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From www.bloodjournal.org by guest on January 12, 2015. For personal use only.
1853
CORRESPONDENCE
DIAGNOSIS OF COBALAMIN DEFICIENCY
To the Editor:
I read with interest the article by Stabler et al’ in which they
described the efficacy of cobalamin therapy in patients with low
serum BIZlevels. Of the 145 patients whom they were able to
follow- up, 59% responded in one or more ways to the therapy, but
it seemed that there was no one single parameter measured that
could predict which patients would respond and which would not.
We have recently begun measuring the biologically important
vitamin B,, carried by transcobalamin I1 (TC 11-B,,), along with
total serum B,,, in all patients referred for estimation of B1, and/or
folate levels, using a modification of the method of Jacob and
Herbert.*In our series, approximately 50% (19 of 37) of all patients
with low total serum B,, ( < 140 pmol/L) also had low or marginally
low levels of TC II-B,z ( < 10 pmoliL).
It may well be that the 59% of Stabler’s patients who responded
to therapy were those in whom a low TC II-B12would have been
found if measured.
Like Stabler et al,’ we are aware of the limitations of the
precision of the assay. Where possible, assays are repeated in those
patients in whom low values of TC 11-B,, are detected. However, at
the time of writing, we have no information on the effect of therapy
on our patients.
However, contrary to the comments of Stabler et all that “(with
few exceptions) all patients with cobalamin deficiency have low
values,” in our series 12% (36 of 309) of patients with normal total
serum B,, had a low or marginally low TC II-B,*.Further, 75% (46
of 74) of patients with low TC 11-B,, had a normal or even high total
Blp The possibility that these patients (all of whom are referred
because of showing one or more symptoms that may be attributed
to the lack of cobalamin) are truly deficient in the vitamin would
not be considered if only the total serum B,, was measured.
ANNE GRIFFITHS
Haematology Department
Royal Hobart Hospital
Hobart, Tasmania
Australia
REFERENCES
1. Stabler S, Allen R, Savage D, Lindenbaum J: Clinical speccyte-related” (TC I and TC 111) and “liver-related” (TC 11) B12
trum and diagnosis of cobalamin deficiency. Blood 762371,1990
binders by instant batch separation using a microfine precipitate of
2. Jacob E, Herbert V Measurement of unsaturated “granulosilica (QUSO G32). J Lab Clin Med 86505,1975
From www.bloodjournal.org by guest on January 12, 2015. For personal use only.
CORRESPONDENCE
1854
RESPONSE
To the Editor:
Dr Griffiths and other investigators' have postulated that measurements of transcobalamin 11-Cbl (TC 11-Cbl) may provide a
better separation of normal subjects and Cbl-deficient patients
than do current measurements of serum total Cbl, which includes
TC I-Cbl, TC 11-Cbl, and TC 111-Cbl. Although this hypothesis is
attractive from a theoretical viewpoint, we do not believe that it is
valid.
A major problem with measurements of serum TC 11-Cbl
concerns the small amount and percentage of Cbl bound to TC I1
and the small but significant imprecision of measuring serum Cbl
where coefficients of variation for replicate assays are in the range
of 5% to 10%. Thus, if one assays the total Cbl in a particular
normal serum sample and obtains a value of 500 pgimL (369
pmolk), one would expect to get values frequently in the range of
450 (332 pmol/L) to 550 pg/mL (406 pmoVL) if the assay were
repeated. Measurements of TC 11-Cbl are obtained indirectly by
adding Quso to serum to adsorb and remove TC 11-Cbl followed by
repeating the standard Cbl assay on the residual serum. If, in our
example with the normal serum, the result is ~ 4 8 p@mL
6
(1359
pmol/L), the subject would be considered to have a normal level of
TC 11-Cbl of 2 1 4 pg/mL ( 2 1 0 pmolk); whereas, if the repeat
value was >486 pg/mL (> 359 pmoVL), the patient would be
considered as having a low value for TC 11-Cbl of < 14 pg/mL ( < 10
pmol/L). Repeat values > 500 pgimL (> 369 pmol/L) are reported
as Ovalues for TC 11-Cbl because negative values have no meaning.
Because the critical level of 486 pg/mL (359 pmol/L) is well within
the range of imprecision, one cannot expect to measure accurately
levels of TC 11-Cbl using this approach. The percentage of Cbl
bound toTC I1 may increase as the total serum Cbl level decreases,
but the imprecision of the assay also increases in a similar manner.
Several years ago, in unpublished studies, one of us (R.H.A.)
used a different technique to measure the amount of TC 11-Cbl in
human serum. The sensitivity of the Cbl assay was increased
approximately sevenfold by using a '"I-labeled Cbl analogue that is
still bound to intrinsic factor with high affinity. Human TC 11-Cbl
was adsorbed from serum using goat antirabbit TC I1 coupled to
Sepharose. The TC 11-Cbl was then eluted at low pH, taken to
dryness by vacuum centrifugation, and assayed directly. With this
approach, it was possible to further increase the sensitivity of the
assay by measuring the TC 11-Cbl present in 0.5 to 1.0 mL of serum
rather than the 0.1 to 0.2 mL used in standard Cbl assays. Using
these methods, it was discovered that the following additional
problems exist with assaying TC 11-Cbl in human serum: (1) Cbl
bound to TC I1 is unstable and is slowly transferred to unsaturated
TC I and TC 111 when serum is stored at 37°C or room temperature
for several hours; and (2) TC 11-Cbl is cleared from serum
pre3umably by white cells, platelets, and red cells after blood has
been drawn and before the cells are removed. Even when these
problems were circumvented by placing blood on ice and centrifuging immediately after collection and keeping the serum on ice, it
was not possible to show that measurements of serum TC 11-Cbl
provided a better separation between normal subjects and Cbldeficient patients than did measurements of total serum Cbl.
In summary, we do not believe that current techniques are
adequate for measuring TC 11-Cbl in serum and believe it is
extremely unlikely that such measurements will provide useful
diagnostic information even if proper assays are used.
SALLY P. STABLER
ROBERT H. ALLEN
Department of Medicine
Universityof Colorado Health Sciences Center
Denver, CO
DAVID SAVAGE
JOHN LINDENBAUM
Department of Medicine
Columbia University
New York. NY
REFERENCE
1. Herbert V, Fong W, Gulle V, Stopler T: Low holotranscoblamin I1 is the earliest serum marker for subnormal vitamin B12
(cobalamin) absorption in patients with AIDS. Am J Heniato
34:132,1990
From www.bloodjournal.org by guest on January 12, 2015. For personal use only.
1991 77: 1853-1854
Diagnosis of cobalamin deficiency [letter; comment]
A Griffiths
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