DIAZYME HOMOCYSTEINE POC TEST KIT
Device Facts
| Record ID | K121053 |
|---|---|
| Device Name | DIAZYME HOMOCYSTEINE POC TEST KIT |
| Applicant | Diazyme Laboratories |
| Product Code | LPS · Clinical Chemistry |
| Decision Date | Jul 31, 2012 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 862.1377 |
| Device Class | Class 2 |
Indications for Use
Diazyme's HCY POC Test Kit is intended to be used with the SMART analyzer in a Point-of-Care setting for the in vitro quantitative determination of total L-homocysteine in serum or plasma. The assay can assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria. For in vitro diagnostic use only.
Device Story
The Diazyme HCY POC Test Kit is an in vitro diagnostic assay for quantitative determination of total L-homocysteine (HCY) in human serum or plasma. It is designed for use with the SMART analyzer, a compact, cuvette-based spectrophotometer. The system utilizes a novel enzyme cycling method: oxidized HCY is reduced to free HCY, which reacts with S-adenosylmethionine (SAM) catalyzed by HCY S-methyltransferase to form methionine and S-adenosylhomocysteine (SAH). SAH is subsequently hydrolyzed into adenosine and HCY, which re-enters the cycle. The resulting adenosine is converted to inosine and ammonia, triggering a glutamate dehydrogenase reaction that converts NADH to NAD+. The concentration of HCY is inversely proportional to the decrease in absorbance at 340nm. The test is performed in point-of-care settings (e.g., physician office laboratories) by nurses or assistants. Users add 20µL of sample to a prefilled reagent cuvette, snap on a prefilled reagent cap, and insert it into the analyzer. The analyzer uses a lot-specific RFID card for calibration and assay parameters. Results assist clinicians in diagnosing and managing hyperhomocysteinemia and homocystinuria.
Clinical Evidence
Bench testing only. Precision evaluated per CLSI EP5-A at the manufacturer site and three physician office laboratories (POL) with multiple users; total CV% ranged from 3.2% to 7.0%. Linearity confirmed from 3-50 µmol/L (R2=0.9992). Method comparison against the predicate (Olympus AU400) showed high correlation (R2=0.9765, n=120). Interference testing confirmed <10% deviation for common endogenous substances. Matrix equivalence established between serum, EDTA plasma, and Li Heparin plasma.
Technological Characteristics
Quantitative enzymatic assay. Reagents: SAM, NADH, TCEP, 2-oxoglutarate, Glutamate dehydrogenase, SAH hydrolase, Adenosine deaminase, HCY S-methyltransferase. Form factor: Single-use cuvettes with pre-filled reagents. Connectivity: RFID card for lot-specific calibration. Measurement: Spectrophotometric (340nm) via SMART analyzer. Traceable to NIST SRM 1955.
Indications for Use
Indicated for in vitro quantitative determination of total L-homocysteine in serum or plasma to assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria. Prescription use only.
Regulatory Classification
Identification
A urinary homocystine (nonquantitative) test system is a device intended to identify homocystine (an analogue of the amino acid cystine) in urine. The identification of urinary homocystine is used in the diagnosis and treatment of homocystinuria (homosystine in urine), a heritable metabolic disorder which may cause mental retardation.
Predicate Devices
- Diazyme Homocysteine Assay (k071971)
Related Devices
- K113793 — HOMOCYSTEINE TEST · Roche · Jun 5, 2012
- K013606 — ACS 180 HOMOCYSTEINE ASSAY · Bayer Diagnostics Corp. · Dec 21, 2001
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE
A. 510(k) Number:
k121053
B. Purpose for Submission:
New device
C. Measurand:
Homocysteine
D. Type of Test:
Quantitative Enzymatic
E. Applicant:
Diazyme Laboratories
F. Proprietary and Established Names:
Diazyme Homocysteine POC Test Kit
G. Regulatory Information:
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| LPS | Class II | 21 CFR 862.1377 | Chemistry |
H. Intended Use:
1. Intended use(s):
See Indications for use below
2. Indication(s) for use:
Diazyme Homocysteine POC Test Kit is intended to be used with the SMART analyzer in a Point-of-Care setting for the in vitro quantitative determination of total L-
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homocysteine in serum or plasma. The assay can assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria. For in vitro Diagnostic Use Only
3. Special conditions for use statement(s):
For prescription use only
The labeling contains a prominent black-box warning:
WARNING: Specimens from patients who are on drug therapy involving S-adenosylmethionine may show falsely elevated levels of homocysteine. Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants and 6-azuridine triacetate may have elevated levels of homocysteine due to their effect on the metabolic pathway.
4. Special instrument requirements:
For use on SMART Analyzer (k092911) only
I. Device Description:
The Diazyme HCY POC Test Kit consists of the following components (1) DRS Cuvettes pre-filled with Reagent 1, (2) DRS Caps Pre-filled with Reagent 2, and (3) one preprogrammed Radio Frequency ID (RFID) card which contains a lot specific calibration curve. Reagent 1 and 2 are comprised of S-adenosylmethionine (SAM) (0.1mM), NADH (>0.2mM), TCEP (>0.5mM), 2- oxoglutarate (5.0mM), Glutamate dehydrogenase (10 KU/L), SAH hydrolase (3.0 KU/L), Adenosine deaminase (5.0 KU/L), and Hcy methyltransferase (5.0 KU/L).
Smart Analyzer (k092911) is a compact cuvette based spectrophotometer machine for point-of-care testing designed to analyze readings from single use reagent cuvettes. The instrument only uses the Diazyme Reagent System (DRS) cuvette and caps and performs the assay with a preprogrammed Radio Frequency ID (RFID) card. The lot specific RFID card contains reagent addition time, mixing time, reading time and calibration curve for estimating Homocysteine concentration.
J. Substantial Equivalence Information:
1. Predicate device name(s):
Diazyme Homocysteine 2 Reagent Enzymatic Assay Kit
2. Predicate 510(k) number(s):
k071971
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3. Comparison with predicate:
| Similarities and Differences | | |
| --- | --- | --- |
| Item | Candidate Device
Diazyme Homocysteine POC
Test Kit | Predicate
k071971 |
| Intended Use/ Indications for Use | Diazyme Homocysteine POC
Test Kit is intended for in vitro
quantitative determination of
total L-homocysteine in serum or
plasma. The assay can assist in
the diagnosis and treatment of
patients suspected of having
hyperhomocysteinemia and
homocystinuria. For in vitro
Diagnostic Use Only | Same |
| Test principle | Indirect measurement of
homocysteine by the
measurement of the cosubstrate
conversion product | Same |
| Type of Test | Quantitative | Same |
| Specimen type | 20μl Human serum or plasma | 13 μl Human serum or
plasma |
| Calibration | Assay Specific Calibrators | RFID card carrying
calibrator information
for the SMART
analyzer |
| Measuring Range | 3 – 50 μmol/L | 2.5 – 50 μmol/L |
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: Statistical Approach
CLSI Guideline EP9-A2: Method Comparison and Bias Estimates Using Patient Samples
CLSI Guideline EP7-A2: Interference Testing in Clinical Chemistry
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# L. Test Principle:
Diazyme Homocysteine POC Test Kit contains reagents intended for use with the SMART analyzer for the quantitative determination of Homocysteine (HCY) in human serum or plasma. Oxidized HCY is first reduced to free HCY which then reacts with a co-substrate, S-adenosylmethionine (SAM) catalyzed by a HCY S-methyltransferase to form methionine (Met) and S-adenosylhomocysteine (SAH). SAH is assessed by coupled enzyme reactions including SAH hydrolase, adenosine (Ado) deaminiase and HCY by SAH hydrolase. The formed HCY that is originated from the co-substrate SAM is cycled into the HCY conversion reaction by HCY S-methyltransferase. This forms a co-substrate conversion product-based enzyme cycling reaction system with significant amplification of detection signals. The formed Ado is immediately hydrolyzed into inosine and ammonia which reacts with glutamate dehydrogenase with concomitant conversion of NADH to $\mathrm{NAD^{+}}$ . The concentration of HCY in the sample is indirectly proportional to the amount of NADH converted to $\mathrm{NAD^{+}}$ which is read spectrophotometrically at $340\mathrm{nm}$ .
# M. Performance Characteristics (if/when applicable):
# 1. Analytical performance:
# a. Precision/Reproducibility:
# Internal Testing
Precision was evaluated according to CLSI EP5-A guideline. In order to evaluate precision three levels of serum controls containing 7.3,11.87 and $29.32\mu \mathrm{mol} / \mathrm{L}$ Homocysteine were tested in duplicate, two runs per day, over ten days (total measurements per sample, $n = 40$ ). The results are summarized in the table below:
| Sample | N | Mean (μmol/L) | Within Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV |
| Control 1 | 40 | 7.5 | 0.24 | 3.2 | 0.26 | 3.4 |
| Control 2 | 40 | 11.8 | 0.22 | 1.8 | 0.42 | 3.5 |
| Control 3 | 40 | 29.0 | 0.82 | 2.8 | 0.95 | 3.3 |
# POC Testing
A precision study was performed at three physician office laboratories by intended users. The study was evaluated at three physician office laboratories (POL). A total of 9 serum samples with HCY levels from $10.26\mu \mathrm{mol} / \mathrm{L}$ to $42.73\mu \mathrm{mol} / \mathrm{L}$ were tested at each site. Total measurements per sample, $n = 20$ . The results are summarized in the table below:
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Site 1
| Sample | N | Mean (μmol/L) | Within Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV |
| Sample 1 | 20 | 11.05 | 0.73 | 6.7 | 0.77 | 7.0 |
| Sample 2 | 20 | 25.82 | 1.60 | 6.2 | 1.38 | 5.3 |
| Sample 3 | 20 | 42.73 | 2.39 | 5.6 | 2.73 | 6.4 |
Site 2
| Sample | N | Mean (μmol/L) | Within Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV |
| Sample 4 | 20 | 10.26 | 0.71 | 6.9 | 0.67 | 6.6 |
| Sample 5 | 20 | 25.18 | 1.32 | 5.2 | 1.39 | 5.5 |
| Sample 6 | 20 | 41.99 | 1.62 | 3.8 | 1.86 | 4.4 |
Site 3
| Sample | N | Mean (μmol/L) | Within Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV |
| Sample 7 | 20 | 11.63 | 0.49 | 4.2 | 0.69 | 6.0 |
| Sample 8 | 20 | 26.34 | 1.98 | 7.5 | 1.79 | 6.8 |
| Sample 9 | 20 | 31.63 | 1.83 | 5.8 | 1.73 | 5.5 |
b. Linearity/assay reportable range:
Percent recovery was assessed across the reportable range of the device (3-50 $\mu$ mol/L). A serum sample containing $50~\mu \mathrm{mol} / \mathrm{L}$ homocysteine was diluted with saline to create 11 sample levels across the measuring range of the device. The samples were measured in triplicate on the SMART Analyzer. The mean of the obtained Homocysteine results (obtained on the SMART analyzer) were plotted against the expected values and an appropriate line fitted by standard linear regression resulted in the following equation:
$$
y = 0.9749x + 0.751; R^2 = 0.9992
$$
Based upon the linearity data and the LoQ study, the sponsor claims a measuring range of 3 to $50~\mu \mathrm{mol} / \mathrm{L}$
The sponsor recommends that all samples $>50\mu \mathrm{M}$ be diluted with deionized water and a 1:1 dilution be performed. In order to support this procedure, the sponsor provided a dilution study in which 4 serum samples were spiked and then diluted 1:1 with water and analyzed on the SMART analyzer using the Homocysteine POC test. Sample range was 62.2-84.2 $\mu \mathrm{mol} / \mathrm{L}$ and $\%$ Recovery was 99.79 - 104.68.
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c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Traceability:
Homocysteine POC test calibration system is traceable to the higher order NIST SRM 1955. Homocysteine POC test utilizes a Calibration Radio Frequency Identification Card (RFID) card that is programmed with a lot specific calibration curve and is supplied in each kit. RFID cards are programmed at the manufacturer site and are subject to the same quality control checks as the reagents and controls. According to the sponsor, the calibration curve is stable until the printed expiration date which is 8 months.
To construct the Diazyme HCY SMART Assay RFID card calibration curve, 5 levels of known calibrator levels are tested with the Diazyme Homocysteine POC Test reagents on three SMART analyzers. The calibrator value and the mean absorbance change are programmed into the RFID card.
Value Assignment (controls): previously cleared in k042448
Detection limit:
The Limit of Blank, Limit of Detection and Limit of Quantitation of Diazyme HCY POC Test kit were determined according to CLSI EP17-A in the following manner:
To calculate the Limit of Blank (LoB) of the Diazyme HCY POC Test kit, the True Blank Sample (water) was tested with 20 replicates daily for three days. LOB was calculated as the mean of the 57th and 58th highest values for the true blanks. Based upon the results, the sponsor claims a LoB=0.06μmol/L.
To calculate the Limit of Detection (LoD) of the Diazyme HCY POC Test kit, five low samples were tested with 4 replicates daily for three days. LoD=LoB + (1.465* SD of Low samples). Based upon the results, the sponsor claims a LoD=0.32 μmol/L.
The lowest level of the calibrator for the HCY RFID card is between 2-3 μmol/L HCY. No values that are below this level are able to be reported by the analyzer.
To calculate the Limit of Quantitation (LoQ) of the Diazyme HCY POC Test kit, 5 serum samples were prepared with HCY values at the lower range (2.1-2.61 μmol/L). 20 replicates of the low samples were run and EP evaluator software was used to estimate the LoQ. Based upon the results and the limitations imposed by the calibrator RFID card (cannot measure samples <2 μmol/L, the sponsor claims a LoQ = 3μmol/L.
The claimed measuring range is 3 - 50 μmol/L based on linearity.
See linearity study in M.1.b of this 510(k) decision summary.
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# e. Analytical specificity:
The level of interference from substances normally present in serum was determined by testing two HCY serum samples (12 $\mu$ M and 29 $\mu$ M) spiked with various concentrations of the interferent. The following substances produced less than $10\%$ deviation when tested at levels equal to the concentrations listed below.
| Endogenous Substances | Concentration |
| --- | --- |
| Unconjugated Bilirubin | 40 mg/dL |
| Conjugated Bilirubin | 40 mg/dL |
| Hemoglobin | 500 mg/dL |
| Triglyceride | 1000 mg/dL |
| Exogenous Substances | Concentration |
| --- | --- |
| Ascorbic Acid | 10 mmol/L |
| Glutathione | 500μM |
| Methionine | 20 μM |
| Cysteine | 1000 μM |
| Pyruvate | 500 μM |
| Cystathionine | 100 μM |
| Hydroxylamine | 1000 μM |
| Carbamazepine | 130 μM |
| Phenytoin | 200 μM |
| 6-azauridine triacetate | 1000 μM |
| S-adenosyl-methionine | 20 μM |
| Carbamazepine-10, 11-epoxide | 60 μM |
| Ethosuximide | 1800 μM |
| Primidone | 200 μM |
| Valproic Acid | 3.5 mM |
| Sodium Nitrate | 500 μM |
| Methotrexate | 2.0 mM |
The labeling states "patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6-azauridine triacetate, may have higher levels of HCY due to metabolic interference with homocysteine metabolism."
# f. Assay cut-off:
Not applicable
# 2. Comparison studies:
a. Method comparison with predicate device:
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# Internal Site Testing
To assess the accuracy of the device, 74 samples (70 natural serum samples and 4 spiked samples were measured using the Diazyme Homocysteine POC test run in parallel with the Diazyme HCY Two Reagent Enzymatic Assay on the Olympus AU400 analyzer. Sample values ranged from 4.17-49.50 μmol/L. Three SMART analyzers and two lots of reagents were used and samples were assayed in singlicate.
The regression results are summarized below:
| Slope | 0.9612 |
| --- | --- |
| Intercept | 0.5246 |
| Correlation Coefficient | 0.9696 |
# POC Site Testing
120 serum samples were tested at three POC sites. Each site tested 40 samples using SMART analyzers. The test results on SMART analyzers were compared to the predicate device on the Olympus AU400 analyzer.
The regression results are summarized below:
| SMART HCY | Site 1 | Site 2 | Site 3 | All sites combined |
| --- | --- | --- | --- | --- |
| n | 40 | 40 | 40 | 120 |
| Slope | 1.0890 | 1.0041 | 1.0600 | 1.0552 |
| Intercept | -0.7438 | -0.6251 | -1.1564 | -0.8860 |
| R² | 0.9830 | 0.9645 | 0.9819 | 0.9765 |
| Range | 5.43-48.95 μmol/L | 3.88-45.43 μmol/L | 4.81-49.86 μmol/L | 3.88-49.86 μmol/L |
# b. Matrix comparison
Forty paired serum and plasma matched sets (EDTAPlasma/Li-Heparin) samples were tested on the SMART analyzer using the Homocytsteine POC test kits. Of the 40 samples that were analyzed 32 were native samples and 8 were spiked samples. Sample range tested was 7.97 - 47.24 μmol/L.
Linear regression analysis is as follows:
EDTA plasma vs. Serum – y=1.0197x-0.5385 R²= 0.9889
Li Heparin vs. Serum – y=0.9632x+0.3177 R²=0.99
Based on the data, the sponsor claims EDTA plasma and Lithium Heparin are acceptable anticoagulants for this assay.
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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:
The labeling states in most of U.S. clinical laboratories, 15mmol.L is used as the cut-off value for normal level of Hcy for adults. However, each laboratory is recommended to establish a range of normal values for the population in their region. $^{1,2,3}$
$^{1}$ Ueland PM, Refsum H, Kvalheim G, et al. Clinical Chem. 39: 263-271. (1993)
$^{2}$ Lussier-Cacan S, Xhignese M, Piolot A, et al. Am J Clin Nutr. 64:587-593. (1996)
$^{3}$ Cotton F, Wautrecht J. Lechevin V, et al. Clin Chem. 49:315-317. (2003)
N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
