What is the diagnostic workup for pernicious (vitamin B12 deficiency) anemia?

From the Guidelines

The workup for pernicious anemia involves a comprehensive approach starting with a complete blood count to identify macrocytic anemia, followed by specific laboratory tests to confirm the diagnosis, including measuring serum vitamin B12 levels and checking for anti-intrinsic factor antibodies 1. To evaluate anemia, there are two common approaches: morphologic and kinetic, with the morphologic approach characterizing anemia based on the mean corpuscular volume (MCV) reported in the initial CBC test, which can indicate microcytic, normocytic, or macrocytic anemia 1. Pernicious anemia is typically characterized by macrocytic anemia, with an MCV greater than 100 fL, indicating vitamin B12 or folate deficiency caused by insufficient uptake or inadequate absorption through lack of intrinsic factor 1. Key laboratory tests for diagnosing pernicious anemia include:

• Measuring serum vitamin B12 levels, which are typically low (<200 pg/mL)
• Checking methylmalonic acid and homocysteine levels, which are elevated in B12 deficiency
• Testing for anti-intrinsic factor antibodies (highly specific for pernicious anemia) and anti-parietal cell antibodies (sensitive but less specific) A peripheral blood smear may show hypersegmented neutrophils and macrocytes, and in some cases, a bone marrow examination might be necessary to rule out other causes of macrocytic anemia 1. Additional tests should include iron studies, folate levels, thyroid function tests, and liver function tests to exclude other potential causes, and once diagnosed, patients should be evaluated for associated autoimmune conditions like thyroid disorders and type 1 diabetes 1. Gastroscopy with biopsy may be performed to assess for atrophic gastritis, which commonly accompanies pernicious anemia due to the autoimmune destruction of gastric parietal cells that produce intrinsic factor necessary for B12 absorption.

From the FDA Drug Label

Hematocrit, reticulocyte count, vitamin B12, folate and iron levels should be obtained prior to treatment. If reticulocytes have not increased after treatment or if reticulocyte counts do not continue at least twice normal as long as the hematocrit is less than 35%, diagnosis or treatment should be reevaluated. Repeat determinations of iron and folic acid may reveal a complicating illness that might inhibit the response of the marrow To work up pernicious anemia, the following steps should be taken:

• Obtain hematocrit, reticulocyte count, vitamin B12, folate, and iron levels prior to treatment 2
• Monitor reticulocyte counts daily from the fifth to seventh days of therapy and then frequently until the hematocrit is normal
• Reevaluate diagnosis or treatment if reticulocytes have not increased after treatment or if reticulocyte counts do not continue at least twice normal as long as the hematocrit is less than 35%
• Repeat determinations of iron and folic acid to reveal any complicating illness that might inhibit the response of the marrow 2

From the Research

Diagnostic Approach

To work up pernicious anemia, the following steps can be taken:

• Perform a full blood count (FBC) to investigate anemia, as the onset and progression of pernicious anemia can be insidious and may not always present with noticeable symptoms 3
• Check serum levels of vitamin B12, as pernicious anemia is characterized by cobalamin deficiency 3, 4, 5, 6
• Detect autoantibodies against intrinsic factor and gastric parietal cells antigen H+/K+ ATPase, which can aid in diagnosis 4, 6
• Consider endoscopic and histological signs of gastritis, as pernicious anemia is associated with atrophic body gastritis 4, 6
• Evaluate for indirect signs of cobalamin deficiency, such as elevated homocysteine and methylmalonic acid, and signs of dyserythropoiesis on the bone marrow aspirate 5

Diagnostic Challenges

• The detection of vitamin B12 levels can be challenging due to interference from intrinsic factor antibodies, which can lead to false normal results 5
• The lack of reliable cobalamin assays and the presence of clinical mimics can make diagnosis difficult 3
• The combined sensitivity and specificity of autoantibodies against gastric parietal cell H+/K+ ATPase and intrinsic factor can aid in diagnosis, but may not be 100% accurate 4

Laboratory Tests

• Multiplex Luminex assay can be used to detect autoantibodies against H+/K+ ATPase and intrinsic factor 4
• Electrochemiluminescence can be used to measure vitamin B12 levels, which is less prone to interference from intrinsic factor antibodies 5
• Protein G SepharoseTM can be used to adsorb immunoglobulins in the sample, confirming the interference of the cobalamin assay with autoantibodies 5