SMART HBA1C ASSAY KIT, MODEL DZ168B-POC, SMART HBA1C ASSAY CONTROL KIT, MODEL DZ168B-CON, SMART HBAC1C ANALYZER

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT COMBINATION TEMPLATE A. 510(k) Number: k092911 B. Purpose for Submission: New device C. Measurand: HbA1c D. Type of Test: Quantitative enzymatic assay E. Applicant: Diazyme Laboratories F. Proprietary and Established Names: SMART HbA1c Assay Reagent Kit; SMART HbA1c Assay Control Kit; SMART Analyzer G. Regulatory Information: | Product Code | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | LCP | II | 21 CFR 864.7470, Glycosylated Hemoglobin Assay | Hematology | | JJX | I, reserved | 21 CFR 862.1660, Quality Control Material | Chemistry | | JJE | I, reserved | 21 CFR 862.2160, Discrete Photometric Chemistry Analyzer for Clinical Use | Chemistry | H. Intended Use: 1. Intended uses: See indications for use below. 2. Indications for use: SMART HbA1c Assay Reagent Kit is intended for use with the SMART analyzer for the quantitative determination of stable HbA1c in human capillary and venous whole blood samples. Measurement of hemoglobin A1c is a valuable indicator for long-term diabetic control. For in vitro diagnostic use only. {1} SMART HbA1c Assay Control Kit is intended for use as quality controls for the SMART HbA1c Assay Reagent Kit. For in vitro diagnostic use only. SMART Analyzer is a compact cuvette based spectrophotometer machine for point-of-care testing, designed to analyze readings for single use SMART reagent cuvette. The SMART HbA1c Analyzer System, consisting of the SMART Analyzer, SMART HbA1c Assay Reagent Kit and SMART HbA1c Assay Control Kit is for in vitro diagnostic use only. 3. Special conditions for use statement(s): For prescription and point of care use only. 4. Special instrument requirements: For use on the SMART Analyzer only. I. Device Description: SMART HbA1c Assay Reagent Kit is composed of the following: - 20 prefilled microtubes containing lysis buffer (CHES pH 8.7, Triton-X100, SDS and Redox agents) - 20 prefilled DRS cuvettes containing Reagent R1a (MES pH 7.0, proteases, Triton-X100 and redox reagents) - 1 brown bottle containing Reagent R1b (MES pH 6.3, redox agent, Triton X100) - 20 prefilled DRS Caps containing Reagent R2 (Tris pH 8.0, FVO enzyme, POD and chromagen) - one RFID card - 40 dual bulb pipettes - 20 20μL capillary tubes. The SMART HbA1c Assay Control Kit consists of two levels of lyophilized control material prepared from human whole blood. Each donor unit of serum in the preparation of the material was tested by FDA-approved methods and found negative for the Human Immunodeficiency Virus Antibody (HIV I/II Ab), Hepatitis B Surface Antigen (HBsAg), and Hepatitis C Virus Antibody (HCV). The SMART analyzer is a compact cuvette based spectrophotometer for point-of-care testing, designed to analyze readings from single use SMART HbA1c reagent cuvettes. J. Substantial Equivalence Information: 1. Predicate device name(s): Diazyme Direct Enzymatic HbA1c Assay 2. Predicate K number(s): k070734 {2} 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Predicate Device (k070734) | Proposed Device | | Intended Use/Indications for use | For use in the quantitative determination of stable HbA1c in human capillary and venous whole blood samples. Measurement of hemoglobin A1c is a valuable indicator for long-term diabetic control. | Same | | Type of test | Quantitative | Same | | Specimen type | Human capillary and venous whole blood | Same | | Differences | | | | --- | --- | --- | | Item | Predicate Device (k070734) | Proposed Device | | Measuring range | 4-16% | 4-12% | | Calibrator | Separately packaged lot specific calibrator kit. User steps needed to use calibrators | Each kit has individual lot specific RFID preprogrammed calibration card. User steps limited to insertion in SMART analyzer | K. Standard/Guidance Document Referenced (if applicable): - CLSI Guideline EP5-A2: Evaluation of Precision Performance of Quantitative Measurement Methods - CLSI Guideline EP6-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach - CLSI Guideline EP9-A2: Method Comparison and Bias Estimation Using Patient Samples - CLSI Protocol EP7-A2: Interference Testing in Clinical Chemistry {3} 4 L. Test Principle: The SMART HbAlc test is an enzymatic assay in which lysed whole blood samples are subjected to extensive protease digestion with Bacillus sp protease. This process releases amino acids including glycated valines from the hemoglobin beta chains. Glycated valines then serve as substrates for specific recombinant fructosyl valine oxidase (FVO) enzyme, produced in E. coli. The recombinant FVO specifically cleaves N-terminal valines and produces hydrogen peroxide. This, in turn, is measured using a horseradish peroxidase (POD) catalyzed reaction and a suitable chromagen. The HbAlc concentration is expressed directly as $\% \mathrm{HbAlc}$ by use of a lot-specific calibration curve that is stored in an RFID card provided with each SMART test kit. M. Performance Characteristics (if/when applicable): 1. Analytical performance: a. Precision/Reproducibility: The precision of the SMART HbA1c Assay was evaluated according to the CLSI Guideline EP5-A: Evaluation of Precision Performance of Quantitative Measurement Methods with modifications. In the study (performed internally), two whole blood specimens containing $5.6\%$ and $7.4\%$ HbA1c and one whole blood HbA1c control containing $11.5\%$ HbA1c were tested during 2 runs per day and with two replicates per run over 10 working days. Reagent Lot 1 was used over the first 5 days and Reagent Lot 2 was used over the remaining 5 days. Both lots of reagents were used on three different SMART analyzers. The mean value (Mean), standard deviation, within run imprecision and total imprecision $\mathrm{CV\%}$ are calculated and summarized in the following tables: Within Run precision CV% | | 5.6% HbA1c | 7.4% HbA1c | 11.5% HbA1c control | | --- | --- | --- | --- | | Total data points | 40 | 40 | 40 | | Mean (%) | 5.4 | 7.4 | 11.4 | | SD (%) | 0.13 | 0.22 | 0.13 | | CV% | 2.4% | 3.0% | 1.2% | Total Precision CV% | | 5.6% HbA1c | 7.4% HbA1c | 11.5% HbA1c control | | --- | --- | --- | --- | | Total data points | 40 | 40 | 40 | | Mean (%) | 5.4 | 7.4 | 11.4 | | SD (%) | 0.15 | 0.19 | 0.15 | | CV% | 2.70% | 2.50% | 1.30% | {4} The precision of the Diazyme SMART HbA1c assay was also evaluated in three physician office laboratories (POL) by nurses and assistants to test systemic and random error on three different SMART HbA1c AssayHbA1c analyzers. Two unaltered EDTA whole blood HbA1c specimens containing $5.4\%$ , $7.7\%$ HbA1c and one whole blood HbA1c control containing $11.5\%$ HbA1c were used. At each POL site, the samples were run in duplicates in two runs per day over 5 days (Sites 1 and 2 used two SMART analyzers; Site 3 used one analyzer). One lot of assay reagents was used at all three sites. Site 1: Result: | | 5.4% HbA1c | 7.7% HbA1c | 11.5% HbA1c control | | --- | --- | --- | --- | | Mean (%) | 5.3 | 7.6 | 11.4 | | SD (%) | 0.13 | 0.15 | 0.09 | | CV% | 2.5% | 2.0% | 0.8% | Site 2: Result: | | 5.4% HbA1c | 7.7% HbA1c | 11.5% HbA1c control | | --- | --- | --- | --- | | Mean (%) | 5.6 | 7.4 | 11.3 | | SD (%) | 0.16 | 0.18 | 0.21 | | CV% | 2.9% | 2.5% | 1.8% | Site 3: Result: | | 5.4% HbA1c | 7.7% HbA1c | 11.5% HbA1c control | | --- | --- | --- | --- | | Mean (%) | 5.5 | 7.3 | 11.4 | | SD (%) | 0.28 | 0.30 | 0.14 | | CV% | 5.0% | 4.0% | 1.2% | Additional estimates of precision: Between instrument precision Two EDTA blood samples with normal and high HbA1c levels were analyzed on four analyzers with six replicates. The mean value (Mean), standard deviation, and imprecision CV% on each analyzer are calculated and summarized in the following table: {5} | Analyzer | Normal Sample (5.5%) | | | High sample (10.7%) | | | | --- | --- | --- | --- | --- | --- | --- | | | HbA1c% (mean) | SD | CV% | HbA1c% (mean) | SD | CV% | | 1 | 5.67 | 0.27 | 4.8% | 10.23 | 0.18 | 1.7% | | 2 | 5.60 | 0.22 | 3.9% | 10.37 | 0.25 | 2.4% | | 3 | 5.55 | 0.24 | 4.4% | 10.33 | 0.29 | 2.8% | | 4 | 5.53 | 0.27 | 4.9% | 10.42 | 0.33 | 3.2% | The overall mean value (Mean), standard deviation, and between-analyzer imprecision CV% are calculated and summarized in the following table: | | Normal sample (5.5%) | High sample (10.7%) | | --- | --- | --- | | mean | 5.59 | 10.34 | | SD | 0.24 | 0.26 | | Overall CV% | 4.3% | 2.5% | - Lot-to-lot variation A panel of 10 blood samples with HbA1c values spanning $5.2 - 11.3\%$ was used in addition to SMART HbA1c Control Set level 1 and Control Set level 2 for lot-to-lot variation study. The blood samples and controls were analyzed in duplicates using three lots of the SMART HbA1c Assay reagents on one single SMART analyzer. During the study, CVs ranged from $1.1 - 4.3\%$ indicating lot-to-lot consistency. # b. Linearity/assay reportable range: Five samples used to obtain linearity data were prepared by dilution of two whole blood samples, DZ Linearity Level 1 and Level 2 with known HbA1c contents of $4.25\%$ and $12.1\%$ respectively. The linearity set was made according to CLSI Guideline EP6-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach and each sample run in duplicate. One reagent lot and one analyzer were used. The results of analysis of the data by linear regression are: $\mathrm{R}^2 = 0.9947$ , slope $= 1.0113$ , and y-intercept $= -0.2981$ The claimed measuring range is $4 - 12\%$ HbA1c for the SMART HbA1c assay. # c. Traceability, Stability, Expected values (controls, calibrators, or methods): The SMART HbA1c assay has met the requirements of the National Glycohemoglobin Standardization Program (NGSP). The NGSP certification requires annual renewal. The current list of NGSP certified methods is available on the NGSP website at: http://www.ngsp.org/prog/index. # Calibration Radio Frequency Identification Card (RFID card) SMART HbA1c Assay utilizes a RFID card that is preprogrammed with a reagent lot {6} specific calibration curve and is supplied in each kit. When RFID card is inserted into SMART analyzer slot (assay procedure step), the lot specific calibration curve is automatically utilized for assay test. RFID cards are programmed at manufacturer site. The assay lot specific calibration curve is generated by experimental data generation using human whole blood calibrator set traced to reference calibrators. RFID calibration curve generation is described in the following steps. 1) Two level human whole blood hemolysates are tested with the Tosoh G7 HPLC method to determine initial %HbA1c values. The Tosoh G7 HbA1c HPLC method is certified by the National Glycohemoglobin Standardization Program (NGSP, University of Missouri School of Medicine). 2) Lot specific SMART HbA1c assay reagents are used with the bi-level calibration material, to test > 30 fresh human whole blood samples and quality control materials. The human whole blood samples are previously tested with the Tosoh's G7 HbA1c HPLC method and other assay methods. Three different instruments are used in the analysis. 3) Calibrator values are adjusted to meet the acceptance criteria and final calibration curve with a point-to-point least squares fit equation. Calibration curve values representing change in absorbance slope versus HbA1c% levels are programmed into cards with the use of a card writer. RFID cards are subject to the same quality system checks as reagents and controls. Values are given in NGSP/DCCT value system. The IFCC values corresponding to NGSP/DCCT values of samples can be determined by use of published conversion formula: NGSP = 0.915 × (IFCC) + 2.15 ## SMART HbA1c Control set The controls are made of human whole blood and are in a lyophilized (freeze-dried) state. The control set are value assigned as follows. 1) Two level human whole blood hemolysates are tested with the Tosoh G7 HPLC method to determine initial %HbA1c values. The Tosoh G7 HbA1c HPLC method is certified by the National Glycohemoglobin Standardization Program (NGSP, University of Missouri School of Medicine). 2) With a reference lot of SMART HbA1c Assay Reagent Kits, the bi-level control material (target ranges are 5.5 – 6.5% for the low control and 7.5 – 9.5% for the high control) is then subjected to extensive replicate analysis using one SMART analyzer to obtain mean values and a range. Range criteria: Deviation from the target value of < 20% for control level 1 (normal) and control level 2 (high). Values are given in NGSP/DCCT value system. The IFCC values corresponding to NGSP/DCCT values of samples can be determined by use of published conversion formula: NGSP = 0.915 × (IFCC) + 2.15 ## Stability Real time stability studies have been conducted. Protocols and acceptance criteria were described and found to be acceptable. When stored unopened at 2-8°C, the 7 {7} SMART HbA1c assay kit is valid for 6 months and the SMART HbA1c control set is valid for 12 months. Reconstituted controls are stable for 14 days when capped tightly and stored at 2-8°C. d. Detection limit: To establish the limit of detection for the SMART assay, a modified protocol was used. The SMART HbA1c Assay measures HbA1c normalized to hemoglobin and the reagents are comprised of redox balanced agents. Sample dilution with water or saline is not acceptable. Therefore, additional simple precision studies were conducted at the lower end of the measuring range (4-12%). In one experiment, a whole blood sample containing 6% HbA1c was diluted by half with redox balanced buffer to 3% HbA1c and tested on SMART analyzer in 12 replicates. The limit of detection is calculated to be the mean + 3SD = 3.92% HbA1c which is below the measuring range (4-12%). Samples below the measuring range are reported as <4% by the SMART Analyzer. To further verify the precision of the SMART HbA1c Assay at the lower end of the measuring range, a blood sample containing 4% HbA1c was tested on SMART analyzer in 10 replicates in a simple precision test. | Mean | 4.02 | | --- | --- | | SD | 0.18 | | CV | 4.51% | e. Analytical specificity: To determine the level of interference from the substances normally present in whole blood, SMART HbA1c Assay was used to test 5.5% and 11.0% HbA1C whole blood samples spiked with various concentrations of substances following CLSI Guideline EP7-A: Interference Testing in Clinical Chemistry dose-response guidelines. The following substances normally present in the blood produced less than 10% deviation when tested at levels equal to the concentrations listed below: | Interference | Concentration | | --- | --- | | Ascorbic Acid | 30 mg/dL | | Bilirubin | 15 mg/dL | | Bilirubin Conjugate | 5 mg/dL | | Uric Acid | 30 mg/dL | | Triglyceride | 4000 mg/dL | | Glucose | 5,000 mg/dL | | Urea | 100 mg/dL | | Labile A1c (glucose) | 1000 mg/dL | | Carbamylated A1c (urea) | 300 mg/dL | {8} In order to evaluate whether SMART HbA1c Assay is interfered by Hemoglobin AD, AE, AS, AC, and AF variants, samples with hemoglobin variants (AD, AE, AS, AC, AF) were obtained from an NGSP reference lab. Expected HbA1c values for these samples were obtained by standard reference methods known to yield true $\% \mathrm{HbA1c}$ values that are unaffected by the variant. Samples were run in duplicate with the SMART HbA1c Assay: | Specimen | Hb Variant | Expected Value | Average % HbA1c Value | Bias | | --- | --- | --- | --- | --- | | 447 | HbAC | 7.9 | 7.9 | 0.00 | | 449 | HbAC | 7.6 | 7.0 | -0.55 | | 451 | HbAC | 5.5 | 6.0 | 0.50 | | 453 | HbAC | 5.5 | 5.8 | 0.30 | | 413 | HbAD | 9.4 | 8.8 | -0.65 | | 418 | HbAD | 7.5 | 7.1 | -0.40 | | 444 | HbAD | 6.3 | 6.5 | 0.20 | | 455 | HbAD | 6.2 | 6.2 | 0.05 | | 414 | HbAE | 5.2 | 5.8 | 0.60 | | 436 | HbAE | 6.8 | 6.3 | -0.50 | | 445 | HbAE | 7.6 | 8.0 | 0.40 | | 448 | HbAE | 10.9 | 10.8 | -0.10 | | 496 | HbAF | 6.0 | 5.5 | -0.50 | | 497 | HbAF | 4.7 | 5.2 | 0.50 | | 498 | HbAF | 8.7 | 8.0 | -0.70 | | 499 | HbAF | 4.9 | 5.3 | 0.40 | | 502 | HbAS | 6.3 | 5.9 | -0.40 | | 503 | HbAS | 6.6 | 6.2 | -0.40 | | 504 | HbAS | 6.6 | 6.2 | -0.35 | | 512 | HbAS | 5.7 | 5.8 | 0.15 | Hemoglobin AD, AE, AS, AC, and AF variants do not significantly interfere with the SMART HbA1c Assay. f. Assay cut-off: Not applicable. 2. Comparison studies: a. Method comparison with predicate device: To demonstrate accuracy, the proposed device was tested with individual blood samples and results compared to a predicate device (Diazyme Direct Enzymatic HbA1c Assay on the Hitachi 917 (k070734)) using CLSI EP9-A2: Method Comparison and Bias Estimation Using Patient Samples as a guideline. The method comparison study was performed internally at Diazyme Laboratories and externally at POL sites. {9} (1) Internal method comparison using EDTA venous whole blood specimens Three SMART analyzers and three lots of reagents were used for the study. A total of 65 EDTA venous whole blood specimens were tested with both proposed device and predicate device. HbA1c values of the samples ranged from $4.5 - 11.1\%$ . One sample containing HbA1c% > 12% was excluded from the analysis. The regression results are summarized in the following table: | n | 64 | | --- | --- | | Slope | 0.955 | | Intercept | 0.30 | | Correlation | 0.94 | | Range of values | 4.5% - 11.1% HbA1c | Passing Bablok regression analysis: $\mathrm{Y} = 0.3478 + 0.9565\mathrm{x}$ Intercept A: 0.3478 $95\%$ CI:0.1000 to 0.7778 Slope B: 0.9565 $95\%$ CI:0.8889 to 1.0000 (2) External method comparison at a POL site using EDTA venous whole blood specimens A total of 42 EDTA venous whole blood specimens were tested with both proposed device and predicate device at a POL site. One lot and three analyzers were used. 3 samples containing greater than $12\%$ HbA1c were excluded from the analysis. $97\%$ of the results (38 out of 39) on the proposed device were within $\pm 0.75\%$ HbA1c of the results obtained for the samples run on the predicate device. | N | 39 | | --- | --- | | Range | 4.7 - 11.9% | | Regression equation | Y = 0.9347x + 0.2249 | | Correlation coefficient | 0.9814 | | Passing Bablok | | | Regression equation | Y = 0.9292x + 0.2390 | | 95% CI for intercept | -0.1481 - 0.5407 | | 95% CI for slope | 0.8814 - 0.9808 | (3) External Comparison of SMART HbA1c Assay using Capillary Blood and Venous Blood Performance for the SMART HbA1c assay was evaluated at three POL sites with a total of three operators. A total of two reagent lots and four SMART analyzers were tested. About 40-50 volunteers were recruited from each POL site for the study. Both {10} capillary finger stick blood and venous blood were drawn from each donor. The capillary finger stick blood was analyzed with Diazyme SMART analyzer; in the mean time, the venous blood was analyzed using the predicate device (Diazyme Enzymatic HbA1c Assay on the Hitachi 917). 2 samples containing greater than 12% HbA1c at Site 1 were excluded from the analysis. 97% of the capillary fingerstick results (124 out of 128) obtained with the proposed device were within ± 0.75% HbA1c of the results obtained the venous samples run on the predicate device. The results of HbA1c% of the capillary finger stick blood obtained with the proposed device and HbA1c% of venous blood using the predicate device were compared using Passing & Bablok regression plot. | N | 128 | | --- | --- | | Range | 5.0 - 11.9% | | Regression equation | Y = 0.94x + 0.4654 | | Correlation coefficient | 0.926 | | Passing Bablok | | | Regression equation | Y = 1.000 x + 0.1000 | | 95% CI for intercept | 0.10 - 0.6375 | | 95% CI for slope | 0.9167 - 1.000 | b. Matrix comparison: SMART HbA1c Assay uses EDTA venous whole blood and finger stick capillary whole blood specimens. See method comparison above. 3. Clinical studies: a. Clinical Sensitivity: Not applicable. b. Clinical specificity: Not applicable. c. Other clinical supportive data (when a. and b. are not applicable): Not applicable. 4. Clinical cut-off: Not applicable. 5. Expected values/Reference range: Non-diabetic individuals have HbA1c values in the range of 4-6% using a DCCT based assay. The American Diabetes Association (ADA) recommends that the primary treatment goal in diabetes should be glycemic control equal to that achieved during the DCCT. ADA states that lowering HbA1c to below or around 7% has been shown to reduce microvascular and neuropathic complications of type 1 and type 2 diabetes. {11} Therefore, for microvascular disease prevention, the HbA1c goal for nonpregnant adults in general is <7% HbA1c.¹ However, each laboratory must establish its own normal range in their country of business taking into account sex, age and ethnicity. ADA recommends that glycemic control goals should be individualized based on duration of diabetes, age/life expectancy, comorbid conditions, known CVD or advanced microvascular complications, hypoglycemia unawareness and individual patient considerations.¹ ¹ American Diabetes Association Clinical Practice Recommendations: Executive Summary: Standards of Medical Care in Diabetes - 2009 Diabetes Care, 32, suppl.1: S6-7 (2009). N. Instrument Name: SMART Analyzer O. System Descriptions: 1. Modes of Operation: The Smart Analyzer is operated exclusively using the Touch Display. All steps necessary for operation are displayed as symbols. To activate the symbols they have to be touched with the fingers or finger nail. To process a test, insert the provided RFID card into the analyzer. This card contains all data necessary to perform the routine tests. No measurement can be started without the RFID test card. The DRS (Diazyme Reagent System) cartridge is inserted in the door that opens automatically upon touch screen input. After input of all requested data and activating the START button on the Touch Display, the lid is automatically closed, and the testing procedure is started. The closed door avoids the intrusion of ambient light, dust, dirt and humidity in the laboratory photometer while the instrument is measuring and when it is not in use. The analyzing process is fully automated after inserting the cartridge into the analyzer; the process starts by closing the analyzer door. Only one cartridge can be run at a time. Once inside the analyzer, the sample and reagent are mixed automatically. As the reaction starts, changes in absorbance are measured at the correct wavelength and referenced to the calibration data preprogrammed in the RFID card. All assay and lot specific information necessary for the analyzer to process a test is read from the RFID card. After the measurement is completed, the door opens automatically, and the test cartridge can now be removed. All SMART HbA1c DRS test cartridge are single use and are designed to be disposed after use. 2. Software: FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types: Yes ☐ or No ☑ 12 {12} 13 3. Specimen Identification: Samples are identified by manually labeling and by logging the patient ID into the analyzer prior to inserting the cartridge into the analyzer, or during the analyzing process. 4. Specimen Sampling and Handling: A specimen in a single use capped DRS cuvette is inserted into the analyzer and read by the spectrophotometer with no liquid/specimen carryover within the analyzer. Once sample is read, cuvette is removed from the analyzer and disposed of. 5. Calibration: Calibration Radio Frequency Identification Card (RFID card) SMART HbA1c Assay utilizes a RFID card that is preprogrammed with a reagent lot specific calibration curve and is supplied in each kit. When RFID card is inserted into SMART analyzer slot (assay procedure step), the lot specific calibration curve is automatically utilized for assay test. RFID cards are programmed at manufacturer site. The assay lot specific calibration curve is generated by experimental data generation using human whole blood calibrator set 6. Quality Control: The user should perform quality control testing to confirm that the SMART HbA1c Analyzer System is working properly and providing reliable results using the SMART HbA1c control set. Additionally, the instrument contains internal checks for detecting electronic, mechanical, and sample problems. Q. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. R. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision.