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                      Vitamin B12 Examination Procedure
**1.Purpose of examination:** 
B12 assay is a Chemiluminescent Microparticle Intrinsic Factor assay for the quantitative determination of vitamin B12 in human serum and plasma. 

**2.Responsibility and Authority:** 
  * Calibration: Technician 
  * Quality Control: Technician 
  * Routine operation: Technician 
  * Overall Monitoring: Quality Manager

**3.Sample Details:** 
  * Type of Sample: Serum,  Plasma
  * Type of container and additives: Plain without any additives 
  * Patient Preparation: As per Primary Sample Collection Manual [[sample_collection_manual|]]
  * Stability: At Room temperature upto 24 hours
  * Storage: 24 hours at 2-8° C

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

**5.Required reagents:**
  * Microparticles Intrinsic Factor (porcine) coated Microparticles in borate buffer with protein (bovine) stabilizers.
  * Conjugate B12 acridinium-labeled Conjugate in MES buffer.
  * Multi Assay manual dilution phosphate buffered saline solution. 
  * Pre Treatment solution B12 Pre-Treatment Reagent 1 containing 1.0 N sodium hydroxide with 0.005% potassium cyanide.
  * B12 Pretreatment Reagent 2 containing alpha monothioglycerol and EDTA.
  * B12 Pretreatment Reagent 3 containing cobinamide dicyanide in a borate buffer with protein (avian) stabilizers.
  * Trigger solution  0.35 N sodium hydroxide.
  * Pre Trigger solution  1.32% (w/v) hydrogen peroxide.
  * Wash-buffer  phosphate buffered saline solution.

**6.Reagent Handling**
  * 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 pipette to remove bubbles
  * Reagent Storage and stability
  * Unopened reagent stable at 15-30°C until expiration date.
  * On board System temperature reagent is stable for 30 days
  * Instability or deterioration should be suspected if there are precipitates, visible signs of leakage or
  * contamination, turbidity, or if calibration or controls do not meet the appropriate criteria.

**7.Calibration Procedure:**
  - ARCHITECT B12 Calibrators
  - Frequency:
  - After calibration expiry period
  - Reagent lot change
  - In case of EQAS  out of range

**Procedure to calibrate parameter:**
  - Start the equipment.WDI abbotte fully.docx
  - Calibrators are ready to use.
  - Put calibrator 15-20 minutes at room temperature  
  - Prior to use, mix the contents by inverting the vial. Do not shake the vial to prevent foam formation. 
  - Take a 150 µl calibrator solution into separate aliquots.
  - Go to the calibration and give the calibration order.
  - Run at least two levels of controls after calibration and verify
  - If control results fall outside acceptable ranges,root cause analysis or recalibration may be necessary.

**8.Quality control Procedure:**
Name: Bio Rad Level 1 , 2 & 3
Frequency: As per Quality Control Procedure

9.Procedure for Reconstitution of IQC 
  - Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette.
  - Leave to stand for 30 min in the dark place.
  - Swirl gently several times during the reconstitution period to ensure that the contents are completely dissolved.
  - 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.
  - Prepare aliquots of 150 µl from the reconstituted QC material.
  - Store these aliquots at -15° C to -20° C.
  - Prior to use, make sure that aliquots should be at room temperature for at least 15 min.

**10.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.

**11.Principle of the procedure used for examinations:**
  - The ARCHITECT B12 assay is a two-step assay with an automated sample pretreatment, for determining the presence of B12 in human serum and plasma using CMIA technology with flexible assay protocols, referred to as Chemiflex.
  - Sample and Pretreatment Reagent 1, Pretreatment Reagent 2, and Pretreatment Reagent 3 are combined.
  - An aliquot of the pre-treated sample is aspirated and transferred into a new Reaction Vessel (RV). The pre-treated sample, assay diluent, and intrinsic factor coated paramagnetic microparticles
are combined. The B12 present in the sample binds to the intrinsic factor coated microparticles.
  - After washing, B12 acridinium-labeled conjugate is added to create a reaction mixture.
  - Following another wash cycle, Pre-Trigger and Trigger Solutions are added to the reaction mixture.
  - he resulting chemiluminescent reaction is measured as relative light units (RLUs). There is an inverse relationship between the amount of B12 in the sample and the RLUs detected by the ARCHITECT iSystem optics.

**12.Sample Preparation:**
Required Sample Volume: 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. 

**13.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

**14.Performance Characteristics:** 
  * Linearity:  0.0038 μIU/mL to 100.0000 μIU/mL.
  * The limit of detection (LOD) :125 pg/mL
  * The limit of quantification(LOQ):125 pg/mL
  * Unit:pg/mL

**Normal ranges:**

**Vitamin B12**
^206-678^pg/mL^

**Acceptable (WHO)**
^>201^pg/mL^


**Deficiency (WHO)**
^<150^pg/mL^


**15.Laboratory Clinical interpretation:**
There are a number of conditions that are associated with low serum B12 levels, 
including iron deficiency, normal near-term pregnancy, vegetarianism,partial gastrectomy/ileal damage, 
celiac disease, use of oral contraception, parasitic competition, pancreatic deficiency,treated epilepsy, and advancing age.  8-11 Disorders associated with elevated serum B12 levels include renal failure, liver disease, and myeloproliferative diseases.

**16.Interference and cross reaction:**
At the concentrations listed below, bilirubin (conjugated and unconjugated), total protein, and triglycerides showed less than 10% interference in the  B12 assay for low samples(concentration range: 150 pg/mL to 250 pg/mL and higher samples (concentration range: > 500 pg/mL)
  * Bilirubin            < 25.1 mg/dL
  * Total Protein        < 12 g/dL
  * Triglycerides        < 3325 mg/dL

**17.Potential source of variation:** 
**Turn around time (TAT):**
**Routine: 6.0 hours
Urgent: 2.0 hours**

**18.Recording of observation:**
  * Software backup 
  * Machine raw data
**19.Storage & Disposal of waste:** Follow storage & discard procedure
**20.Environmental & Safety control:**
**21.Prevention:**
  * Wash hands thoroughly after handling.
  * Wear protective gloves / protective
  * clothing / eye protection.
**22.Response:**
  * IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
  * If eye irritation persists: Get medical advice / attention.
  * IF ON SKIN: Wash with plenty of water.
  * If skin irritation occurs: Get medical advice / attention.
  * Take off contaminated clothing and wash it before reuse.

**23.References:**
  - Beuerlein FJ. Testing strategies for anemias. Lab Mgmnt 1988; Dec:23-29.
  - Pennypacker LC, Allen RH, Kelly JP et al. High Prevalence of Cobalamin Deficiency in Elderly Outpatients. J Am Geriatr Soc 1992;40:1197-1204.
  - Obeid R, Herrmann W. Holotranscobalamin in laboratory diagnosis of cobalamin deficiency compared to total cobalamin and methylmalonic acid. Clin Chem Lab Med 2007; 45(12):1746-1750.
  - Klee GG. Cobalamin and folate evaluation: measurement of methylmalonic acid and homocysteine vs vitamin B12 and folate. Clin Chem 2000;46:1277-1283.
  - Snow CF. Laboratory Diagnosis of Vitamin B12 and Folate Deficiency: A Guide for the Primary Care Physician.

|**Printed copy of this document is considered uncontrolled.** It should be compared with controlled electronic copy before use|

^ Name of Laboratory : Laboratory Services Sir T. Hospital (LSSTH),Bhavnagar ^^^^
^Document No.1^**Document Name**: Vitamin B12 Examination Procedure^**Unique ID**:LSSTH /BIOCHEM/ SOP-5^^
^Issue No. : 01^Issue Date :30/04/2024^Page No.^^
^Amend No.^ Amend Date ^Prepared by: Section Incharge^Approved & Issued by: HOD,Biochemistry^