Oral Presentation for clinical placement 3.pptx

Placement presentation

1. Identifying operational issues with TOTAL B12 and resolving them by implementing active B12 Place: Flinders Medical Centre (FMC) department: AUTOMATED CORE LAB – BIOCHEMISTRY DATE: 15.09.25 – 10.10.25 – 140hrs By : Arockia Peter Francis Student: 11762771 Vitamin B12

2. overview Importance of vitamin B12 (COBALAMIN) Measuring total B12 Concerns with total B12 Overcome by active B12 Active B12 work-ups Implemented active B12 testing (SA PATHOLOGY) Conclusion References

3. VITAMIN B12 (COBALAMIN) Water-soluble micronutrient Unable to synthesize by the body ( Hannibel et al., 2009) Carbohydrate, fat and protein metabolism Nucleic acid synthesize Act as a coenzyme for methionine synthase (MS) and methylmalonyl -CoA mutase (MCM), deficiency in B12 leads to accumulation of Homocystein and Methylmalonic acid. Formation and regeneration of red blood cells Deficiency: Haematological disorder – megaloblastic anemia Neurological disorders – demyelination and cognitive decline Bone disorder and optic neuropathy – but less frequent Causes: Malabsorption due to pernicious anemia (autoimmune), aging, poor dietary intake Genetic disorders https://ods.od.nih.gov/factsheets/VitaminB12-HealthProfessional/

4. Measuring total B12 The most direct assessment to determine Vitamin B12 status ( Hannibel et al.,2016) Widely available in clinical chemistry laboratories Ranges: normal (>250 pmol /L) low (150–249 pmol /L) acute deficiency (<149 pmol /L ( Mirkazemi , C et al., 2012)

5. Concerns with total B12 Limitations: 1. This assay measures total circulating B12, of which 80% - bound to haptocorrin – not bioavailable for cell uptake 20% - bound to bioactive protein – holo-transcobalamin (Holo-TC) – available for cell uptake The serum B12 does not represent the cellular B12 status ( Lysne, V et al., 2016) A patient with a genetic error for B12 metabolism can represent a normal to low total B12, but actually be deficient in the cellular B12 level ( Hannibel et al., 2016) Total B12 assay is not a reliable biomarker for vitamin B12 status by itself ( Hannibel et al.,2016)

6. SOLUTION By implementing active B12 (Holo-TC) Active B12 measures only vitamin B12 which were bound to Holo-TC. After absorption, vitamin B12 binds to Holo-TC and is distributed to all the cells. Cells uptake Holo-TC by the receptor called t ranscobalamin receptor ( TCblR ; CD320)(Quadros et al.,2009) Holo-TC is the successive marker for bioactive Vitamin B12 ( Noxo et al.,2000) The normal range of holo-TC in healthy individuals is 20–125 pmol /L (Valente et al.,2011). SA Pathology Interpretation

7. Active B12 test implementation plan and steps 1. Preparation of the validation/Verification plan for active B12. We started making a validation plan for FMC using template VAL-753 2. Preparation of FMC QC date We downloaded active B12 into line 2 (Roche Cobas e602), calibrated and ran QC every day for 20 days.  3. Running correlation samples from RAH RAH (Royal Adelaide Hospital) is the only lab currently running active B12 in SA pathology. So we requested some active B12 patient samples for a correlation study. 4. Testing external QAP samples We enrolled in the active B12 program on the RCPA QAP website. Since we have already enrolled in special endocrine tests, such as PTH and Cortisol, we freeze the QAP samples each month, and they remain stable for up to 12 months. We retrieved those frozen samples, thawed them, and ran them for active B12.

8. 5. Running dilution series A dilution series is conducted to demonstrate the linearity of an assay. Reading 1, 2, and 3 should be performed on different days to account for data variability. 6. Functional sensitivity A functional sensitivity ( LoQ ) is the lowest concentration at which the analyte can be reliably detected. "Functional sensitivity” has been set to a predefined concentration that yields a coefficient of variation (CV) of 20%. 7. Submitting data for approval All workups have been submitted in Q Pulse for pathologist approval. 8. IT changes and trial run Once approved, my supervisor contacted the IT team and scheduled a date for the IT changeover. The trial patient was entered and checked for transferring results from the middleware to the LIS. 9. Sent email to lab staff My supervisor sent a memo to all lab staff regarding the transition of the active B12 changeover. 10. Successful transition to active B12 

9. Implemented active B12 in sa pathology Communication was passed to all the staff for the changeover. Change to B12 testing requests Trainer: Skye Rumbelow Introduction/Description Transition from Vitamin B12 testing to Holo-TC (Active B12) on Roche Cobas e602 Applicability FMC Core Lab - Biochemistry New Test/Laboratory Change/Information   Holotranscobalamin (Active B12) is the biologically active fraction of Vitamin B12 and it is proposed to be a more diagnostically accurate and clinically relevant indicator of vitamin B12 deficiency than total serum vitamin B12.   From the 1 st July 2025, the Department of Health amended the MBS items for vitamin B12 testing (66838 and 66839) and created a new item for vitamin B12 testing (66842). To reduce costs and provide the most clinically relevant results for our patients SA Pathology is transitioning to testing Holo-TC (active B12) only . Total B12 testing will continue to be offered only at the RAH on the e801 platform for specific patients with inborn errors of metabolism. Given the higher cost of Holo-TC testing, testing will be consolidated to the following laboratories: RAH, FMC, LMH, Mount Gambier, Modbury, tQEH and Noarlunga.   Reagent: Roche Active B12 Storage: Cold Room 2 (4°) Dr Jill Lipsett Clinical Service Director SA Pathology

10. OTHER Limitations and Solutions 2. Time-consuming: Roche COBAS test inserts, Total B12 - 28 mins Active B12 – 18 mins 3. Sustainability: Total B12 – 2 separate cassettes (pre-treatment and testing) Active B12 – 1 cassette Less waste 4. Stability: Onboard stability Total B12 – 35 days Active B12- 48 days Reagent packs: Left- Total B12, Right – Active B12

11. Conclusion Vitamin B12 - A very essential water-soluble micronutrient Total vitamin B12 measurement is a widely available assay for vitamin B12 status. Limitation: overall circulating Vitamin B12, but not the cellular availability level. Solution: Active B12 (Holo-TC) provides a clinically reliable interpretation for vitamin B12 deficiency. FMC validation of active B12 has been successfully conducted (QC, Correlation tests, Dilution assay, Sensitivity tests, RCPA QAP samples) and passed in all. OUR NEXT GOAL, UNDER CONSTRUCTION NOW…… After implementing active B12, the next stage of progress for this analyte will be adding a reflex homocysteine test for active B12-deficient patients (<25 pmol /L).

12. REFERENCE Green R., Allen L. H., Bjørke-Monsen A. L., Brito A.,  Guéant J.-L., Miller J. W., Molloy A. M., Nexo E., Stabler S., Toh B.-H., Ueland P. M., and  Yajnik C. (2017). Vitamin B12 deficiency,  Nature Reviews Disease Primers,  3, no. 1, article 17040,  https://doi.org/10.1038/nrdp.2017.40 , 2-s2.0-85021656925. Greibe E, Andreasen BH, Lildballe DL, et al. Uptake of cobalamin and markers of cobalamin status: a longitudinal study of healthy pregnant women. Clin Chem Lab Med 2011;49:1877-1882. Hannibal, L., Lysne, V., Behringer, S., Grünert , S. C., Spiekerkoetter , U., Jacobsen, D. W., & Blom, H. J. (2016). Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency. Frontiers in Molecular Biosciences , 3 , 205004. https://doi.org/10.3389/fmolb.2016.00027 Harrington, D. J. (2018). Methods for assessment of Vitamin B12. Laboratory Assessment of Vitamin Status , 265-299. https://doi.org/10.1016/B978-0-12-813050-6.00012-7 Lysne, V., Strand, E., Svingen, G. F. T., Bjørndal, B., Pedersen, E. R., Midttun, Ø ., et al. (2016). Peroxisome proliferator-activated receptor activation is associated with altered plasma one-carbon metabolites and B-vitamin status in rats.  Nutrients  8:26. doi : 10.3390/nu8010026 Mirkazemi , C., Peterson, G. M., Tenni , P. C., and Jackson, S. L. (2012). Vitamin B 12 deficiency in Australian residential aged care facilities.  J. Nutr . Health Aging 16, 277–280. doi : 10.1007/s12603-011-0348-2 Nexo, E., Christensen, A. L., Petersen, T. E., and Fedosov, S. N. (2000). Measurement of transcobalamin by ELISA.  Clin. Chem.  46, 1643–1649. Quadros, E. V., Nakayama, Y., and Sequeira, J. M. (2009). The protein and the gene encoding the receptor for the cellular uptake of transcobalamin-bound cobalamin.  Blood 113, 186–192. doi : 10.1182/blood-2008-05-158949 Solomon, L. R. (2015). Functional cobalamin (vitamin B12) deficiency: role of advanced age and disorders associated with increased oxidative stress.  Eur. J. Clin. Nutr .  69, 687–692. doi : 10.1038/ejcn.2014.272 Stabler S. P., Vitamin B12 deficiency,  The New England Journal of Medicine . (2013) 368, no. 2, 149-160,  https://doi.org/10.1056/NEJMcp1113996 , 2-s2.0-84872061155,  23301732 Valente, E., Scott, J. M., Ueland, P. M., Cunningham, C., Casey, M., and Molloy, A. M. (2011). Diagnostic accuracy of holotranscobalamin , methylmalonic acid, serum cobalamin, and other indicators of tissue vitamin B(1)(2) status in older people.  Clin. Chem.  57, 856–863. doi : 10.1373/clinchem.2010.158154

13. Thank you Skye Rumbelow Darren Scott