Advice
Evidence review
Evidence review
Clinical and technical evidence
Regulatory bodies
A search of the Medicines and Healthcare Products Regulatory Agency website revealed no manufacturer Field Safety Notices or Medical Device Alerts for this device. A search of the US Food and Drug Administration (FDA) database: Manufacturer and User Device Facility Experience (MAUDE) identified 2 adverse event reports. One related to a case of falsely elevated Active‑B12 results for 5 patients using the ARCHITECT i2000SR analyser. No impact on patient management was reported. The second adverse event report related to a case of falsely elevated Active‑B12 results while using the ARCHITECT Active‑B12 assay. No adverse impact on patient management was reported.
Clinical evidence
Twelve studies were identified, 3 of which were excluded because they only compared the results of Active‑B12 with alternative measures of either total vitamin B12 or holotranscobalamin (holoTC) but did not provide information specifically on diagnostic accuracy (Al‑Aisari et al. 2010; Brady et al. 2008; Fragasso et al. 2012). One study (Greibe and Nexo 2011) was excluded because it specifically evaluated the use of the Active‑B12 test in relation to the CobaSorb test for vitamin B12 absorption, rather than evaluating diagnostic accuracy. The remaining 8 studies were considered relevant to this briefing (Bamonti et al. 2010; Heil et al. 2012; Lee et al. 2009; Obeid and Herrmann 2007; Remacha et al. 2014; Sobczynska‑Malefora et al. 2014; Valente et al. 2011; Woo et al. 2010; see Appendix for search strategy and selection criteria). These studies investigated the diagnostic accuracy of the Active‑B12 assay against other markers of vitamin B12 deficiency in clinical populations.
The study by Bamonti et al. (2010; tables 1 and 2) aimed to establish a cut‑off threshold for holoTC for identifying vitamin B12 deficiency using the Active‑B12 assay (AxSYM analyser) and to evaluate the analytical performance of the Active‑B12 assay.
The correlation between holoTC and total vitamin B12, folate, creatinine and homocysteine (Hcy) levels was assessed using routine blood specimens from 250 people with serum total vitamin B12 concentrations of less than 221 pmol/l. Cut‑off points were derived for holoTC, and sensitivity and specificity calculated compared with total vitamin B12 levels. For defining vitamin B12 deficiency using holoTC, a cut‑off threshold of 40 pmol/l was chosen. For total vitamin B12, a cut‑off threshold of <139 pmol/l was chosen to define 'low' values. Qualitative agreement between holoTC and total vitamin B12 was 65.2% (p<0.05). Of the 250 people tested, 84 people were identified as having 'normal' levels of both total vitamin B12 and holoTC, and 79 people as having 'abnormal' (low) levels on both tests. In 33 people, total vitamin B12 values were low and holoTC was normal, and in 54 people the opposite was found.
Using this cut‑off threshold and comparing total vitamin B12 levels with a lower reference interval of <139 pmol/l, holoTC showed a sensitivity of 0.74 (95% confidence interval [CI] 0.62 to 0.86) and a specificity of 0.52 (95% CI 0.38 to 0.66). The area under the curve (AUC) in receiver operating curve (ROC) analysis for the Active‑B12 assay was 0.75 (95% CI 0.63 to 0.87), which was higher than those for folate, Hcy and creatinine.
Agreement between holoTC and folate was 55.2% (p<0.001) and with Hcy 51.6% (p<0.001). The authors noted that the Active‑B12 assay was easy to use because of the simplicity of the pre‑analytical phase and the automation of the analyser. The authors concluded that the results confirmed the reliability of the Active‑B12 assay and stated that it is adequate for routine use in assessing cobalamin deficiency in populations with reduced total vitamin B12 values.
The study by Heil et al. (2012; tables 3 and 4) aimed to validate the diagnostic accuracy of holoTC using the Active‑B12 assay (AxSYM analyser) as a screening test for vitamin B12 deficiency. ROC analysis was done on 360 samples collected from patients for whom vitamin B12 testing was requested.
Samples were measured for serum holoTC (using the Active‑B12 assay) and total vitamin B12 levels. MMA levels were used as a reference standard to define metabolic vitamin B12 deficiency, with 3 pre‑defined cut‑off levels to define vitamin B12 deficiency: >0.32 μmol/l (90th percentile), >0.45 μmol/l (97.5th percentile), and >0.77 μmol/l (99th percentile). ROC decision plots were generated for each cut‑off.
Using the ROC curve to evaluate different cut‑off values for screening (using an MMA threshold of 0.45 μmol/l as the reference standard) the authors agreed that the cut‑off offering highest sensitivity with acceptable specificity (>50%) should lie between 19 pmol/l to 36 pmol/l. The authors concluded that holoTC showed better test performance than vitamin B12 and could replace vitamin B12 testing in detecting vitamin B12 deficiency.
The study by Lee et al. (2009; tables 5 and 6) aimed to compare the diagnostic performance of the Active‑B12 for holoTC (AxSYM analyser) with total vitamin B12 levels in patients after gastrectomy. Hcy and mean cell volume (MCV) were also measured. They studied 128 patients who had a gastrectomy after a diagnosis of gastric cancer. Reference values were obtained from 100 healthy people.
HoloTC was measured using the Active‑B12 assay (AxSYM analyser, Abbott Diagnostics), total vitamin B12 was measured using the Abbott ARCHITECT B12 kit (Abbott Diagnostics), and serum Hcy was measured using the AxSYM Hcy kit (AxSYM analyser, Abbott Diagnostics). Cut‑off values for abnormal total vitamin B12 were <189 pg/ml with the borderline range of 189–400 pg/ml. Cut‑off values for abnormality were >14.05 µmol/l for Hcy and >95 fl for MCV.
Serum holoTC was low in 32 patients (25%), whereas total vitamin B12 was low in 10 patients (7.8%) and borderline in 50 patients (39.1%). HoloTC was low in all patients with low total vitamin B12 and normal in all patients with normal total vitamin B12. In the 50 patients with borderline total vitamin B12, 44% were classified as having a low level of holoTC.
Patients with both low holoTC and low total vitamin B12 had significantly higher Hcy levels than patients with normal values for either total vitamin B12, holoTC or both (p<0.001). The authors concluded that serum holoTC was more sensitive than total serum vitamin B12 for detecting vitamin B12 deficiency and was therefore a more effective marker.
The study by Obeid and Herrmann (2007; tables 7 and 8) aimed to evaluate the usefulness of measuring serum holoTC compared with total vitamin B12 in assessing vitamin B12 status in routine laboratory analysis. The authors assessed serum samples from 1018 patients referred to their laboratory in Germany for total cobalamin testing. Levels of total vitamin B12, holoTC, and MMA were measured, and associations between these markers were analysed. Concentrations of serum holoTC were measured using the Active‑B12 assay on an AxSYM analyser. MMA was measured using gas chromatography‑mass spectrometry. The test for measuring total B12 was not reported in the paper. MMA ≥300 nmol/l was used as a cut‑off value to define metabolic cobalamin deficiency.
In patients with normal renal function (number of patients unclear), ROC curve analysis of the holoTC and total vitamin B12 tests in detecting a serum concentration of MMA ≥300 nmol/l showed that the AUC was larger for holoTC (0.71) than for total vitamin B12 (0.60). This indicated a better diagnostic sensitivity and specificity for the holoTC test compared with the total vitamin B12 test. A sensitivity of 72% could be expected by using a cut‑off of 35 pmol/l for holoTC and 243 pmol/l for total vitamin B12. Statistical significance was not reported for this analysis.
The authors concluded that compared with total vitamin B12, the holoTC assay was better in detecting elevated concentrations of MMA in patients with normal renal function and that holoTC levels can be used as a first‑line parameter in detecting vitamin B12 deficiency.
The study by Remacha et al. (2014; tables 9 and 10) evaluated holoTC levels measured using the Active‑B12 assay and conducted a concordance analysis with MMA and Hcy levels in patients with low or borderline levels of serum cobalamin. Serum vitamin B12 levels and red cell folate (RCF) were evaluated using the Elecsys immunoassay (Roche Diagnostics). Serum MMA levels were assessed using a mass spectrometer. High MMA was considered when levels were greater than 0.4 nmol/l. Forty‑five healthy people without anaemia, 106 patients with low levels of serum vitamin B12 (≤200 pmol/l), and 27 patients with folate deficiency (RCF <500 nmol/l and vitamin B12 >200 pmol/l) were included. In addition to the lower level of the reference interval for holoTC, several other cut‑off points were tested. HoloTC was compared with Hcy and MMA by concordance analysis.
In 71% of patients with low total vitamin B12, serum holoTC was below the cut‑off value of 33.5 pmol/l. Of 31 samples with low cobalamin but normal holoTC, MMA or Hcy levels were high in 13 patients, indicating likely cobalamin deficiency.
Concordance analysis was done for 2 cut‑off levels of total vitamin B12: ≤200 pmol/l and 150 pmol/l. At the ≤200 pmol/l cut‑off, concordance between holoTC and MMA levels was not statistically significant. Concordance between Hcy and holoTC was 62% (Kappa index 0.245, p=0.006). At the ≤150 pmol/l cut‑off, concordance between holoTC and MMA levels was not statistically significant. Concordance between holoTC and Hcy was 74% but was not statistically significant (Kappa index 0.215, p=0.08).
The authors concluded that holoTC levels were low in patients with low total vitamin B12 levels and folate levels, but that concordance of holoTC with MMA and Hcy levels in this group was poor. They suggest that these data do not support holoTC as the earliest marker of cobalamin deficiency.
The study by Sobczynska‑Malefora et al. (2014; tables 11 and 12) evaluated a service that had substituted serum vitamin B12 measurement with holoTC supported with MMA when holoTC levels were indeterminate. Over 4000 samples received for the assessment of vitamin B12 status in a large London NHS hospital in a 4 ‑month period were included. Serum holoTC was measured using the Abbott Active‑B12 assay (AxSYM analyser). Serum MMA was measured by liquid chromatography‑tandem mass spectrometry.
The study categorised samples as 'indeterminate' for holoTC between 25–50 pmol/l. Samples with holoTC in this range also had MMA analysis if the glomerular filtration rate was ≥60 ml/min/1.73m2 or not available. An MMA concentration >280 nmol/l was considered to be elevated. The frequency of elevated MMA when holoTC was indeterminate was assessed in patients with known and unknown renal function.
Of the 4175 samples, 1019 (24%) were in the indeterminate range. Of these, 802 had MMA analysis, because renal function was normal or unknown. Of these, 244 samples had elevated MMA and 534 samples did not show elevated MMA. For samples with indeterminate holoTC, MMA was elevated in 31%. The authors concluded that MMA levels should be tested when samples fall in the indeterminate range.
The study by Valente et al. (2011; tables 13 and 14) investigated the ability of holoTC (measured using the Active‑B12 assay), total Hcy, MMA, serum and RCF and other haematological variables to determine vitamin B12 deficiency in an older population.
Non‑fasting blood samples and information on diet, lifestyle and medical history were provided by 700 consecutive outpatients attending an outpatient memory clinic in a geriatric unit in Dublin, Ireland. A separate reference population of 120 healthy volunteers was recruited from employees of the Active‑B12 assay manufacturer and medical students at the local hospital. This group was used to determine a reference interval for the red cell cobalamin, holoTC and total serum cobalamin assays.
Total serum cobalamin was assessed using the Ciba‑Corning radioassay and RCF concentration was assessed by microbiological assays. MMA was assessed using mass spectrometry. Total Hcy and holoTC (Active‑B12) were assessed using the AxSYM analyser. Cut‑off values were 20 pmol/l for holoTC, 123 pmol/l for serum cobalamin, 0.36 µmol/l for MMA, 15 µmol/l for Hcy, <6.8 nmol/l for serum folate and <340 nmol/l for RCF. The reference standard for cobalamin deficiency was red cell cobalamin <33 pmol/l.
In the outpatient group the prevalence of low red cell cobalamin was 9.6% and the prevalence of low holoTC was 8.1%. The prevalence of raised MMA and Hcy was 41.7% and 52.2% respectively. In the group characterised as vitamin B12 deficient by the reference standard, holoTC, serum cobalamin, serum folate and RCF were all significantly lower and MMA and total Hcy were significantly higher compared with the non‑deficient group. The AUC analysis showed that holoTC was the best indicator of tissue vitamin B12 status (AUC 0.90). Differences in AUC between holoTC and serum cobalamin (0.80), MMA (0.78) and total Hcy (0.75) were statistically significant (p<0.001).
The study by Woo et al. (2010; tables 15 and 16) aimed to test the association between levels of holoTC measured using the Active‑B12 assay with serum vitamin B12 levels, the precision of the assay, and its diagnostic value. The study included 45 samples from patients, for whom a vitamin B12 test had been requested. It also included 139 samples from patients with normocytic or macrocytic anaemia, who were admitted to a hospital in South Korea.
Serum vitamin B12 levels were measured, as well as holoTC, folate and Hcy levels. Low holoTC was seen in 7 samples (4%) across the 2 groups (184 samples). Of these, only 1 had low serum vitamin B12, 4 were in the borderline range and 2 in the normal range of vitamin B12. In 2 samples with low holoTC, Hcy was high while folate levels were normal. In 10 samples with low folate levels, holoTC levels were within normal range. The authors concluded that holoTC levels were more sensitive than serum vitamin B12, although the study did not use a formal reference standard.
Recent and ongoing studies
No ongoing or in‑development trials for the Active‑B12 assay were identified from searches of clinical trials registers. According to the manufacturer, several evaluations of Active‑B12 are ongoing at various NHS trusts. Data will be available and published by the end of 2015.
Costs and resource consequences
Use of the technology could replace total vitamin B12 as the standard test for vitamin B12 deficiency. It is possible that the increased test accuracy might reduce the number of indeterminate results, which may reduce the need for confirmatory tests such as methylmalonic acid. The use of the Active‑B12 assay would not eliminate the need for a second confirmatory test, MMA, if results fall within the indeterminate range (Sobczynska‑Malefora et al. 2014).
Vitamin B12 testing is currently recommended when clinical signs suggest possible deficiency (Devalia et al. 2014). Use of holoTC testing instead of vitamin B12 testing could result in earlier diagnosis of vitamin B12 deficiency; this could prevent complications of the deficiency and save costs associated with complications.
According to the manufacturer, the ARCHITECT Active‑B12 assay does not need sample pre‑treatment so it can be run at 200 tests per hour. The ARCHITECT Total B12 test can only be run at 100 tests per hour. Introducing the Active‑B12 assay to replace the Total B12 test may therefore have a positive impact on workload, through increased capacity and reduced total analytical time.
A senior NHS laboratory worker advised that total vitamin B12 tests in the hospital setting typically cost from £10−£15. One specialist commentator reported that the NHS laboratory price charged to general practitioners for the total vitamin B12 test is £2.68. This includes reagent cost, labour and overheads. They also noted that private laboratory prices are usually higher, and the primary care price may be discounted or be part of a block contract. According to the manufacturer, the expected cost of the ARCHITECT Active‑B12 is around £3.50 including VAT, depending on the number of samples and order size. The manufacturer states that there are about 215 ARCHITECT i2000SR or i1000SR analysers in use in the UK, with around 50% of major hospital laboratories having access to 1 of these analysers.
Pricing arrangements for the Active‑B12 test appear to vary depending on whether the test is ordered in a primary or secondary care setting, the laboratory providers, primary care contractual arrangements, and discounts. For this reason and in the absence of formal research into the economic consequences of using the assay, the cost implications of adopting the Active‑B12 assay to replace total vitamin B12 testing are unclear.
Strengths and limitations of the evidence
From the perspective of evaluation of the Active‑B12 assay as a diagnostic test, although holoTC is considered to measure bioavailable vitamin B12, there is no clear reference standard and relevant studies have included a variety of comparator tests. Reference intervals and cut‑off thresholds for establishing 'low' holoTC varied across studies as did thresholds for other markers of vitamin B12.
Four studies compared the diagnostic performance of holoTC levels, measured with the Active‑B12 assay with other markers of vitamin B12 deficiency (Bamonti et al. 2010; Heil et al. 2012; Valente et al. 2011; Obeid and Herrmann, 2007). In the 4 studies, holoTC consistently showed better diagnostic performance. Of these, 1 study suggested that the test showed better performance than folate, Hcy and creatinine (Bamonti et al. 2010), none of which are first‑line tests of vitamin B12 deficiency. In the remaining 3 studies, the holoTC assay showed superior accuracy to serum vitamin B12 measures, against different reference standards for vitamin B12 deficiency. One study (Valente et al. 2011) showed that holoTC measurement was better than MMA level testing in an older population.
The study by Remacha et al. (2014) suggests that in a selection of samples with low or borderline total cobalamin 29% might return normal holoTC readings, but only half have normal MMA and Hcy. This raises the possibility that using holoTC may lead to false negatives although firm conclusions are limited by the lack of an accepted reference standard, definition of deficiency and the potential for false positives with MMA and Hcy testing (Devalia et al. 2014).
Several studies were small, resulting in very low numbers of positive test results, and some recruited specific clinical subgroups such as patients who had gastrectomies (Lee et al. 2009) and older people (Valente et al. 2011). There was little detail in all of the studies about the sampling frame, available population or the people recruited, which limits the generalisability of the findings.
No diagnostic intervention studies were found that might give information on the effect of the Active‑B12 assay on clinical decision‑making or outcomes. No studies had data on assay throughput, time to test results or laboratory factors including workforce requirements.
Of the included studies, 2 declared support from the manufacturer of the Active‑B12 assay (Bamonti et al. 2010; Valente et al. 2011), of which 1 study (Valente et al. 2011) included authors who were employed by or consultants to the manufacturer. Two studies (Heil et al. 2012; Obeid and Herrmann, 2007) declared that assays used in the studies were provided by the manufacturer.