TO THE EDITOR: Measurement of vitamin B12 levels is the gold standard for the diagnosis of vitamin B12 deficiency. In current practice, total serum vitamin B12 measurements are performed in the clinical laboratory with competitive binding luminescence assays, the results of which may not always accurately reflect actual vitamin B12 stores [1]. Here, we report a case in which a competitive binding luminescence assay led to a falsely increased vitamin B12 result for a patient presenting with classic hematologic and biochemical features of pernicious anemia.

A 45-year-old woman presented with complaints of nausea, weight loss, fatigue, and dizziness that was present for 1 month without any known systemic disease. She ate a good, well-balanced diet and was not taking any medication. On clinical examination, pallor was the only significant finding. Laboratory examination results are shown in Table 1. In the peripheral blood smear, anisopoikilocytosis, macroovalocytes, rare schistocytes, teardrop forms, microspherocytes, and hypersegmented neutrophils were observed. She received four units of red blood cells within a 1-month period. Bone marrow examination showed markedly hypercellular marrow with marked erythroid hyperplasia and megaloblastic hemopoiesis. The hematologic and biochemical features of the blood test results were inconsistent with the diagnosis of any disease.

The assays for vitamin B12 were performed in our laboratory using the UniCelR DxI 800 Cbl assay (Beckman Coulter, Brea, CA, USA), and another assay was performed in a different laboratory using the Elecsys E170 Cbl assay (Roche Diagnostics Corp, Indianapolis, IN, USA). Despite high vitamin B12 levels in repeated assays owing to a very strong suspicion of pernicious anemia, further investigation was performed to establish vitamin B12 deficiency (Table 1), and parenteral vitamin B12 replacement was initiated. Cyanocobalamin was administered by intramuscular injection at an initial dose of 1,000 mcg once per day for 1 week and followed by 1,000 mcg once per week. Approximately 2 weeks after the supplementation was initiated, clinical and hematological recovery was observed (Fig. 1).

Holotranscobalamin (holo-TC), also known as active B12, is the only form of vitamin B12 that is taken up and used by the cells in the body. It accounts for approximately 10% of the circulating vitamin B12 and is the earliest marker showing vitamin B12 depletion [1,2].

Vitamin B12 deficiency is generally suspected based on related symptoms, clinical findings, and laboratory results and is confirmed by measuring vitamin B12 levels. However, current vitamin B12 measurement methods may miss the lack of vitamin B12 in some cases. These methods, based on competitive binding luminescence assays, have been used since 1990. The assay uses binding to intrinsic factor (IF) following dissociation from the binding proteins, with a readout based on the remaining amount of unbound IF. The main problem with these assays is caused by the presence of IF antibodies in the test sample. IF antibodies may bind the test IF reagent and if there is a failure in the denaturation step intended to denature IF-blocking antibodies, spuriously normal or increased vitamin B12 levels can be measured [3,4]. Low vitamin B12 levels can be measured as false normal or false high, especially in pernicious anemia, due to excessive amounts of anti-intrinsic factor antibodies present in the serum [5,6,7].

In the light of data from the available literature, a normal or high vitamin B12 measurement does not exclude vitamin B12 deficiency in cases when vitamin B12 deficiency is suspected. In such an instance, holo-TC and/or metabolic tests, such as homocysteine or methylmalonic acid, may be considered for patients for whom there is a high suspicion of pernicious anemia in the absence of a low vitamin B12 level. Additionally, an alternate approach involves providing vitamin B12 treatment and confirming or eliminating vitamin B12 deficiency according to the response status.

Hematological recovery with supplementation of vitamin B12. ^a)2U of red blood cells replacement. ^b)Supplementation of vitamin B12 initiated.

Table 1 Laboratory findings of the patient before injection of vitamin B12.