International Journal of Laboratory Hematology (Blackwell) Volume 36 Issue 4 2014 [Doi...

8/19/2019 International Journal of Laboratory Hematology (Blackwell) Volume 36 Issue 4 2014 [Doi 10.1111_ijlh.12158] Topra…

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Vitamin B12 and folate deficiency: should we use a different

cutoff value for hematologic disorders?

B. TOPRAK, H. Z. YALCIN, A. COLAK

Department of Clinical

Biochemistry, Tepecik Training

and Research Hospital, Izmir,

Turkey

Correspondence:

Burak Toprak, Department of

Clinical Biochemistry, TepecikTraining and Research Hospital,

Gaziler Cad., No:468, Yenisehir,

Izmir 35183, Turkey.

Tel.: +90-232-4696969;

Fax: +90-232-4330756;

E-mail: [email protected]

doi:10.1111/ijlh.12158

Received 27 August 2013;

accepted for publication 12 Sep-

tember 2013

Keywords

Anemia, B12, cutoff value,

folate, macrocytosis

S U M M A R Y

Introduction: Anemia and macrocytosis are well-defined expected

hematologic findings of vitamin B12 and folate deficiency; how-

ever, some previous studies did not show a significant association

of subnormal B12 with anemia and macrocytosis.

Methods: We retrospectively analyzed 17 713 laboratory patientrecords to evaluate vitamin B12 and folate levels in relation to

anemia and macrocytosis.

Results: In an age- and sex-adjusted logistic regression model, low

B12 status but not marginal B12 status was significantly associated

with anemia [ORs respectively, 1.291 (95% CI, 1.182 – 1.410),

1.022 (95% CI, 0.943 – 1.108)] and macrocytosis [ORs, respectively,

3.853 (95% CI, 3.121 – 4.756), 1.031 (95% CI, 0.770 – 1.381)]. Also

low folate status but not marginal folate status was significantly

associated with anemia [adjusted ORs, respectively, 1.819 (95% CI,

1.372 – 2.411), 1.101 (95% CI, 0.931 – 1.301)] and macrocytosis

[adjusted ORs, respectively, 2.945 (95% CI, 1.747 – 4.965), 1.228

(95% CI, 0.795 – 1.898)].

Conclusion: Our results show that increased anemia and macrocyto-

sis are observed at values below commonly used B12 lower-refer-

ence thresholds. Determining a hematologic cutoff value may help

physicians in clinical practice.

IN T R O D U C T IO N

Vitamin B12 and folate are important cofactors in the

DNA synthesis. Deficiency of both of these water solu-

ble vitamins is frequent health problems particularly

in the elderly [1, 2]. Common causes of cobalamin

deficiency are food-cobalamin malabsorbtion, perni-

cious anemia, and dietary deficiency [3 – 6]. A number

of biomarkers have been used for cobalamin defi-

ciency diagnosis, including serum cobalamin,

methylmalonic acid (MMA), homocystein, and holo-

transcobalamin II. The most common biomarker used

for diagnosis is serum cobalamin. Older studies used

lower cobalamin cutoff values


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therefore, usage of higher cobalamin cutoff values

was suggested by some authors for improvement of

sensitivity [9]. Vitamin B12 deficiency is associated

with hematologic and neurologic abnormalities.

Hematologic manifestations of cobalamin deficiencyinclude anemia, macrocytosis, pancytopenia and neu-

trophil hypersegmentation [10 – 14]. The effect of

cobalamin deficiency on hematopoiesis is documented

in the literature [15, 16], and the association between

pernicious anemia and cobalamin deficiency is well

known, but studies about hematologic response to

B12 treatment is inconsistent [10, 17 – 24]. Some stud-

ies failed to demonstrate a significant hematologic

response to cobalamin therapy [17 – 22].

The reasons for folate deficiency are reduced

intake, malabsorbtion, increased metabolism, and

increased requirements. Folate deficiency is associated

with megaloblastic anemia mood disorders and neural

tube defects [25]. The lower limit of the reference

range for serum folate is usually set at about 6.8 nM

(3 ng/mL) [26]. Some studies defined 200 pg/mL). Folate

levels were classified as low (≤2.2 ng/mL), marginal(2.2 – 3 ng/mL) and normal (>3 ng/mL). World Health

Organization criteria (hemoglobin 200 pg/mL (148 pM) and folate >3 ng/mL

(6.8 nM) were considered as reference and age- and

gender-adjusted ORs of marginal and low b12, and

folate status for anemia and macrocytosis was esti-

mated. To investigate the effect of age on vitamin B12

and folate deficiency, age groups (18 – 50, 51 – 60,

61 – 70, 71 – 80, 80 – 85) were generated. ORs of age

groups for low cobalamin and folate status were esti-

mated using logistic regression analyses. The age

group 18 – 50 was reference.

RESULTS

A total of 17 713 vitamin B12, folate, hemoglobin,

and MCV results were investigated. There were12 870 female and 4843 male subjects. The results

show that 14.7% of the subjects have vitamin

B12 ≤ 150 pg/mL (111 pM) and 34.1% have vitamin

B12 ≤ 200 pg/mL (148 pM). Only 1.1% of the subjects

have folate ≤2.2 ng/mL (5 nM) and 4.7% have folate

≤3.0 ng/mL (6.8 nM). There were 35% anemic sub-

jects, and 2.5% of the subjects have macrocytosis

(Table 1).

In an age- and sex-adjusted logistic regression

model, low B12 status but not marginal B12 status

was significantly associated with anemia [ORs, respec-

tively, 1.291 (95% CI, 1.182 – 1.410), 1.022 (95% CI,

0.943 – 1.108)] and macrocytosis [ORs, respectively,

3.853 (95% CI, 3.121 – 4.756), 1.031 (95% CI, 0.770 –

1.381)]. Low B12 status and marginal B12 status were

significantly related to male sex [age-adjusted ORs,

respectively, 1.300 (95% CI, 1.183 – 1.427), 1.134

(95% CI, 1.041 – 1.237)] (Table 2).

Also low folate status but not marginal folate status

was significantly associated with anemia [adjusted

© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 409–414

410 B. TOPRAK, H. Z. YALCIN AND A. COLAK | VITAMIN B12 AND FOLATE DEFICIENCY


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ORs, respectively, 1.819 (95% CI, 1.372 – 2.411), 1.101

(95% CI, 0.931 – 1.301)] and macrocytosis [adjusted

ORs, respectively, 2.945 (95% CI, 1.747 – 4.965), 1.228

(95% CI, 0.795 – 1.898)]. Both low and marginal folate

status were related to male sex [age-adjusted ORs,

respectively, 2.026 (95% CI, 1.720 – 2.386), 1.804

(95% CI, 1.350 – 2.409)] (Table 3).

Age over 70 was significantly associated with low

vitamin B12 status [ORs, 1.338 (95% CI, 1.183 –

1.513) for 71 – 80 age group, 1.500 (95% CI, 1.221 –

1.843) for 81 – 85 age group] and low folate status

[ORs, 1.725 (95% CI, 1.187 – 2.507) for 71 – 80 age

group, 2.705 (95% CI, 1.606 – 4.556) for 81 – 85 age

group] (Table 4).

D IS C U S S IO N

In this study, we evaluated vitamin B12 and folate

levels in relation to anemia and macrocytosis which

are major hematologic manifestations of vitamin B12and folate deficiency. Our data showed that low b12

status was significantly associated with increased ane-

mia and macrocytosis, but marginal B12 status was

not associated with increased anemia and macrocyto-

sis. Although it is well known that vitamin b12 defi-

ciency causes pernicious anemia, observational

studies reported inconsistent results about vitamin

b12 deficiency and anemia relation [30 – 33]. More-

over, intervention studies investigating response to

vitamin b12 treatment reported conflicting results

[17 – 21, 23, 24]. Some of the studies did not demon-

strate a significant hemoglobin increase and MCV

decrease to vitamin b12 therapy. The data of previ-

ous intervention studies show that subjects who

responded to vitamin b12 treatment with an eleva-

tion of hemoglobin or decrease of MCV have lower

vitamin b12 levels than nonresponders [17, 19, 23].

This evidence is consistent with our finding. Mar-

ginal vitamin B12 status (151 – 200 pg/mL) has very

limited nonsignificant effect on anemia and macrocy-

tosis. This suggests that the appropriate vitamin B12

cutoff value to expect hematologic consequences as

anemia or macrocytosis is lower than the frequentlyused cutoff 200 pg/mL (148 pM). This also may

explain the inconsistency between intervention stud-

ies. Studies that included subjects with very low

Table 2. Anemia and macrocytosis in the three vitamin B12 groups

Vitamin B12 status

Normal

(n = 11 675)

Marginal

(n = 3439)

Low

(n = 2599)

(%) (%) OR (95% CI) P (%) OR (95% CI) P

Male 26.1 28 1.134 (1.041 – 1.237)* 0.004 32.1 1.300 (1.183 – 1.427)†


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vitamin b12 levels demonstrated significant response

to cobalamin therapy. Possibly subjects with subnor-

mal or marginal vitamin b12 levels did not showed

any hematologic response to therapy and caused

some studies to report nonsignificant results. Diag-

nosing vitamin B12 deficiency may be a complicated

issue in some patients. Anemia and macrocytosis are

often absent in cobalamin deficiency; in addition,

deficiency may be subtle or only with minor

neuropsychiatric symptoms [34–36]. MMA and hom-

ocystein are sensitive biomarkers and may be

increased in many patients with normal and subnor-

mal vitamin B12 concentrations. Using a higher cut-

off (>200 pg/mL) value for defining deficiency is

useful for diagnosing patients with neuropsychiatric

disorders or elevated MMA and homocystein concen-

trations, but we think that the cutoff value for he-

matologic disorders should be different and lower

from the value which is useful for diagnosing subtle

deficiency states. Anemia is a frequently encountered

problem in medical practice. The major causes of

anemia are iron deficiency and chronic inflammation

[37]. To determine whether anemia is caused by

cobalamin deficiency or not may be problematic. In

clinical practice, a subject with anemia and subnor-

mal vitamin B12 concentrations may be regarded as

vitamin b12 deficiency anemia although the underly-

ing cause was different in many cases. Using a

higher cutoff value may lead to an overdiagnosis of

vitamin B12 deficiency and an increase in the num-

ber of misdiagnosed anemic patients. We think that

laboratories should provide a hematologic cutoff

value to expect anemia and macrocytosis and a dif-

ferent higher cutoff value for subtle or neurologic

deficiency states. Cascade testing was proposed by

some authors [19, 26]. We agree with cascade testing

Table 3. Anemia and macrocytosis in the three folate groups

Folate status

Normal(n = 16 872)

Marginal

(n = 640)

Low

(n = 201)

(%) (%) OR (95% CI) P (%) OR (95% CI) P

Male 26.6 42 2.026 (1.720 – 2.386)*


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approach, but that approach did not define a distinct

hematologic cutoff value.

Low folate status but not marginal folate status was

significantly associated with anemia and macrocytoses.

In the present study, odds ratios of marginal folatestatus for anemia and macrocytosis were higher than

marginal vitamin b12 status. Intervention studies

about hematologic response to folate therapy in sub-

jects with subnormal folate concentrations are limited.

We think that there is not enough evidence to deter-

mine a hematologic cutoff for folate deficiency. The

usage of higher folate cutoff values may improve

sensitivity to detect deficiency.

We found that age over 70 was significantly associ-

ated with low vitamin B12 and folate status. Our

results are consistent with several studies which

reported that vitamin b12 deficiency and folate

deficiency were related with increasing age [1, 2, 38].

This retrospective study has some limitations.

Firstly, medical history of the subjects was not avail-

able; therefore, confounding factors may exist. Our

results reflect an outpatient population, and it is not

representative of general population. Biomarkers as

homocystein and MMA which can be used to distin-

guish between deficiency and nondeficiency states

were not available in our study. We were not able to

evaluate the association of these biomarkers with ane-mia and macrocytosis when used together with vita-

min B12 and folate. Lack of standardization between

B12 and folate assays is another limitation which

makes difficult to use a uniform reference value.

As a conclusion, current study that is based on a

large data set suggests that vitamin B12 relation with

anemia and macrocytosis should be reconsidered.

Determining a hematologic cutoff value may help

physicians in clinical practice.

A U T H O R C O N T R IB U T IO N SBT designed the study and wrote the paper. HZY

obtained data, drafted the paper, and provided critical

revisions. AC substantially contributed to analyses and

interpretation of the data.

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