Laboratory testing for cobalamin deficiency in megaloblastic anemia

J_ID: JOH Customer A_ID: JOH23421 Cadmus Art: JOH23421 Ed. Ref. No.: 13-0043.R1 Date: 5-March-13 Laboratory testing for cobalamin deficiency in megaloblastic anemia Cobalamin (vitamin B12) deficiency is a common cause of megaloblastic anemia in Western populations.
Laboratory evaluation of megaloblastic anemia frequently includes the assessment of patient cobalaminand folate status. Current total serum cobalamin measurements are performed in the clinical laboratorywith competitive binding luminescence assays, whose results may not always accurately reflect actualcobalamin stores. Surrogate markers of cobalamin deficiency such as methylmalonic acid and homocys-teine have been utilized to improve diagnostic accuracy; however, the specificity of these tests by them-selves is rather low. Measurement of the biologically active fraction of cobalamin, holotranscobalamin, hasbeen proposed as a replacement for current total cobalamin assays. Although holotranscobalamin meas-urements appear to have slighter better sensitivity, the specificity of this assay remains to be determined.
The relative merits and demerits of commonly available methods to assess cobalamin deficiency in patientswith suspected megaloblastic anemia are discussed. Am. J. Hematol. 00:000–000, 2013.
cytoplasm, cobalamin is a cofactor for methionine synthe- Megaloblastic anemia is characterized by distinctive tase, which catalyzes the reduction of homocysteine (HCY) hematopoietic cell morphology, ineffective hematopoiesis, to methionine. Deficiency of cobalamin results in inhibition and is frequently mediated by underlying biochemical defi- of methionine synthetase activity and an increase HCY lev- ciencies in cobalamin and/or folate. Megaloblastic anemia els. In mitochondria, cobalamin is a required cofactor in the can also result from congenital disorders (e.g., orotic acidu- methylmalonyl CoA mutase-catalyzed production of succi- ria, Lesch–Nyhan syndrome, and congenital dyserythro- nyl CoA from methylmalonyl CoA. Deficiencies of cobala- poietic anemia), as a consequence of myelodysplastic min will lead to increased levels of methylmalonic acid syndrome, or from acquired disorders of DNA synthesis (MMA) owing to a block at this step. As serum levels of seen in the settings of chemotherapy. It is important to MMA and HCY are increased with cobalamin deficiency, note that megaloblastic anemia is a morphologic diagnosis these metabolites are utilized clinically as surrogate based on the cytologic and histologic features seen on the peripheral smear, bone marrow aspirate, and bone marrow Ultimately, cobalamin deficiency leads to inhibited con- core biopsy. However, as cobalamin deficiency is a com- version of deoxyuridine monophosphate to deoxythymidine mon cause of megaloblastic anemia in Western popula- monophosphate. The resulting elevated deoxyuridine tri- tions, a biochemical diagnosis of megaloblastic anemia phosphophate (dUTP) levels lead to misincorporation of owing to cobalamin deficiency based on the results of clini- dUTP into nascent DNA. Normally, DNA uracil glycosylase cal chemistry assays, without correlative bone marrow eval- excises dUTP residues from nascent DNA strands, but uation, frequently dictates therapy choices. This article will because there is no deoxythymidine triphosphate available review the various clinical laboratory assays utilized to eval- for replacement, DNA strand breaks occur and there can uate cobalamin deficiency and present their potential pit- be significant DNA fragmentation. Presumably, this is the falls in the assessment of megaloblastic anemia.
biochemical underpinning of the morphologic features seenin the nuclei of hematopoietic precursors that are diagnos-tic of megaloblastic anemia.
Ineffective DNA synthesis in hematopoietic progenitor cells is the underlying mechanism that leads to megalo- Clinical Consequences of Cobalamin Deficiency blastic anemia. The consequent dyssynchrony between Cobalamin deficiency results in both neuropsychiatric nuclear and cytoplasmic development is most apparent in and hematological deficits (for review, see [1,2]). A classic Wright-stained hematopoietic precursors from bone marrow and specific finding in patients with advanced cobalamin aspirates. Despite peripheral cytopenias, the bone marrow deficiency is subacute combined degeneration of the dorsal is hypercellular, frequently with a relative erythroid hyper- and lateral spinal columns owing to myelinopathy of these plasia. Erythroid precursors have nuclei that are larger than neural tracts. These changes are irreversible. Other normal and appear immature relative to cytoplasmic devel-opment with open sieve-like nuclear chromatin patterns. Inthe neutrophil lineage, giant band nuclei are characteristic.
Department of Pathology and Laboratory Medicine, University of Wisconsin The development of megakaryocytes is also affected and School of Medicine and Public Health, Madison, WI reflected by peripheral thrombocytopenia. Morphologically *Correspondence to: David T. Yang, Department of Pathology and Labora- characteristic findings in the peripheral blood include mac- tory Medicine, University of Wisconsin School of Medicine and Public roovalocytic anemia with anisopoikilocytosis and hyperseg- Health, Box 8550 Clinical Science Center, 600 Highland Avenue, Madison,WI 53792. E-mail: dtyang@wisc.edu Received for publication 16 January 2013; Revised 8 February 2013;Accepted 14 February 2013 Biochemical Consequences of Cobalamin Deficiency Cobalamin is an obligate cofactor for two specific intra- cellular metabolic reactions required to produce the basic http://wileyonlinelibrary.com/cgi-bin/jhome/35105 Path: N:/3b2/JOH#/Vol00000/130042/APPFile/JW-JOH#130042 J_ID: JOH Customer A_ID: JOH23421 Cadmus Art: JOH23421 Ed. Ref. No.: 13-0043.R1 Date: 5-March-13 affected neurological functions can include cerebellar changes that result in ataxia and cognitive decline. Neuro- There is no universally agreed upon gold standard assay logical manifestations of cobalamin deficiency may occur in for determining cobalamin levels in humans. In fact, surro- the absence of hematological findings. As cobalamin defi- gate biomarkers (MMA and HCY) of cobalamin deficiency ciency progresses, peripheral cytopenias in all three line- are widely utilized in clinical medicine to improve diagnostic ages become increasingly pronounced and manifest the sensitivity, despite poor specificity. Elevated levels of MMA unique morphologic features that are described above.
are often utilized as a gold standard with which to comparenew cobalamin testing platforms despite published data which show that increased levels of MMA by themselves As cobalamin is not synthesized by plants, humans do not necessarily correlate with clinically evident cobala- depend chiefly on foods of animal origin to maintain adequate cobalamin stores. To ensure efficient absorption Historically, the first widely used clinical assay for cobala- of cobalamin, our gut has a complex process that includes min was a microbiologic assay. This assay utilized strains (1) freeing cobalamin from food by proteases and acids, Lactobacillus leichmannii or Euglena gracilis that depended (2) binding free cobalamin with salivary haptocorrin in the on exogenously added cobalamin for growth. Cobalamin stomach, (3) digestion of haptocorrin in the proximal small from a patient serum sample was extracted and incubated bowel and transfer of cobalamin to intrinsic factor (IF) with the bacterium and growth was proportional to the secreted by gastric parietal cells, (4) attachment of cobala- amount of cobalamin present. These assays suffered from min–IF to specific receptors in the ileum, and (5) endo- several drawbacks including an extended incubation time of cytosis of the cobalamin–IF complex followed by release several days, bacterial growth that could be affected by a number of interferences such as antibiotics [7], and the fact Once absorbed in the ileum, cobalamin is largely bound that microbiological assays are difficult to standardize to two proteins in the serum. In total, 70–90% is biologically inactive and bound to haptocorrin (transcobalamin I), Subsequently, a radiodilution assay was developed and whereas the remainder is bound to transcobalamin II and widely adopted in the 1970s. Here, cobalamin was termed holotranscobalamin. Transcobalamin II is required extracted from patient serum, converted to cyanocobala- for B12 transport to cells and congenital deficiency of trans- min, and then mixed with radiolabeled57cyanocobalamin.
cobalamin II results in the typical neurologic and hemato- The level of radiolabeled cyanocobalamin binding to puri- logic findings seen in cobalamin deficiency [3]. Haptocorrin fied IF was measured, and from this, the amount of patient deficiency does not appear to result in clinically apparent cyanocobalamin was calculated. This test also suffered cobalamin deficiencies [4]. Transcobalamin II–cobalamin several limitations, not the least of which was the use of complexes are endocytosed after binding to transcobalamin radiolabeled isotopes in the clinical laboratory. In addition, II receptors on target cells. Once in the cell, cobalamin is it had been noted by the late 1970s that this assay may released from transcobalamin II, reduced, and bound as a give falsely normal values of serum cobalamin levels in cofactor for methylmalonyl-CoA mutase (mitochondria) or patients with pernicious anemia [8]. The failure of the assay was ascribed to cobalamin analogues present in affectedpatients and the use of an impure form of IF which boundthese analogues.
Another cobalamin-related test of historical note is the Interference of any of the requirements for ingestion, Schilling test, first introduced in 1954, but not utilized today absorption, distribution, or utilization can result in clinically in part because of the need for radiolabeled isotopes. The evident cobalamin deficiency. Diets lacking in an exoge- Schilling test is a multistep assay that is capable of assess- nous source of cobalamin will, over time, result in defi- ing the etiology of cobalamin malabsorption, but because ciency although it may take 2–5 years for cobalamin pernicious anemia is the most common cause of cobalamin deficiency to manifest clinically [5]. Cobalamin deficiency deficiency in Western populations, it has largely been can be precipitated by any malabsorption syndrome or sur- replaced by assays for the IF-blocking antibodies and anti- gical alteration to the gastrointestinal tract such as ileal parietal cell antibodies that are associated with pernicious bypass. Pancreatic exocrine insufficiency can also result in malabsorption owing to the failure of enzymatic degradationof cobalophilin–cobalamin complexes and release of cobal-amin for subsequent binding to IF.
Pernicious anemia is an autoimmune disease character- Modern laboratory testing for total plasma cobalamin lev- ized by megaloblastic anemia that is a direct consequence els commonly involves a competitive binding chemilumines- of autoantibody production which targets gastric parietal cence assay which has the advantage of easy scalability to cells or IF, leading to malabsorption of cobalamin. Medica- a high-throughput automated procedure. The sensitivity of tions may also affect the release of IF (H2 blockers) or these tests for the detection of frank cobalamin deficiency block absorption of cobalamin–IF complexes (neomycin (<200 pg/mL) in patients with clinical manifestations, such and metformin). In some instances, cobalamin deficiency as megaloblastic anemia, is estimated to exceed 90–95%, may result from infection with the fish tapeworm, Diphyllo- with some notable exceptions discussed below. The speci- bothrium latum, which establishes itself in the small intes- ficity of cobalamin measurements relating to clinical defi- ciency have not been formally determined but have been estimated to be <80%. The sensitivity of detection of sub- Finally, as mentioned above, inherited defects in factors clinical cobalamin deficiency is notably less, with estimates required for absorption or processing of cobalamin have ranging from 40 to 80% [9]. Each automated platform offers been shown to result in the characteristic neurologic and a proprietary variation on the following theme which results hematologic findings seen in B12 dietary deficiency, which in a chemiluminescence output measurement: has contributed to the delineation of the physiological mole-cules responsible for in vivo cobalamin transport and 1. Total vitamin B12 is liberated from protein binders in Path: N:/3b2/JOH#/Vol00000/130042/APPFile/JW-JOH#130042 J_ID: JOH Customer A_ID: JOH23421 Cadmus Art: JOH23421 Ed. Ref. No.: 13-0043.R1 Date: 5-March-13 2. The free B12 is allowed to then compete with exoge- haptocorrin deficiency [21] and folate deficiency [22]. In nously added labeled-B12 for binding to a limited cases of folate deficiency, cobalamin levels can normalize with folate therapy. Others have reported spuriously low- 3. IF is bound to a solid phase (or IF is initially coated on measured cobalamin levels associated with multiple mye- paramagnetic beads), unbound ligand is washed away, loma [23], HIV [24], pregnancy, and oral contraceptive use and a conjugate to the labeled-B12 is added. After [25]. In these cases, decreased production of serum cobal- addition of substrate, the conjugate creates chemilumi- amin-binding proteins is thought to be responsible (Table I).
nescence that is proportional to the amount of labeled- A measurement of cobalamin levels above the upper limit B12 present. Thus, there is an inverse relationship of the reference range has been associated with occult between the quantity of patient B12 present in the malignancy including myeloproliferative neoplasms that serum and the amount of luminescence generated.
cause increased haptocorrin levels. In addition, a recentstudy found associations between high levels of measured Despite multiple variations on this theme, a common fac- cobalamin and alcoholic liver disease, solid malignancies, tor to all the competitive binding luminescence assays and renal disease [26]. In part owing to these variables (CBLAs) is the use of purified IF as a means to specifically contributing to significant intraindividual temporal variation bind vitamin B12. In contrast to the previous use of impure in measured cobalamin levels, some authors have sug- forms of IF that were implicated in spuriously elevated gested that cobalamin testing is an unreliable indicator of cobalamin levels owing to nonspecific binding to cobalamin analogs in the serum, current CBLAs utilize a highly purifiedIF that has low affinity for cobalamin analogues [10]. How- Measurement and Clinical Utility of MMA and HCY ever, the use of purified IF may render the CBLA-based tests particularly susceptible to interfering anti-IF antibodies.
Elevated levels of MMA and HCY are commonly used as adjuvant diagnostics to confirm a suspected diagnosis of Technical Causes of Spuriously Measured Cobalamin cobalamin deficiency. Although many studies have used serum MMA as a “gold standard” for evaluating cobalamin Because of the inverse relationship between patient assays, this practice has been subjected to controversy.
cobalamin levels and assay output of chemiluminescence, The sensitivity of elevated serum MMA measurements in any substance that interferes with the chemiluminescence detecting patients with overt cobalamin deficiency is production will spuriously elevate cobalamin levels. This is reported to be >95%; however, the specificity of this test especially problematic in patients with pernicious anemia has not been determined [9,27]. For example, Hvas et al.
who have IF-blocking antibodies that may bind the test IFreagent. This assay failure has been reported by multipleinstitutions over the last decade with multiple CBLA-based TABLE I. Performance Characteristics of Clinical Assays Used for Detecting testing platforms [11–17]. The largest of these studies uti-lized patient serum from patients with documented anti-IF antibodies, who all had clinically expressed cobalamin defi-ciency. Total cobalamin levels were determined by radioim- significant diagnostic error [15]. All of these errors were false-negative results that reported normal cobalamin levels when, in fact, the patients were significantly deficient.
CBLAs commonly have an antibody denaturation step intended to denature IF-blocking antibodies and failure in this step has been implicated in spurious elevation of coba- lamin levels [11]. In addition, it has been argued that CBLA assays are imprecise upon repeated testing [18] although whether this is owing to temporal physiologic fluctuations of cobalamin, or CBLA assay imprecision is subjected to Finally, cutoff values to define cobalamin deficiency con- tinue to be controversial. It has been estimated that defin- ing cobalamin deficiency based on the common cutoff of 200 pg/mL may falsely identify 30% of elderly patients who have no clinical or metabolic signs of cobalamin deficiency as cobalamin deficient [5]. On the other hand, subclinical cobalamin deficiency is also a genuine concern in the same elderly population and increasing the cutoff value may exacerbate this problem. Readers are referred to a review of this area [20]. Supplementation of cobalamin test- ing with MMA and HCY will increase the specificity of test- ing, and borderline cases often warrant a trial of cobalamin Physiologic Causes of Spuriously Measured Physiologic conditions that have been reported to cause low-measured cobalamin levels without associated clinical signs of cobalamin deficiency include mild to severe Path: N:/3b2/JOH#/Vol00000/130042/APPFile/JW-JOH#130042 J_ID: JOH Customer A_ID: JOH23421 Cadmus Art: JOH23421 Ed. Ref. No.: 13-0043.R1 Date: 5-March-13 [6] did not find any correlation between elevated plasma holotranscobalamin levels were more likely to have higher levels of MMA and clinical manifestations of cobalamin defi- concentrations of MMA. The conclusion was that utilization ciency even after controlling for patients with renal disease.
of both tests would be superior than either one alone.
Moreover, this study of 432 untreated patients with mod- Unfortunately, they were unable to control for potential con- estly elevated MMA levels, who did not exhibit signs or founding variables other than renal insufficiency.
symptoms of cobalamin deficiency showed MMA levels that Two other studies examined the relative ability of holo- spontaneously declined in 44% and increased in 16%, transcobalamin and total cobalamin to identify patients with demonstrating considerable biological and temporal vari- elevated MMA levels and found that holotranscobalamin ability. Thus, caution is required when interpreting MMA performed more robustly than total cobalamin [33,34]. Simi- levels that are near commonly used cutoff points, and larly, serum holotranscobalamin was superior to total cobal- MMA should never be a sole laboratory criterion for deter- amin and MMA levels in predicting red cell cobalamin mining cobalamin deficiency. Measurement of MMA is levels although the significance of red cell cobalamin levels largely restricted to larger reference laboratories because it with regards to cobalamin deficiency remains to be deter- is most commonly quantified by isotope dilution technology with gas chromatography-mass spectrometry.
Conversely, when holotranscobalamin, MMA, or HCY lev- In addition to cobalamin deficiency, there are multiple els were used to asses which was a better a predictor of factors that can affect MMA levels. Patients who are hypo- response to vitamin B12 therapy in clinically cobalamin- volemic or who have renal insufficiency may have elevated deficient patients, none of the tested metrics performed levels of MMA unrelated to cobalamin status. Patients with better than low total cobalamin in predicting who would congenital metabolic defects such as methylmalonic acidu- ria have elevated levels of MMA. In addition, it has been Although initial studies of the sensitivity of holotranscoba- reported that MMA (and HCY) levels are elevated in lamin for detecting cobalamin deficiency appears to slightly patients with neurodegenerative disorders such as amyo- improve on that of direct total cobalamin measurements, studies have suggested that the specificity of holotransco- HCY levels are also elevated in cases of cobalamin defi- balamin measurements to detect cobalamin deficiencies ciency with a similar sensitivity to that of MMA [27]; how- remains low [33,37]. Problems remain in defining a gold ever, elevated HCY is less specific for cobalamin deficiency standard test and definition of cobalamin deficiency with than MMA. Elevated HCY levels are also associated with which to compare new assays to. As pointed out by Carmel folate deficiency, renal insufficiency, hypovolemia, hypothyr- [29], factors unrelated to cobalamin levels that affect holo- oidism, psoriasis, congenital metabolic defects, and neuro- transcobalamin levels remain to be elucidated. In addition, degenerative disease (Table I). Medications such as in vivo transient variation patterns of holotranscobalamin methotrexate, theophylline, phenytoin, hydrochlorothiazide, levels in cobalamin-replete individuals remain to be deter- mined. For example, can a brief reduction in dietary cobal- amin transiently reduce holotranscobalamin levels and leadto erroneous conclusions regarding cobalamin status? Although preliminary data suggest that the measurement of holotranscobalamin may provide a modest improvement over total cobalamin for the detection of cobalamin defi- It has long been suggested that serum holotranscobala- ciency, there is insufficient evidence to support the whole- min may be a better indicator of B12-deficiency states than sale adoption of holotranscobalamin testing in routine serum cobalamin because it represents the biologically active fraction of cobalamin in humans and may bedepleted first in subclinical cobalamin deficiency. However,there is concern that the noncobalamin-related determi- nants of physiologic and pathologic holotranscobalamin var- Advanced cobalamin deficiency can result in hematologic iations have yet to be fully elucidated [29]. For example, and neurologic manifestations where diagnosis commonly there is some limited data, suggesting that transcobalamin revolves around laboratory testing of total cobalamin levels.
levels are affected by liver disease, macrophage activation, Current testing for total cobalamin levels is based on and transcobalamin autoantibody generation [30].
CBLAs which provide good sensitivity and reasonable Traditionally, holotranscobalamin has been measured specificity in clinically overt cobalamin deficiency. Adjuvant through a modified radioimmunoassay. More recently, reli- assessment of MMA and HCY levels can further improve able monoclonal antibody-based assays for determining the specificity of testing in patients with adequate renal serum levels of holotranscobalamin have become available function. However, it is important to be aware that failure of [31]. Several studies utilizing this assay suggest that serum CBLA assays to detect cobalamin deficiency can occur, holotranscobalamin measurements are more sensitive in especially in cases of pernicious anemia where IF-blocking detecting cobalamin deficiency than tradition total serum antibodies have been implicated in causing assay interfer- ence. In addition, early or subclinical cases of cobalamin Miller et al. [32] compared total serum cobalamin and deficiency may not be detected with current testing plat- serum holotranscobalamin levels in a population of patients forms and standard reference ranges. Given these caveats, who were 60 years old, utilizing MMA and HCY levels as when laboratory measurements are incongruous with the gold standards for cobalamin deficiency. They found that clinical impression, alternative evaluations such as review cobalamin and holotranscobalamin measurements were of the peripheral blood smear, can provide valuable and essentially equivalent in their ability to identify cobalamin- reliable information to support the clinical impression.
deficient patients. Significantly, only about half of the Finally, holotranscobalamin testing appears to show prom- patients with elevated MMA and HCY levels were identified ise as a superior assay for detecting cobalamin deficiency; as cobalamin deficient, implying a poor sensitivity for either however, more work needs to be done to identify physio- method in this population when cobalamin deficiency is logic and pathologic variations of holotranscobalamin levels defined as elevated MMA and HCY levels. They also found at the population level before its potential incorporation into that patients identified with both low total cobalamin and Path: N:/3b2/JOH#/Vol00000/130042/APPFile/JW-JOH#130042 J_ID: JOH Customer A_ID: JOH23421 Cadmus Art: JOH23421 Ed. Ref. No.: 13-0043.R1 Date: 5-March-13 19. Carmel R, Solomon LR. Is testing for clinical cobalamin deficiency truly unreli- able? Blood 2005;106:1136–1138; author reply 1137–1138.
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