In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on Roche/Hitachi cobas c systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co‑substrate conversion product instead of assessing co‑substrate or Hcy conversion products. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal. The formed Ado is immediately hydrolyzed into inosine and ammonia. In the last step, the enzyme glutamate dehydrogenase (GLDH) catalyzes the reaction of ammonia with 2‑oxoglutarate and NADH to form NAD+. The concentration of Hcy in the sample is directly proportional to the amount of NADH converted to NAD+ (ΔA340 nm).
|
ADA | ||||
Ado |
| Inosine + NH3 |
GLDH | ||||
NH3 + NADH + 2‑Oxoglutarate |
| Glutamate + NAD+ + H2O |
Measuring range
3‑50 µmol/L
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Lower limits of measurement
Limit of Blank, Limit of Detection and Limit of Quantitation
Limit of Blank | = 3 µmol/L |
Limit of Detection | = 3 µmol/L |
Limit of Quantitation | = 5.5 µmol/L |
The Limit of Blank, Limit of Detection and Limit of Quantitation were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A2 requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 %.
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %).
The Limit of Quantitation is the lowest analyte concentration that can be reproducibly measured with a total error of 30 %. It has been determined using low concentration homocysteine samples.
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut‑off value for normal levels of Hcy in adults.
In European laboratories, 12 μmol/L is used as the cut‑off value for normal levels of Hcy in adults.
Group | Folate supplemented | Nonsupplemented |
Pregnancy | 8 | 10 |
Children < 15 years | 8 | 10 |
Adults 15‑65 years | 12 | 15 |
Elderly > 65 years | 16 | 20 |
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 10 % of initial value for analyte concentrations > 15 µmol/L or ± 1.5 µmol/L for analyte concentrations ≤ 15 µmol/L.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Exceptions: 0.5 mmol/L Glutathione, 100 μmol/L Cystathionine, 0.5 mmol/L Pyruvate.
Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference. However, SAH is only detectable at sub‑nmol/L concentrations in normal plasma, and should not cause concern.
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay can not use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry‑over is available via the cobas link. The latest version of the carry‑over evasion list can be found with the NaOHD/SMS/SCCS Method Sheet for information. For further instructions refer to the operator’s manual.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
08057826190 | Homocysteine Enzymatic Assay (cobas c pack 1), 100 tests | System‑ID 2070 011 | cobas c 303, cobas c 503 |
Homocysteine Enzymatic Assay (cobas c pack 2), 100 tests | System‑ID 2070 012 |
Materials required (but not provided):
05385504190 | HCYS Calibrator Kit (2 x 3 mL) | Code 20590 | |
05142423190 | HCYS Control Kit Control 1 (2 x 3 mL) | Code 20254 | |
HCYS Control Kit Control 2 (2 x 3 mL) | Code 20255 | ||
08063494190 | Diluent NaCl 9 % (123 mL) | System‑ID 2906 001 |
HCYS: ACN 20700
Ready for use
Test definition | |||
Reporting time | 10 min | ||
Wavelength (sub/main) | 700/340 nm | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 132 µL | – | |
R2 | 21 µL | – | |
R3 | 15 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 10.5 µL | – | – |
Decreased | 10.5 µL | 25.0 µL | 100 µL |
Increased | 10.5 µL | – | – |
For further information about the assay test definitions refer to the application parameters setting screen of the corresponding analyzer and assay.
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 4 weeks |
Do not freeze.
Calibrators | S1‑5: HCYS Calibrator Kit | |
Calibration mode | Non‑linear | |
Calibration frequency | Automatic full calibration Full calibration |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the analyzers are given below. These data represent the performance of the analytical procedure itself.
Results obtained in individual laboratories may differ due to heterogenous sample materials, aging of analyzer components and mixture of reagents running on the analyzer.
Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP05‑A3 requirements with repeatability (n = 84) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days).
Repeatability | Mean | SD | CV |
Homocysteine Control 1 | 13.2 | 0.149 | 1.1 |
Homocysteine Control 2 | 37.2 | 0.462 | 1.2 |
Human serum 1 | 9.52 | 0.135 | 1.4 |
Human serum 2 | 12.1 | 0.195 | 1.6 |
Human serum 3 | 15.0 | 0.297 | 2.0 |
Human serum 4 | 26.2 | 0.433 | 1.7 |
Human serum 5 | 45.3 | 0.704 | 1.6 |
Intermediate precision | Mean | SD | CV |
Homocysteine Control 1 | 13.2 | 0.374 | 2.8 |
Homocysteine Control 2 | 37.3 | 0.763 | 2.0 |
Human serum 1 | 9.55 | 0.330 | 3.5 |
Human serum 2 | 12.1 | 0.437 | 3.6 |
Human serum 3 | 15.4 | 0.447 | 2.9 |
Human serum 4 | 26.2 | 0.653 | 2.5 |
Human serum 5 | 45.3 | 0.945 | 2.1 |
Hcy values for human serum samples obtained on the cobas c 503 analyzer (y) were compared with those determined using the corresponding reagent on a cobas c 501 analyzer (x).
Sample size (n) = 74
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 0.984x + 0.122 µmol/L | y = 0.979x + 0.215 µmol/L |
τ = 0.979 | r = 0.999 |
The sample concentrations were between 4.11 and 48.4 µmol/L. |
Sample size (n) = 82
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 1.014x - 0.407 µmol/L | y = 1.010x - 0.319 µmol/L |
τ = 0.951 | r = 0.997 |
The sample concentrations were between 3.53 and 48.7 µmol/L. |
Homocysteine (Hcy) is a thiol‑containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein‑bound and free.
Elevated levels of tHcy has emerged as an important risk factor in the assessment of cardiovascular disease.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta‑synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism. Elevated levels of tHcy are also linked with Alzheimer’s disease
R1 | NADH reagent S‑adenosylmethionine 0.1 mmol/L, TCEP* > 0.5 mmol/L, 2‑oxoglutarate < 5.0 mmol/L, NADH > 0.2 mmol/L, buffer, pH 9.1 (25 °C), preservative, stabilizer |
R2 | Enzyme reagent Homocysteine S‑methyltransferase (HMTase) 5.0 kU/L, glutamate dehydrogenase (GLDH) 10 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, detergent |
R3 | Start reagent Adenosine deaminase (bovine) 5.0 kU/L, S‑adenosyl‑homocysteine hydrolase (SAHase) 3.0 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, stabilizer |
*Tris(2‑carboxyethyl)phosphine
Cat. No. 08057826190 consists of 2 cobas c packs: 1 x R1 + R2 and 1 x R3. R1 is in position B and R2 is in position C of cobas c pack 1. R3 is in position C of cobas c pack 2.
For in vitro diagnostic use for health care professionals. Exercise the normal precautions required for handling all laboratory reagents.
Infectious or microbial waste:
Warning: handle waste as potentially biohazardous material. Dispose of waste according to accepted laboratory instructions and procedures.
Environmental hazards:
Apply all relevant local disposal regulations to determine the safe disposal.
Safety data sheet available for professional user on request.
For quality control, use control materials as listed in the “Order information” section. In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. It is recommended to perform quality control always after lot calibration and subsequently at least every 4 weeks.
Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li‑heparin, K2‑EDTA and K3‑EDTA plasma.
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolysed or turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at -20 °C |
Centrifuge samples containing precipitates before performing the assay.
See the limitations and interferences section for details about possible sample interferences.
In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on Roche/Hitachi cobas c systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co‑substrate conversion product instead of assessing co‑substrate or Hcy conversion products. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal. The formed Ado is immediately hydrolyzed into inosine and ammonia. In the last step, the enzyme glutamate dehydrogenase (GLDH) catalyzes the reaction of ammonia with 2‑oxoglutarate and NADH to form NAD+. The concentration of Hcy in the sample is directly proportional to the amount of NADH converted to NAD+ (ΔA340 nm).
ADA | ||||
Ado |
| Inosine + NH3 |
GLDH | ||||
NH3 + NADH + 2‑Oxoglutarate |
| Glutamate + NAD+ + H2O |
Measuring range
3‑50 µmol/L
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Lower limits of measurement
Limit of Blank and Limit of Detection
Limit of Blank | = 3 µmol/L |
Limit of Detection | = 3 µmol/L |
The Limit of Blank and Limit of Detection were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A2 requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 % (empirical value determined on the
cobas c 503 analyzer: 0.1 µmol/L).
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %). (Empirical value determined on the cobas c 503 analyzer: 0.3 µmol/L.)
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut‑off value for normal levels of Hcy in adults.
In European laboratories, 12 μmol/L is used as the cut‑off value for normal levels of Hcy in adults.
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 10 % of initial value for analyte concentrations > 15 µmol/L or ± 1.5 µmol/L for analyte concentrations ≤ 15 µmol/L.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Triglycerides: No significant interference from triglycerides up to a concentration of 1790 mg/dL.
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Additional drugs tested include: Glutathione at 0.5 mmol/L, Cystathionine at 100 μmol/L, and Pyruvate at 0.5 mmol/L. No interference was found.
Note: Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference. However, SAH is only detectable at sub‑nmol/L concentrations in normal plasma, and should not cause concern.
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay can not use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry‑over is available via the cobas link. The latest version of the carry‑over evasion list can be found with the NaOHD/SMS/SCCS
|
| Analyzer(s) on which cobas c pack(s) can be used | |
08057826190 | Homocysteine Enzymatic Assay (cobas c pack 1), 100 tests | System‑ID 2070 011 | |
Homocysteine Enzymatic Assay (cobas c pack 2), 100 tests | System‑ID 2070 012 | ||
05385504190 | HCYS Calibrator Kit (2 x 3 mL) | Code 20590 | |
05142423190 | HCYS Control Kit Control 1 (2 x 3 mL) | Code 20254 | |
HCYS Control Kit Control 2 (2 x 3 mL) | Code 20255 | ||
08063494190 | Diluent NaCl 9 % (123 mL) | System‑ID 2906 001 |
HCYSX: ACN 20701
Ready for use
Test definition | |||
Reporting time | 10 min | ||
Wavelength (sub/main) | 700/340 nm | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 132 µL | – | |
R2 | 21 µL | – | |
R3 | 15 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 10.5 µL | – | – |
Decreased | 10.5 µL | 25.0 µL | 100 µL |
Increased | 10.5 µL | – | – |
For further information about the assay test definitions refer to the application parameters setting screen of the corresponding analyzer and assay.
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 4 weeks |
Do not freeze.
Calibrators | S1‑5: HCYS Calibrator Kit | |
Calibration mode | Non‑linear | |
Calibration frequency | Full calibration |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the analyzers are given below. These data represent the performance of the analytical procedure itself.
Results obtained in individual laboratories may differ due to heterogenous sample materials, aging of analyzer components and mixture of reagents running on the analyzer.
Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP05‑A3 requirements with repeatability (n = 84) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days).
Repeatability | Mean | SD | CV |
Homocysteine Control 1 | 13.2 | 0.149 | 1.1 |
Homocysteine Control 2 | 37.3 | 0.462 | 1.2 |
Human serum 1 | 9.52 | 0.135 | 1.4 |
Human serum 2 | 12.1 | 0.195 | 1.6 |
Human serum 3 | 15.0 | 0.297 | 2.0 |
Human serum 4 | 26.2 | 0.433 | 1.7 |
Human serum 5 | 45.3 | 0.704 | 1.6 |
Intermediate precision | Mean | SD | CV |
Homocysteine Control 1 | 13.2 | 0.374 | 2.8 |
Homocysteine Control 2 | 37.3 | 0.763 | 2.0 |
Human serum 1 | 9.55 | 0.330 | 3.5 |
Human serum 2 | 12.1 | 0.437 | 3.6 |
Human serum 3 | 15.4 | 0.447 | 2.9 |
Human serum 4 | 26.2 | 0.653 | 2.5 |
Human serum 5 | 45.3 | 0.945 | 2.1 |
Hcy values for human serum samples obtained on the cobas c 503 analyzer (y) were compared with those determined using the corresponding reagent on a cobas c 501 analyzer (x).
Sample size (n) = 74
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 0.984x + 0.122 µmol/L | y = 0.979x + 0.215 µmol/L |
τ = 0.979 | r = 0.999 |
The sample concentrations were between 4.11 and 48.4 µmol/L. |
Sample size (n) = 82
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 1.014x - 0.407 µmol/L | y = 1.010x - 0.319 µmol/L |
τ = 0.951 | r = 0.997 |
The sample concentrations were between 3.53 and 48.7 µmol/L. |
Homocysteine (Hcy) is a thiol‑containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein‑bound and free.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta‑synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism.
Excess Hcy is related to a higher risk of coronary heart disease, stroke, and peripheral vascular disease (fatty deposits in peripheral arteries). Excess Hcy in the blood stream may cause injuries to arterial vessels due to its irritant nature, and result in inflammation and plaque formation, which may eventually cause blockage of blood flow to the heart. Homocysteine levels may be reduced with treatment, but that does not necessarily reduce the occurrence of cardiovascular disease (CVD) events such as stroke and heart attack. Because findings from studies that evaluated the association between the increase in Homocysteine levels and CVD have been inconsistent, the American Heart Association has not yet called hyperhomocysteinemia (elevated Hcy in the blood) a major risk factor for CVD.
Guidelines for tHcy determination in clinical laboratories have recently been established.
R1 | NADH reagent S‑adenosylmethionine 0.1 mmol/L, TCEP* > 0.5 mmol/L, 2‑oxoglutarate < 5.0 mmol/L, NADH > 0.2 mmol/L, buffer, pH 9.1 (25 °C), preservative, stabilizer |
R2 | Enzyme reagent Homocysteine S‑methyltransferase (HMTase) 5.0 kU/L, glutamate dehydrogenase (GLDH) 10 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, detergent |
R3 | Start reagent Adenosine deaminase (bovine) 5.0 kU/L, S‑adenosyl‑homocysteine hydrolase (SAHase) 3.0 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, stabilizer |
*Tris(2‑carboxyethyl)phosphine
Cat. No. 08057826190 consists of 2 cobas c packs: 1 x R1 + R2 and 1 x R3. R1 is in position B and R2 is in position C of cobas c pack 1. R3 is in position C of cobas c pack 2.
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
For USA: Caution: Federal law restricts this device to sale by or on the order of a physician.
For quality control, use control materials as listed in the \"Order information\" section.
In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. It is recommended to perform quality control always after lot calibration and subsequently at least every 4 weeks.
Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li‑heparin, K2‑EDTA and K3‑EDTA plasma.
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolysed samples should not be used for this assay. Turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at -20 °C |
Centrifuge samples containing precipitates before performing the assay.
See the limitations and interferences section for details about possible sample interferences.
Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.
In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on Roche/Hitachi cobas c systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co‑substrate conversion product instead of assessing co‑substrate or Hcy conversion products. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal. The formed Ado is immediately hydrolyzed into inosine and ammonia. In the last step, the enzyme glutamate dehydrogenase (GLDH) catalyzes the reaction of ammonia with 2‑oxoglutarate and NADH to form NAD+. The concentration of Hcy in the sample is directly proportional to the amount of NADH converted to NAD+ (ΔA340 nm).
|
ADA | ||||
Ado |
| Inosine + NH3 |
GLDH | ||||
NH3 + NADH + 2‑Oxoglutarate |
| Glutamate + NAD+ + H2O |
Measuring range
3‑50 µmol/L
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Lower limits of measurement
Limit of Blank, Limit of Detection and Limit of Quantitation
Limit of Blank | = 3 µmol/L |
Limit of Detection | = 3 µmol/L |
Limit of Quantitation | = 5.5 µmol/L |
The Limit of Blank, Limit of Detection and Limit of Quantitation were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A2 requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 %.
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %).
The Limit of Quantitation is the lowest analyte concentration that can be reproducibly measured with a total error of 30 %. It has been determined using low concentration homocysteine samples.
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut‑off value for normal levels of Hcy in adults.
In European laboratories, 12 μmol/L is used as the cut‑off value for normal levels of Hcy in adults.
Group | Folate supplemented | Nonsupplemented |
Pregnancy | 8 | 10 |
Children < 15 years | 8 | 10 |
Adults 15‑65 years | 12 | 15 |
Elderly > 65 years | 16 | 20 |
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 10 % of initial value for analyte concentrations > 15 µmol/L or ± 1.5 µmol/L for analyte concentrations ≤ 15 µmol/L.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Exceptions: 0.5 mmol/L Glutathione, 100 μmol/L Cystathionine, 0.5 mmol/L Pyruvate.
Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference.
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay can not use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry‑over is available via the cobas link. The latest version of the carry‑over evasion list can be found with the NaOHD/SMS/SCCS Method Sheet for information. For further instructions refer to the operator’s manual.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
08057826 190 | Homocysteine Enzymatic Assay (cobas c pack 1), 100 tests | System‑ID 2070 011 | cobas c 503 |
Homocysteine Enzymatic Assay (cobas c pack 2), 100 tests | System‑ID 2070 012 |
Materials required (but not provided):
05385504 190 | HCYS Calibrator Kit (2 x 3 mL) | Code 20590 | |
05142423 190 | HCYS Control Kit Control 1 (2 x 3 mL) | Code 20254 | |
HCYS Control Kit Control 2 (2 x 3 mL) | Code 20255 | ||
08063494 190 | Diluent NaCl 9 % (123 mL) | System‑ID 2906 001 |
HCYS: ACN 20700
Ready for use
Test definition | |||
Reporting time | 10 min | ||
Wavelength (sub/main) | 700/340 nm | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 132 µL | – | |
R2 | 21 µL | – | |
R3 | 15 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 10.5 µL | – | – |
Decreased | 10.5 µL | 25.0 µL | 100 µL |
Increased | 10.5 µL | – | – |
For further information about the assay test definitions refer to the application parameters setting screen of the corresponding analyzer and assay.
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 4 weeks |
Do not freeze.
Calibrators | S1‑5: HCYS Calibrator Kit | |
Calibration mode | Non‑linear | |
Calibration frequency | Automatic full calibration Full calibration |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the analyzers are given below. These data represent the performance of the analytical procedure itself.
Results obtained in individual laboratories may differ due to heterogenous sample materials, aging of analyzer components and mixture of reagents running on the analyzer.
Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP05‑A3 requirements with repeatability (n = 84) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). The following results were obtained:
Repeatability | Mean | SD | CV |
Homocysteine Control 1 | 13.2 | 0.149 | 1.1 |
Homocysteine Control 2 | 37.2 | 0.462 | 1.2 |
Human serum 1 | 9.52 | 0.135 | 1.4 |
Human serum 2 | 12.1 | 0.195 | 1.6 |
Human serum 3 | 15.0 | 0.297 | 2.0 |
Human serum 4 | 26.2 | 0.433 | 1.7 |
Human serum 5 | 45.3 | 0.704 | 1.6 |
Intermediate precision | Mean | SD | CV |
Homocysteine Control 1 | 13.2 | 0.374 | 2.8 |
Homocysteine Control 2 | 37.3 | 0.763 | 2.0 |
Human serum 1 | 9.55 | 0.330 | 3.5 |
Human serum 2 | 12.1 | 0.437 | 3.6 |
Human serum 3 | 15.4 | 0.447 | 2.9 |
Human serum 4 | 26.2 | 0.653 | 2.5 |
Human serum 5 | 45.3 | 0.945 | 2.1 |
Hcy values for human serum samples obtained on the cobas c 503 analyzer (y) were compared with those determined using the corresponding reagent on a cobas c 501 analyzer (x).
Sample size (n) = 74
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 0.984x + 0.122 µmol/L | y = 0.979x + 0.215 µmol/L |
τ = 0.979 | r = 0.999 |
The sample concentrations were between 4.11 and 48.4 µmol/L. |
Homocysteine (Hcy) is a thiol‑containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein‑bound and free.
Elevated levels of tHcy has emerged as an important risk factor in the assessment of cardiovascular disease.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta‑synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism. Elevated levels of tHcy are also linked with Alzheimer’s disease
R1 | NADH reagent S‑adenosylmethionine 0.1 mmol/L, TCEP* > 0.5 mmol/L, 2‑oxoglutarate < 5.0 mmol/L, NADH > 0.2 mmol/L, buffer, pH 9.1 (25 °C), preservative, stabilizer |
R2 | Enzyme reagent Homocysteine S‑methyltransferase (HMTase) 5.0 kU/L, glutamate dehydrogenase (GLDH) 10 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, detergent |
R3 | Start reagent Adenosine deaminase (bovine) 5.0 kU/L, S‑adenosyl‑homocysteine hydrolase (SAHase) 3.0 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, stabilizer |
*Tris(2‑carboxyethyl)phosphine
Cat. No. 08057826190 consists of 2 cobas c packs: 1 x R1 + R2 and 1 x R3. R1 is in position B and R2 is in position C of cobas c pack 1. R3 is in position C of cobas c pack 2.
For in vitro diagnostic use for health care professionals. Exercise the normal precautions required for handling all laboratory reagents.
Infectious or microbial waste:
Warning: handle waste as potentially biohazardous material. Dispose of waste according to accepted laboratory instructions and procedures.
Environmental hazards:
Apply all relevant local disposal regulations to determine the safe disposal.
Safety data sheet available for professional user on request.
For quality control, use control materials as listed in the “Order information” section. In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. It is recommended to perform quality control always after lot calibration and subsequently at least every 4 weeks.
Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li‑heparin, K2‑EDTA and K3‑EDTA plasma.
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolysed or turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at -20 °C |
Centrifuge samples containing precipitates before performing the assay.
See the limitations and interferences section for details about possible sample interferences.
In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on Roche/Hitachi cobas c systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co‑substrate conversion product instead of assessing co‑substrate or Hcy conversion products of Hcy. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal. The formed Ado is immediately hydrolyzed into inosine and ammonia. In the last step, the enzyme glutamate dehydrogenase (GLDH) catalyzes the reaction of ammonia with 2‑oxoglutarate and NADH to form NAD+. The concentration of Hcy in the sample is directly proportional to the amount of NADH converted to NAD+ (ΔA340 nm).
|
ADA | ||||
Ado |
| Inosine + NH3 |
GLDH | ||||
NH3 + NADH + 2‑Oxoglutarate |
| Glutamate + NAD++ H2O |
Measuring range
3‑50 µmol/L
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Lower limits of measurement
Limit of Blank, Limit of Detection and Limit of Quantitation
Limit of Blank | = 3 µmol/L |
Limit of Detection | = 3 µmol/L |
Limit of Quantitation | = 5.5 µmol/L |
The Limit of Blank and Limit of Detection were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 %.
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %).
The Limit of Quantitation is the lowest analyte concentration that can be reproducibly measured with a total error of 30 %. It has been determined using low concentration homocysteine samples.
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut‑off value for normal levels of Hcy in adults.
In European laboratories, 12 μmol/L is used as the cut‑off value for normal levels of Hcy in adults:
Group | Folate supplemented | Nonsupplemented |
Fasting/basal tHcy, µmol/L | ||
Pregnancy | 8 | 10 |
Children < 15 years | 8 | 10 |
Adults 15‑65 years | 12 | 15 |
Elderly > 65 years | 16 | 20 |
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 10 % of initial value for analyte concentrations > 15 µmol/L or ± 1.5 µmol/L for analyte concentrations ≤ 15 µmol/L.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Exceptions: 0.5 mmol/L Glutathione, 100 μmol/L Cystathionine, 0.5 mmol/L Pyruvate.
Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to metabolic interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference.
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay can not use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas c systems. The latest version of the carry‑over evasion list can be found with the NaOHD-SMS-SmpCln1+2-SCCS Method Sheets. For further instructions refer to the operator’s manual. cobas c 502 analyzer: All special wash programming necessary for avoiding carry‑over is available via the cobas link, manual input is required in certain cases.
Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
05385415 190 | Homocysteine Enzymatic Assay 100 tests | System‑ID 07 7487 1 | cobas c 311, cobas c 501/502 |
Materials required (but not provided):
05385504 190 | HCYS Calibrator Kit (2 x 3 mL) | Code 590 | |
05142423 190 | HCYS Control Kit Control 1 (2 x 3 mL) | Code 254 | |
HCYS Control Kit Control 2 (2 x 3 mL) | Code 255 | ||
04489357 190 | Diluent NaCl 9 % (50 mL) | System‑ID 07 6869 3 |
For cobas c 311/501 analyzers:
HCYS: ACN 778
For cobas c 502 analyzer:
HCYS: ACN 8778
Ready for use
cobas c 311 test definition | |||
Assay type | 2‑Point End | ||
Reaction time / Assay points | 10 / 36‑57 | ||
Wavelength (sub/main) | 700/340 nm | ||
Reaction direction | Decrease | ||
Units | µmol/L | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 176 µL | – | |
R2 | 28 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 14 µL | – | – |
Decreased | 14 µL | 30 µL | 120 µL |
Increased | 14 µL | – | – |
cobas c 501/502 test definition | |||
Assay type | 2‑Point End | ||
Reaction time / Assay points | 10 / 51‑70 | ||
Wavelength (sub/main) | 700/340 nm | ||
Reaction direction | Decrease | ||
Units | µmol/L | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 176 µL | – | |
R2 | 28 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 14 µL | – | – |
Decreased | 14 µL | 30 µL | 120 µL |
Increased | 14 µL | – | – |
HCYS | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 4 weeks |
Diluent NaCl 9 % | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 12 weeks |
Do not freeze.
Calibrators | S1‑5: HCYS Calibrator Kit | |
Multiply the lot-specific HCYS Calibrator Kit calibrator value by the factors below to determine the standard concentrations for the 5‑point calibration curve: | ||
S1: 0.050 | S4: 0.500 | |
S2: 0.100 | S5: 1.00 | |
S3:0.250 | ||
Calibration mode | RCM | |
Calibration frequency | Full calibration |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the analyzers are given below. Results obtained in individual laboratories may differ.
Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP5 requirements with repeatability (n = 21) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). The following results were obtained:
Repeatability | Mean µmol/L | SD µmol/L | CV % |
Homocysteine Control 1 | 12.2 | 0.2 | 1.5 |
Homocysteine Control 2 | 39.1 | 0.7 | 1.8 |
Human serum 1 | 8.26 | 0.16 | 2.0 |
Human serum 2 | 13.1 | 0.2 | 1.8 |
Human serum 3 | 30.0 | 0.4 | 1.4 |
Human serum 4 | 44.4 | 0.9 | 2.0 |
Intermediate precision | Mean µmol/L | SD µmol/L | CV % |
Homocysteine Control 1 | 12.2 | 0.3 | 2.1 |
Homocysteine Control 2 | 39.1 | 0.8 | 2.0 |
Human serum 1 | 8.26 | 0.19 | 2.3 |
Human serum 2 | 13.1 | 0.3 | 2.1 |
Human serum 3 | 30.0 | 0.5 | 1.8 |
Human serum 4 | 44.4 | 1.0 | 2.2 |
Hcy values for human serum samples obtained on the cobas c 501 analyzer (y) were compared with those determined using the corresponding reagent on a COBAS INTEGRA 400 analyzer (x).
Sample size (n) = 56
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 0.962x + 0.248 µmol/L | y = 0.993x - 0.175 µmol/L |
τ = 0.971 | r = 0.999 |
The sample concentrations were between 3.03 and 47.2 µmol/L. |
Homocysteine (Hcy) is a thiol‑containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein‑bound and free.
Elevated levels of tHcy has emerged as an important risk factor in the assessment of cardiovascular disease.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta‑synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism. Elevated levels of tHcy are also linked with Alzheimer’s disease
R1 | NADH reagent S‑adenosylmethionine 0.1 mmol/L, TCEP* > 0.5 mmol/L, 2‑oxoglutarate < 5.0 mmol/L, NADH > 0.2 mmol/L, buffer, pH 9.1 (25 °C), preservative, stabilizer |
R2 | Enzyme reagent Homocysteine S‑methyltransferase (HMTase) 5.0 kU/L, glutamate dehydrogenase (GLDH) 10 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, detergent |
R3 | Start reagent Adenosine deaminase (bovine) 5.0 kU/L, S‑adenosyl‑homocysteine hydrolase (SAHase) 3.0 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, stabilizer |
*Tris(2‑carboxyethyl)phophine
R1 is in postion A, R2 is in postition B and R3 is in postion C.
For in vitro diagnostic use for health care professionals. Exercise the normal precautions required for handling all laboratory reagents.
Infectious or microbial waste:
Warning: handle waste as potentially biohazardous material. Dispose of waste according to accepted laboratory instructions and procedures.
Environmental hazards:
Apply all relevant local disposal regulations to determine the safe disposal.
Safety data sheet available for professional user on request.
For quality control, use control materials as listed in the \"Order information\" section.
In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li‑heparin, K2‑EDTA and K3‑EDTA plasma.
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolysed or turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at -20 °C |
Centrifuge samples containing precipitates before performing the assay.
See the limitations and interferences section for details about possible sample interferences.
In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on Roche/Hitachi cobas c systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co-substrate conversion product instead of assessing co-substrate or Hcy conversion products of Hcy. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal. The formed Ado is immediately hydrolyzed into inosine and ammonia.
ADA | ||
Ado |
| Inosine + NH 3 |
GLDH | ||
NH3 + NADH + |
| Glutamate + NAD+ + H2O |
Measuring range
3‑50 µmol/L
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Lower limits of measurement
Limit of Blank and Limit of Detection
Limit of Blank | = 0.3 µmol/L |
Limit of Detection | = 0.7 µmol/L |
Results for Limit of Blank and Limit of Detection were determined on the cobas c 501 analyzer and met the predetermined specification of ≤ 3 µmol/L for both Limit of Blank and Limit of Detection.
The Limit of Blank and Limit of Detection were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 %.
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %).
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut‑off value for normal levels of Hcy in adults.
European laboratories, 12 μmol/L is used as the cut‑off value for normal levels of Hcy in adults.
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 10 % of initial value.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Triglycerides: No significant interference from triglycerides up to a concentration of 1790 mg/dL.
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Additional drugs tested include Glutathione at 0.5 mmol/L, Cystathionine at 100 μmol/L, and Pyruvate at 0.5 mmol/L; no interference was found.
Note: Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to metabolic interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference. However, SAH is
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay cannot use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas c systems. The latest version of the carry‑over evasion list can be found with the NaOHD-SMS-SmpCln1+2-SCCS Method Sheets. For further instructions refer to the operator’s manual. cobas c 502 analyzer: All special wash programming necessary for avoiding carry‑over is available via the cobas link, manual input is required in certain cases.
Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
05385415 190 | Homocysteine Enzymatic Assay (100 tests) | System-ID 07 7487 1 | |
05385504 190 | HCYS Calibrator Kit (2 x 3 mL) | Code 590 | |
05142423 190 | HCYS Control Kit |
| |
04489357 190 | Diluent NaCl 9 % (50 mL) | System-ID 07 6869 3 |
For cobas c 311/501 analyzers:
HCYS: ACN 778
For cobas c 502 analyzer:
HCYS: ACN 8778
Ready for use
cobas c 311 test definition | |||
Assay type | 2-Point End | ||
Reaction time / Assay points | 10 / 36‑57 | ||
Wavelength (sub/main) | 700/340 nm | ||
Reaction direction | Decrease | ||
Units | µmol/L | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 176 µL | – | |
R2 | 28 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 14 µL | – | – |
Decreased | 14 µL | 30 µL | 120 µL |
Increased | 14 µL | – | – |
cobas c 501/502 test definition | |||
Assay type | 2-Point End | ||
Reaction time / Assay points | 10 / 51‑70 | ||
Wavelength (sub/main) | 700/340 nm | ||
Reaction direction | Decrease | ||
Units | µmol/L | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 176 µL | – | |
R2 | 28 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 14 µL | – | – |
Decreased | 14 µL | 30 µL | 120 µL |
Increased | 14 µL | – | – |
HCYS | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 4 weeks |
Diluent NaCl 9 % | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 12 weeks |
Do not freeze.
Calibrators | S1-5: HCYS Calibrator Kit | |
Multiply the lot-specific HCYS Calibrator Kit calibrator value by the factors below to determine the standard concentrations for the 5-point calibration curve: | ||
S1: 0.050 | S4: 0.500 | |
S2: 0.100 | S5: 1.00 | |
S3: 0.250 | ||
Calibration mode | RCM | |
Calibration frequency | Full calibration
|
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the cobas c 501 analyzer(s) are given below. Results obtained in individual laboratories may differ.
Repeatability and intermediate precision were determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP5 requirements (2 aliquots per run, 2 runs per day, 21 days). The following results were obtained:
Repeatability | Mean | SD | CV |
|---|---|---|---|
Homocysteine Control 1 | 12.2 | 0.2 | 1.5 |
Homocysteine Control 2 | 39.1 | 0.7 | 1.8 |
Human serum 1 | 8.26 | 0.16 | 2.0 |
Human serum 2 | 13.1 | 0.2 | 1.8 |
Human serum 3 | 30.0 | 0.4 | 1.4 |
Human serum 4 | 44.4 | 0.9 | 2.0 |
Intermediate precision | Mean | SD | CV |
|---|---|---|---|
Homocysteine Control 1 | 12.2 | 0.3 | 2.1 |
Homocysteine Control 2 | 39.1 | 0.8 | 2.0 |
Human serum 1 | 8.26 | 0.19 | 2.3 |
Human serum 2 | 13.1 | 0.3 | 2.1 |
Human serum 3 | 30.0 | 0.5 | 1.8 |
Human serum 4 | 44.4 | 1.0 | 2.2 |
Hcy values for human serum samples obtained on a cobas c 501 analyzer (y) were compared
Sample size (n) = 102
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 0.999x - 0.696 µmol/L | y = 0.997x - 0.571 µmol/L |
τ = 0.959 | r = 0.996 |
The sample concentrations were between 4.76 and 46.59 µmol/L. |
Homocysteine (Hcy) is a thiol‑containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein‑bound and free.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta‑synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism.
Excess Hcy is related to a higher risk of coronary heart disease, stroke, and peripheral vascular disease (fatty deposits in peripheral arteries). Excess Hcy in the blood stream may cause injuries to arterial vessels due to its irritant nature, and result in inflammation and plaque formation, which may eventually cause blockage of blood flow to the heart. Homocysteine levels may be reduced with treatment, but that does not necessarily reduce the occurrence of cardiovascular disease (CVD) events such as stroke and heart attack. Because findings from studies that evaluated the association between the increase in Homocysteine levels and CVD have been inconsistent, the American Heart Association has not yet called hyperhomocysteinemia (elevated Hcy in the blood) a major risk factor for CVD.
Guidelines for tHcy determination in clinical laboratories have recently been established.
R1 | NADH reagent S‑adenosylmethionine 0.1 mmol/L, TCEP FREFTris(2carboxyethyl)phosphine > 0.5 mmol/L, 2‑oxoglutarate < 5.0 mmol/L, NADH > 0.2 mmol/L, buffer, pH 9.1 (25 °C), preservative, stabilizer |
R2 | Enzyme reagent Homocysteine S‑methyltransferase (HMTase) 5.0 kU/L, glutamate dehydrogenase (GLDH) 10 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, detergent |
R3 | Start reagent Adenosine deaminase (bovine) 5.0 kU/L, S‑adenosyl‑ homocysteine hydrolase (SAHase) 3.0 kU/L, casein (bovine) ≤ 0.2 %, buffer, pH 7.2 (25 °C), preservative, stabilizer |
R1 is in position A, R2 is in position B and R3 is in position C.
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
For USA: Caution: Federal law restricts this device to sale by or on the order of a physician.
For quality control, use control materials as listed in the \"Order information\" section.
In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li-heparin, K2‑EDTA and K3‑EDTA plasma.
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolyzed samples should not be used for this assay. Turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at ‑20 °C |
See the limitations and interferences section for details about possible sample interferences.
Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.
Centrifuge samples containing precipitates before performing the assay.
In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on COBAS INTEGRA systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co‑substrate conversion product instead of assessing co‑substrate or Hcy conversion products of Hcy. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal. The formed Ado is immediately hydrolyzed into inosine and ammonia. In the last step, the enzyme glutamate dehydrogenase (GLDH) catalyzes the reaction of ammonia with 2‑oxoglutarate and NADH to form NAD+. The concentration of Hcy in the sample is directly proportional to the amount of NADH converted to NAD+ (ΔA340 nm).
|
Ado | ADA | Inosine + NH3 |
NH3 + NADH + 2‑Oxoglutarate | GLDH | Glutamate + NAD++ H2O |
3‑50 µmol/L
The lower and the upper limit of the measuring range depends on the actual calibrator value.
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Limit of Blank, Limit of Detection and Limit of Quantitation
Limit of Blank | = 3 µmol/L |
Limit of Detection | = 3 µmol/L |
Limit of Quantitation | = 5.5 µmol/L |
The Limit of Blank, Limit of Detection and Limit of Quantitation were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 %.
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %).
The Limit of Quantitation is the lowest analyte concentration that can be reproducibly measured with a total error of 30 %. It has been determined using low concentration homocysteine samples.
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut-off value for normal levels of Hcy in adults.
In European laboratories, 12 μmol/L is used as the cut-off value for normal levels of Hcy in adults:
Group | Folate supplemented | Nonsupplemented |
Fasting/basal tHcy, µmol/L | ||
Pregnancy | 8 | 10 |
Children < 15 years | 8 | 10 |
Adults 15‑65 years | 12 | 15 |
Elderly > 65 years | 16 | 20 |
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 1.5 µmol/L of initial values of samples ≤ 15 µmol/L and within ± 10 % for samples > 15 µmol/L.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to metabolic interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference. However, SAH is only detectable at sub-nmol/L concentrations in normal plasma, and should not cause concern.
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay can not use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on COBAS INTEGRA analyzers.
Where required, special wash/carry-over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzers on which cobas c pack can be used | |
05385415 190 | Homocysteine Enzymatic Assay (100 tests) | System-ID 07 7487 1 | COBAS INTEGRA 400 plus |
Materials required (but not provided):
05385504 190 | HCYS Calibrator Kit (2 × 3 mL) | System-ID 07 7493 6 | |
05142423 190 | HCYS Control Kit Control 1 (2 × 3 mL) | System-ID 07 7490 1 | |
20756350 322 | NaCl Diluent 9 % (6 × 22 mL) | System-ID 07 5635 0 |
Test HCYS, test ID 0‑006
Ready for use
Measuring mode | Absorbance |
Abs. calculation mode | Kinetic |
Reaction mode | R1/R2-S-SR |
Reaction direction | Decrease |
Wavelength A/B | 340/659 nm |
Calc. first/last | 50/62 |
Unit | µmol/L |
Diluent (H2O) | ||
R1 | 175 µL | – |
R2 | 27 µL | – |
Sample | 14 µL | – |
SR | 18 µL | – |
Total volume | 234 µL |
Measuring mode | Absorbance |
Abs. calculation mode | Kinetic |
Reaction mode | R1/R2-S-SR |
Reaction direction | Decrease |
Wavelength A/B | 340/659 nm |
Calc. first/last | 73/95 |
Unit | µmol/L |
Diluent (H2O) | ||
R1 | 175 µL | – |
R2 | 27 µL | – |
Sample | 14 µL | – |
SR | 18 µL | – |
Total volume | 234 µL |
Shelf life at 2‑8 °C | See expiration date on cobas c pack label | |
COBAS INTEGRA 400 plus system | ||
On-board in use at 10‑15 °C | 4 weeks | |
COBAS INTEGRA 800 system | ||
On-board in use at 8 °C | 4 weeks |
Calibrators | HCYS Calibrator Kit |
Calibration mode | Logit/log 5 |
Calibration replicate | Duplicate recommended |
Calibration frequency | Full calibration
|
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the analyzers are given below. Results obtained in individual laboratories may differ.
Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP5 requirements with repeatability (n = 21) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). The following results were obtained:
Repeatability | Mean | SD | CV |
|---|---|---|---|
Homocystein Control 1 | 12.2 | 0.1 | 1.0 |
Homocystein Control 2 | 38.9 | 0.5 | 1.3 |
Human serum 1 | 8.47 | 0.09 | 1.1 |
Human serum 2 | 13.5 | 0.1 | 0.9 |
Human serum 3 | 31.2 | 0.3 | 0.9 |
Human serum 4 | 45.5 | 0.6 | 1.4 |
Intermediate precision | Mean | SD | CV |
|---|---|---|---|
Homocystein Control 1 | 12.2 | 0.2 | 1.4 |
Homocystein Control 2 | 38.9 | 0.6 | 1.5 |
Human serum 1 | 8.47 | 0.11 | 1.3 |
Human serum 2 | 13.5 | 0.2 | 1.4 |
Human serum 3 | 31.2 | 0.5 | 1.4 |
Human serum 4 | 45.5 | 0.8 | 1.7 |
Hcy values for human serum samples obtained on a COBAS INTEGRA 800 analyzer (y) were compared with those determined using the same reagent on a COBAS INTEGRA 400 analyzer (x).
Sample size (n) = 56
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 1.00x + 0.144 µmol/L | y = 1.04x - 0.224 µmol/L |
τ = 0.967 | r = 0.998 |
The sample concentrations were between 3.39 and 46.8 µmol/L.
Homocysteine (Hcy) is a thiol-containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein-bound and free.
Elevated levels of tHcy has emerged as an important risk factor in the assessment of cardiovascular disease.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta-synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism. Elevated levels of tHcy are also linked with Alzheimer’s disease
R1 | NADH reagent FREFTris(2‑carboxyethyl)phosphine > 0.5 mmol/L; 2‑oxoglutarate < 5.0 mmol/L; NADH > 0.2 mmol/L; buffer, pH 9.1 (25 °C); preservative; stabilizer |
R2 | Enzyme reagent |
SR | Start reagent |
R1 is in position A, R2 is in position B and SR is in position C.
For in vitro diagnostic use for health care professionals. Exercise the normal precautions required for handling all laboratory reagents.
Infectious or microbial waste:
Warning: handle waste as potentially biohazardous material. Dispose of waste according to accepted laboratory instructions and procedures.
Environmental hazards:
Apply all relevant local disposal regulations to determine the safe disposal.
Safety data sheet available for professional user on request.
For quality control, use control materials as listed in the \"Order information\" section. In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum
Plasma: Li-heparin, K2-EDTA and K3–EDTA plasma
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolysed or turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
Centrifuge samples containing precipitates before performing the assay.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at ‑20 °C |
In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on COBAS INTEGRA systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co‑substrate conversion product instead of assessing co‑substrate or Hcy conversion products of Hcy. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal.
Ado | ADA | Inosine + NH3 |
NH3 + NADH + 2‑Oxoglutarate | GLDH | Glutamate + NAD++ H2O |
3‑50 µmol/L
The lower and the upper limit of the measuring range depends on the actual calibrator value.
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Limit of Blank, (LoB) and Limit of Detection (LoD)
Limit of Blank | = 0.63 µmol/L |
Limit of Detection | = 1.40 µmol/L |
Results for LoB and LoD were determined on the COBAS INTEGRA 800 analyzer and met the predetermined specification of ≤ 3 µmol/L for both LoB and LoD.
The Limit of Blank and Limit of Detection were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 %.
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %).
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut-off value for normal levels of Hcy in adults.
In European laboratories, 12 μmol/L is used as the cut-off value for normal levels of Hcy in adults.
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 10 % of initial value.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Triglycerides: No significant interference from
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Note: Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to metabolic interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference. However, SAH is only detectable at sub-nmol/L concentrations in normal plasma, and should not cause concern.
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay cannot use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on COBAS INTEGRA analyzers. Refer to the CLEAN Method Sheet for further instructions and for the latest version of the Extra wash cycle list.
Where required, special wash/carry-over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which cobas c pack can be used | |
05385415 190 | Homocysteine Enzymatic Assay (100 tests) | System-ID 07 7487 1 | |
05385504 190 | HCYS Calibrator Kit (2 × 3 mL) | System-ID 07 7493 6 | |
05142423 190 | HCYS Control Kit Control 1 (2 × 3 mL) | System-ID 07 7490 1 | |
20756350 322 | NaCl Diluent 9 % (6 × 22 mL) | System-ID 07 5635 0 |
Test HCYS, test ID 0‑006
Ready for use
Measuring mode | Absorbance |
Abs. calculation mode | Kinetic |
Reaction mode | R1/R2-S-SR |
Reaction direction | Decrease |
Wavelength A/B | 340/659 nm |
Calc. first/last | 50/62 |
Unit | µmol/L |
Diluent (H2O) | ||
R1 | 175 µL | – |
R2 | 27 µL | – |
Sample | 14 µL | – |
SR | 18 µL | – |
Total volume | 234 µL |
Shelf life at 2‑8 °C | See expiration date on cobas c pack label |
4 weeks |
Calibrators | HCYS Calibrator Kit |
Calibration mode | Logit/log 5 |
Calibration replicate | Duplicate recommended |
Calibration frequency | Full calibration |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the analyzers are given below. Results obtained in individual laboratories may differ.
Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP5 requirements with repeatability and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). The following results were obtained:
Repeatability | Mean | SD | CV |
|---|---|---|---|
Homocysteine Control 1 | 12.2 | 0.1 | 1.0 |
Homocysteine Control 2 | 38.9 | 0.5 | 1.3 |
Human serum 1 | 8.47 | 0.09 | 1.1 |
Human serum 2 | 13.5 | 0.1 | 0.9 |
Human serum 3 | 31.2 | 0.3 | 0.9 |
Human serum 4 | 45.5 | 0.6 | 1.4 |
Intermediate precision | Mean | SD | CV |
|---|---|---|---|
Homocysteine Control 1 | 12.2 | 0.2 | 1.4 |
Homocysteine Control 2 | 38.9 | 0.6 | 1.5 |
Human serum 1 | 8.47 | 0.11 | 1.3 |
Human serum 2 | 13.5 | 0.2 | 1.4 |
Human serum 3 | 31.2 | 0.5 | 1.4 |
Human serum 4 | 45.5 | 0.8 | 1.7 |
Hcy values for human serum samples obtained on a COBAS INTEGRA 800 analyzer (y) were compared with those determined using the Diazyme reagent on a COBAS INTEGRA 400 analyzer (x).
Sample size (n) = 57
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 1.00x + 0.144 µmol/L | y = 1.04x - 0.224 µmol/L |
τ = 0.967 | r = 0.998 |
The sample concentrations were between 3.39 and 46.8 µmol/L.
Homocysteine (Hcy) is a thiol‑containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein‑bound and free.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta‑synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism.
Excess Hcy is related to a higher risk of coronary heart disease, stroke, and peripheral vascular disease (fatty deposits in peripheral arteries). Excess Hcy in the blood stream may cause injuries to arterial vessels due to its irritant nature, and result in inflammation and plaque formation, which may eventually cause blockage of blood flow to the heart. Homocysteine levels may be reduced with treatment, but that does not necessarily reduce the occurrence of cardiovascular disease (CVD) events such as stroke and heart attack. Because findings from studies that evaluated the association between the increase in Homocysteine levels and CVD have been inconsistent, the American Heart Association has not yet called hyperhomocysteinemia (elevated Hcy in the blood) a major risk factor for CVD.
Guidelines for tHcy determination in clinical laboratories have recently been established.
R1 | NADH reagent FREFTris(2carboxyethyl)phosphine > 0.5 mmol/L; 2‑oxoglutarate < 5.0 mmol/L; NADH > 0.2 mmol/L; buffer, pH 9.1 (25 °C); preservative; stabilizer |
R2 | Enzyme reagent |
SR | Start reagent |
R1 is in position A, R2 is in position B and SR is in position C.
Pay attention to all precautions and warnings listed in Section 1 / Introduction of this Method Manual.
For USA: Caution: Federal law restricts this device to sale by or on the order of a physician.
For quality control, use control materials as listed in the “Order information” section. In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum
Plasma: Li-heparin, K2-EDTA and K3-EDTA plasma
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolysed samples should not be used for this assay. Turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
See the limitations and interferences section for details about possible sample interferences.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at ‑20 °C |
Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.
Centrifuge samples containing precipitates before performing the assay.
In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on Roche/Hitachi cobas c systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co‑substrate conversion product instead of assessing co‑substrate or Hcy conversion products of Hcy. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal.
ADA | ||||
Ado |
| Inosine + NH3 |
GLDH | ||||
NH3 + NADH + |
| Glutamate + NAD++ H2O |
Measuring range
3‑50 µmol/L
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Lower limits of measurement
Limit of Blank and Limit of Detection
Limit of Blank | = 0.3 µmol/L |
Limit of Detection | = 0.7 µmol/L |
Results for Limit of Blank and Limit of Detection were determined on the cobas c 501 analyzer and met the predetermined specification of ≤ 3 µmol/L for both Limit of Blank and Limit of Detection.
The Limit of Blank and Limit of Detection were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 %.
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %).
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut‑off value for normal levels of Hcy in adults.
In European laboratories, 12 μmol/L is used as the cut‑off value for normal levels of Hcy in adults.
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 10 % of initial value.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Additional drugs tested include Glutathione at 0.5 mmol/L, Cystathionine at 100 μmol/L, and Pyruvate at 0.5 mmol/L; no interference was found.
Note: Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to metabolic interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference. However, SAH is
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay cannot use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry‑over is available via the cobas link, manual input is required in certain cases. The latest version of the carry‑over evasion list can be found with the NaOHD/SMS/SmpCln1+2/SCCS Method Sheet and for further instructions refer to the operator’s manual.
Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
06542921 190 | Homocysteine Enzymatic Assay 200 tests | System‑ID 03 7487 1 | |
System‑ID 04 7487 1 | |||
05385504 190 | HCYS Calibrator Kit (2 x 3 mL) | Code 590 | |
05142423 190 | HCYS Control Kit Control 1 (2 x 3 mL) | Code 254 | |
HCYS Control Kit Control 2 (2 x 3 mL) | Code 255 | ||
05172152 190 | Diluent NaCl 9 % (119 mL) | System‑ID 08 6869 3 |
HCYS: ACN 8778
Ready for use
cobas c 701/702 test definition | |||
Assay type | 2‑Point End | ||
Reaction time / Assay points | 10 / 28‑38 | ||
Wavelength (sub/main) | 700/340 nm | ||
Reaction direction | Decrease | ||
Units | µmol/L | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 176 µL | – | |
R2 | 28 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 14 µL | – | – |
Decreased | 14 µL | 30 µL | 120 µL |
Increased | 14 µL | – | – |
HCYS | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 4 weeks |
On‑board on the reagent manager: | 1 hour |
Diluent NaCl 9 % | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | |
On‑board on the reagent manager: | 24 hours |
Do not freeze.
Calibrators | S1‑5: HCYS Calibrator Kit | |
Multiply the lot‑specific HCYS Calibrator Kit calibrator value by the factors below to determine the standard concentrations for the 5‑point calibration curve: | ||
S1: 0.050 | S4: 0.500 | |
S2: 0.100 | S5: 1.00 | |
S3: 0.250 | ||
Calibration mode | RCM | |
Calibration frequency | Full calibration |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the analyzers are given below. Results obtained in individual laboratories may differ.
Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP5 requirements with repeatability (n = 21) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). The following results were obtained:
Repeatability | Mean | SD | CV |
|---|---|---|---|
Homocysteine Control 1 | 12.3 | 0.2 | 1.3 |
Homocysteine Control 2 | 38.9 | 0.4 | 1.1 |
Human serum A | 6.15 | 0.13 | 2.1 |
Human serum B | 16.9 | 0.2 | 1.4 |
Human serum C | 23.3 | 0.3 | 1.3 |
Intermediate precision | Mean | SD | CV |
|---|---|---|---|
Homocysteine Control 1 | 12.2 | 0.3 | 2.1 |
Homocysteine Control 2 | 39.1 | 0.8 | 2.0 |
Human serum 1 | 8.26 | 0.19 | 2.3 |
Human serum 2 | 13.1 | 0.3 | 2.1 |
Human serum 3 | 30.0 | 0.5 | 1.8 |
Human serum 4 | 44.4 | 1.0 | 2.2 |
Results for intermediate precision were obtained on the master system cobas c 501 analyzer.
Hcy values for human serum samples obtained on the
Sample size (n) = 62
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 0.979x + 0.267 µmol/L | y = 0.974x + 0.212 µmol/L |
τ = 0.974 | r = 0.997 |
The sample concentrations were between 3.65 and 49.0 µmol/L. |
Homocysteine (Hcy) is a thiol‑containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein‑bound and free.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta‑synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism.
Excess Hcy is related to a higher risk of coronary heart disease, stroke, and peripheral vascular disease (fatty deposits in peripheral arteries). Excess Hcy in the blood stream may cause injuries to arterial vessels due to its irritant nature, and result in inflammation and plaque formation, which may eventually cause blockage of blood flow to the heart. Homocysteine levels may be reduced with treatment, but that does not necessarily reduce the occurrence of cardiovascular disease (CVD) events such as stroke and heart attack. Because findings from studies that evaluated the association between the increase in Homocysteine levels and CVD have been inconsistent, the American Heart Association has not yet called hyperhomocysteinemia (elevated Hcy in the blood) a major risk factor for CVD.
Guidelines for tHcy determination in clinical laboratories have recently been established.
R1 | NADH reagent |
R2 | Enzyme reagent |
R3 | Start reagent |
*Tris(2‑carboxyethyl)phosphine
Cat. No. 06542921190 consists of 2 cobas c packs: 1 x R1 + R2 and 1 x R3.
R1 is in position B and R2 is in position C of cobas c pack 1. R3 is in position C of cobas c pack 2.
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
For USA: Caution: Federal law restricts this device to sale by or on the order of a physician.
For quality control, use control materials as listed in the \"Order information\" section.
In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li‑heparin, K2‑EDTA and K3‑EDTA plasma.
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolysed samples should not be used for this assay. Turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at -20 °C |
See the limitations and interferences section for details about possible sample interferences.
Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.
Centrifuge samples containing precipitates before performing the assay.
In vitro test for the quantitative determination of total L‑homocysteine in human serum and plasma on Roche/Hitachi cobas c systems. The assay can assist in the diagnosis of patients suspected of having hyperhomocysteinemia or homocystinuria.
Homocysteine Enzymatic Assay is based on a novel enzyme cycling assay principle that assesses the co‑substrate conversion product instead of assessing co‑substrate or Hcy conversion products of Hcy. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co‑substrate, S‑adenosylmethionine (SAM), to form methionine (Met) and S‑adenosylhomocysteine (SAH), catalyzed by a Hcy S‑methyltransferase. SAH is assessed by coupled enzyme reactions where SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase, and Hcy is cycled into the Hcy conversion reaction to form a reaction cycle that amplifies the detection signal. The formed Ado is immediately hydrolyzed into inosine and ammonia. In the last step, the enzyme glutamate dehydrogenase (GLDH) catalyzes the reaction of ammonia with 2‑oxoglutarate and NADH to form NAD+. The concentration of Hcy in the sample is directly proportional to the amount of NADH converted to NAD+ (ΔA340 nm).
|
ADA | ||||
Ado |
| Inosine + NH3 |
GLDH | ||||
NH3 + NADH + 2‑Oxoglutarate |
| Glutamate + NAD++ H2O |
Measuring range
3‑50 µmol/L
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.
Lower limits of measurement
Limit of Blank, Limit of Detection and Limit of Quantitation
Limit of Blank | = 3 µmol/L |
Limit of Detection | = 3 µmol/L |
Limit of Quantitation | = 5.5 µmol/L |
The Limit of Blank and Limit of Detection were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the concentration below which analyte‑free samples are found with a probability of 95 %.
The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low concentration samples.
The Limit of Detection corresponds to the lowest analyte concentration which can be detected (value above the Limit of Blank with a probability of 95 %).
The Limit of Quantitation is the lowest analyte concentration that can be reproducibly measured with a total error of 30 %. It has been determined using low concentration homocysteine samples.
In most of the U.S. clinical laboratories, 15 µmol/L is used as the cut‑off value for normal levels of Hcy in adults.
In European laboratories, 12 μmol/L is used as the cut‑off value for normal levels of Hcy in adults:
Age, pregnancy, and renal function are important. The intake of folic acid as either supplement or through fortification of foods must also be considered:
Group | Folate supplemented | Nonsupplemented |
Fasting/basal tHcy, µmol/L | ||
Pregnancy | 8 | 10 |
Children < 15 years | 8 | 10 |
Adults 15‑65 years | 12 | 15 |
Elderly > 65 years | 16 | 20 |
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
Criterion: Recovery within ± 10 % of initial value for analyte concentrations > 15 µmol/L or ± 1.5 µmol/L for analyte concentrations ≤ 15 µmol/L.
Icterus:
Hemolysis:
Lipemia (Intralipid):
Drugs: No interference was found at therapeutic concentrations using common drug panels.
Exceptions: 0.5 mmol/L Glutathione, 100 μmol/L Cystathionine, 0.5 mmol/L Pyruvate.
Patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6‑azuridine triacetate, may have higher levels of Hcy due to metabolic interference with Hcy metabolism.
S‑Adenosylhomocysteine (SAH) will cause a significant positive interference.
Addition of 3‑deazaadenosine to inhibit Hcy production in red cells has been suggested. However, the Homocysteine Enzymatic Assay can not use samples containing 3‑deazaadenosine since it inhibits one of the key enzymes used in the assay.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry‑over is available via the cobas link, manual input is required in certain cases. The latest version of the carry‑over evasion list can be found with the NaOHD/SMS/SmpCln1+2/SCCS Method Sheet and for further instructions refer to the operator’s manual.
Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which cobas c pack(s) can be used | |
06542921 190 | Homocysteine Enzymatic Assay (200 tests) | System‑ID 03 7487 1 | cobas c 701/702 |
System‑ID 04 7487 1 |
Materials required (but not provided):
05385504 190 | HCYS Calibrator Kit (2 x 3 mL) | Code 590 | |
05142423 190 | HCYS Control Kit Control 1 (2 x 3 mL) | Code 254 | |
HCYS Control Kit Control 2 (2 x 3 mL) | Code 255 | ||
05172152 190 | Diluent NaCl 9 % (119 mL) | System‑ID 08 6869 3 |
HCYS: ACN 8778
Ready for use
cobas c 701/702 test definition | |||
Assay type | 2‑Point End | ||
Reaction time / Assay points | 10 / 28‑38 | ||
Wavelength (sub/main) | 700/340 nm | ||
Reaction direction | Decrease | ||
Units | µmol/L | ||
Reagent pipetting | Diluent (H2O) | ||
R1 | 176 µL | – | |
R2 | 28 µL | – | |
R3 | 20 µL | – | |
Sample volumes | Sample | Sample dilution | |
Sample | Diluent (NaCl) | ||
Normal | 14 µL | – | – |
Decreased | 14 µL | 30 µL | 120 µL |
Increased | 14 µL | – | – |
HCYS | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 4 weeks |
On‑board on the reagent manager: | 1 hour |
Diluent NaCl 9 % | |
Shelf life at 2‑8 °C: | See expiration date on cobas c pack label. |
On‑board in use and refrigerated on the analyzer: | 4 weeks |
On‑board on the reagent manager: | 24 hours |
Do not freeze.
Calibrators | S1‑5: HCYS Calibrator Kit | |
Multiply the lot‑specific HCYS Calibrator Kit calibrator value by the factors below to determine the standard concentrations for the 5‑point calibration curve: | ||
S1: 0.050 | S4: 0.500 | |
S2: 0.100 | S5: 1.00 | |
S3: 0.250 | ||
Calibration mode | RCM | |
Calibration frequency | Full calibration |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against NIST SRM 1955 reference material.
Representative performance data on the analyzers are given below. Results obtained in individual laboratories may differ.
Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP5 requirements with repeatability (n = 21) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). The following results were obtained:
Repeatability | Mean µmol/L | SD µmol/L | CV % |
Homocysteine Control 1 | 12.3 | 0.2 | 1.3 |
Homocysteine Control 2 | 38.9 | 0.4 | 1.1 |
Human serum A | 6.15 | 0.13 | 2.1 |
Human serum B | 16.9 | 0.2 | 1.4 |
Human serum C | 23.3 | 0.3 | 1.3 |
Intermediate precision | Mean µmol/L | SD µmol/L | CV % |
Homocysteine Control 1 | 12.2 | 0.3 | 2.1 |
Homocysteine Control 2 | 39.1 | 0.8 | 2.0 |
Human serum 1 | 8.26 | 0.19 | 2.3 |
Human serum 2 | 13.1 | 0.3 | 2.1 |
Human serum 3 | 30.0 | 0.5 | 1.8 |
Human serum 4 | 44.4 | 1.0 | 2.2 |
Results for intermediate precision were obtained on the master system cobas c 501 analyzer.
Hcy values for human serum samples obtained on the
Sample size (n) = 62
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 0.979x + 0.267 µmol/L | y = 0.974x + 0.212 µmol/L |
τ = 0.974 | r = 0.997 |
The sample concentrations were between 3.65 and 49.0 µmol/L. |
Homocysteine (Hcy) is a thiol‑containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein‑bound and free.
Elevated levels of tHcy has emerged as an important risk factor in the assessment of cardiovascular disease.
Elevated tHcy levels are caused by four major factors, including:
genetic deficiencies in enzymes involved in Hcy metabolism such as cystathionine beta‑synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
nutritional deficiency in B vitamins such as B6, B12 and folate;
renal failure for effective amino acid clearance; and
drug interactions, such as with nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolism. Elevated levels of tHcy are also linked with Alzheimer’s disease
R1 | NADH reagent |
R2 | Enzyme reagent |
R3 | Start reagent |
*Tris(2‑carboxyethyl)phosphine
Cat. No. 06542921 190 consists of 2 cobas c packs: 1 x R1 + R2 and 1 x R3.
R1 is in position B and R2 is in position C of cobas c pack 1. R3 is in position C of cobas c pack 2.
For in vitro diagnostic use for health care professionals. Exercise the normal precautions required for handling all laboratory reagents.
Infectious or microbial waste:
Warning: handle waste as potentially biohazardous material. Dispose of waste according to accepted laboratory instructions and procedures.
Environmental hazards:
Apply all relevant local disposal regulations to determine the safe disposal.
Safety data sheet available for professional user on request.
For quality control, use control materials as listed in the \"Order information\" section.
In addition, other suitable control material can be used.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.
Follow the applicable government regulations and local guidelines for quality control.
For specimen collection and preparation only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li‑heparin, K2‑EDTA and K3‑EDTA plasma.
It is important to centrifuge blood samples immediately after collection to separate the plasma from the blood cells. If immediate centrifugation is not possible, collected blood specimens should be kept on ice and centrifuged within an hour. Hemolysed or turbid specimens or severely lipemic specimens are not recommended for the Hcy assay.
The sample types listed were tested with a selection of sample collection tubes that were commercially available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.
Stability: LREFRefsum H, Smith AD, Ueland PM, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion. Clin Chem 2004;50(1):3-32. ,LREFFiskerstrand T, Refsum H, Kvalheim G, et al. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability. Clin Chem 1993 Feb;39(2):263-271. ,LREFRasmussen K and Moller J. Total homocysteine measurement in clinical practice. Ann Clin Biochem 2000;37:627-648. | 4 days at 15‑25 °C |
4 weeks at 2‑8 °C | |
10 months at -20 °C |
Centrifuge samples containing precipitates before performing the assay.
See the limitations and interferences section for details about possible sample interferences.
Homocysteine Enzymatic Assay
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