Differential diagnosis of nutritional anemias prior to nutrient
supplementation is imperative for prevention of progressive neurological damage, as in the case for untreated vitamin B12 deficiency, or prevention of
the undesirable side-effects of unnecessary iron supplementation (e.g.
GI distress, iron overload in hemochromatosis gene carriers, increased risk
of infection during acute stress). The following table classifies nutritional anemias and anemia of chronic inflammation according to red cell parameters.
| Red Cell Index | Iron or Copper Deficiency Anemia (microcytic hypochromic) | Folate or Vitamin B12 Deficiency Anemia (macrocytic normochromic) | Anemia of Chronic Inflmmation (normocytic normocromic) |
| MCV | low | high | normal |
| MCHC | low | normal | normal |
| MCH | low | high | normal |
To confirm iron deficiency as a cause of microcytic anemia it is recommended to first evaluate the zinc protoporphyrin/heme ratio (ZPP/H). ZPP/H is an inexpensive screening test for iron deficiency. Zinc protoporphyrin is produced during heme synthesis in the developing erythron when iron availability is limited, thus iron deficiency is characterized by an increase in the ZPP/H. Confirmation of iron deficiency following an elevated ZPP/H (>80 umol/mol) requires determination of serum ferritin. Ferritin concentration <20 ng/mL is considered iron deficient. Both ZPP/H and serum ferritin can be elevated by chronic inflammation. Under conditions of chronic inflammation a serum ferritin <70 ng/mL may be considered iron deficient. ZPP/H can also be elevated in lead toxicity and protoporphyria (a rare congenital disease). Serum iron, total iron binding capacity (TIBC) and % transferrin saturation may also be used in the diagnosis of iron deficiency, however, these tests are less predictive of iron stores. Percent saturation is very useful in evaluation of iron overload. The following table outlines the changes in laboratory test results reflecting changes in iron status.
Indices of Iron Status
| Test | Iron Overload | Normal | Iron Depletion | Iron-deficient erythropoiesis | Iron-deificiency anemia |
| Bone Marrow Fe | 4+ | 2-3+ | 0-1+ | 0 | 0 |
| Serum FE (ug/dL) | >175 | 115±50 | 115 | <60 | <40 |
| TIBC (ug/dL) | <300 | 300±30 | 360 | 390 | 410 |
| % Transferrin Saturation | >60 | 35±15 | 30 | <15 | <15 |
| Ferritin (ug/L) | >300 | 100±60 | 20 | 10 | <10 |
| % Sidero-blasts | 40-60 | 40-60 | 40-60 | <10 | <10 |
| ZPP/H (umol/mol) | <80 | <80 | 80 | >80 | >80 |
Macrocytosis requires evaluation of both folate and vitamin B12 nutriture. The effect of either nutrient deficiency is impairment of genesis and maturation of red blood cells causing large, nucleated cells to be released into the circulation. Both nutrients arrest DNA synthesis by preventing the formation of thymidine monophosphate although folate and vitamin B12 are used at different steps of the synthetic pathway. Folate supplementation can compensate for vitamin B12 deficiency in DNA synthesis reversing macrocytic anemia and thereby masking vitamin B12 deficiency. Undiagnosed vitamin B12 deficiency will result in progressive permanent neurological damage.
One of the earliest clinical signs of both folate and vitamin B12 deficiency is hyper-segmentation of neutrophils. Deficiency should be suspected when >5% of cells have five or more lobes or when any six lobed cells are seen within a random sample of 100 cells. Hyper-segmentation may also occur in uremia, myeloproliferative disorders, myelofibrosis, and as a congenital lesion in 1% of the population.
Plasma vitamin B12 is the available test for evaluating vitamin B12 stores. Plasma vitamin B12 will be elevated in myeloproliferative disorders and hepatic tissue damage. A low plasma vitamin B12 is almost always indicative of deficiency. The cause of vitamin B12 deficiency must be determined for appropriate treatment. Vitamin B12 deficiency can result from either dietary deficiency or impaired absorption. Pernicious anemia, inadequate secretion of intrinsic factor, achlorhydria, a history of gastric or ileal resections, or diseases associated with malabsorption (e.g. Crohn's disease) may cause impaired vitamin B12 absorption. The Schilling test can be used to distinguish insufficient secretion of intrinsic factor from malabsorption syndromes. In this test radioactive B12 is taken orally, and its urinary excretion is measured over 24 hours. A flushing dose of unlabeled B12 is given with the labeled B12 to saturate liver storage and enhance labeled B12 excretion. Normally, >7% of the labeled B12 is recovered in the urine. If absorption is low, it is necessary to repeat the test with administration of intrinsic factor. The following table outlines the changes in laboratory test results reflecting changes in vitamin B12 status.
Red cell folate is the preferred test for evaluation of folate stores. Falsely elevated concentrations are seen in patients with raised reticulocyte counts and low levels occur in vitamin B12 deficiency. Plasma folate can be used to assess status however, it is affected by recent folate intake. The following table outlines t he changes in laboratory test results reflecting changes folate status.
Vitamin B12
| Test | Normal | Negative vitamin B12 balance | Vitamin B12 depletion | Vitamin B12 deficient erythro-poiesis | Vitamin B12 deficiency anemia |
| Hypersegmentation | No | No | No | Yes | Yes |
| Serum B12 | 200-900 pg/mL | 150-200 pg/mL | 100-150 pg/mL | 80-100 pg/mL | <80 pg/mL |
| RBC folate | >160 | >160 | >160 | <140 | <140 |
| MCV | Normal | Normal | Normal | Normal | Elevated |
Folate
| Test | Normal | Negative folate balance | Folate depletion | Folate deficient erythro-poiesis | Folate deficiency anemia |
| Serum folate (ng/mL) | >5 | <3 | <3 | <3 | <3 |
| RBC folate (ng/mL) | >200 | >200 | <160 | <120 | <100 |
| Lobe average | <3.5 | <3.5 | <3.5 | >3.5 | >3.5 |
| MCV | Normal | Normal | Normal | Normal | Elevated |
| Hb | >12 | >12 | >12 | >12 | <12 |