Diagnostic accuracy of holotranscobalamin, methylmalonic acid, serum cobalamin, and other indicators of tissue vitamin B₁₂ status in the elderly.
ABSTRACT Vitamin B₁₂ deficiency is common among the elderly, and early detection is clinically important. However, clinical signs and symptoms have limited diagnostic accuracy and there is no accepted reference test method.
In elderly subjects (n = 700; age range 63-97 years), we investigated the ability of serum cobalamin, holotranscobalamin (holoTC), total homocysteine (tHcy), methylmalonic acid (MMA), serum and erythrocyte folate, and other hematologic variables to discriminate cobalamin deficiency, defined as red blood cell cobalamin <33 pmol/L.
Serum holoTC was the best predictor, with area under the ROC curve (95% CI) 0.90 (0.86-0.93), and this was significantly better (P ≤ 0.0002) than the next best predictors; serum cobalamin, 0.80 (0.75-0.85), and MMA, 0.78 (0.72-0.83). For these 3 analytes, we constructed a 3-zone partition of positive and negative zones and a deliberate indeterminate zone between. The boundaries were values of each test that resulted in a posttest probability of deficiency of 60% and a posttest probability of no deficiency of 98%. The proportion of indeterminate observations for holoTC, cobalamin, and MMA was 14%, 45%, and 50%, respectively. Within the holoTC indeterminate zone (defined as 20-30 pmol/L), discriminant analysis selected only erythrocyte folate, which correctly allocated 65% (58/89) of the observations. Renal dysfunction compromised the diagnostic accuracy of MMA but not holoTC or serum cobalamin.
This study supports the use of holoTC as the first-line diagnostic procedure for vitamin B₁₂ status.
Article: Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis. 1988.Nutrition 11(2):181; discussion 180, 182. · 3.03 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: To better estimate how frequently patients with low serum cobalamin (Cbl) levels in current clinical practice are truly deficient in Cbl and to determine the incidence of atypical or nonclassic presentations of Cbl deficiency, we prospectively studied 300 unselected consecutive patients with serum Cbl concentrations less than 200 pg/mL seen at two medical centers over a 2-year period. Baseline hematologic, neuropsychiatric, and biochemical measurements were obtained, followed by a course of parenteral Cbl therapy and reassessment. A response to Cbl therapy was defined as one or more of the following: (1) an increase in hematocrit of 0.05 or more; (2) a decrease in mean cell volume of 5 fL or more; (3) a clearing of hypersegmented neutrophilis and macroovalocytes from the peripheral blood smear; and (4) an unequivocal and prompt improvement of neuropsychiatric abnormalities. Of the 300 patients with serum Cbl levels less than 200 pg/mL, 86 had one or more responses to Cbl therapy and 59 had no response. In 155, insufficient data was available. In the Cbl-responsive patients, normal values were found for the following tests: hematocrit, 44%; mean cell volume less than or equal to 100 fL, 36%; white blood cell count, 84%; platelet count, 79%; serum lactic dehydrogenase, 43%; and serum bilirubin, 83%. Peripheral blood smears were nondiagnostic in 6% when reviewed by the investigators, but 33% as reported by routine laboratories. Serum Cbl levels in the 100 to 199 pg/mL range were present in 38%. Neuropsychiatric abnormalities were noted in 28%, often in the absence of anemia, macrocytosis, or both. Serum levels of methylmalonic acid and/or total homocysteine were elevated greater than 3 SDs above the mean for normal subjects in 94% of the Cbl-responsive patients. We conclude that Cbl deficiency should be considered and investigated in patients with unexplained hematologic or neuropsychiatric abnormalities of the kind seen in Cbl deficiency, even if anemia, an elevated mean cell volume, a marked depression of the serum Cbl, or other classic hematologic or biochemical abnormalities are lacking. Levels of serum methylmalonic acid and total homocysteine are useful as ancillary diagnostic tests in the diagnostis of Cbl deficiency.Blood 10/1990; 76(5):871-81. · 9.90 Impact Factor
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ABSTRACT: We reviewed 153 episodes of cobalamin deficiency involving the nervous system that occurred in 143 patients seen over a recent 17-year period at 2 New York City hospitals. Pernicious anemia was the most common underlying cause of the deficiency. Neurologic complaints, most commonly paresthesias or ataxia, were the first symptoms of Cbl deficiency in most episodes. The median duration of symptoms before diagnosis and treatment with vitamin B12 was 4 months, although long delays in diagnosis occurred in some patients. Diminished vibratory sensation and proprioception in the lower extremities were the most common objective findings. A wide variety of neurologic symptoms and signs were encountered, however, including ataxia, loss of cutaneous sensation, muscle weakness, diminished or hyperactive reflexes, spasticity, urinary or fecal incontinence, orthostatic hypotension, loss of vision, dementia, psychoses, and disturbances of mood. Multiple neurologic syndromes were often seen in a single patient. In 42 (27.4%) of the 153 episodes, the hematocrit was normal, and in 31 (23.0%), the mean corpuscular volume was normal. Neutropenia and thrombocytopenia were unusual even in anemic patients. In nonanemic patients in whom diagnosis was delayed, neurologic progression frequently occurred although the hematocrit remained normal. In 27 episodes, the serum cobalamin concentration was only moderately decreased (in the range of 100-200 pg/ml) and in 2 the serum level was normal. Neurologic impairment, as assessed by a quantitative severity score, was judged to be mild in 99 episodes, moderate in 39 and severe in 15. Severity of neurologic dysfunction before treatment was clearly related to the duration of symptoms prior to diagnosis. In addition, the hematocrit correlated significantly with severity, independent of the longer duration of symptoms in nonanemic patients. Four patients experienced transient neurologic exacerbations soon after beginning treatment with cyanocobalamin, with subsequent recovery. Followup evaluation was adequate to assess the neurologic response to vitamin B12 therapy in 121 episodes. All patients responded, and in 57 (47.1%), recovery was complete, with no remaining symptoms or findings on examination. The severity score was reduced by 50% or greater after treatment in 91% of the episodes. Residual long-term moderate or severe neurologic disability was noted following only 7 (6.3%) episodes. The extent of neurologic involvement after treatment was strongly related to that before therapy as well as to the duration of symptoms. The percent improvement over baseline neurologic status after treatment was inversely related to duration of symptoms and hematocrit. Some evidence of response was always seen during the first 3 months of treatment.(ABSTRACT TRUNCATED AT 400 WORDS)Medicine 08/1991; 70(4):229-45. · 4.35 Impact Factor
Diagnostic Accuracy of Holotranscobalamin,
Methylmalonic Acid, Serum Cobalamin, and Other
Indicators of Tissue Vitamin B12Status in the Elderly
Edward Valente,1*John M. Scott,2Per-Magne Ueland,3Conal Cunningham,4
Miriam Casey,4and Anne M. Molloy2
the elderly, and early detection is clinically important.
nostic accuracy and there is no accepted reference test
years), we investigated the ability of serum cobalamin,
holotranscobalamin (holoTC), total homocysteine
rocyte folate, and other hematologic variables to dis-
criminate cobalamin deficiency, defined as red blood
cell cobalamin ?33 pmol/L.
was significantly better (P ? 0.0002) than the next best
predictors; serum cobalamin, 0.80 (0.75–0.85), and
MMA, 0.78 (0.72–0.83). For these 3 analytes, we con-
aries were values of each test that resulted in a posttest
45%, and 50%, respectively. Within the holoTC indeter-
minate zone (defined as 20–30 pmol/L), discriminant
analysis selected only erythrocyte folate, which correctly
tion compromised the diagnostic accuracy of MMA but
CONCLUSIONS: This study supports the use of holoTC as
© 2011 American Association for Clinical Chemistry
Vitamin B12deficiency is considered to be a serious
public health issue among elderly populations; how-
ever, there is no consensus on diagnosis. The classic
signs of deficiency, such as macrocytosis, anemia, and
an abnormal low serum vitamin B12(cobalamin) con-
centration, are quite often absent (1). Furthermore,
neurological rather than hematologic symptoms may
be the only clinical indications (2), and a relationship
between low concentrations of cobalamin and neuro-
logical symptoms has been reported (3, 4). In clinical
practice, however, many elderly patients present with
diffuse nonspecific symptoms, and cobalamin defi-
ciency is only one of several differential diagnoses (5).
disease on the basis of signs and symptoms alone.
Serum cobalamin measures cobalamin bound to
the 2 circulating binding proteins, haptocorrin and
20%–30% of cobalamin bound to TC (i.e., holotrans-
cobalamin, the holoTC fraction) for which there is a
receptor-mediated cellular uptake. The function of
haptocorrin is currently unknown, and low haptocor-
rin concentrations, found in approximately 15% of
(6). Thus the cobalamin assay is recognized to be po-
tentially unreliable (7). Methylmalonic acid (MMA)
and total homocysteine (tHcy) concentrations are in-
sidered more sensitive indicators of vitamin B12status
than serum cobalamin, but questions of specificity
have been raised, particularly among the elderly, in
11). HoloTC has been postulated to be the earliest
last few years, reliable methods for estimating holoTC
1Axis-Shield Diagnostics, Dundee, UK;2School of Biochemistry and Immunology,
Trinity College Dublin, Dublin, Republic of Ireland;3Section for Pharmacology,
Institute of Medicine, University of Bergen, and Laboratory of Clinical Biochem-
istry, Haukeland University Hospital, Bergen, Norway;4Mercers Institute for
Research on Aging, St James Hospital, Dublin, Republic of Ireland.
* Address correspondence to this author at: Axis-Shield Diagnostics, The Tech-
nology Park, Dundee DD2 1XA, UK. E-mail email@example.com.
Received October 18, 2010; accepted March 28, 2011.
Previously published online at DOI: 10.1373/clinchem.2010.158154
5Nonstandard abbreviations: TC, transcobalamin; holoTC, holotranscobalamin;
MMA, methylmalonic acid; tHcy, total homocysteine; eGFR(CG), estimated
glomerular filtration rate by Cockcroft–Gault equation; PPV, positive predictive
value; NPV, negative predictive value; Hct, hematocrit; Hb, hemoglobin; AUC,
area under the curve; MCV, mean corpuscular volume; LR, likelihood ratio.
Clinical Chemistry 57:6
Endocrinology and Metabolism
have become available (13, 14). The true performance
of these tests is still debated, however, and it is neces-
by these newer markers over existing procedures and
how they might be used in clinical practice.
Vitamin B12is an essential component in erythro-
poiesis and is rapidly incorporated into immature
erythrocytes (15). The red cell cobalamin concentra-
tion provides an estimate of tissue vitamin B12status
over the past 120 days, analogous to the red cell folate
concentration. Harrison (16–18) studied red cell co-
reticulocyte concentration as a result of treatment. A
between holoTC concentrations and red cell cobala-
min concentrations and that holoTC was a better indi-
cator of red cell B12depletion than plasma cobalamin
loTC, MMA, tHcy, folate, and hematologic indices
were evaluated against a reference standard of red cell
cobalamin concentrations in a population of elderly
subjects. Our aim was to establish a diagnostic strategy
for this common clinical condition.
to collect environmental, medical, metabolic, and ge-
notype data on 2000 individuals ?60 years old with
mild to moderate cognitive impairment. The first 700
subjects recruited comprised the study population
(December 2008 to October 2009). Nonfasting blood
lifestyle, and medical history. The study protocol was
approved by the Dublin Federated Hospitals Research
Ethics committee, and all individuals gave written in-
A separate reference population of 120 healthy
volunteers (64 women, with 27 using oral contracep-
tives, and 56 men) was recruited among the employees
of Axis-Shield and medical students at the local hospi-
tal, age range 18–62 years (October 2009 to March
2010). This population was used to determine a refer-
ence interval for the red cell cobalamin assay. We also
chose to establish reference intervals for holoTC, be-
cause it is a relatively novel marker, and for the micro-
biological total serum cobalamin assay used, because
there were no reports in the literature. Institutional
tained for all volunteers.
All blood samples were processed within 3 h of sam-
pling. MMA determination was performed at the Uni-
versity of Bergen; all other biochemical measurements
were carried out at Trinity College, Dublin. Red cell
cobalamin, red cell folate, and MMA/tHcy concentra-
ples maintained at 4 °C. For determination of holoTC,
cobalamin, and serum folate, bloods were collected
into clotting tubes. After processing, serum, plasma,
til assayed. Hematologic and renal function data were
glomerular filtration rate [eGFR(CG)] was calculated
by use of the Cockcroft–Gault equation.
man et al. (19) and had interassay CVs of ?11.7% in
measured cobalamin in 4 pooled washed red cell sam-
experiments, and CVs of ?5.9% in their extracted su-
pernatants stored at ?80 °C for 1 week before assay.
trations noted above were ?5.1%. Interassay CVs of 2
pooled washed red cell samples at 66 and 91 pmol/L
over 40 assays during the course of the study were
14.9% and 14.6%, respectively (see Supplemental De-
scription 1 in the Data Supplement, which accompa-
nies the online version of this article at http://
We determined serum and red cell folate
concentrations by microbiological assay using a
chloramphenicol-resistant strain of Lactobacillus casei
and intraassay CVs were ?11.0%. We measured total
cobalamin in serum by microbiological assay using a
colistin sulfate–resistant strain of L. leichmanii (del-
brueckii) (NCIB 12519, ATCC 43787) (21, 22). Inter-
using a GC-MS method based on methylchlorofor-
mate derivatization with a between-day CV of ?5%
(23). We measured tHcy and holoTC on the Abbott
AxSYM (14, 24). The between-assay CVs were ?5.2
and 11.1%, respectively.
Cutoff values for the metabolites tHcy and MMA,
in relation to possible vitamin B12deficiency, are not
15 ?mol/L for tHcy (25). For holoTC, serum cobala-
the 95% central reference interval as established in our
reference population. Serum folate and red cell folate
Accuracy of Indicators of Tissue B12Status
Clinical Chemistry 57:6 (2011)
The values for red cell cobalamin, serum cobalamin,
holoTC, tHcy, MMA, serum folate, and red cell folate
in the elderly population were positively skewed and
were natural log–transformed before analysis. We
ric means with 2.5–97.5 percentiles. A 2-sample t-test
was used to test for differences in the means. We used
cobalamin to dichotomize red cell cobalamin concen-
ables. The associations between the test variables and
red cell cobalamin concentrations were further evalu-
ated by stepwise multiple linear regression analysis.
Possible violations of the model assumptions were as-
sessed. Stepwise linear discriminant functions, using
the same variables as in the stepwise regression model
(except holoTC), were developed for observations be-
tween holoTC values of 20–30 pmol/L. The discrimi-
nant function used to predict vitamin B12deficiency
was assessed by sensitivity, specificity, and proportion
of results correctly allocated. Stepwise models had a
P value to enter of 0.05 and removal of 0.10. For the
best-performing markers at specific cutoff points, we
assessed sensitivity, specificity, positive predictive
value (PPV), and negative predictive value (NPV) in
the whole population and in subgroups defined by
eGFR(CG) status. Data were analyzed with Analyze-It
and SPSS version 18.
The reference population was between 18 and 62 (me-
Multivitamin or B vitamin supplements were used by
cell cobalamin determination. The mean (SD) of the
red cell cobalamin concentration in the 118 eligible
subjects was 97.2 (32.8) pmol/L. The 95% central ref-
erence intervals for serum holoTC, serum cobalamin
(both natural log–transformed), and red cell cobala-
min (not transformed) in the reference population
were determined parametrically (see online Supple-
mental Table 1). Using these data, the cutoffs for defi-
ciency were defined as 20 pmol/L for holoTC, 123
pmol/L for serum cobalamin, and 33 pmol/L for red
A description of the elderly population is pre-
sented in Table 1. Of the 700 participants in the study,
63–97) years. Vitamin B12– or folate-containing sup-
plements were used by 6%. The prevalences of low red
cell cobalamin, holoTC, and total cobalamin were
9.6%, 8.1%, and 8.0%, respectively. The prevalences
for abnormal MMA and tHcy were 41.7% and 52.2%,
respectively, and for serum and red cell folate, 1.7%
and 1.4%. There were strong positive correlations (see
online Supplemental Table 2) between red cell cobala-
Table 1. Descriptive characteristics of the elderly population.
2.5th to 97.5th
Women, n (%)
Red cell cobalamin, pmol/L
Serum total cobalamin, pmol/L
Serum folate, nmol/L
Red cell folate, nmol/L
Clinical Chemistry 57:6 (2011)
min and holoTC and cobalamin (r ? 0.63 and 0.51,
respectively). Red cell folate was also positively corre-
?0.45) and tHcy (r ? ?0.33), and there were no sig-
nificant associations with either creatinine (r ? 0.05)
or eGFR(CG) (r ? ?0.02).
Table 2 compares the means of test results and
other variables in individuals who had red cell cobala-
min concentrations above and below the cutoff of 33
pmol/L packed red cells. There were no significant dif-
ferences in age, creatinine, eGFR(CG), or hematocrit
globin (Hb) ?13 g/dL (130 g/L) for men and 12 g/dL
(120 g/L) for women, 38 of 66 (58%) and 266 of 633
(42%) were anemic in each group. All serum markers
of vitamin B12status as well as red cell folate were
highly significantly different (P ? 0.0001 for all), and
serum folate was also different (P ? 0.018).
We used the red cell cobalamin lower limit of 33
onstrate that holoTC is the best-performing indicator
in AUC between holoTC and serum cobalamin (0.80),
MMA (0.78), and tHcy (0.75) were significant (P ?
0.0002) (see Fig. 1).
MULTIPLE LINEAR REGRESSION
volume (MCV)]. The multiple regression model (see
Table 2. Mean values (95% CI) in participants with and without vitamin B12deficiency.a
Variablen DeficientNNondeficient 2-tailed P
Red cell cobalamin, pmol/Lc
Serum cobalamin, pmol/Lc
Serum folate, nmol/Lc
Red cell folate, nmol/Lc
82 (80 to 83)
0.38 (0.37 to 0.39)
12.0 (11.7 to 12.4)
90 (88 to 92)
23.4 (21.9 to 25.0)
18.2 (15.6 to 21.2)
139 (119 to 162)
23.5 (18.6 to 29.6)
712 (621 to 816)
0.651 (0.536 to 0.790)
22.5 (20.1 to 25.1)
85 (79 to 91)
48 (44 to 53)
81 (80 to 81)
0.39 (0.38 to 0.39)
12.5 (12.4 to 12.7)
92 (91 to 92)
71.7 (69.4 to 74.1)
51.4 (49.0 to 53.9)
270 (258 to 284)
30.8 (28.7 to 32.9)
1040 (998 to 1084)
0.325 (0.311 to 0.338)
15.7 (15.4 to 16.1)
90 (87 to 92)
48 (47 to 50)
aVitamin B12deficiency was defined as red cell cobalamin ?33 pmol/L packed red cells.
bFor SI value (g/L), multiply by 10.
ensitivity (true posit
Total cobalamin (pmol/L)
1 − Specificity (false positives)
Fig. 1. ROC plots for serum total cobalamin, holoTC,
and MMA for vitamin B12deficiency, defined as red
cell cobalamin <33 pmol/L.
Total sample size, 699; number of individuals B12deficient,
67. The AUC for holoTC (0.90) was significantly different
(P ? 0.0002) from those of both serum total cobalamin
(0.80) and MMA (0.78).
Accuracy of Indicators of Tissue B12Status
Clinical Chemistry 57:6 (2011)
online Supplemental Table 4) containing only log(ho-
loTC) accounted for 43.8% of the variance in red cell
cobalamin (r2? 0.44). The order of addition to the
model was log(red cell folate), MCV, log(MMA), log-
Age, weight, sex, creatinine, eGFR(CG), Hct, Hb, and
tHcy were not in the model. All except tHcy had a low
univariate r value, and tHcy was highly correlated with
other variables, especially MMA. The residuals for the
model with 6 variables were normally distributed
of the heterogeneity assumption.
PERFORMANCE OF SELECTED TESTS TO DIAGNOSE
The performance of the 3 best predictor markers
(holoTC, MMA, and serum cobalamin) to diagnose
vitamin B12deficiency in the elderly population at cut-
specificity at these cutoff concentrations.
By use of Bayes’s theorem, a pretest probability of
9.6%, desirable posttest probabilities of 60% (for
ruling-in deficiency) and 2% (for ruling-out defi-
ciency), a desirable positive likelihood ratio (LR?) of
14, and a negative likelihood ratio (LR?) of 0.23 were
deduced. The resulting analyte decision thresholds for
and proportion of samples that would fall into the re-
spective indeterminate zones were holoTC 13.7% (96/
699), serum total cobalamin 44.7% (313/700), and
MMA 49.8% (349/700).
Eighty-nine samples had holoTC values between 20
linear discriminant function (Wilks ? P ? 0.042), and
all 89 observations had a corresponding log(red cell
folate) value. Observations with red cell folate values
gory, correctly allocating 65% (sensitivity 65%, speci-
Using eGFR(CG) levels to categorize kidney function,
we then estimated the performance of holoTC, serum
total cobalamin, and MMA, at defined cutoffs, for de-
tecting vitamin B12deficiency. We found that the PPV
for diagnosing deficiency using the defined cutoffs was
higher for holoTC than for serum total cobalamin or
MMA regardless of kidney function (Table 5).
amin as an index of tissue B12stores. Our results dem-
all other indicators in this elderly population. Red cell
Table 3. Diagnostic performance of holoTC, serum cobalamin, and MMA at a single cutoff.
Table 4. Three-zone decision thresholds for holoTC, serum cobalamin, and MMA based on LR? of 14 and
LR? of 0.23.
Serum cobalamin, pmol/L
Clinical Chemistry 57:6 (2011)
cobalamin concentrations have previously been used
to indicate tissue vitamin B12levels (16, 28–30). Only
the immature red cells (reticulocytes) incorporate the
vitamin in a receptor-mediated process (31), and
therefore red cell cobalamin is a reflection of status in
ing a very early marker, since holoTC predicts 44% of
the variation in red blood cell cobalamin in the multi-
ple linear regression model. The model suggests that
red cell cobalamin has a direct relationship with ho-
loTC, red cell folate, MCV, and serum cobalamin and
an inverse relationship with MMA and serum folate.
The direction of the MCV and serum folate relation-
ships are perhaps unexpected, but MCV has low diag-
nostic value in the detection of vitamin B12deficiency
(32), and serum folate could be confounded by recent
intake or supplementation. Hb concentrations did not
enter the regression model.
The prevalence of low red cell cobalamin in this
loTC (8.1%) and serum total cobalamin (8.0%). In
contrast, and as reported by others (8), the prevalence
of abnormal MMA and tHcy was much higher
(?40%). In our elderly population, there was a good
correlation (r ? 0.63) between holoTC and red cell
cobalamin, similar to that shown by Tisman et al. (19)
for estimation of holoTC. We found an even stronger
correlation in our healthy reference population (r ?
There is some variation in the literature regarding
previously published estimates, but the lower limit of
the reference interval we established from these data
(33 pmol/L) was somewhat lower than previous pub-
lished estimates (16, 30). The reference interval esti-
mate for holoTC of 20–125 pmol/L was similar to that
published by Brady et al. (14) (19–134 pmol/L).
HoloTC was superior (P ? 0.0002) to both serum
total cobalamin and MMA for diagnosing tissue B12
deficiency in the ROC analysis, whereas MMA and se-
rum total cobalamin were essentially equivalent (AUC
0.90, 0.80, and 0.78, respectively). A previous study in
an elderly group concluded that holoTC had modestly
better diagnostic accuracy than total serum cobalamin
(33). This was based on using MMA as the reference
standard, which has known limitations in an elderly
population (11). Hematologic indicators of anemia
had the lowest AUC, consistent with reports that neu-
rological lesions may be more common among elderly
individuals (1, 2). Serum folate (0.61), red cell folate
(0.71), and tHcy (0.75) gave intermediate AUCs.
As with most diagnostic tests, there is not perfect
discrimination between disease and nondisease. Al-
though the performance of any of these 3 circulating
markers at a single cutoff may not be sufficient for
on a 3-zone partition with an intentional gray zone
60% would be sufficient to rule in the disease, on the
basis that the treatment is effective and safe, and that a
out deficiency. This approach also allows for a direct
Table 5. Performance of holoTC, serum cobalamin, and MMA to detect B12 deficiency in eGFR(CG) ranges.a
eGFR(CG) rangenPPV %NPV %Meanb
?60226 (225 for holoTC)
aVitamin B12deficiency was defined as red cell cobalamin ?33 pmol/L packed red cells. Cutoffs as in Table 3.
Accuracy of Indicators of Tissue B12Status
Clinical Chemistry 57:6 (2011)
For holoTC, around 14% of samples tested would fall
within this gray zone, whereas for serum total cobala-
min and MMA the proportions were 45% and 50%,
suggesting that these would not be useful tests under
It has been long recognized that the serum total
cobalamin assay has a wide gray zone. In a study of
the Framingham elderly population, a cutoff point
of 258 pmol/L was suggested (35). On the other
hand, many physicians react only to very low cobal-
pg/mL) (36). It is tempting to suggest that these are 2
is to maximize positive or negative predictive value. We
found that a serum total cobalamin cutoff of 79 pmol/L
as follows: ?133 pmol/L, 22.2%; ?118 pmol/L, 30%;
?96 pmol/L, 40%; and ?74 pmol/L, 50%, results very
Most reports comment that MMA concentrations
and we found that an MMA concentration of 0.31
diagnosis of B12deficiency with high probability would
not be reached until very high concentrations of MMA.
a diagnostic tool for vitamin B12deficiency in Denmark,
which found that physicians did not tend to react until
onstrate that serum total cobalamin and MMA provide
be initiated. For values ?30 pmol/L, no treatment is re-
test probability (for example using symptoms and his-
tory) may allow diagnosis using holoTC gray-zone
likelihood ratios. In this study, for holoTC values of 22,
24, and 26 pmol/L, these were 10.2, 7.6, and 5.9 for LR?
Of the biochemical markers, only red cell folate
concentrations were useful for further discrimination.
Observations with red cell folate values ?778 nmol/L
were allocated to the B12-deficient category, correctly
allocating 65% of observations (sensitivity 65%, speci-
ficity 65%). A possible explanation for this finding is
the “methylfolate trap” hypothesis (39). Whatever the
cause, this observation is consistent with reports that
60% of patients with pernicious anemia have low red
blood cell folate concentrations (40).
We partitioned all subjects according to kidney
function. The mean concentration of holoTC, serum
cobalamin, and MMA was increased in the lowest
eGFR(CG) range, but there was no evidence of com-
promised performance for holoTC or serum cobala-
min to detect true vitamin B12deficiency as kidney
function decreased. HoloTC was the best-performing
marker, and even in the most compromised individu-
PPV of MMA showed a steady decline (23%–20% to
12%) as renal function decreased, suggesting that the
diagnostic performance of MMA is, in part, con-
founded by renal function.
The strengths of this study are that all indicators
enough to give adequate power for AUC differences in
the ROC analysis.
Although the concentration of red cell cobalamin
probably depends on body content of vitamin B12, our
knowledge of other determinants is limited. Factors
that influence erythrocyte production may affect mea-
sured concentrations (16–18). However, we observe
ance in red cell cobalamin concentrations, providing
reassurance that the results are valid. It is possible that
the relationship between holoTC and red cell cobala-
min is greater than we report here in elderly individu-
als, since the relationship was considerably stronger in
our healthy reference population. We did not exclude
serum folate–deficient subjects from our multiple re-
uncertain in practice whether these subjects also were
cobalamin deficient. There were 12 serum folate–
deficient patients, 2 of whom had low red cell cobala-
min concentrations, and there were 4 observations in
the holoTC indeterminate zone with a serum folate
red cell cobalamin concentrations.
In conclusion, the results of the present study of
elderly individuals support the use of holoTC as the
first-line diagnostic procedure for vitamin B12status,
holoTC gray zone, the use of red cell folate concentra-
tions provides additional discriminative information
The practicality of such a strategy, however, would de-
pend on standardization of red cell folate determina-
tion and using suitably reliable methodology.
the intellectual content of this paper and have met the following 3 re-
quirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Clinical Chemistry 57:6 (2011)
uscript submission, all authors completed the Disclosures of Potential
Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: E. Valente, Scientific Marketing, Axis-
A.M. Molloy, Axis-Shield.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: The elderly participants in this study were re-
cruited as part of an ongoing observational cohort study funded by
the Irish Department of Agriculture, Fisheries and Food and the
(FHRI). J.M. Scott, Axis-Shield Diagnostics; A.M. Molloy, Axis-
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the
of data, or preparation or approval of manuscript.
Acknowledgments: The authors thank Karen Creevey and Re-
gina Dempsey for excellent technical assistance and Harry Staines
for constructive discussions and assistance with statistical
1. Carmel R. Pernicious anemia: the expected find-
ings of very low serum cobalamin levels, anemia
and macrocytosis are often lacking. Arch Intern
2. Lindenbaum J, Healton EB, Savage DG, Brust JC,
Garrett TJ, Podell ER, et al. Neuropsychiatric dis-
orders caused by cobalamin deficiency in the
absence of anemia or macrocytosis. N Engl J Med
3. Stabler SP, Allen RH, Savage DG, Lindenbaum
J. Clinical spectrum and diagnosis of cobalamin
deficiency. Blood 1990;76:871–81.
4. Healton EB, Savage DG, Brust CM, Garrett TJ,
Lindenbaum J. Neurologic aspects of cobalamin
deficiency. Medicine 1991;70:229–45.
5. Stabler SP, Lindenbaum J, Allen RH. Vitamin B12
deficiency in the elderly: current dilemmas. Am J
Clin Nutr 1997;66:741–9.
6. Carmel R. Mild transcobalamin I (haptocorrin)
deficiency and low serum cobalamin concentra-
tions. Clin Chem 2003;49:1367–74.
7. Solomon LR. Cobalamin-responsive disorders in
the ambulatory care setting: unreliability of co-
balamin, methylmalonic acid, and homocysteine
testing. Blood 2005;105:978–85.
8. Joosten E, van den Berg A, Riezler R. Metabolic
evidence that deficiencies of vitamin-B12(cobal-
amin), folate and vitamin-B6occur commonly in
elderly people. Am J Clin Nutr 1993;58:468–76.
9. Chanarin I, Metz J. Diagnosis of cobalamin
deficiency: the old and the new. Br J Haematol
10. Loikas S, Koskinnen P, Irjala K, Löppönen M,
Isoaho R, Kivela ¨ SL, Pelliniemi TT. Renal impair-
ment compromises the use of total homocysteine
and methylmalonic acid but not total vitamin B12
and holotranscobalamin in screening for vitamin
B12deficiency in the aged. Clin Chem Lab Med
11. Vogiatzoglou A, Oulhaj A, Smith DA, Nurk E,
Drevon CA, Ueland PM, et al. Determinants of
plasma methylmalonic acid in a large population:
implications for assessment of vitamin B12status.
Clin Chem 2009;55:2198–206.
12. Herzlich B, Herbert V. Depletion of serum holo-
transcobalamin II: an early sign of negative vita-
min B12balance. Lab Invest 1988;58:332–7.
13. Nexø E, Christensen AL, Hvas AM, Petersen TE,
Fedosov SN. Quantification of holotranscobala-
min, a marker of vitamin B12deficiency. Clin
14. Brady J, Wilson L, McGregor L, Valente E, Orning
L. Active B12: a rapid, automated assay for holo-
transcobalamin on the Abbott AxSYM analyzer.
Clin Chem 2008;54:567–73.
15. Retief FP, Gottlieb CW, Herbert V. Mechanism of
vitamin B12uptake by erythrocytes. J Clin Invest
16. Harrison RJ. Vitamin B12levels in erythrocytes in
normal subjects and in pernicious anaemia. J Clin
17. Harrison RJ. Vitamin B12levels in erythrocytes in
hypochromic anaemia. J Clin Pathol 1971;24:
18. Harrison RJ. Vitamin B12levels in erythrocytes in
anaemia due to folate deficiency. Br J Haematol
19. Tisman G, Vu T, Amin J, Luszko G, Brenner M,
Ramos M, et al. Measurement of red blood cell-
vitamin B12: a study of the correlation between
intracellular B12content and concentrations of
plasma holotranscobalamin II. Am J Hematol
20. Molloy AM, Scott JM. Microbiological assay for
serum, plasma, and red cell folate using cryopre-
served, microtiter plate method. Methods Enzy-
21. Kelleher BP, Walshe KG, Scott JM, O’Broin SD.
Microbiological assay for vitamin B12with use of
a colistin-sulfate-resistant organism. Clin Chem
22. Kelleher BP, O’Broin SD. Microbiological assay for
vitamin B12 performed in 96-well microtitre
plates. J Clin Pathol 1991;44:592–5.
23. Windelberg A, Arseth O, Kvalheim G, Ueland PM.
Automated assay for the determination of meth-
ylmalonic acid, total homocysteine, and related
amino acids in human serum or plasma by means
of methylchloroformate derivatization and gas
chromatography-mass spectrometry. Clin Chem
24. La’Ulu S, Rawlins ML, Pfeiffer CM, Zhang M,
Roberts WL. Performance characteristics of six
homocysteine assays. Am J Clin Pathol 2008;130:
25. Clarke R, Refsum H, Birks J, Evans JG, Johnston C,
Sherliker P, et al. Screening for vitamin B-12 and
folate deficiency in older persons. Am J Clin Nutr
26. Pfeiffer CM, Caudill SP, Gunter EW, Osterloh J,
Sampson EJ. Biochemical indicators of B vitamin
status in the US population after folic acid
fortification: results from the National Health and
Nutrition Examination Survey 1999–2000. Am J
Clin Nutr 2005;82:442–50.
27. Daly LE, Kirke PN, Molloy A, Weir DG, Scott JM.
Folate levels and neural tube defects: implica-
tions for prevention. JAMA 1995;274:1698–702.
28. Das KC, Manusselis C, Herbert V. Determination
of vitamin B12(cobalamin) in serum and erythro-
cytes by radioassay, and of holotranscobalamin II
(holoTC-II) and holohaptocorrin (holoTC-I and -III)
in serum by adsorbing holoTC II on microfine
silica. J Nutr Biochem 1991;2:455–64.
29. Herbert V. Staging vitamin B-12 (cobalamin) sta-
tus in vegetarians. Am J Clin Nutr 1994;59
30. Omer A, Finlayson NDC, Shearman DJC, Samson
RR, Girdwood RH. Erythrocyte vitamin B12activity
in health, polycythemia, and in deficiency of vi-
tamin B12and folate. Blood 1970;35:73–82.
31. Reteif FP, Gottlieb CW, Herbert V. Delivery of
Co57B12to erythrocytes from alpha and beta
globulin of normal, B12deficient, and chronic
myeloid leukemia serum. Blood 1967;29:837–51.
32. Oosterhuis WP, Niessen RW, Bossuyt PM, Sanders
GT, Sturk A. Diagnostic value of the mean corpus-
cular volume in the detection of vitamin B12defi-
ciency. Scan J Clin Lab Invest 2000;60:9–18.
33. Clarke R, Sherliker P, Hin H, Nexo E, Hvas A-M,
Schneede J, et al. Detection of vitamin B12defi-
ciency in older people by measuring vitamin B12
or the active fraction of vitamin B12, holotrans-
cobalamin. Clin Chem 2007;53:963–70.
34. Pauker SG, Kassirer JP. The threshold approach to
clinical decision making. N Engl J Med 1980;302:
35. Lindenbaum J, Rosenberg IH, Wilson PW, Stabler
SP, Allen RH. Prevalence of cobalamin deficiency
in the Framingham elderly population. Am J Clin
36. Matchar DB, McCrory DC, Millington DS, Feuss-
ner JR. Performance of the serum cobalamin as-
say for diagnosis of cobalamin deficiency. Am J
Med Sci 1994;308:276–83.
37. Hvas A-M, Nexo E. Diagnosis and treatment of
vitamin B12deficiency: an update. Haematologica
38. Hvas A-M, Vestergaard H, Gerdes LU, Nexo E.
Physicians’ use of plasma methylmalonic acid as
a diagnostic tool. J Intern Med 2000;247:311–7.
39. Shane B, Stokstad EL. Vitamin B12-folate interre-
lationships. Ann Rev Nutr 1985;5:115–41.
40. Chanarin I. The megaloblastic anaemias. Oxford:
Blackwell Scientific Publishing; 1979.
Accuracy of Indicators of Tissue B12Status
Clinical Chemistry 57:6 (2011)