxyl terminal amino acids, that preferred anchor residues for class-I MHC molecules.
• TAP deficiency can lead to a disease syndrome that has both immune-deficiency and auto-immunity aspects.

iii. Assembly of peptides with class-I MHC molecule:

• Like other proteins, the α-chain and β 2 microglobulin components of the class-I MHC molecule are synthesized on polysome along the rough endoplasmic reticulum.
• Assembly of these components into stable class-I MHC molecule that can exit the RRE require binding of peptides into peptide binding groove of class-I MHC molecules.
• The assembly process involves several steps and needs help of molecular chaperone.
• The first molecular chaperone involved in assembly of class-I MHC is calnexin.
• It is a resident membrane protein of RER.
• Calnexin associated with free class-I α-chain and promotes its folding.
• When β 2 -microglobulin binds class-I α-chain, calnexin is released and class-I MHC associates with another chaperone calreticulin and tapasin (TAP-associated protein).
• Tapasin brings TAP transporter carrying peptides to the proximity with class-I MHC molecule and allows to acquire the antigenic peptides.
• An additional protein with enzymatic activity, ERp57, form disulfide bond to tapasin and non-covalently associates with calreticulin to stabilize the interaction and allows release of MHC-I-class after acquiring antigenic peptides.
• As a consequence, the productive peptide binding with MHC of class-I releases from the complex of calreticulin, tapasin and ERp57, exit from RER and displays on the cell surface via golgi complex.

Endocytic pathway of antigen processing and presentation:

• The endocytic pathway processed and present the exogenous Ag. i.e. antigens generated outside the cells. E.g. Bacteria.
• At first APC phagocytosed, endocytosed or both, the antigen.
• Macrophage and dendritic cells internalize the antigen by both the process.
• While other APCs are non-phagocytic or poorly phagocytic. E.g. B cell internalize the antigen by receptor mediated endocytosis.
• Then antigen is processed and presented on the cell surface along with class-II MHC molecules which are recognized by CD4 + TH cell.

Steps involved in endocytic pathway:

• Peptide generation from internalized molecules (Ag) in endocytic vesicles.
• Transport of class-II MHC molecule to endocytic vesicles.
• Assembly of peptides with Class-II MHC molecules.

i. Peptide generation from internalized molecules (Ag) in endocytic vesicles:

• Once an antigen is internalized, it is degraded into peptides within compartments of endocytic processing pathway.
• The endocytic pathway appears to involve three increasingly acidic compartments, early endosomes (pH 6-6.5), late endosomes or endo-lysosome (pH 5-6) and lysosomes (pH 4.5-5).
• The internalized antigens move from early to late endosomes and finally to lysosomes, encountering hydrolytic enzymes and a lower pH in each compartment.
• Within the compartment, antigen is degraded into oligopeptides of about 13-18 residues.
• The mechanism by which internalized Ag moves from one endocytic compartment to next has not been clearly demonstrated.
• It has been suggested that early endosome move from periphery to inward to become late endosome and finally lysosomes.
• Alternatively, small transport vesicles may carry Ag from one compartment to next.

ii. Transport of class-II MHC molecule to endocytic vesicles:

• When class-II MHC molecules are synthesized within RER, three pairs of class-II αβ- chains associated with a pre-assembled trimer of a protein called invariant chain (Li, CD74).
• This trimeric protein prevents any endogenously antigen to bind to the cleft.
• The invariant chain consists of sorting signals in its cytoplasmic tail.
• It directs the transport of class-II MHC molecule to endocytic compartments from the trans-golgi network.

iii. Assembly of peptides with class-II MHC molecules:

• Class-II MHC-invariant chain complexes are transported from RER through golgi complex and golgi-network and through endocytic compartment, moving from early endosome to late endosome and finally to lysosome.
• The proteolytic activities increase in each compartment, so the invariant is slowly degraded.
• However, a short fragment of invariant chain remained termed as CLIP (Class-II associated invariant chain).
• CLIP physically occupies the peptide binding, cleft of class-II MHC molecule, presumably preventing any premature binding of antigenic peptides.
• A non-classical class-II MHC molecule known as HLA-DM is required to catalyze the exchange of CLIP with antigenic peptides.
• The reaction between HLA-DO, which binds to HLA-DM and lessens the efficiency of the exchange reactions.
• Conditions of higher acidity in endocytic compartment weakens the association of DM/DO and increase the possibility of antigenic peptide binding despite of DO.
• As with class-I MHC molecule, peptide binding is required to maintain the structure and stability of class-II MHC molecules.
• Once a peptide has bound the peptide-class II MHC complex is transported to the plasma membrane where neutral pH enables the complex to assume the compact and stable form.

• Antigen processing and presentationCytosolic and Endocytic pathway

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Antigen Processing and Presentation

• specialized antigen presenting cells (APCs) can activate the immune system
• killer T-cells can monitor the intracellular contents of all cells
• helper T-cells can be alerted to both intracellular and extracellular antigens
• endogenous antigens are proteins produced by the cell
• exogenous antigens are proteins that are taken up by the cell
• they can be stably exported to the cell surface
• they can be recognized specifically by T-cells
• recognize antigens loaded onto MHC class II
• recognize antigens loaded onto MHC class I
• degradation of proteins into component peptides
• translocation of the peptides into the appropriate compartment
• loading of the peptides onto MHC proteins
• the location from where the antigen originated
• the type of response that is required

• T-cell receptors can bind specifically to the peptide
• CD4 and CD8 can bind specifically to the corresponding MHC
• Integrins can bind APCs
• specific detection of antigens
• activation of T-cells

• - Antigen Processing and Presentation

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A guide to antigen processing and presentation

Affiliations.

• 1 Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
• 2 Society of Fellows, Harvard University, Cambridge, MA, USA.
• 3 Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
• 4 Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. [email protected].
• PMID: 35418563
• DOI: 10.1038/s41577-022-00707-2

Antigen processing and presentation are the cornerstones of adaptive immunity. B cells cannot generate high-affinity antibodies without T cell help. CD4 + T cells, which provide such help, use antigen-specific receptors that recognize major histocompatibility complex (MHC) molecules in complex with peptide cargo. Similarly, eradication of virus-infected cells often depends on cytotoxic CD8 + T cells, which rely on the recognition of peptide-MHC complexes for their action. The two major classes of glycoproteins entrusted with antigen presentation are the MHC class I and class II molecules, which present antigenic peptides to CD8 + T cells and CD4 + T cells, respectively. This Review describes the essentials of antigen processing and presentation. These pathways are divided into six discrete steps that allow a comparison of the various means by which antigens destined for presentation are acquired and how the source proteins for these antigens are tagged for degradation, destroyed and ultimately displayed as peptides in complex with MHC molecules for T cell recognition.

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• Review Article
• Published: 18 September 2024

Antigen presentation for central tolerance induction

• Ludger Klein 1 &
• Elisabetta Petrozziello 1  

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• Adaptive immunity
• Antigen-presenting cells

The extent of central T cell tolerance is determined by the diversity of self-antigens that developing thymocytes ‘see’ on thymic antigen-presenting cells (APCs). Here, focusing on insights from the past decade, we review the functional adaptations of medullary thymic epithelial cells, thymic dendritic cells and thymic B cells for the purpose of tolerance induction. Their distinct cellular characteristics range from unconventional phenomena, such as promiscuous gene expression or mimicry of peripheral cell types, to strategic positioning in distinct microenvironments and divergent propensities to preferentially access endogenous or exogenous antigen pools. We also discuss how ‘tonic’ inflammatory signals in the thymic microenvironment may extend the intrathymically visible ‘self’ to include autoantigens that are otherwise associated with highly immunogenic peripheral environments.

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L.K. receives funding through the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) under SFB 1054/3 Project A01 (210592381) and SFB-TRR 355/1 Project B01 (490846870). L.K. and E.P. receive DFG funding under Project number 456882036.

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B cells that emerge early in life, are primarily found in the peritoneal cavity and have distinctive functions in innate immunity and the production of natural antibodies.

Conventional B cells found in secondary lymphoid organs, where they are crucial for adaptive immunity and antibody production.

Macroautophagy or microautophagy shuttles cytoplasmic material into lysosomal compartments and thereby intersects with the MHC class II loading pathway, so that peptides from endogenously expressed proteins can be loaded onto MHC class II.

A growth hormone predominantly secreted in the stomach, but also known to counteract age-dependent thymus involution.

Cells embedded in the epithelial lining of the intestine that endocytose luminal antigens and transport them to intraepithelial dendritic cells, macrophages and B cells.

The repertoires of peptides that are bound by MHC molecules.

(TRAs). An arbitrary term to describe genes with a confined expression pattern restricted to at most five peripheral cell types (in distinction from ubiquitously expressed self-antigens such as housekeeping genes).

Chemosensory and IL-25-expressing cells that are embedded in the epithelial lining of the intestine. Their name refers to characteristic brush-like microvilli.

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Klein, L., Petrozziello, E. Antigen presentation for central tolerance induction. Nat Rev Immunol (2024). https://doi.org/10.1038/s41577-024-01076-8

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Chapter 5 antigen presentation to t lymphocytes.

In an adaptive immune response, antigen is recognized by two distinct sets of highly variable receptor molecules—the immunoglobulins that serve as antigen receptors on B cells and the antigen-specific receptors of T cells. As we saw in Chapter 3, T cells recognize only antigens that are displayed on cell surfaces. These antigens may derive from pathogens that replicate within cells, such as viruses or intracellular bacteria, or from pathogens or their products that cells internalize by endocytosis from the extracellular fluid. T cells can detect the presence of intracellular pathogens because infected cells display on their surface peptide fragments derived from the pathogens' proteins. These foreign peptides are delivered to the cell surface by specialized host-cell glycoproteins, the MHC molecules, which are also described in Chapter 3. The MHC glycoproteins are encoded in a large cluster of genes that were first identified by their potent effects on the immune response to transplanted tissues. For that reason, the gene complex was termed the major histocompatibility complex (MHC) . We now know that within this region of the genome, in addition to those genes encoding the MHC molecules themselves, are many genes whose products are involved in the production of the MHC:peptide complexes.

We will begin by discussing the mechanisms of antigen processing and presentation, whereby protein antigens are degraded into peptides inside cells and the peptides are then carried to the cell surface bound to MHC molecules. We will see that the two different classes of MHC molecule, known as MHC class I and MHC class II, deliver peptides from different cellular compartments to the surface of the infected cell. Peptides from the cytosol are bound to MHC class I molecules and are recognized by CD8 T cells, whereas peptides generated in vesicles are bound to MHC class II molecules and recognized by CD4 T cells. The two functional subsets of T cells are thereby activated to initiate the destruction of pathogens resident in these two different cellular compartments. Some CD4 T cells activate naive B cells that have internalized specific antigen, and thus also stimulate the production of antibodies to extracellular pathogens and their products.

In the second part of this chapter we will see that there are several genes for each class of MHC molecule: that is, the MHC is polygenic. Each of these genes has many variants: that is, the MHC is also highly polymorphic. Indeed, the most remarkable feature of the MHC class I and II genes is their genetic variability. MHC polymorphism has a profound effect on antigen recognition by T cells, and the combination of polygeny and polymorphism greatly extends the range of peptides that can be presented to T cells by each individual and each population at risk from an infectious pathogen.

• The generation of T-cell receptor ligands
• The major histocompatibility complex and its functions
• Summary to Chapter 5
• General references
• Section references
• Cite this Page Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. Chapter 5, Antigen Presentation to T Lymphocytes.

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Microbiology Devices; Reclassification of Antigen, Antibody, and Nucleic Acid-Based Hepatitis B Virus Assay Devices

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• Document Details Published Content - Document Details Agencies Department of Health and Human Services Food and Drug Administration Agency/Docket Number Docket No. FDA-2024-N-3533 CFR 21 CFR 866 Document Citation 89 FR 78265 Document Number 2024-21932 Document Type Proposed Rule Pages 78265-78278 (14 pages) Publication Date 09/25/2024 Published Content - Document Details
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Supplementary information:, i. background—regulatory authorities, ii. regulatory history of the devices, a. qualitative hbv antigen assays, b. hbv antibody assays (including qualitative and quantitative anti-hbs), c. quantitative hbv nucleic acid-based assays, iii. device description, iv. proposed reclassification and summary of reasons for reclassification, v. public health benefits and risks to health, vi. summary of data upon which the reclassification is based, vii. proposed special controls, viii. analysis of environmental impact, ix. paperwork reduction act of 1995, x. proposed effective date, xi. codification of orders, xii. references, list of subjects in 21 cfr part 866, part 866—immunology and microbiology devices.

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Microbiology Devices; Reclassification of Antigen, Antibody, and Nucleic Acid-Based Hepatitis B Virus Assay Device

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The Food and Drug Administration (FDA, the Agency, or we) is proposing to reclassify qualitative hepatitis B virus (HBV) antigen assays, qualitative HBV antibody assays and quantitative assays that detect anti-HBs (antibodies to HBV surface antigen (HBsAg)), and quantitative HBV nucleic acid-based assays, all of which are postamendments class III devices, into class II (general controls and special controls), subject to premarket notification. FDA is also proposing three new device classification regulations along with the special controls that the Agency believes are necessary to provide a reasonable assurance of safety and effectiveness for each device.

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Maria Ines Garcia, Center for Devices and Radiological Health, Food and Drug Administration, 10903 New Hampshire Ave., Bldg. 66, Rm. 3104, Silver Spring, MD 20993, 301-796-7017, [email protected] .

The Federal Food, Drug, and Cosmetic Act (FD&C Act), as amended, establishes a comprehensive system for the regulation of medical devices intended for human use. Section 513 of the FD&C Act ( 21 U.S.C. 360c ) established three categories (classes) of devices, reflecting the regulatory controls needed to provide reasonable assurance of their safety and effectiveness. The three categories of devices are class I (general controls), class II (general controls and special controls), and class III (general controls and premarket approval).

Section 513(a)(1) of the FD&C Act defines the three classes of devices. Class I devices are those devices for which the general controls of the FD&C Act (controls authorized by or under sections 501, 502, 510, 516, 518, 519, or 520 ( 21 U.S.C. 351 , 352 , 360 , 360f , 360h , 360i , or 360j ) or any combination of such sections) are sufficient to provide reasonable assurance of safety and effectiveness; or those devices for which insufficient information exists to determine that general controls are sufficient to provide reasonable assurance of safety and effectiveness or to establish special controls to provide such assurance, but because the devices are not purported or represented to be for a use in supporting or sustaining human life or for a use which is of substantial importance in preventing impairment of human health, and do not present a potential unreasonable risk of illness or injury, are to be regulated by general controls (section 513(a)(1)(A) of the FD&C Act). Class II devices are those devices for which general controls by themselves are insufficient to provide reasonable assurance of safety and effectiveness, and for which there is sufficient information to establish special controls to provide such assurance, including the issue of performance standards, postmarket surveillance, patient registries, development and dissemination of guidelines, recommendations, and other appropriate actions the Agency deems necessary to provide such assurance (section 513(a)(1)(B) of the FD&C Act). Class III devices are those devices for which insufficient information exists to determine that general controls and special controls would provide a reasonable assurance of safety and effectiveness, and are purported or represented to be for a use in supporting or sustaining human life or for a use which is of substantial importance in preventing impairment of human health, or present a potential unreasonable risk of illness or injury (section 513(a)(1)(C) of the FD&C Act).

Devices that were not in commercial distribution before May 28, 1976 (generally referred to as “postamendments devices”) are automatically classified by section 513(f)(1) of the FD&C Act into class III without any FDA rulemaking process. Those devices remain in class III and require premarket approval, unless, and until: (1) FDA reclassifies the device into class I or II, or (2) FDA issues an order finding the device to be substantially equivalent, in accordance with section 513(i) of the FD&C Act, to a predicate device that does not require premarket approval. The Agency determines whether new devices are substantially equivalent to predicate devices by means of the premarket notification procedures in section 510(k) of the FD&C Act and part 807, subpart E ( 21 CFR part 807, subpart E ) of FDA's regulations.

A postamendments device that has been initially classified in class III under section 513(f)(1) of the FD&C Act may be reclassified into class I or class II under section 513(f)(3) of the FD&C Act. Section 513(f)(3) of the FD&C Act provides that FDA, acting by administrative order, can reclassify the device into class I or class II on its own initiative, or in response to a petition from the manufacturer or importer of the device. To change the classification of the device, the proposed new class must have sufficient regulatory controls to provide reasonable assurance of the safety and effectiveness of the device for its intended use.

FDA relies upon “valid scientific evidence”, as defined in section 513(a)(3) of the FD&C Act and 21 CFR 860.7(c)(2) , in the classification process to determine the level of regulation for devices. To be considered in the reclassification process, the “valid scientific evidence” upon which the Agency relies must be publicly available (see section 520(c) of the FD&C Act). ( print page 78267) Publicly available information excludes trade secret and/or confidential commercial information, e.g., the contents of a pending premarket approval application (PMA) (see section 520(c) of the FD&C Act).

In accordance with section 513(f)(3) of the FD&C Act, FDA is issuing this proposed order to reclassify qualitative HBV antigen assays intended for qualitative detection of HBV antigens as an aid in the diagnosis of acute or chronic HBV infection in specific populations, HBV antibody assays (including qualitative and quantitative anti-HBs) intended for use in the detection of antibodies to HBV, and quantitative HBV nucleic acid-based assays intended for use in the detection of HBV nucleic acid in specimens from individuals with antibody evidence of HBV infection, all of which are postamendments class III devices, into class II (general controls and special controls) subject to premarket notification, under three new device classification regulations with the names “Qualitative Hepatitis B Virus Antigen Assays,” “Hepatitis B Virus Antibody Assays,” and “Hepatitis B Virus Nucleic Acid-Based Assays.” FDA believes the standard in section 513(a)(1)(B) of the FD&C Act is met as there is sufficient information to establish special controls, which, in addition to general controls, will provide reasonable assurance of the safety and effectiveness of these devices. [ 1 ]

Section 510(m) of the FD&C Act provides that FDA may exempt a class II device from the premarket notification requirements under section 510(k) of the FD&C Act, if FDA determines that premarket notification is not necessary to provide reasonable assurance of the safety and effectiveness of the device. FDA has determined that premarket notification is necessary to provide a reasonable assurance of the safety and effectiveness of HBV antigen assays, HBV antibody assays, and HBV nucleic acid-based assays for their intended uses, therefore, the Agency does not intend to exempt these proposed class II devices from the requirement for premarket notification (510(k)) submission as provided under section 510(m) of the FD&C Act. If this proposed order is finalized, persons who intend to market this type of device must submit to FDA a premarket notification under section 510(k) of the FD&C Act prior to marketing the device.

Under section 513(f)(1) of the FD&C Act, qualitative HBV antigen assays, HBV antibody assays (including qualitative and quantitative anti-HBs), and quantitative HBV nucleic acid-based assays are automatically classified into class III because they were not introduced or delivered for introduction into interstate commerce for commercial distribution before May 28, 1976, and have not been found substantially equivalent to a device placed in commercial distribution after May 28, 1976, which was subsequently classified or reclassified into class II or class I. Therefore, they are subject to PMA requirements under section 515 of the FD&C Act ( 21 U.S.C. 360e ). Qualitative HBV antigen assays and HBV antibody assays (including qualitative and quantitative anti-HBs) are prescription devices and assigned product code LOM. Quantitative HBV nucleic acid-based assays are prescription devices and assigned product code MKT.

The first proposed device reclassification action applies to qualitative HBV antigen assay devices that are prescription in vitro diagnostic devices intended for qualitative detection of HBV antigens as an aid in the diagnosis of acute or chronic HBV infection in specific populations. On February 8, 2001, FDA approved its first HBV antigen assay (DiaSorin's ETI-EBK PLUS) for use in the qualitative detection of hepatitis Be antigen (HBeAg) in human serum or plasma (ethylenediaminetetraacetic acid (EDTA), citrate, or heparin) as indicative of a laboratory diagnosis of HBV infection through its PMA process under section 515 of the FD&C Act. On June 1, 2001, FDA approved its first HBV surface antigen (HBsAg) assay (Roche Elecsys HBsAg Immunoassay, Elecsys HBsAg Confirmatory, and Precicontrol HBsAg) for the qualitative detection of HBsAg in human serum or plasma (heparin, EDTA, sodium citrate) in adult pregnant and non-pregnant individuals. In a May 22, 2002, Federal Register notice ( 67 FR 36009 ), FDA announced the approval order and the availability of the Summary of Safety and Effectiveness Data (SSED) for these devices. Since the first approval order for an HBV antigen assay issued on February 8, 2001, FDA has approved 16 additional original PMAs for qualitative HBV antigen assays that are prescription devices intended for the detection of HBV antigens. These assays are intended as an aid in the diagnosis of acute or chronic HBV infection in conjunction with clinical findings and other diagnostic procedures ( e.g., HBV serology and antigen testing, liver function, etc.). These assays are not intended for use in screening of blood, plasma, cells, or tissue donors.

A review of the medical device reporting (MDR) databases indicates that there were 625 reported events for qualitative HBV antigen assays as of June 2024. Of these reported events, a significant majority of these were determined to be of no known impact or consequence to the patient. Events reported included false reactive results, false non-reactive results, incorrect or inadequate assay results, incorrect/inadequate/imprecise readings, improper or incorrect procedure or method, device operates differently than expected, and adverse event without identified device or use problems. Where incorrect results were obtained, it was not clear what the correct result should have been. As of June 2024, there have been no class III recalls, six class II recalls, and no class I recalls  [ 2 ] involving qualitative HBV antigen assays. The class II recalls occurred since 2006 due to defective caps, device design, no marketing application, signal for reactive results, and biased results for biotin concentrations that were lower than indicated. No patient harm was identified. These facts, coupled with the low number of reported events that caused patient harm, indicate a good safety record for this device class. These recall events reflect the risks to health identified in section V below, and FDA believes the special controls proposed herein, in addition to general controls, can effectively mitigate the risks identified in these recalls.

The second type of devices this proposed reclassification order applies to are qualitative HBV antibody assays and quantitative anti-HBs assays that are prescription in vitro diagnostic devices intended for use in the detection of antibodies to HBV. These devices are intended to aid in the diagnosis of HBV infection in persons with signs and symptoms of hepatitis and in persons at risk for HBV infection. On September 29, 2000, FDA approved its first qualitative HBV antibody assay (Ortho-Clinical Diagnostics, Inc.'s Vitros ( print page 78268) Immunodiagnostic Products: Anti-HBS Reagent Pack/Anti-HBS Calibrators) for the qualitative in vitro determination of total antibody to hepatitis B surface antigen (anti-HBs) in human serum as an aid in determining susceptibility to HBV infection for individuals prior to or following HBV vaccination, or where vaccination status is unknown, and for use with other HBV serological markers for the laboratory diagnosis of HBV disease associated with HBV infection, through its PMA process under section 515 of the FD&C Act. In a March 12, 2001, Federal Register notice ( 66 FR 14390 ), FDA announced the approval order and the availability of the SSED for this device. On July 22, 2002, FDA approved its first quantitative Anti-HBs (Siemens Healthcare Diagnostics Products Ltd.'s Immulite 2000 XPI Anti-HBs) for the quantitative measurement of total antibodies to the hepatitis B surface antigen (anti-HBs) in human serum and plasma (heparinized or EDTA) as an aid in the determination of susceptibility to HBV infection for individuals prior to or following HBV vaccination, or where vaccination status is unknown, or for use with other HBV serological markers for the laboratory diagnosis of HBV disease associated with HBV infection, through its PMA process under section 515 of the FD&C Act.

Since the first approval order of a qualitative HBV antibody assay on September 29, 2000, FDA has approved 31 additional original PMAs for qualitative HBV antibody assays for the detection of antibodies to HBV. FDA has also approved six assays for quantitative anti-HBs detection. Qualitative HBV antibody assays and quantitative anti-HBs assays are intended to aid in the diagnosis of HBV infection in persons with signs and symptoms of hepatitis and in persons at risk for HBV infection in conjunction with clinical findings and other diagnostic procedures ( e.g., HBV serology and antigen testing, liver function, etc.). These assays are not intended for use in screening of blood, plasma, cells, or tissue donors.

A review of the MDR databases indicates that there were 1,107 reported events for HBV antibody assays between years 2001 and June 2024. Of these reported events, a significant majority of these were of no known impact to the patient, and only four resulted in impact to patients such as misdiagnosis or viral infection. Events reported included adverse events without identified device or use problem, disconnection/low assay results, false non-reactive results, false reactive results, false high assay results (for example, the first assay result had a low signal to cutoff (s/co) value and repeat testing produced a higher s/co value), incorrect assay results, inadequate assay results, and low assay results (for example, the first assay result was in the equivocal zone, repeat testing produced a non-reactive result, and testing with an alternate device produced a reactive result). In numerous cases, it was not possible to determine what the correct result should have been (further testing was not performed, insufficient sample volume, different assays were used). As of June 2024, FDA is aware of 4 class III recalls, 12 class II recalls, and no class I recalls for these devices. The class II recalls occurred in 2007, 2008, 2009, 2011, 2012, 2013, 2014, 2018, and 2019, and were related to issues such as false reactive results, false high assay results, defective caps, and errors in labeling, packaging, or software. No patient harm has been identified. These facts, coupled with the low number of reported events that impacted the patient, indicate a good safety record for this device class. These recall events reflect the risks to health identified in section V below, and FDA believes the special controls proposed herein, in addition to general controls, can effectively mitigate the risks identified in these recalls.

Finally, the third type of device this proposed reclassification order applies to are quantitative HBV nucleic acid-based assay devices for use as a prescription in vitro diagnostic device intended for use in the detection of HBV nucleic acid in specimens from individuals with antibody evidence of HBV infection. On September 4, 2008, FDA approved its first quantitative HBV nucleic assay (Roche Molecular Systems, Inc.'s COBAS TaqMan HBV Test For Use With The High Pure System), an in vitro nucleic acid amplification assay for the quantitation of HBV deoxyribonucleic acid (DNA) in human serum or plasma (EDTA) intended for use as an aid in the management of patients with chronic HBV infection undergoing antiviral therapy, through its PMA process under section 515 of the FD&C Act.

Since the first approval order, FDA has approved four additional original PMAs for quantitative HBV nucleic acid-based assays for the quantitative detection of HBV DNA. The detection of HBV DNA is used for management of patients undergoing antiviral therapy for assessing response to treatment and not as a diagnostic for HBV infection.

The following section provides examples of the different technologies used. The different technologies begin with specimen lysis and HBV DNA through hybridization with magnetic particles. The differences in the technologies occur with the method of amplification:

• In one technology, the target HBV DNA sequence is amplified. The presence of HBV amplification products is detected by measuring the fluorescence of the HBV probe that binds to the target. Similarly, the presence of the internal control amplification product is detected. In the absence of HBV or internal control target sequences, probe fluorescence is quenched. In the presence of HBV or internal control target, the HBV or internal control probes bind to their target.
• In another technology, target amplification occurs via transcription-based nucleic acid amplification by fluorescent labeled probes (torches). More torches hybridize when more amplicon is present creating a higher fluorescent signal. The time taken for the fluorescent signal to reach a threshold proportional to the starting HBV DNA concentration is measured in relation to internal controls.

A review of the MDR databases indicates that as of June 2024 there were 13 reported events for nucleic acid-based HBV DNA assays since the first reported event in 2009. MDRs were for the following reasons: (1) incorrect, inadequate, or imprecise result or readings; (2) high readings; and (3) non-reproducible results. Of these, two had no known impact or consequence to the patient and two occurred when the patient had no signs, symptoms, or conditions. As of June 2024, FDA is aware of one class III recall, five class II recalls, and no class I recalls for these devices. The class II recalls occurred between 2005 and 2022 and were related to issues such as misquantitation of high results for negative samples (carryover from a high positive sample tested adjacent to a negative sample may produce an incorrect positive result), liquid level detection of reagent cassette, under filled and over filled enzyme reagent vials in assay kits, software, and low level of recombinant HBV DNA found in one lot of reagent. These facts, coupled with the low number of reported events that impacted the patient, indicate a good safety record for this device class. These recall events reflect the risks to health identified in section V below, and FDA believes the special controls proposed herein, in addition to general controls, can effectively mitigate the risks identified in these recalls. ( print page 78269)

The HBV assays that are the subject of this proposed order are postamendments prescription in vitro diagnostic devices classified into class III under section 513(f)(1) of the FD&C Act.

A qualitative HBV antigen assay is a prescription in vitro diagnostic device intended for use in the qualitative detection of HBV antigens and for use as an aid in the diagnosis of HBV infection in specific populations. HBV antigen assays aid in the diagnosis of acute or chronic HBV infection. HBV antigen assays typically detect the presence of Hepatitis B surface antigen (HBsAg) or Hepatitis B e antigen (HBeAg). HBV antigens (HBsAg and HBeAg), when present in samples, bind to anti-HBs or anti-HBe antibodies to form a complex that is bound to a solid phase ( e.g., microparticles, microtiter plate or other technology). Detection of the complexes can be performed using different methods which measure the presence/absence of anti-HBs or anti-HBe antibodies in the sample.

Diagnosis of HBV infection should not be established based on a single assay result but should be determined in conjunction with clinical findings and other diagnostic procedures ( e.g., HBV serology and antigen testing, liver function, etc.). These assays are not intended for use in screening of blood, plasma, cells, or tissue donors.

A qualitative HBV antibody assay is a prescription in vitro diagnostic device intended for use in the qualitative detection of antibodies to HBV and for use as an aid in the diagnosis of HBV infection in specific populations. HBV antibody assays aid in the diagnosis of HBV infection in persons with signs and symptoms of hepatitis and in persons at risk for HBV infection. Antibody assays typically detect the presence of antibodies to HBsAg (anti-HBs), Hepatitis B core antigen (anti-HBc), or HBeAg (anti-HBe). Diagnosis of HBV infection should not be established based on a single assay result, but should be determined in conjunction with clinical findings and other diagnostic procedures ( e.g., HBV serology and antigen testing, liver function, etc.). These assays are not intended for use in screening of blood, plasma, cells, or tissue donors.

A quantitative assay that detects anti-HBs (antibodies to HBV surface antigen (HBsAg)) is a prescription in vitro diagnostic device that is intended for quantitative use to aid in the diagnosis of HBV infection in persons with signs and symptoms of hepatitis and in persons at risk for HBV infection. Detection of anti-HBs indicates a present or past infection with HBV and can be used in conjunction with clinical findings such as other HBV serological markers (detection of other HBV antigens and antibodies to HBV) for diagnosis of HBV infection. Anti-HBs assay results may be used as an aid in the determination of susceptibility to HBV infection in individuals prior to vaccination or when vaccination status is unknown.

In some device designs, HBV antibodies, when present in the sample, bind to HBV antigens to form a complex that is bound to a solid phase ( e.g., microparticles, microtiter plate, or other technology). Detection of complexes can be performed using different methods that measure the presence/absence of HBV antibodies in the sample.

A quantitative HBV nucleic acid-based assay is a prescription in vitro diagnostic device intended for use in the detection of HBV nucleic acid in specimens from individuals with antibody evidence of HBV infection. In these devices, the detection of HBV nucleic acid is used for management of patients undergoing antiviral therapy for assessing response to treatment and NOT as a diagnostic for HBV infection.

FDA is proposing to reclassify qualitative HBV antigen, HBV antibody assays (including qualitative and quantitative anti-HBs), and quantitative HBV nucleic acid-based assays from class III (general controls and premarket approval) to class II (general controls and special controls) and to establish new names for the device types that will be within the classification regulations. FDA proposes to revise 21 CFR part 866 to create three new device classification regulations with the names “Qualitative Hepatitis B Virus Antigen Assays,” “Hepatitis B Virus Antibody Assays,” and “Hepatitis B Virus Nucleic Acid-Based Assays.” FDA believes that these names and proposed identification language most accurately describe these devices.

• A Qualitative Hepatitis B Virus (HBV) Antigen Assay is tentatively identified as an in vitro diagnostic device intended for prescription use for qualitative use with human serum, plasma, or other matrices that aids in the diagnosis of chronic or acute HBV infection. HBV surface antigen (HBsAg) is also used for screening of HBV infection in pregnant women to identify neonates who are at risk of acquiring hepatitis B during perinatal period. The assay is not intended for screening of blood, plasma, cells, or tissue donors.
• A Hepatitis B Virus (HBV) Antibody Assay is tentatively identified as an in vitro diagnostic device intended for prescription use in the detection of antibodies to HBV in human serum and plasma, or other matrices, and or as an aid in the diagnosis of HBV infection in persons with signs and symptoms of hepatitis and in persons at risk for hepatitis B infection. In addition, anti-HBc IgM (IgM antibodies to core antigen) assay is indicative of recent HBV infection. Anti-HBs (antibodies to surface antigen) assay results may be used as an aid in the determination of susceptibility to HBV infection in individuals prior to or following HBV vaccination or when vaccination status is unknown. The assay is not intended for screening of blood, plasma, cells, or tissue donors. The assay is intended as an aid in diagnosis in conjunction with clinical findings and other diagnostic procedures.
• A Hepatitis B Virus (HBV) Nucleic Acid-Based Assay is tentatively identified as an in vitro diagnostic device intended for prescription use in the detection of HBV nucleic acid in specimens from individuals with antibody evidence of HBV infection. In these devices, the detection of HBV nucleic acid is used as an aid in the management of HBV-infected individuals. The assay is intended for use with human serum or plasma (and other matrices as applicable) from individuals with HBV. The assay is not intended for use as a donor screening assay for the presence of HBV nucleic acids in blood, blood products, plasma, cells, or tissue donors.

Based upon our review experience and consistent with the FD&C Act and FDA's regulations in 21 CFR 860.134 , FDA believes that these devices should be reclassified from class III into class II with special controls because there is sufficient information to establish special controls that, along with general controls, can provide reasonable assurance of the devices' safety and effectiveness.

FDA is proposing to reclassify the HBV assays that are the subject of this proposed order. On September 7, 2023, the Microbiology Devices Panel (Panel) of the Medical Devices Advisory Committee convened to discuss and make recommendations regarding the ( print page 78270) reclassification of HBV assays from class III (general controls and premarket approval) to class II (general controls and special controls) ( https://www.fda.gov/​media/​173609/​download ). Panel members unanimously agreed that special controls, in addition to general controls, are necessary and sufficient to mitigate the risks to health of patients presented by these devices and to provide reasonable assurance of the safety and effectiveness of these devices (Refs. 1 and 2). The Panel agreed with FDA-identified risks and identified additional risk(s) and benefit(s) to include in the overall risk assessment. The Panel also discussed potential mitigation measure(s)/control(s) FDA should consider for each of the identified risks and recommended that, as part of any reclassification, the expected performance for these devices should remain the same. Notably, the performance of approved HBV antigen assays has generally been at least 97 percent sensitivity and 99 percent specificity. For approved anti-HBs, anti-Hbe, and anti-HBc total assays the sensitivity has generally been at least 95 percent, for approved anti-HBc IgM assays the sensitivity has been at least 86 percent, and for all HBV approved antibody assays the specificity has generally been above 97 percent.

FDA believes that at this time, sufficient data and information exist such that the risks identified in section V below can be mitigated by establishing special controls, and that these special controls, together with general controls, are necessary to provide a reasonable assurance of the safety and effectiveness of these HBV assays and therefore proposes these devices to be reclassified from class III (general controls and premarket approval) to class II (general controls and special controls). In accordance with section 513(f)(3) of the FD&C Act and 21 CFR part 860, subpart C , FDA is proposing to reclassify qualitative HBV antigen assays, HBV antibody assays (including qualitative and quantitative anti-HBs), and quantitative HBV nucleic acid-based assays from class III into class II, subject to premarket notification (510(k)) requirements. FDA believes that there is sufficient information available to FDA through FDA's accumulated experience with these devices from reviewing the PMAs for these HBV assays, and the Panel considerations and recommendations regarding the proposed special controls that FDA believes would effectively mitigate the risks to health identified in section V. Absent the special controls identified in this proposed order, general controls applicable to the devices are insufficient to provide reasonable assurance of the safety and effectiveness of the devices. FDA expects that the reclassification of these devices would enable more manufacturers to develop these assays such that patients would benefit from increased access to safe and effective tests.

FDA is proposing to create three separate classification regulations for HBV assays that will be reclassified from class III to class II. HBV assays are prescription in vitro diagnostic devices, and under this proposed order, if finalized, these devices will be identified as prescription in vitro diagnostic devices. As such, the devices must satisfy prescription labeling requirements for in vitro diagnostic products (see 21 CFR 809.10(a)(4) and (b)(5)(ii) ). In this proposed order, if finalized, FDA has identified the special controls under section 513(a)(1)(B) of the FD&C Act that, together with general controls, will provide a reasonable assurance of the safety and effectiveness of these assays.

FDA is also proposing to create a new product code for HBV antibody assays (including qualitative and quantitative anti-HBs) that will be assigned upon any finalization of this proposed order. Qualitative HBV antigen assays will continue to be assigned the product code LOM upon any finalization of this proposed order.

Section 510(m) of the FD&C Act provides that FDA may exempt a class II device from the premarket notification requirements under section 510(k) of the FD&C Act, if FDA determines that premarket notification is not necessary to provide reasonable assurance of the safety and effectiveness of the device. For these HBV assays, FDA has determined that premarket notification is necessary to provide a reasonable assurance of the safety and effectiveness of these devices. Therefore, the Agency does not intend to exempt these proposed class II devices from 510(k) requirements. If this proposed order is finalized, persons who intend to market a new HBV assay will no longer need to have a PMA for these devices but can instead submit to FDA a 510(k) and receive clearance prior to marketing the device. A 510(k) typically results in a shorter premarket review timeline compared to a PMA, which ultimately provides more timely access of these types of devices to patients.

FDA is providing a substantive summary of the valid scientific evidence concerning the public health benefits of the use of HBV assays (see also https://www.fda.gov/​media/​171770/​download ), and the nature (and if known, the incidence) of the risks of the devices (see further discussion of the special controls being proposed to mitigate these risks in section VII of this proposed order).

HBV infection represents a significant global public health burden. According to the World Health Organization (WHO), in 2019 there were approximately 296 million people chronically infected people worldwide, with 1.5 million new HBV infections each year. [ 3 ] It is estimated by the Centers for Disease Control and Prevention (CDC) that chronic HBV infection in the United States affects at least between 580,000 to 1.17 million people with HBV infection in the United States; two-thirds of whom may be unaware of their infection. [ 4 ] HBV infection can be asymptomatic, and accordingly, many HBV-infected individuals are unaware of their HBV infection. Approximately 95 percent of adult patients with acute infection, defined as the first 6 months after infection, recover completely, and 5 percent of adults develop chronic HBV. [ 5 ] Infants born to women who are HbsAg-positive are at high risk of HBV infection. In absence of treatment, infants infected with HBV have a 90 percent risk of progression to chronic HBV and up to 25 percent of infants who acquire chronic HBV infection will die prematurely from HBV-related hepatocellular carcinoma or cirrhosis. [ 6 ] Patients who are tested and become aware that they are HBV infected may modify risk behaviors to prevent transmission to others and can be referred for treatment. Patients with chronic HBV infection have a risk of developing liver damage, liver cancer, or liver failure. They can also spread their infection to others. HBV can be reactivated in patients receiving immunosuppressive therapies, resulting in serious risk of liver failure or liver-associated death (Ref. 3). HBV is a vaccine-preventable liver infection.

With the initiation of the WHO Viral Hepatitis Elimination Plan  [ 7 ] and the Department of Health and Human Services (HHS) Viral Hepatitis National ( print page 78271) Strategic Plan for the United States, [ 8 ] it is important for individuals to know their HBV infected status, to link HBV infected individuals to care, and to eliminate virus transmission. Therefore, diagnosis of patients with HBV infection through devices such as HBV antibody and antigen assays is essential to ensure that patients are linked to the appropriate care. Current CDC HBV Screening and Testing Recommendations include testing of the following groups: all adults 18 and older at least once in their lifetime using a triple panel test, pregnant women during pregnancy, people who are at ongoing risk for exposure, and anyone who requests HBV testing. [ 9 ]

FDA considered our accumulated experience with the regulation of these HBV assays, input from the Panel meeting, and postmarket information regarding these HBV assays, i.e., information from FDA's publicly available MDR, Manufacturer and User Facility Device Experience (MAUDE), and Medical Device Recall databases.

These HBV assays provide a benefit to the public health by informing individuals of their HBV infected status, linking HBV infected individuals to appropriate care, and aiding in eliminating virus transmission. Once an individual is tested and diagnosed as HBV infected, HBV nucleic acid testing is performed to inform treatment decisions. While HBV infection is treatable, it is not curable, which means that most people who start HBV antiviral treatment must continue it for life. The goal of current treatment is to suppress the virus and reduce the likelihood of long-term complications and transmission (Refs. 3 and 4). Thus, identifying individuals who are HBV infected, linking them to care, and managing their HBV infection to alleviate development of liver damage, liver cancer, liver failure, and potential HBV transmission would not only greatly impact public health but also go a long way towards helping the United States achieve HBV elimination.

Probable risks to health associated with the use of HBV assays include risks related to the risk of false results (false positives, false negatives, inaccurate low assay results, inaccurate high assay results, false reactive results, or false non-reactive results), failure to correctly interpret assay results, and failure to correctly operate the device. For HBV antigen and antibody assays, false positive results are generally referred to as false reactive results and false negative results are generally referred to as false non-reactive results. False results can lead to uninfected individuals receiving unnecessary further testing and treatment or infected individuals remaining undiagnosed and untreated. Undiagnosed and untreated individuals are likely to experience increases in morbidity and mortality and can spread the infection to others. FDA has identified the following additional specific risks to health associated with each of the HBV assays listed below.

Factors that may cause decreased assay sensitivity and/or an increased rate of false non-reactive results include, but are not limited to, the presence of interfering substances in the sample, acute infection at a stage that is too early for a device to detect the infection, and antigen concentrations that are too low to be detected by the device. Factors that may lead to false reactive results include device contamination from reactive samples, cross-reactivity with other antigens, or misinterpretation of invalid results as reactive.

• A false reactive assay result for HbeAg. Incorrectly interpreting the assay results as a reactive assay result or failing to correctly operate the assay causing a false reactive assay result may lead to continued treatment for hepatitis B with antiviral medication when it otherwise would not be indicated. Antiviral medication has risks including toxicity and more rarely allergic reactions. Over time, viral resistance in patients who are co-infected but undiagnosed with other viruses that are treated with the same antiviral medication, such as HIV, can lead to viral resistance.
• A false reactive assay result for HbsAg. Incorrectly interpreting the assay results as a reactive assay result or failing to correctly operate the assay causing a false reactive assay result may contribute to unnecessary additional testing, potentially delaying diagnosis of alternative causes of liver disease when present and may impact the psychological well-being of the patient. Factors that may increase the rate of false reactive assay reporting include cross-reactivity with antigens from other microorganisms or other disease conditions.
• A false non-reactive result for HbeAg. Incorrectly interpreting the assay results as a non-reactive assay result or failing to correctly operate the assay causing a false non-reactive assay result may lead to missing the opportunity for treatment of an HBV infected individual with antiviral medication or premature discontinuation of antiviral treatment when continuation of treatment is otherwise indicated should a clinician be falsely led to determine a patient has seroconverted HbeAg to anti-Hbe. Premature discontinuation of antiviral medication could result in adverse effects on patient health, such as cirrhosis, liver cancer, and liver damage, all of which are known to contribute to patient morbidity and mortality, or may contribute to public health risk by leading to virus transmission.
• A false non-reactive assay result for HbsAg. Incorrectly interpreting the assay results as a non-reactive assay result or failing to correctly operate the assay causing a false non-reactive assay result may delay or prevent a patient with HBV infection from being identified and linked to care. Missed identification of patients with chronic HBV infection could lead to adverse effects on patient health such as cirrhosis, liver cancer, and liver damage, all of which are known to contribute to patient morbidity and mortality. A false non-reactive HbsAg assay incorrectly interpreted as non-reactive also may contribute to public health risk by leading to virus transmission.

Factors that may cause decreased assay sensitivity and/or an increased rate of false non-reactive results include, but are not limited to, the presence of interfering substances in the sample, acute infection at a stage that is too early for a device to detect the infection, and antibody concentrations that are too low to be detected by the device. They also can be caused by misinterpretation of invalid results as non-reactive. Factors that may lead to false reactive results include device contamination from reactive samples, cross-reactivity with other antibodies, or misinterpretation of invalid results as reactive.

• A false reactive assay result for anti-HBs and anti-HBc. Incorrectly interpreting the assay results as a reactive assay result or failing to correctly operate the assay causing a false reactive assay result may lead to improper patient management. A false reactive antibody assay result could result in the unnecessary continuation of antiviral treatment. Antiviral medication has risks including toxicity and more rarely allergic reactions. Over time, viral resistance in patients who are co-infected but undiagnosed with other viruses that are treated with the same antiviral medication, such as HIV, can ( print page 78272) lead to viral resistance. Consequently, repeatedly false reactive results have the potential to lead to inappropriate patient management decisions.
• A false reactive assay result for anti-HBs. Incorrectly interpreting the assay results as a reactive assay result or failing to correctly operate the assay causing a false reactive assay result when the device is used as an aid in the determination of susceptibility to HBV infection in individuals prior to or following HBV vaccination or where vaccination status is unknown may cause a patient to be considered previously exposed and therefore immune to HBV or that the patient was successfully vaccinated. A false reactive result may cause the patient to not receive a vaccine, vaccine booster, hyperimmune globulin, and would be at higher risk of infection if exposed to HBV.
• A false reactive assay result for anti-Hbe. Incorrectly interpreting the assay results as a reactive assay result, or failing to correctly operate the assay causing a false reactive assay result may lead to missing the opportunity for treatment of HBV infection with antiviral medications in a subset of individuals for whom treatment would otherwise be indicated, or premature discontinuation of antiviral treatment when continuation of treatment is otherwise indicated should a clinician be falsely led to determine a patient has seroconverted HbeAg to anti-Hbe. Premature discontinuation of antiviral medication could result in adverse effects on patient health such as cirrhosis, liver cancer, and liver damage, all of which are known to contribute to patient morbidity and mortality, or may contribute to public health risk by leading to inadvertent transmission of virus by an infected individual.
• A false non-reactive assay result for anti-HBc. When the device is used as an aid in the diagnosis of HBV infection in patients with symptoms of hepatitis or who may be at risk for HBV infection, incorrectly interpreting the assay results as non-reactive assay result, or failing to correctly operate the assay causing a false non-reactive assay result may lead to non-diagnosis or a delay in diagnosis of HBV infection with an associated delay in therapy and potentially increased risk of HBV-related morbidity or mortality. Patients with active infection may unknowingly continue to infect others. False non-reactive results can also lead to unnecessary diagnostic evaluation if alternative etiologies of hepatitis are pursued. False non-reactive assay results may occur if the level of antibody in a specimen is below the limit of detection of the assay.
• A false non-reactive assay result for anti-HBs. When the device is used as an aid in the determination of susceptibility to HBV infection in individuals prior to or following HBV vaccination or where vaccination status is unknown, incorrectly interpreting the assay results as a non-reactive assay result or failing to correctly operate the assay causing a false non-reactive assay result may lead to unnecessary repeated vaccination for HBV.
• A false non-reactive assay result for anti-Hbe. Incorrectly interpreting the assay results as non-reactive assay result or failing to correctly operate the assay causing a false non-reactive assay result may lead to improper patient management, including continued treatment for HBV with antiviral medication. Antiviral medication has risks including toxicity and more rarely allergic reactions. Over time, viral resistance in patients who are co-infected but undiagnosed with other viruses using the same antiviral medication, such as HIV, can lead to viral resistance.

Decreased assay sensitivity and/or an increased rate of false negative assay reporting may occur with patient samples that contain different genotypes or rare de novo mutations in HBV genomic regions targeted by the device. In these situations, HBV viral load can transiently decrease and/or become undetectable in samples before the virus enters chronic replication.

• A false positive or falsely elevated quantitative HBV nucleic acid assay result. Incorrectly interpreting the assay results as a positive assay result or failing to correctly operate the assay causing a false positive assay result may negatively influence patient management decisions. Such decisions may include the administration or continuation of unnecessary antiviral treatment in patients with chronic HBV infection with its known toxicities and more rarely allergic reactions. Certain patients with falsely elevated HBV nucleic acid assay results may not undergo liver biopsy to investigate other causes of liver disease when the biopsy would otherwise be indicated for certain patients.
• A false negative or falsely decreased quantitative HBV nucleic acid assay result. Incorrectly interpreting the assay results as a negative assay result, or failing to correctly operate the assay causing a false negative assay result may negatively influence patient management decisions for patients with chronic HBV infection, including the withholding of treatment, failure to treat, or premature discontinuation of treating HBV infection when antiviral treatment is otherwise indicated or the choice of an inappropriate treatment. This could lead to adverse effects on patient health such as progressive liver disease, cirrhosis and/or hepatocellular carcinoma, and other cancers. Patients with active HBV replication also risk spreading the virus to others. Certain patients with falsely low HBV nucleic acid assay results may undergo liver biopsy to investigate other causes of liver disease.

The safety and effectiveness of these device types has become well established since the initial approval of the first qualitative HBV antibody assay in 2000, the first HBV antigen assay in 2001, and the first quantitative HBV nucleic acid-based assay in 2008. FDA has considered and analyzed the following information: (1) accumulated experience regulating these HBV assays, (2) input from the Panel meeting, and (3) postmarket information regarding HBV assays, i.e., information from FDA's publicly available MDR, MAUDE, and Medical Device Recall databases. The available evidence demonstrates that there are public health benefits derived from the use of HBV assays indicated for use to aid in diagnosis of HBV infection and/or for use to aid in the management of HBV infected patients, or as an aid in the determination of susceptibility to HBV infection (anti-HBs). In addition, the nature of the associated risks to health are known, and special controls can be established to sufficiently mitigate these risks.

Based on our review of the information described above, FDA has determined that special controls, in addition to general controls, are necessary to provide a reasonable assurance of safety and effectiveness for HBV assays, and that sufficient information exists to establish such special controls. Therefore, FDA, on its own initiative, is proposing to reclassify these postamendments devices from class III (general controls and premarket approval) into class II (general controls and special controls), subject to premarket notification (510(k)) requirements.

FDA believes that these devices can be classified into class II with the establishment of special controls. FDA believes that the following proposed special controls would mitigate each of ( print page 78273) the risks to health described in section V and that these special controls, in addition to general controls, would provide a reasonable assurance of safety and effectiveness for HBV assays. Tables 1 through 3 below demonstrate how FDA believes each risk to health described in section V would be mitigated by the proposed special controls for each device type.

The risk of inaccurate interpretation of assay results can be mitigated by special controls requiring certain labeling, including providing clearly stated warnings and limitations and information on principles of operation and procedures in performing the assay.

Risks associated with false results ( e.g., false non-reactive and false reactive assay results) and with the failure to correctly operate the device can be mitigated through a combination of special controls, including certain labeling requirements, certain design verification and validation information, and performance studies. Examples of verification and validation information to be included in the design of the device include documentation of performance specifications including analytical and clinical performance criteria. In addition, design verification and validation activities must include documentation of a complete device description, critical reagents, risk analysis strategies, lot release criteria, stability studies, and protocols. Required statements in labeling can aid in mitigating the failure of the device to perform as indicated, for example including a statement that use of the assay with specimen types other than those specifically identified for use with this device may cause inaccurate assay results. Special controls requiring additional labeling to provide a brief summary of the instructions for use can also mitigate these risks.

Table 1—Risks to Health and Mitigation Measures for Qualitative HBV Antigen Assays

Identified risks to health Mitigation measures False reactive/non-reactive assay result Certain labeling information, including limitations, explanation of procedures, and results interpretation information.   Certain design verification and validation information, including certain device description information, risk analysis strategies, lot release criteria, stability studies and protocols, and performance criteria including analytical studies and clinical studies. Failure to correctly interpret the assay results Certain labeling information, including warnings, limitations, results interpretation information, and explanation of procedures.   Certain design verification and validation information, including certain device description information, critical reagent information, risk analysis strategies, lot release criteria, and stability studies and protocols. Failure to correctly operate the device Certain labeling information, including warnings, limitations, results interpretation information, and explanation of procedures.   Certain design verification and validation information, including certain device description, critical reagent information, risk analysis strategies, lot release criteria, and stability studies and protocols.

The risk of falsely reactive, non-reactive, elevated, or lowered assay results can be mitigated by special controls requiring certain labeling, including providing clearly stated warnings and limitations and information on principles of operation and procedures in performing the assay.

Risks associated with the failure of the device to perform as indicated ( e.g., false non-reactive and false reactive assay results) can be mitigated through a combination of special controls, including certain labeling requirements, certain design verification and validation information, and performance studies. Examples of verification and validation information to be included in the design of the device include documentation of performance specifications including analytical and clinical performance criteria. In addition, design verification and validation activities must include documentation of a complete device description, critical reagents, risk analysis strategies, lot release criteria, stability studies, and protocols. Required statements in labeling can aid in mitigating the failure of the device to perform as indicated; for example, including a statement that use of the assay with specimen types other than those specifically identified for use with this device may cause inaccurate assay results.

Table 2—Risks to Health and Mitigation Measures for HBV Antibody Assays (Including Qualitative and Quantitative Anti-HBs)

Identified risks to health Mitigation measures False reactive/false non-reactive assay result. In addition, for quantitative assays: Falsely elevated/falsely lowered assay result Certain labeling information, including limitations, explanation of procedures, and results interpretation information. Certain design verification and validation information including certain device description information, risk analysis strategies, lot release criteria, stability studies and protocols, and performance criteria including analytical studies and clinical studies. Failure to correctly interpret the assay results Certain labeling information, including warnings, limitations, results interpretation information, and explanation of procedures.   Certain design verification and validation information including certain device description, critical reagent information, risk analysis strategies, lot release criteria, and stability studies and protocols. ( print page 78274) Failure to correctly operate the devices Certain labeling information, warnings, limitations, results interpretation information, and explanation of procedures.   Certain design verification and validation information including certain device description, critical reagent information, risk analysis strategies, lot release criteria, and stability studies and protocols.

The risk of falsely positive, negative, elevated, or lowered assay results can be mitigated by special controls requiring certain labeling, including providing clearly stated warnings and limitations, device description information, and detailed instructions in the device labeling regarding the interpretation of assay results and principles of operation and procedures in performing the assay.

Risks associated with the failure of the device to perform as indicated ( e.g., inaccurately low or high results, false negative results, and false positive assay results) can be mitigated through a combination of special controls related to certain labeling requirements, design verification and validation activities, and performance studies. Examples of verification and validation information to be included in the design of the device include documentation of a complete device description, calibrators, critical reagents, traceability, and lot release criteria. In addition, design verification and validation must include documentation of performance specifications, including analytical and clinical performance criteria. Required statements in labeling can aid in mitigating the occurrence of inaccurate results. The risks of false positive/false negative/falsely elevated/falsely lowered results due to decreased assay sensitivity can be mitigated by special controls related to certain labeling, design verification and validation activities, risk analysis strategies, and performance studies.

Table 3—Risks to Health and Mitigation Measures for Quantitative HBV Nucleic Acid-Based Assays

Identified risks to health Mitigation measures False positive/false negative/falsely elevated/falsely lowered result Certain labeling information, including limitations, explanation of procedures, and results interpretation information. Certain design verification and validation information, including certain device description information, risk analysis strategies, lot release criteria, stability studies and protocols, and performance criteria including analytical studies and clinical studies. Failure to correctly interpret the assay results Certain labeling information, including warnings, limitations, results interpretation information, and explanation of procedures.   Certain design verification and validation information, including certain device description, critical reagent information, risk analysis strategies, lot release criteria, and stability studies and protocols. Failure to correctly operate the device Certain labeling warnings, limitations, results interpretation information, and explanation of procedures.   Certain design verification and validation information including certain device description, critical reagent information, risk analysis strategies, lot release criteria, and stability studies and protocols.

If this proposed order is finalized, qualitative HBV antigen assays, HBV antibody assays (including qualitative and quantitative anti-HBs), and quantitative HBV nucleic acid-based assays will be reclassified into class II (general controls and special controls) and would be subject to premarket notification requirements under section 510(k) of the FD&C Act. Firms submitting a 510(k) of the FD&C Act for such devices will be required to comply with the particular mitigation measures set forth in the special controls. FDA believes that adherence to the special controls, in addition to the general controls, is necessary to provide a reasonable assurance of safety and effectiveness of HBV assays.

We have determined under 21 CFR 25.34(b) that this action is of a type that does not individually or cumulatively have a significant effect on the human environment. Therefore, neither an environmental assessment nor an environmental impact statement is required.

While this proposed order contains no new collections of information, it does refer to previously approved FDA collections of information. The previously approved FDA collections of information are subject to review by the Office of Management and Budget (OMB) under the Paperwork Reduction Act of 1995 (PRA) ( 44 U.S.C. 3501-3521 ). The collections of information in 21 CFR part 820 have been approved under OMB control number 0910-0073; the collections of information in part 807, subpart E, have been approved under OMB control number 0910-0120; and the collections of information in 21 CFR parts 801 and 809 have been approved under OMB control number 0910-0485.

FDA proposes that any final order based on this proposed order become effective 30 days after the date of its publication in the Federal Register .

Under section 513(f)(3) of the FD&C Act, FDA may issue final orders to reclassify devices. FDA will continue to codify classifications and ( print page 78275) reclassifications in the Code of Federal Regulations (CFR). Changes resulting from final orders will appear in the CFR as newly codified orders. Therefore, under section 513(f)(3) of the FD&C Act, in the proposed order, we are proposing to codify qualitative hepatitis B virus antigen assays in the new § 866.3178, hepatitis B virus antibody assays (including qualitative and quantitative anti-HBs) in the new § 866.3179, and quantitative hepatitis B virus nucleic acid-based assays in the new § 866.3180, under which these HBV assays would be reclassified from class III into class II.

The following references marked with an asterisk (*) are on display at the Dockets Management Staff (see ADDRESSES ) and are available for viewing by interested persons between 9 a.m. and 4 p.m., Monday through Friday; they also are available electronically at https://www.regulations.gov . References without asterisks are not on public display at https://www.regulations.gov because they have copyright restriction. Some may be available at the website address, if listed. References without asterisks are available for viewing only at the Dockets Management Staff. Although FDA verified the website addresses in this document, please note that websites are subject to change over time.

*1. Summary Minutes Prepared for the September 7, 2023, Meeting of the Microbiology Devices Panel (available at https://www.fda.gov/​media/​173610/​download ).

*2. Meeting Transcript Prepared for the September 7, 2023, Meeting of the Microbiology Devices Panel (available at https://www.fda.gov/​media/​173609/​download ).

3. Terrault, N.A., A.S.F. Lok, B.J. McMahon, et al., “Update on Prevention, Diagnosis, and Treatment of Chronic Hepatitis B: AASLD 2018 Hepatitis B Guidance.” Hepatology, 67(4): 1560-1599, 2018.

4. CDC, “Clinical Testing and Diagnosis for Hepatitis B,” https://www.cdc.gov/​hepatitis-b/​hcp/​diagnosis-testing/​index.html . Accessed July 11, 2024.

• Laboratories
• Medical devices

Therefore, under the Federal Food, Drug, and Cosmetic Act, and under authority delegated to the Commissioner of Food and Drugs, it is proposed that 21 CFR part 866 be amended as follows:

1. The authority citation for part 866 continues to read as follows:

Authority: 21 U.S.C. 351 , 360 , 360c , 360e , 360j , 360 l, 371.

2. Add § 866.3178 to subpart D to read as follows:

(a) Identification. A qualitative hepatitis B virus (HBV) antigen assay is identified as an in vitro diagnostic device intended for prescription use for qualitative use with human serum, plasma, or other matrices that aids in the diagnosis of chronic or acute HBV infection. HBV surface antigen (HbsAg) is also used for screening of HBV infection in pregnant women to identify neonates who are at risk of acquiring hepatitis B during perinatal period. The assay is not intended for screening of blood, plasma, cells, or tissue donors.

(b) Classification. Class II (special controls). The special controls for this device are:

(1) The labeling required under § 809.10(b) of this chapter must include:

(i) A prominent statement that the assay is not intended for the screening of blood, plasma, cells, or tissue donors.

(ii) A detailed explanation of the principles of operation and procedures for performing the assay.

(iii) A detailed explanation of the interpretation of results.

(iv) Limitations, which must be updated to reflect current clinical practice and disease presentation and management. The limitations must include statements that indicate:

(A) The specimen types for which the device has been cleared, and that use of this assay with specimen types other than those specifically cleared for this device may result in inaccurate assay results.

(B) When appropriate, performance characteristics of the assay have not been established in populations of immunocompromised or immunosuppressed patients or other populations where assay performance may be affected.

(C) Diagnosis of hepatitis B infection should not be established on the basis of a single assay result but should be determined by a licensed healthcare professional in conjunction with the clinical presentation, history, and other diagnostic procedures.

(D) Detection of HBV antigens indicates a current infection with hepatitis B virus but does not differentiate between acute or chronic infection. False reactive HbsAg result may occur for up to 2 weeks after vaccination with HbsAg containing vaccine.

(E) Current methods for the detection of hepatitis B antigens may not detect all potentially infected individuals. A non-reactive assay result does not exclude the possibility of exposure to or infection with hepatitis B virus. A non-reactive assay result in individuals with prior exposure to hepatitis B may be due to but not limited to antigen levels below the detection limit of this assay or lack of antigen reactivity to the antibodies in this assay. HBV mutants lacking the ability to produce antigens have been reported. These may occur as “escape” mutants in the presence of anti-HBV antibodies and such patients may be infectious.

(F) Results obtained with this assay may not be used interchangeably with results obtained with a different manufacturer's assay.

(2) Design verification and validation must include the following:

(i) A detailed device description, including all parts that make up the device, ancillary reagents required but not provided, an explanation of the device methodology, design of the capture antibody(ies), external controls, and computational path from collected raw data to reported result ( e.g., how collected raw signals are converted into a reported signal and result), as applicable to the detection method and device design.

(ii) For devices with assay calibrators, the design and composition of all primary, secondary, and subsequent quantitation standards used for calibration as well as their traceability to a standardized reference material that FDA has determined is appropriate ( e.g., a recognized consensus standard). In addition, analytical testing must be performed following the release of a new lot of the standard material that was used for device clearance or approval, or when there is a transition to a new calibration standard.

(iii) Documentation and characterization ( e.g., supplier, determination of identity, purity, and stability) of all critical reagents (including description of the capture antibody(ies)), and protocols for maintaining product integrity throughout its labeled shelf life.

(iv) Risk analysis and management strategies, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on assay performance.

(v) Final release criteria to be used for manufactured assay lots with appropriate evidence that lots released ( print page 78276) at the extremes of the specifications will meet the identified analytical and clinical performance characteristics as well as stability.

(vi) Stability studies for reagents must include documentation of an assessment of real-time stability for multiple reagent lots using the indicated specimen types and must use acceptance criteria that ensure that analytical and clinical performance characteristics are met when stability is assigned based on the extremes of the acceptance range.

(vii) All stability protocols, including acceptance criteria.

(viii) Final release assay results for each lot used in clinical studies.

(ix) Reproducibility study data that includes the testing of three independent production lots.

(x) Detailed documentation of analytical performance studies conducted, as appropriate to the technology, specimen types tested, and intended use of the device, including, the limit of blank (LoB), limit of detection (LoD), cutoff, precision (reproducibility) including lot-to-lot and/or instrument-to-instrument precision, interference, cross reactivity, carryover, hook effect, seroconversion panel testing, matrix equivalency, prominent mutants/variants detection ( e.g., for HbsAg), specimen stability, reagent stability, and cross-genotype antigen detection sensitivity, when appropriate.

(xi) Analytical sensitivity of the assay is the same or better than that of other cleared or approved assays.

(xii) For devices with associated software or instrumentation, documentation must include a detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.

(xiii) Detailed documentation and results from a clinical study. Performance must be analyzed relative to an FDA cleared or approved HBV antigen assay or a comparator that FDA has determined is appropriate. This study must be conducted using appropriate patient samples, with an appropriate number of HBV reactive and non-reactive samples in applicable risk and disease categories, and any applicable confirmatory testing. Additional relevant patient groups must be validated as appropriate. The samples must include prospective (sequential) samples for each identified specimen type and, as appropriate, additional characterized clinical samples. Samples must be sourced from geographically diverse areas. This study must be conducted in the appropriate settings by the intended users to demonstrate clinical performance.

3. Add § 866.3179 to subpart D to read as follows:

(a) Identification. A hepatitis B virus (HBV) antibody assay is identified as an in vitro diagnostic device intended for prescription use in the detection of antibodies to HBV in human serum, plasma, or other matrices, and as a device that aids in the diagnosis of HBV infection in persons with signs and symptoms of hepatitis and in persons at risk for hepatitis B infection. In addition, results from an anti-HBc IgM (IgM antibodies to core antigen) assay indicating the presence of anti-HBc IgM are indicative of recent HBV infection. Anti-HBs (antibodies to surface antigen) assay results may be used as an aid in the determination of susceptibility to HBV infection in individuals prior to or following HBV vaccination or when vaccination status is unknown. The assay is not intended for screening of blood, plasma, cells, or tissue donors. The assay is intended as an aid in diagnosis in conjunction with clinical findings and other diagnostic procedures.

(A) When appropriate, performance characteristics of the assay have not been established in populations of immunocompromised or immunosuppressed patients or other special populations where assay performance may be affected.

(B) Detection of HBV antibodies to a single viral antigen indicates a present or past infection with hepatitis B virus, but does not differentiate between acute, chronic, or resolved infection.

(C) The specimen types for which the device has been cleared, and that use of the assay with specimen types other than those specifically cleared for this device may result in inaccurate assay results.

(D) Diagnosis of hepatitis B infection should not be established on the basis of a single assay result but should be determined by a licensed healthcare professional in conjunction with the clinical presentation, history, and other diagnostic procedures.

(E) A non-reactive assay result may occur early during acute infection, prior to development of a host antibody response to infection, or when analyte levels are below the limit of detection of the assay.

(v) For devices intended for the quantitative detection of HBV antibodies (anti-HBs), in addition to the special controls listed in paragraphs (b)(1) and (2) of this section, labeling required under § 809.10(b) of this chapter must include:

(A) The assay calibrators' traceability to a standardized reference material that FDA has determined is appropriate ( e.g., a recognized consensus standard) and the limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), linearity, and precision to define the analytical measuring interval.

(B) Performance results of the analytical sensitivity study testing a standardized reference material that FDA has determined is appropriate ( e.g., a recognized consensus standard).

(i) Detailed device description, including all parts that make up the device, ancillary reagents required but not provided, an explanation of the device methodology, and design of the antigen(s) and capture antibody(ies) sequences, rationale for the selected epitope(s), degree of amino acid sequence conservation of the target, and the design and composition of all primary, secondary and subsequent standards used for calibration.

(ii) Documentation and characterization ( e.g., supplier, determination of identity, and stability) of all critical reagents (including description of the antigen(s) and capture antibody(ies)), and protocols for maintaining product integrity throughout its labeled shelf life.

(iii) Risk analysis and management strategies, such as Failure Modes Effects ( print page 78277) Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on assay performance.

(iv) Final release criteria to be used for manufactured assay lots with appropriate evidence that lots released at the extremes of the specifications will meet the identified analytical and clinical performance characteristics as well as stability.

(v) Stability studies for reagents must include documentation of an assessment of real-time stability for multiple reagent lots using the indicated specimen types and must use acceptance criteria that ensure that analytical and clinical performance characteristics are met when stability is assigned based on the extremes of the acceptance range.

(vi) All stability protocols, including acceptance criteria.

(vii) When applicable, analytical sensitivity of the assay is the same or better than that of other cleared or approved assays.

(viii) Analytical performance studies and results for determining the limit of blank (LoB), limit of detection (LoD), cutoff, precision (reproducibility), including lot-to-lot and/or instrument-to-instrument precision, interference, cross reactivity, carryover, hook effect, seroconversion panel testing, matrix equivalency, specimen stability, reagent stability, and cross-genotype antibody detection sensitivity, when appropriate.

(ix) For devices intended for the detection of antibodies for which a standardized reference material (that FDA has determined is appropriate) is available, the analytical sensitivity study and results testing the standardized reference material. Detailed documentation of that study and its results must be provided, including the study protocol, study report, testing results, and all statistical analyses.

(x) For devices with associated software or instrumentation, documentation must include a detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.

(xi) Detailed documentation of clinical performance testing from a clinical study with an appropriate number of HBV reactive and non-reactive samples in applicable risk categories and conducted in the appropriate settings by the intended users. Performance must be analyzed relative to an FDA cleared or approved HBV antibody assay or a comparator that FDA has determined is appropriate. Additional relevant patient groups must be validated as appropriate. The samples must include prospective (sequential) samples for each identified specimen type and, as appropriate, additional characterized clinical samples. Samples must be sourced from geographically diverse areas.

(3) For any HBV antibody assay intended for quantitative detection of anti-HBV antibodies, the following special controls, in addition to those special controls listed in paragraphs (b)(1) and (2) of this section, also apply:

(i) Detailed documentation of the metrological calibration traceability hierarchy to a standardized reference material that FDA has determined is appropriate.

(ii) Detailed documentation of the following analytical performance studies conducted, as appropriate to the technology, specimen types tested, and intended use of the device, including upper and lower limits of quantitation (UloQ and LloQ, respectively), linearity using clinical samples, and an accuracy study using the recognized international standard material.

4. Add § 866.3180 to subpart D to read as follows:

(a) Identification. A nucleic acid-based hepatitis B virus (HBV) assay is identified as an in vitro diagnostic device intended for prescription use in the detection of HBV nucleic acid in specimens from individuals with antibody evidence of HBV infection. In these devices, the detection of HBV nucleic acid is used as an aid in the management of HBV-infected individuals. The assay is intended for use with human serum or plasma (and other matrices as applicable) from individuals with HBV. The assay is not intended for use as a donor screening assay for the presence of HBV nucleic acids in blood, blood products, plasma, cells, or tissue donors, or as a diagnostic assay to confirm the presence of HBV infection.

(1) Labeling required under § 809.10(b) of this chapter must include:

(i) A prominent statement that the assay is not intended for use as a screening assay for the presence of HBV DNA in blood or blood products, plasma, cells, or tissue donors, or as a diagnostic assay to confirm the presence of HBV infection.

(iv) Limitations, which must be updated to reflect current clinical practice and disease presentation and/or management. These limitations must include statements that indicate:

(A) Management of patients undergoing hepatitis B virus treatment should not be established on the basis of a single assay result but should be determined by a licensed healthcare professional in conjunction with the clinical presentation, history, and other diagnostic procedures, e.g., HBV serologic testing, liver function assays, liver elastography, etc.

(B) The specimen types for which the device has been cleared, and that use of this assay with specimen types other than those specifically cleared for this device may result in inaccurate assay results.

(C) The results obtained with this assay may not be used interchangeably with results obtained with a different manufacturer's assay.

(i) Detailed device description, including the device components, ancillary reagents required but not provided, and an explanation of the device methodology. Additional information appropriate to the technology must be included such as design of primers and probes, rationale for the selected gene targets, specifications for amplicon size, and degree of nucleic acid sequence conservation.

(iii) Documentation and characterization ( e.g., determination of the identity, supplier, purity, and stability) of all critical reagents (including nucleic acid sequences for primers and probes) and protocols for maintaining product integrity.

(iv) Risk analysis and management strategies demonstrating how risk ( print page 78278) control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on assay performance.

(v) Final release criteria to be used for manufactured assay lots with appropriate evidence that lots released at the extremes of the specification will meet the identified analytical and clinical performance characteristics as well as stability.

(viii) Detailed documentation of analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including limit of detection (LoD), linearity, precision, endogenous and exogenous interferences, cross-reactivity, carryover, matrix equivalency, sample and reagents stability, and as applicable, upper and lower limits of quantitation (ULoQ and LLoQ, respectively). Samples selected for use must be from subjects with clinically relevant circulating genotypes in the United States. Cross-reactivity studies must include samples from HBV nucleic acid negative subjects with other viral or non-viral causes of liver disease, including autoimmune hepatitis, alcoholic liver disease, chronic hepatitis C virus (HCV), primary biliary cirrhosis, and nonalcoholic steatohepatitis, when applicable. The effect of each identified nucleic-acid isolation and purification procedure on detection must be evaluated.

(ix) For devices with associated software or instrumentation, documentation must include a detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.

(x) Detailed documentation of performance from a clinical study with a design and number of clinical samples (appropriately statistically powered) that is appropriate for the intended use of the device as well as conducted in the appropriate settings by the intended users. The samples must include prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. Samples must be sourced from geographically diverse areas.

Dated: September 20, 2024.

Lauren K. Roth,

Associate Commissioner for Policy.

1.  FDA notes that the “ACTION” caption for this proposed order is styled as “Proposed amendment; proposed order,” rather than “Proposed order.” Beginning in December 2019, this editorial change was made to indicate that the document “amends” the Code of Federal Regulations. The change was made in accordance with the Office of the Federal Register's (OFR) interpretations of the Federal Register Act ( 44 U.S.C. chapter 15 ), its implementing regulations ( 1 CFR 5.9 and parts 21 and 22 ), and the Document Drafting Handbook.

2.  Class I, II, and III recalls are defined in 21 CFR 7.3(m) .

3.   https://www.who.int/​news-room/​fact-sheets/​detail/​hepatitis-b . Accessed on July 12, 2024.

4.  Centers for Disease Control and Prevention—Clinical Overview of Hepatitis B (Available at https://www.cdc.gov/​hepatitis-b/​hcp/​clinical-overview/​index.html ). Accessed on July 12, 2024.

5.  Ibid.

6.  Ibid.

7.   https://www.who.int/​health-topics/​hepatitis/​elimination-of-hepatitis-by-2030#tab=​tab_​1 . Accessed on July 12, 2024.

8.   https://www.hhs.gov/​sites/​default/​files/​Viral-Hepatitis-National-Strategic-Plan-2021-2025.pdf . Accessed on July 12, 2024.

9.   https://www.cdc.gov/​hepatitis/​hbv/​index.htm . Accessed on July 12, 2024.

[ FR Doc. 2024-21932 Filed 9-24-24; 8:45 am]

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