CK-MB Examination Procedure

1.Purpose of examination: CK-MB estimation from serum or plasma by IFCC Method/Immunoinhibition method.

2.Responsibility and Authority:

• Calibration: Technician
• Quality Control: Technician
• Routine operation: Technician
• Overall Monitoring: Quality Manager

3.Sample Details:

1. Type of Sample: Serum, Plasma
2. Type of container and additives: Plain without any additives
3. Patient Preparation: As per Primary Sample Collection Manual sample_collection_manual
4. Stability: At Room temperature 18°–28°C (64°–82°F) stability ≤ 24 hours
5. Handling and transport: As per Primary Sample collection manual
6. Storage: 24 hours at 2-8° C

4.Required Equipment: Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, FullyAuto Chemistry Analyzer

5.Required reagents: R1:

• Imidazole buffer (pH 6.1)125 mmol/L
• Glucose 25 mmol/L
• N-acetyl-L-cysteine 25 mmol/L
• Hexokinase (phosphorylating) ≥ 52 μkat/L
• EDTA 2.5 mmol/L
• Magnesium acetate 12.5 mmol/L
• NADP 2.5 mmol/L
• Anti-CK-M monoclonal antibodies (mouse) ≥ 2,000 U/L

R2:

• Creatine phosphate (pH 9.5) 100 mmol/L
• ADP 10 mmol/L
• AMP 25 mmol/L
• Diadenosine 5-phosphate 0.05 mmol/L
• G6P-DH ≥ 125 μkat/L

6.Reagent Handling

1. Remove any air bubbles present in the reagents with a new applicator stick, or allow the reagents to settle at the appropriate storage temperature to allow the bubbles to dissipate. To minimize volume depletion, do not use a transfer pipette to remove bubbles
2. Reagent Storage and stability
3. Unopened reagent stable at 2-8°C until expiration date.
4. On board System temperature reagent is stable for 42 days.
5. Instability or deterioration should be suspected if there are precipitates, visible signs of leakage or
6. contamination, turbidity, or if calibration or controls do not meet the appropriate criteria.

7.Calibration Procedure:

• CK MB Calibrator
• Frequency:

1. Reagent lot change
2. QC out of range
3. After service or maintenance
4. Replacement in any parts of Instrument

Procedure:

1. Start the equipment.WDI abbotte fully.docx
2. Calibrators are ready to use.
3. Put calibrator 15-20 minutes at room temperature
4. Prior to use, mix the contents by inverting the vial. Do not shake the vial to prevent foam formation.
5. Take a 150 µl calibrator solution in separate aliquots.
6. Go to the calibration and give the calibration order.
7. Verify calibration with at least two levels of controls.
8. If control results fall outside acceptable ranges,root cause analysis or recalibration may be necessary.

8.Quality control Procedure: Name: Cardiac Control Level 1 , 2 & 3 Frequency: As per Quality Control Procedure Procedure for Reconstitution of IQC

1. Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette.
2. Leave to stand for 30 min in the dark place.
3. Swirl gently several times during the reconstitution period to ensure that the contents are completely dissolved.
4. Prior to use, mix the contents by inverting the vial. Do not shake the vial as the information of foam should be avoided. Ensure that no lyophilized material remains un-reconstituted.
5. Prepare aliquots of 150 µl from the reconstituted QC material.
6. Store these aliquots at -15° C to -20° C.
7. Prior to use, make sure that aliquots should be at room temperature for at least 15 min.

Procedure to run IQC

• Press Control order
• Select Assay / Panel, to be run.
• Select the control/s and its level/s
• Give Carrier Number and Position number
• Press F3 / Add order
• Put respected carrier in RSH rack
• Check IQC results, in case outliers call residents.

9.Principle of the procedure used for examinations:

• Creatine kinase catalyzes the reaction between creatine phosphate and ADP with formation of creatine and ATP. The ATP formed, in the presence of glucose and hexokinase (HK), yields ADP and glucose-6-phosphate. The glucose-6-phosphate formed in the presence of glucose-6-phosphate dehydrogenase (G6P-DH) reacts with β-NADP+ forming 6-phosphogluconate and β-NADPH. The presence of mouse antibodies that inhibit CK-MM activity in the reaction mixture allows the determination of the residual activity of CK-B isoenzymes (CK-MBand CK-BB). The CK-MB activity is obtained by multiplying the CK-B activity by two. By measuring the variation of the absorbance due to transformation of β-NADP+ into β-NADPH in a time interval at 340 nm,it is possible to calculate the residual activity in the examined sample.

10.Sample Preparation:

• Required SampleVolume: 150 µl of the sample
• Temperature: 37°

Take 150-200µl of the sample from Primary tube to the secondary aliquot. Write the sample ID on the aliquot. Procedure to run Patient sample

• Press Patient order
• Select Assay /Panel, to be run.
• Give Carrier Number and Position number
• Press F3 / Add order
• Put respected carrier in RSH rack

11.Performance Characteristics:

• Linearity: up to 2000 U/L
• The limit of detection (LOD):3 U/L
• Unit: U/L

Normal and critical ranges:

12.Laboratory Clinical interpretation: Elevated CK values are due to muscle damage and associated pathologies. CK determination, usually performed with CK2 (also called CK-MB), is used for the diagnosis and follow-up of AMI (acute myocardial infarction) and some muscle diseases.

Interference and cross reaction:

• No interference from Bilirubin up to 6.6 mg/dL
• No interference from Intralipid up to 600 mg/dL
• No interference from Sulfapyrdine up to 300mg/dl
• No interference from Temozolomyde up to 20 mg/dl

13.Potential source of variation: Turn around time (TAT):

• Routine: 6.0 hours
• Urgent: 2.0 hours

14.Recording of observation:

• Software backup
• Machine raw data

15.Storage & Disposal of waste: Follow storage & discard procedure

16.Environmental & Safety control: Reagent R1 contains imidazole and sodium azide that Causes irritations eye Damage ,may damage fertility or the unborn child exposure organ contact with acid liberate very toxic gas. 17.Precautions:

1. Wear protective gloves / protective clothing / eye protection
2. Do not breathe mist / vapors / spray
3. Wash hands thoroughly after handling
4. Keep only in original container
5. IF ON SKIN (or hair): Take off immediately all contaminated clothing. Rinse skin with water / shower.

18.References:

1. US department of labor,Occupational safety and health administration.
2. US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories.
3. World health organization. Biosafety manual,3rd edition
4. ARCHITECT CK MB 6K25-30 307233/R0