Principle: | Certification: | Clinical Significance: | Specimen: | Materials: | Reagents: | Standardization: | Procedure: | Limitations: | Results Derivation: | Expected Result(s) | Quality Control: | Reference:
Both the concentration of Hemoglobin A1c specifically and the concentration of total hemoglobin are measured, and the ratio reported as percent hemoglobin A1c. For the measurement of total hemoglobin, potassium ferricyanide is used to oxidize hemoglobin in the sample to methemoglobin. The methemoglobin then complexes with thiocyanate to form thiocyan-methemoglobin, the colored species that is measured. The extent of color development at 531 nm is proportional to the concentration of total hemoglobin in the sample.
For the measurement of specific HbA1c, an inhibition of latex agglutination assay is used. An agglutinator (synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c) causes agglutination of latex coated with HbA1c specific mouse monoclonal antibody. This agglutination reaction causes increased scattering of light, which is measured as an increase in absorbance at 531 nm. HbA1c in whole blood specimens competes for the limited number of antibody-latex binding sites causing an inhibition of agglutination and a decreased scattering of light. The decreased scattering is measured as a decrease in absorbance at 531 nm. The HbA1c concentration is then quantified using a calibration curve of absorbance versus HbA1c concentration. The percent HbA1c in the sample is then calculated as follows:
% HbA1c = [HbA1c] / [Total Hemoglobin] X 100
All measurements and calculations are performed automatically by the DCA 2000+ Analyzer, and the screen displays percent HbA1c at the end of the assay. The DCA 2000+ HbA1c method is NGSP certified. The final reportable result is traceable to the Diabetes Control and Complications Trial (DCCT)
1. Orientation, authorization and competency of staff and licensed practitioners performing POCT are provided by the director’s (named on the CLIA certificate) designee as indicated in the “Point of Care (POCT) Program” procedure.
2. Whole Blood Hemoglobin A1c Testing will be performed only by certified operators.
A. To be certified, an operator must successfully complete the requirements of the Whole Blood Hemoglobin A1c Testing; POCT – Operation Certification Record.
B. If the individual seeking certification is unable to demonstrate acceptable testing technique, he/she must be retrained and successfully retested for authorization to perform POCT for whole blood Hemoglobin A1c.
C. Recertification is required annually:
a. If the previously certified operator has successfully performed and documented the test two times or less in the last 12 months, he/she will:
1. Perform a quality control test on the DCA Vantage Analyzer.
2. Review the Hemoglobin A1c; Siemens DCA Vantage: POCT procedure.
3. Complete the Hemoglobin A1c; Siemens DCA Vantage POCT self-instructional/competency documentation packet.
b. If the certified operator has successfully performed the test more than twice in the last 12 months, he/she will review the current policy/procedure, and complete the Hemoglobin A1c; Siemens DCA Vantage POCT self-instructional/competency documentation packet but will not have to demonstrate proficiency with another authorized operator.
D. The certification record of operators and instructors will be maintained at the site.
Hemoglobin A1c is formed by the non-enzymatic glycation of the N-terminus of the β-chain of hemoglobin Ao. The level of hemoglobin A1c is proportional to the level of glucose in the blood over a period of approximately two months. Thus, hemoglobin A1c is accepted as an indicator of the mean daily blood glucose concentration over the preceding two months. The clinical values obtained through regular measurement of hemoglobin A1c leads to changes in diabetes treatment and improvement of metabolic control as indicated by a lowering of hemoglobin A1c values.
1. 1 µl of capillary (by fingerstick) or venous whole blood.
2. Acceptable anticoagulants: EDTA and Heparin
3. Once the glass capillary is filled with a 1 µl blood sample, begin analysis within five minutes. If the blood remains in the glass capillary for more than five minutes before beginning the test, reject the sample and recollect.
4. Store EDTA and heparin whole blood at -70° to 5° C for up to two weeks.
A. Do not refreeze previously frozen blood samples.
B. Do not store blood samples in a self-defrosting freezer.
C. Allow blood to reach room temperature naturally.
D. Mix blood samples thoroughly before use.
1. DCA Vantage Analyzer
2. Capillary Holders, included in each box of reagent
Capillary holders are stored at room temperature. Unused capillary holders may be saved and used with any lot of reagent cartridges.
3. Lint free tissue (McKesson #MK020)
1. DCA Systems HbA1c Control Kit (McKesson)
A. Controls are stable until the last day of the expiration month printed on the label, stored at 2 - 8°C. Do not use if moisture is present in the vial prior to reconstitution.
B. Remove control vial from the refrigerator just prior to reconstitution.
a. Gently tap the bottom of the vial on the counter to collect as much material as possible on the bottom of the vial.
b. Carefully remove the cap from the control vial.
c. Holding the Reconstitution Fluid dropper bottle vertically, add six (6) drops of fluid to the control bottle.
Note: Discard the first drop to ensure a constant volume of drops thereafter.
d. Carefully replace the cap, not the eyedropper, and swirl the control bottle several times. Let stand at room temperature for 15 minutes.
e. After 15 minutes, coat all surfaces by rotating and inverting the vial. Continue mixing until the solution is homogeneous and all lyophilized material is dissolved.
f. Remove and discard the cap. Replace with the Eyedropper Cap Assembly (snap it onto the vial). Use the reconstituted controls within 30 minutes or refrigerate to store for later use.
C. Reconstituted control solutions should not be frozen. Leave the bottle cap on when not in use. Controls may remain at room temperature for a period of 30 minutes when testing, and should be stored refrigerated at all other times. Store the controls refrigerated in an upright position and tightly capped. Discard any reconstituted control solution appearing turbid or obviously contaminated. The reconstituted control is stable for 3 months from the date of reconstitution when stored refrigerated.
2. DCA Systems Hemoglobin A1c Reagent Cartridges (McKesson)
A. Reagent cartridges can be stored for up to 3 months at room temperature.
B. Store stock refrigerated at 2 - 8° C.
C. After opening the foil pouch, the cartridge must be used within 1 hour. Do not use the reagent cartridge if the cartridge is damaged, the flexible pull-tab is loose or missing, the desiccant is missing, or if loose desiccant particles are found inside the foil pouch.
D. Upon removal from refrigerated storage, allow the reagent cartridge to warm naturally at room temperature for 10 minutes in unopened foil pouch or five minutes if removed from pouch.
Standardization is performed at least every six months and each time a new lot of reagent is used. Values for reagent calibration parameters are encoded onto the calibration card provided with each lot of reagent cartridges. Prior to use of reagent cartridges, the calibration bar code must be scanned. This accesses the appropriate calibration curve for the particular lot number of reagent in use. If the calibration curve has not been scanned into the DCA Vantage memory for the particular lot number of cartridges in use, the instrument prompts the user to scan the calibration card.
Note: The instrument can store two calibrations from two different reagent lots.
1. Locate the dot on the instrument next to the bar code track.
2. Locate the bar code on the Calibration Card.
3. Hold the card so that the bar code faces right.
4. Insert the card into the bar code track (above dot.) Hold card gently against the right side of track.
5. Slide the card down past the dot.
A. A beep sounds to signal a successful bar code calibration scan.
B. If no beep sounds, repeat the procedure. If a beep repeatedly fails to sound, refer to the Operating Manual, Troubleshooting.
6. To view the calibration status of the DCA Vantage Analyzer:
A. Press the MENU key
B. Press the MENU/NEXT key until (VIEW CALIBRATION STATUS) is displayed.
C. Press ENTER to view the most recent calibration.
D. Press NEXT to recall the second stored calibration.
E. To exit the menu, press ESCAPE twice or scan a bar code.
1. Patient Sample Analysis: Set the instrument power switch to ON. Allow time for the instrument to warm up (1 to 8 minutes). [READY: SCAN BAR CODE] is displayed and a beep is heard when the instrument is ready.
A. Assign a sample or a patient's sequence number.
a. Press the MENU/NEXT key until [SET SEQUENCE NUMBER?] is displayed.
b. Press the UP arrow or the DOWN arrow key to select the digit above the cursor.
c. Press the NEXT key to move the cursor to the next digit. Repeat this procedure to select the second and third digits.
d. Press ENTER to save your changes.
e. Press ESCAPE to exit the menu. To display the next menu item, press NEXT.
B. Fill the capillary with blood as follows:
a. VENOUS SAMPLE:
1. Mix sample well.
2. Remove stopper from blood collection tube.
3. Hold the capillary holder at an angle.
4. Touch only the tip of the capillary to blood sample.
b. CAPILLARY SAMPLE:
1. After performing a fingerstick, wipe away the first drop that forms.
2. Hold the capillary holder at an angle.
3. Touch ONLY the tip of the capillary to a drop of blood on the finger until the capillary is filled.
Note: Any blood touching the plastic of the capillary holder can cause an invalid A1c result or possibly an error message. If blood contacts the plastic part of the capillary holder, discard the capillary holder and start over.
C. Using a lint-free tissue carefully wipe the outside of the glass capillary. Use caution to prevent the tissue from touching the open end of the glass capillary. Contact with the open end of the capillary could result in loss of specimen. If the sample loss is obvious, discard the capillary holder; then repeat the procedure using a new capillary holder.
D. Inspect the glass capillary tube for the presence of bubble(s). If bubbles are obvious, discard the capillary holder; then repeat procedure using a new capillary holder.
E. Carefully insert the capillary holder into the reagent cartridge until the holder snaps into place.
2. Scan/Load/ Pull:
A. Locate the dot (on the instrument) next to the bar code track.
B. Locate the bar code on the Reagent Cartridge.
C. Hold the cartridge so that the bar code faces right.
D. Insert the cartridge into the bar code track (above the dot). Hold the cartridge gently against the right side of the track.
E. Slide the cartridge down past the dot.
a. A beep sounds to signal a successful scan.
b. If no beep sounds, repeat the procedure. If a beep repeatedly fails to sound, refer to Troubleshooting, Section Six of the Operating Manual.
F. At the display prompt, [LOAD CARTRIDGE OR CONTROL, PULL-TAB, CLOSE DOOR], open the cartridge compartment door.
a. Hold the cartridge so that the bar code faces RIGHT.
b. Insert the cartridge into the compartment until a subtle snap is heard/felt.
G. Using a smooth, slow, continuous motion pull the flexible plastic pull-tab completely out of the reagent cartridge. Close the door and dispose of the plastic pull-tab. Five seconds after the door is closed, a beep sounds and the assay begins.
Note: If you accidentally close the door before you pull the flexible tab, you have five seconds to re-open the door; the display returns to "LOAD CARTRIDGE". You may now pull the tab or correct the existing problem.
H. Record the displayed result on the patient requisition before removing the reagent cartridge.
I. To remove the reagent cartridge:
a. Open the cartridge compartment door. If the door is opened within 15 minutes after assay completion, the test result is displayed for only 30 seconds. If the door is not opened, the test result will remain displayed for 15 minutes. At 15 minutes an audible tone (error buzz) sounds and the display changes to "READY: REMOVE TEST".
Note: If the displayed test result was not recorded, use the MENU to recall up to 16 results.
b. Locate the button on the right side of the cartridge compartment.
c. Push the button and hold it down with your right hand.
d. With your left hand, gently push the tab on the cartridge to right; this action releases (unlocks) the cartridge.
e. Pull the reagent cartridge out of the compartment and dispose of in a biohazard container.
3. Control Analysis:
Set the instrument power switch to ON. Allow time for the instrument to warm up (1 to 8 minutes). (READY: SCAN BAR CODE) is displayed and a beep is heard when the instrument is ready.
Run the Normal and Abnormal controls for each new Reagent Kit opened.
A. From the Hemoglobin A1c Reagent Kit, remove a capillary holder from the plastic package.
B. Unscrew the control bottle eyedropper cap assembly. Avoid introducing air bubbles into the sample. While applying only slight pressure to the bulb, insert the tip of the eyedropper into the control solution (tilt bottle as necessary). Release pressure on bulb to aspirate a very small amount of control solution.
C. Hold the glass capillary tube to the control solution collected in the eyedropper and completely fill the 1 μL tube. Touch ONLY the tip of the tube to the control solution. If an air bubble(s) is present in the filled tube, discard the capillary holder and refill a new one.
Note: Use caution to prevent the control solution from coming in contact with the plastic part of the capillary holder. If the control solution comes in contact with the plastic part of the capillary holder, discard the capillary holder.
D. Avoid touching the eyedropper to any other surfaces. Squeeze any excess control solution out of the eyedropper back into the control solution bottle. Carefully replace and screw the eyedropper cap assembly back onto control bottle.
E. Using a lint-free tissue, carefully wipe any control solution off the sides of the glass capillary tube. Avoid allowing the tissue to touch the open end of the tube. Contact with the open end could result in loss of specimen. If sample loss is obvious, discard the capillary holder and refill a new one.
F. Insert the capillary holder into a reagent cartridge until the holder gently snaps into place.
G. Use the DCA Systems Hemoglobin A1c Control Card (one side for Normal and one side for Abnormal) found in the control kit for the set-up procedure.
a. Locate the dot on the instrument next to the bar code track.
b. Locate the bar code on the Control Card.
c. Hold the card so that the control bar code faces right.
d. Insert the card into the bar code track (above dot). Hold card gently against the right side of track.
e. Quickly slide the card down past the dot. A beep sounds to signal a successful scan. If no beep sounds, repeat the procedure. If a beep repeatedly fails to sound, refer to the Operating Manual, Troubleshooting.
H. When the display prompts [CONTROL C1 OR CONTROL C2], press ENTER to confirm.
I. Scan the reagent cartridge and proceed with the test as described in Patient Sample Analysis (Section SCAN/LOAD/PLUNGE/PULL above).
Note: The instrument will automatically indicate an out-of-range control value by displaying [CONTROL OUT OF RANGE], press ESCAPE to display the out-of-range control result.
J. Record the control value on DCA Vantage log before removing the cartridge.
K. To recall up to 16 control values:
a. Press the MENU key.
b. Press the NEXT key until the display prompts [RECALL CONTROL RESULTS?]
c. Press ENTER to view the most recently assayed control result.
d. Press NEXT to recall up to 16 control results.
e. Press ESCAPE twice or scan a bar code to exit the menu.
4. Procedure Notes:
A. Refer to the DCA Vantage Analyzer Operating Manual for:
a. Instrument Set-up
c. Troubleshooting errors.
B. When the instrument is not in use the power may be turned OFF without the loss of stored results. However, when the power is subsequently restored, a warm up period of 1 to 8 minutes is required.
C. When the instrument is not in use for more than 5 minutes, the display will change to a block-shaped moving cursor. If the screen saver is on, press any key to return to a normal display before performing any other steps.
D. The current time is displayed using a "blinking" colon. When the colon does not blink, the time displayed is the time that the assay began.
1. The DCA Vantage A1c assay gives accurate and precise results over a range of total hemoglobin of 7 to 24 g/dl. Patient specimens should be assayed by a test employing a different assay principle if their total hemoglobin concentrations are outside this range.
2. Samples containing high amounts of Hemoglobin F (>10%) may yield lower than expected Hemoglobin A1c results with this test. Hemoglobin A1c results for such patients should be run on another method.
3. Highly lipemic blood samples stored for long periods of time and/or refrozen should not be assayed using this method.
4. Conditions such as hemolytic anemia, polycythemia, homozygous HbS and HbC, can result in decreased life span of the red blood cells, which causes HbA1c results to be lower than expected.
1. The DCA Vantage A1c linear range is 2.5% to 14.0%.
2. A result displayed as "<" indicates a concentration below the lower limit (2.5%). Report result as <2.5 % A1c.
3. A result displayed as ">" indicates a concentration above the upper limit (14.0%). Report result as >14.0 % A1c.
1. Nondiabetic ≥ 1.5 yr: 4.0 - 6.0%
Well controlled: <7.0%
Poorly controlled: >8.0%
1. Endogenous Control:
To assure quality of both testing procedures and patient results for HbA1c, the DCA Vantage System performs 48 optical, electronic, mechanical and reagent system checks during the course of each specimen assay. These checks include calibration verification during every test. Should an assay or system error occur during any individual measurement, the system automatically reports an error message, preventing the reporting of erroneous patient results.
2. Exogenous Control:
A. CMS "Waived" status.
B. The Low and High HbA1c Controls will be run for each new reagent kit lot number and shipment.
C. All DCA Systems Control results should be within the acceptable ranges specified in the control package insert. When the bar code on the Control Card is scanned prior to running the control, the instrument will automatically indicate on the display screen whether the control results are within or out of limits.
Note: Control ranges are also listed in the control package insert.
D. If a control fails to generate an acceptable result, do not use the kit; complete the following procedure:
a. Repeat the QC test.
b. Document the failed test result and repeat test in the designated test log, notify the Site Supervisor and the office of the UCL Technical Director immediately.
c. Indicate in the designated test log who was contacted along with the date and time of the contact.
3. Criteria for accepting or rejecting a run is stated in the “Quality Control; General” protocol in the Quality Assurance manual.
4 Analytical Range: HbA1c analytical range is 2.5 - 14%.
5. Calibrator: Calibration card is included in each kit.
6. Calibration Frequency: Calibration is performed for each new lot number of reagent.
7. Calibration Verification: Calibration Verification is not applicable for Waived tests.
8. Control Ranges: Acceptable control ranges are obtained from the manufacturer's control package insert.
1. NCCLS Document GP2-A3, Vol. 16, No. 15, NCCLS, Wayne, PA.
2. DCA 2000+ Analyzer Operating Manual. Bayer HealthCare LLC, Subsidiary of Bayer Corporation., Mishawaka, IN 46544. Rev. 6/03.
3. Package insert: DCA 2000 Hemoglobin A1c Reagent Kit. Bayer Corp., Elkhart IN 46515. Rev. 6/03.
3. Package insert: DCA 2000 Hemoglobin A1c Normal and Abnormal Control Kit. Bayer HealthCare LLC, Subsidiary of Bayer Corp., Mishawaka, IN 46544. Rev. 6/03.
4. Diabetes Care 2008;31:1473-8
5. DCA Vantage Analyzer Operator’s Guide. Siemens. Tarrytown, NY. Rev. A. 2007/05.
6. DCA Systems Hemoglobin A1c Reagent Kit package insert. Siemens. Tarrytown, NY. Rev. B. 2008/08.
i. August 2007 M. Muir
ii. October 2008 S. Raymond (Revised: IX.4; XI.5; added)
iii. March 2009 L. McGovern (Revised: for DCA Vantage Analyzer)
iv. October 2009 L. McGovern, S. Raymond (Revised: III. added)
Representative: Crescent Com. Health Ctr.
Interim Review: January 2011 L. McGovern (no changes)