Diagnosis of Megaloblastic Anemia

As mentioned above the definitive diagnosis requires identification of the presence of megaloblasts in bone marrow aspirate. The taking of such an aspirate (usually from the hip bone) involves some discomfort for the patient and must be performed by an appropriately trained practitioner. Frequently, the routine diagnosis of unexplained macrocytic anemia falls to general physicians or general practitioners. In this situation, where there is clear evidence that the macrocytic anemia is due to deficiency of vitamin B12 or folate, it is not necessary to obtain a bone marrow aspirate to confirm megaloblastic changes. However, when bone marrow is not examined initially and a patient is treated for deficiency of vitamin B12 or folic acid, it is essential to verify that their response to treatment includes correction of anemia and macrocytosis. If there is any doubt, a bone marrow aspirate must be performed to exclude other possible underlying hematological disorders.

The first stage of diagnosis is based on the result of a full blood count (FBC) (also called complete blood count (CBC) in some countries) using an automatic instrument such as a Coulter counter. An FBC is done on virtually every patient admitted to hospital. Frequently, an FBC would also form part of an outpatient work-up or might be ordered by a GP through an associated hospital or laboratory. Where the hemoglobin level is below the reference value with respect to sex and age indicating anemia, the mean corpuscular volume (MCV) is assessed. This parameter essentially gives a mean of the size of red blood cells in the circulation. Mega-loblastic anemia usually results in larger than normal red cells in the circulation and thus a raised MCV; however, sometimes quite advanced stages of megaloblastic anemia can be accompanied by a normal and, infrequently, even below normal MCV. This can arise because of the concomitant presence of iron deficiency. A raised MCV accompanying the anemia seen in the FBC (macrocytic anemia) moves the diagnosis to being one of megaloblastic anemia, although other causes of macrocytosis such as hypothyroidism or excess alcohol consumption may need to be considered also. Conventionally, the next step is to carry out a bone marrow aspirate to verify if megaloblasts are present, but, as mentioned earlier, this step can be omitted if the diagnosis of vitamin B12 or folate deficiency can be made rapidly and accurately. After a positive bone marrow aspirate, or in its absence if this step is omitted, the next analysis would be the determination of circulatory levels of folate and vitamin B12. If only one of the vitamin levels is in the deficient range, most clinicians would embark upon the regimen of therapy discussed below. As mentioned above, anti-folate or anti-DNA drugs, such as methotrexate, 5-fluorouracil, or cyclophosphamide, will also arrest DNA biosynthesis and cause megaloblastic anemia; however, it is usually known when patients are on such anticancer chemotherapy.

The circulating levels of folate and vitamin B12 can be measured in serum or plasma samples by a number of methods. Most regard microbiological assays using Lactobacillus casei for folate and Lactobacillus leichmannii for vitamin B12 as the 'gold standard.' However, these assays are difficult to perform and most laboratories use methods based on enzyme linked immunosorbent assays (ELISA) or competitive binding assays using a natural binder such as intrinsic factor for vitamin B12 or ^-lactoglobulin for folate. While very low plasma or serum levels of <2.0 mgl 1 (4.5 nM) for folate and <120ngl-1 (88 pM) for vitamin B12, are considered as being diagnostic of deficiency, there is a gray area for both assays 2.0-2.7mgl-1 (4.5-6.1 nM) for serum folate and 120-200 ngl-1 (88-148 pM) for vitamin B12 indicating possible deficiency. Values above 2.7 mgl 1 (6.1 nM) for folate or 200ngl-1 (148pM) for vitamin B12 usually indicate the absence of deficiency.

Some laboratories also offer red cell folate levels. The red cell during its maturation in the bone marrow incorporates a level of folate commensurate with what is present in the circulation during that period. When the red cell passes from the bone marrow into the circulation it can neither take up nor lose folate until the end of its life, usually 120 days later. Thus, the circulatory red cells give an average of the folate level over the previous 4 months. Unlike the plasma or serum level the red cell folate level is not influenced by recent fluctuation in dietary intake. Thus, low red cell folate levels of <100 mgl1 (226nM) are a very good indication of folate deficiency with a range of 100-150mgl-1 (226-340nM) where there is possible deficiency and values above 150mgl-1 (340 nM) generally indicating the absence of folate deficiency. While red cell folate levels have significant advantages over serum folate levels they have one very significant drawback. Red cell folate levels are also significantly reduced in vitamin B12 deficiency. This is because the bone marrow cells take up the predominant circulating form of folate, namely 5-methyl THF. However, this form, which has just a single glutamate, is not retained by the cells unless it is converted into a predominant cellular form of folate with on average five glutamate residues. The enzyme that adds these glutamates does not use 5-methyl THF as a substrate; therefore, 5-methyl THF must be converted to THF before it can be converted to a polyglutamate. The only enzyme in the cell that converts 5-methyl THF to THF is the vitamin B12-dependent methionine synthase. As mentioned above, its activity is reduced or absent in vitamin B12-deficient bone marrow. Thus, such cells have an inability to conjugate and retain the circulating form of folate and as a result have reduced red cell folate levels. Thus, a low red cell folate level may lead to the misdiagnosis of vitamin B12 deficiency as folate deficiency, a circumstance which for the reasons discussed later must be avoided at all costs. Conseqently, it is always necessary to measure the level of plasma or serum folate . If it is also low or deficient and accompanied by a low red cell folate this is indicative of folate rather than vitamin B12 deficiency. This is because the circulating folate levels tends to back up in the serum resulting in higher rather than lower serum folate levels in vitamin B12 deficiency.

Before therapy, further investigations could be undertaken. These largely depend upon the availability of such tests in any particular clinical context. Elevated plasma homocysteine levels occur in both vitamin B12 and folate deficiency and raised homo-cysteine does not establish which vitamin is deficient. This is because such elevation is due to a reduction in the flux of homocysteine back to methionine as part of the methylation cycle (Figure 1). The enzyme that is compromised is methionine synthase, which uses vitamin B12 as a cofactor (Figure 2) and 5-methyl-tetrahydrofolate (Figure 3) and homocysteine as its substrates. This enzyme, and consequently the methylation cycle, thus requires both a normal folate and a normal vitamin B12 status for optimum activity. Thus reduction in the status of either vitamin is always accompanied by an elevation of plasma homocys-teine. Homocysteine is also elevated in other circumstances, most notably in impaired renal function. This can, to some extent, be corrected for the creatinine level. Homocysteine is also elevated in vitamin B6 deficiency and common C!T677 MTHFR polymorphism. Thus, while elevated plasma homocys-teine confirms the presence of megaloblastic anemia, establishing which vitamin is deficient still relies on measurement of the circulating levels of the vitamins involved.

The measurement of plasma, serum, or urine MMA is very helpful in confirming a diagnosis of vitamin B12 deficiency. This analyte is elevated due





N — Co^-N

to a reduction in the activity of methylmalonyl CoA mutase, the other vitamin B12-dependent enzyme in man (Figure 4). It appears that it is not possible to be functionally deficient in vitamin B12 without a concomitant elevation in MMA, and so a false negative result is not really an issue. However, MMA like plasma homocysteine is also elevated during renal impairment, and while this can to some extent be corrected for by a raised creatinine, it cannot be assumed that elevation of MMA is due to vitamin B12 deficiency. While the estimation of plasma homocysteine is widely available the estimation of MMA requires gas chromatography mass spectroscopy (GC-MS) and has very limited availability in practice. Newer methods to measure vitamin B12 on its transport protein TC II are under development.

For the reasons given above, it is essential that vitamin B12 deficiency is not confused with folate deficiency. As mentioned previously, both conditions present with a morphologically indistinguishable megaloblastic anemia. The inappropriate treatment of vitamin B12 deficiency with folic acid is to be avoided at all costs (see below). Apart from using biochemical assays to measure circulatory levels of the two vitamins and looking for an elevation of the biomarkers plasma homocysteine and MMA, further tests can also implicate vitamin B12 malabsorption, the most common type of severe vitamin B12 deficiency. These include the Schilling test and the detection of antibodies against either intrinsic factor or the parietal cells that manufacture it.

In practice, if vitamin B12 deficiency cannot be ruled out, many clinicians will treat patients with vitamin B12 if uncertain about the diagnosis. If this is followed by a reticulocyte response and complete disappearance of the anemia, it confirms a diagnosis of vitamin B12 deficiency. The appropriate treatment regimen can then be implemented (see later). If treatment with vitamin B12 does not result in improvement of the anemia then the patient is treated for folic acid deficiency, but only after vitamin B12 deficiency has been excluded by all means at the clinician's disposal.

OH: hydroxycobalamin CH3: methylcobalamin Ado: 5'deoxyadenosylcobalamin VCN: cyanocobalamin

Figure 2 The structure of naturally occurring vitamin B12 (hydroxycobalamin), its synthetic form cyanocobalamin, and its two cofactor forms methylcobalamin and 5'deoxyadenosylcobalamin. Hydroxycobalamin, X = Co-hydroxide; cyanocobalamin, X = Co-cyanide; methylcobalamin, X = Co-CH3; deoxyadenosyl-cobalamin, X = Co5'deoxyadenosyl.

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