IFCC seminar – Berlin 16 th May
Diagnostic Accuracy of Holotranscobalamin, Methylmalonic Acid, Serum Cobalamin and other indicators of tissue vitamin B 12 status in the elderly Professor John Scott School of Biochemistry and Immunology Trinity College Dublin Dublin 2 Ireland e-mail: jscott@tcd.ie 2
Vitamin B 12 Deficiency [Cobalamin] Dietary Malabsorption Vegans Vegetarians Poor Animal Products Pernicious Gastric Atrophy Anaemia Many elderly have diets poor in B 12 and varying levels of gastric atrophy 3
Neuropathy (diagnosis difficult) Methylated (+ CH 3 ) DNA, Protein, Lipids Methyl (-CH 3 ) Transferases Methionine synthase Methyl Tetrahydrofolate Vitamin B 12 DNA Cell Division Anaemia (diagnosis easy) 4
red cells R 20% Active B 12 Receptor (R) (Holotranscobalamin II) Transport R all cells Total B 12 (100%) Haptocorrin (TC I + TC III) Inactive 80% (proteases) 5
Traditional Diagnosis; Total serum B 12 New Diagnosis; Active B 12 Active B 12 out performs Total B 12 (1) RC-B 12 assay reflects tissue B 12 (2) Comparison of ROC plots (3) Assay Monoclonal Specific to Active B 12 (will not bind to Apotranscobalamin) (4) Model: Active B 12 in model has superior specificity and sensitivity than Total B 12 6
EVIDENCE Red blood cell vitamin B 12 (Tissue B 12 ) Reference population (n = 118) mean 97.2 (32.8) ρ mol/L - 95% central reference interval - cut off for deficiency 33 < ρ mol/L red cell B 12 Elderly (69-92 years) study population (n = 700) Red cell B 12 < 33 ρ mol/L n = 67 deficient > 33 ρ mol/L n = 633 non-deficient 7
ROC plots for serum total cobalamin, holoTC and MMA for vitamin B12 deficiency, defined as red cell cobalamin <33 pmol/L 1 0.9 0.8 Sensitivity (true positives) 0.7 No discrimination 0.6 Active B12 pmol/L 0.5 S.B12 pmol/L 0.4 MMA umol/l 0.3 0.2 0.1 0 0 0.2 0.4 0.6 0.8 1 1 - Specificity (false positives)
Performance of markers at a single cut-off Marker Cutoff Sensitivity% Specificity% PPV% NPV% holoTC <20pmol/L 55 (43 to 67) 96 (94 to 97) 56 (45 to 70) 95 (93 to 97) Serum Cbl <123pmol/L 33 (22 to 45) 95 (93 to 96) 39 (26 to 53) 93 (91 to 95) MMA <0.36µmpl/L 81 (69 to 89) 63 (59 to 66) 19 (14 to 24) 97 (95 to 98) HoloTC was superior to MMA and Cobalamin as predictor of B12 deficiency
3-zone partition with deliberate grey-zone Any result above the test The “grey” or indeterminate value B has a very high zone, test values is this region probability of no B12 have neither a high or low deficiency, set at 98% for a probability for deficiency. negative result Further evaluation of the patient would be required. We would be interested to estimate how wide this “grey- B Any result below the zone” is in terms of percentage test value A has a very of patients whose results would GZ high probability of B12 be within this area. A deficiency, set at 60% for a positive result Note: this is written for total B12 and Active B12 where low values indicate deficiency- the same principle applies for MMA, but in this case high values indicate deficiency
Proportion of samples in grey-zone MMA (uM) Active B12 (pM) B12 (pM) LR+ =14 1.40 19.6 79 LR- = 0.23 0.31 29.9 238 Samples in Grey-Zone 349/700 96/699 313/700 (N) Samples in Grey-Zone 50 14 45 (%) For MMA and total vitamin B12 a very high proportion of the population would be unclassified, restricting the utility of these tests. HoloTC measurement has clearly superior clinical utility with only 14% of samples in the grey-zone.
Performance of markers in relation to kidney function (glomerular filtration rate). 90 Positive Predictive Value (PPV) % 80 • HoloTC had highest PPV for diagnosing 70 B12 deficiency even in 60 Active-B12 most compromised 50 Serum cobalamin patients with no 40 MMA relationship to renal 30 function 20 10 • PPV of MMA shows 0 a steady decline � 60 30-59 15-29 suggesting the eGFR(CG) range diagnostic performance of MMA may be impacted by renal function
Suggested testing algorithm in holoTC screening Subjects at risk of B 12 deficiency holoTC 20-30 pmol/L holoTC <20 pmol/L holoTC >30 pmol/L ~14% of samples Treatment Measure HoloTC No treatment every 6 months until status resolved
ACKNOWLEDGEMENTS Diagnostic Accuracy of Holotranscobalamin, Methylmalonic Acid, Serum Cobalamin and other indicators of tissue vitamin B 12 status in the elderly Edward Valente, John Scott, Per-Magne Ueland, Conal Cunningham, Miriam Casey and Anne Molloy From Clinical Chemistry 57:6, 2011 14
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