Immunoenzymatic/immunohistochemical (IHC) staining techniques allow for the qualitative identification of tissue antigens. Antigens are visualized via the sequential application of a specific antibody to the antigen (primary antibody) and a detection system. Immunohistochemical detection systems usually consist of a secondary antibody to the primary antibody (link antibody), an enzyme complex and a chromogenic substrate. The enzymatic activation of the chromogen yields a visible reaction product at the antigen site. Results aid in the diagnosis of pathophysiological processes which may or may not be associated with a particular antigen.

Device Description

The DAKO LSAB® 2 Kit, HRP (Code No. K0677) is based on a modified labeled avidin-biotin (LAB) technique in which a biotinylated secondary antibody forms a complex with peroxidase-conjugated streptavidin molecules. This kit is very similar to the original DAKO LSAB® Kit, HRP (K0680) previously cleared in submission K924726. Refinements to the LSAB® 2 Kit include the elimination of a separate protein blocking reagent incubation and reconfigured substrate-chromogen reagents.

The DAKO Envision™ Systems, HRP, contains a Peroxidase Blocking Reagent similar to the Hydrogen Peroxide provided with the LSAB® and LSAB® 2 Kit, the Envision™ System does not require a protein blocking reagent incubation. The Envision™ System also eliminates the sequential applications of link antibody and streptavidin common to the LSAB® 2 Kits. A polymer, labeled with goat anti-mouse and goat anti-rabbit immunoglobulins conjugated to horseradish peroxidase eliminates these steps. The DAKO Envision™ System offers the user a choice of two protocols. Protocol #1 consecutively incubates the primary antibody and Peroxidase Labeled Polymer for ten (10) minutes. Protocol #2 increases the staining intensity of the Envision™ System by lengthening the incubations of the primary antibody and the labeled polymer to thirty (30) minutes. The DAKO Envision™ System. HRP, is available with DAB (Code No. K1390) or Ready-to-Use AEC substrate-chromogen (Code No. K1391). Both systems use the same Peroxidase Blocking Reagent and Peroxidase Labeled Polymer.

Concentrated primary rabbit/mouse antibodies or DAKO® Ready-to-Use N-Series Primary Antibodies and Negative Control Reagents are suitable for use with the DAKO LSAB® 2 Kit and DAKQ Envision™ Systems. Primary antibodies are not included with the DAKO LSAB® 2 Kit, HRP or DAKO Envision™ Systems, and must be purchased separately by the user.

AI/ML Overview

The provided text describes the DAKO LSAB® 2 Kit, HRP, and DAKO Envision™ Systems, HRP, which are immunohistochemical (IHC) staining systems used for qualitative identification of tissue antigens. The document is a Summary of Safety and Effectiveness for a K951965 submission.

Here's an analysis of the provided information against the requested categories:

1. A table of acceptance criteria and the reported device performance

Acceptance Criteria (Implicit)	Reported Device Performance
Equivalent interpretive results to predicate device (DAKO LSAB® Kit, HRP)	Equivalent interpretive results observed in all test specimens.
Equivalent interpretive results to DAKO LSAB® 2 Kit, HRP (when comparing to Envision™ System)	Equivalent interpretive results observed in all test specimens (for both AEC and DAB substrate-chromogens).
Equivalent results for different tissue fixation methods (formalin-fixed, paraffin embedded vs. acetone-fixed, frozen tissues, blood smears)	Equivalent results observed.
Protocol #2 in Envision™ System increases staining intensity and allows for greater primary antibody dilution compared to Protocol #1.	Results support package insert claims: Protocol #2 increases staining intensity. Concentrated primary antibodies used with Protocol #2 may be diluted up to twenty times the optimal dilution used with Protocol #1.
LSAB2 with ready-to-use AEC performs similarly or better than LSAB2 with 2-component AEC.	Same level of staining for N1545 and N1520. Slightly more intense staining (3-3.5 vs 4+) for ready-to-use AEC with N1514. Background staining evaluated as negative.

2. Sample sizes used for the test set and the data provenance (e.g., country of origin of the data, retrospective or prospective)

Sample Size: The document repeatedly mentions "all test specimens" and specific tissue types like "tonsil" and "melanoma", and "liver," "kidney," and "lymphoid tissues." However, no specific numerical sample sizes are provided for any of the comparative tests. The sample size for the comparison of LSAB2 with 2-component AEC versus ready-to-use AEC for specific antibodies (LCA, UCHL1, HMB45) is denoted by "N1514," "N1520," and "N1545" in parentheses next to the antibody names, implying these might refer to specific tissue samples or an internal numbering system, but the actual number of specimens is not listed.
Data Provenance: The document does not specify the country of origin of the data. The data appears to be retrospective in nature, as it describes comparisons of the new devices against existing products using various tissue types.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g., radiologist with 10 years of experience)

The document does not specify the number of experts used to establish the ground truth or interpret the results.
It also does not provide any qualifications for the individuals who performed the interpretation/evaluation ("Equivalent interpretive results were observed," "Results showed that the same level of staining was obtained," "Background staining... evaluated as negative").

4. Adjudication method (e.g., 2+1, 3+1, none) for the test set

The document does not describe any formal adjudication method for the test set. The evaluations appear to be direct comparisons of staining results.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No MRMC comparative effectiveness study was performed.
This device is an IHC staining kit, not an AI or imaging diagnostic device. Therefore, the concept of "human readers improve with AI vs. without AI assistance" is not applicable here.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

This question is not applicable as the device is an IHC staining kit and not an algorithm or AI system.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

The ground truth appears to be based on visual interpretation of staining results by unspecified individuals, likely experienced researchers or pathologists within DAKO, evaluating "equivalent interpretive results," "level of staining," and "background staining." This is implicitly based on established immunohistochemical principles and the expected reactivity of the primary antibodies on the tested tissues.

8. The sample size for the training set

The document describes comparative testing of new kits against existing kits and variations within the new kits. It does not mention a "training set" in the context of machine learning or algorithm development, as these are chemical kits. All described tests appear to be evaluations of the final products.

9. How the ground truth for the training set was established

As there is no "training set" in the context of this device type, this question is not applicable. The "ground truth" for the performance evaluations (test sets) was established by visual assessment of staining consistency and intensity by presumably qualified personnel.

Summary

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K951965

Summary of Safery and Effectiveness

DAKO LSA8® 2 Kit, HRP DAKO ENVISION™ SYSTEMS, HRP

1. Introduction

lmmunoenzymatic/immunohistochemical (IHC) staining techniques allow for the qualitative identification of tissue antigens. Antigens are visualized via the sequential application of a specific antibody to the antigen (primary antibody) and a detection system. Immunohistochemical detection systems usually consist of a secondary antibody to the primary antibody (link antibody), an enzyme complex and a chromogenic substrate. The enzymatic activation of the chromogen yields a visible reaction product at the antigen site. Results aid in the diagnosis of pathophysiological processes which may or may not be associated with a particular antigen.

II. Device Descriptions

III. Experimental Data

Concurrent testing was done with the DAKO LSAB® Kit, HRP and the DAKO LSAB® 2 Kit, HRP using formalin-fixed, paraffin embedded tissues. Equivalent interpretive results were observed in all test specimens.

Summary of Safety & Effectiveness/ rev. C/ p. 1

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Experimental Data, continued III.

Similar concurrent testing was done with the DAKO LSAB® 2 Kit, HRP and the DAKO Envision™ System, HRP with Ready-To-Use AEC substrate-chromogen (K1391) using formalin-fixed, paraffin embedded tissues. Equivalent interpretive results were observed in all test specimens.

This test procedure was repeated using the DAKO LSAB® 2 Kit, HRP and the DAKO Envision™ System, HRP with liguid DAB substrate-chromogen (K1390). Equivalent interpretive results were observed in all test specimens.

Additional testing was done with the DAKO LSAB® 2 Kit, HRP and the DAKO Envision™ System, HRP with Ready-To-Use AEC substrate-chromogen (K1391) using acetone-fixed, frozen tissues and blood smears. Results were equivalent.

Results obtained from comparative testing of the DAKO Envision™ System, HRP with Ready-to-Use AEC substrate-chromogen using Protocols #1 and #2 support package insert claims for Protocol #2. Protocol #2 increases the staining intensity of the DAKO Envision™ System. Concentrated Primary antibodies used with protocol #2 may be diluted up to twenty tmes the optimal dilution used with Protocol #1.

Comparison testing of LSAB2 with 2 component AEC versus ready-to-use AEC was performed using LCA (N1514), UCHL1 (N1520), and HMB45 (N1545). The tissues tested included tonsil (N1514, and N1520) and melanoma (N1545). Results showed that the same level of staining was obtained with both product configurations for N1545 and N1520. Slightly more intense staining (2 component AEC staining = 3-3.5 vs ready-to-use AEC staining = 4 + ) was noted for ready-to-use AEC with N1514. Background staining of both negative controls and positively stained tissues was evaluated as negative i.e., no staining. Thus, the LSAB2 with the ready-to-use AEC is similar or better in performance to LSAB2 with the 2-component AEC.

IV. Product Specific Limitations

DAKO LSAB® 2, Kit (K0677):

Endogenous avidin-binding activity (EABA) has been noted in frozen sections of liver (entire hepatic nodule) and kidney (tubular epithelium), as well as in frozen and formalin-fixed lymphoid tissues (paracortical histiocytes). EABA can be suppressed by sequential 20-minute incubations, first with 0.1% avidin and then with 0.01% biotin in 0.05 M Tris-HC1 buffer, pH 7.2-7.6 prior to the application of the primary antibody, or use DAKO® Biotin Blocking System (code no. X0590).

DAKO Envision™ System, HRP (K1390, K1391):

Some primary antibodies may require proteolytic digestion and target retrieval to achieve optimal staining results. Protocol #2 is recommended for use. Contact DAKO Technical Services at 800/424-0021 for additional information.

Endogenous peroxidase or pseudoperoxidase activity can be found in hemoproteins such as hemoglobin, myoglobin, cytochrome, and catalase as well as in eosinophils. This activity can be inhibited by incubating specimens with Peroxidase Blocking Reagent Bottle #1 of the DAKO Envision™ System, HRP for five (5 ± 1) minutes prior to the application of the primary antibody. Blood and bone marrow smears and frozen tissues can also be treated with this reagent. However, this procedure does not abolish the reddish-brown pigment of hemoproteins. Alternatively, a solution of methanol-hydrogen peroxide can be used. Some antigens may become denatured with this procedure.

Regulation Number and Section

§ 866.5550 Immunoglobulin (light chain specific) immunological test system.

(a)
Identification. An immunoglobulin (light chain specific) immunological test system is a device that consists of the reagents used to measure by immunochemical techniques both kappa and lambda types of light chain portions of immunoglobulin molecules in serum, other body fluids, and tissues. In some disease states, an excess of light chains are produced by the antibody-forming cells. These free light chains, unassociated with gamma globulin molecules, can be found in a patient's body fluids and tissues. Measurement of the various amounts of the different types of light chains aids in the diagnosis of multiple myeloma (cancer of antibody-forming cells), lymphocytic neoplasms (cancer of lymphoid tissue), Waldenstrom's macroglobulinemia (increased production of large immunoglobulins), and connective tissue diseases such as rheumatoid arthritis or systemic lupus erythematosus.(b)
Classification. Class II (performance standards).