What is the recommended diagnostic work‑up for iron‑deficiency anemia?

Diagnostic Work-up for Iron Deficiency Anemia

Begin with a complete blood count including red cell indices (MCV, MCH, RDW), reticulocyte count, serum ferritin, transferrin saturation (calculated as serum iron × 100 ÷ TIBC), and C-reactive protein to simultaneously confirm anemia, assess iron status, and detect inflammation. 1, 2

Initial Laboratory Panel

Minimum Required Tests

• Order CBC with MCV, MCH, and RDW to identify microcytosis (MCV < 80 fL), hypochromia (MCH < 27 pg), and elevated RDW (> 14%), which together strongly suggest iron deficiency rather than thalassemia trait. 1, 2, 3

• Measure serum ferritin as the single most powerful test for diagnosing iron deficiency; ferritin < 30 µg/L confirms depleted iron stores in patients without inflammation. 2, 3, 4

• Calculate transferrin saturation (TSAT) because TSAT < 20% is the primary confirmatory marker for iron deficiency, especially when ferritin lies between 30–100 µg/L or may be falsely elevated by inflammation. 1, 2

• Obtain C-reactive protein (CRP) or ESR to identify inflammation, which can elevate ferritin up to 100 µg/L despite true iron deficiency. 1, 2

• Count reticulocytes to distinguish inadequate marrow response (low or normal reticulocytes in iron deficiency) from hemolysis (elevated reticulocytes). 1, 2

Interpreting Ferritin Thresholds

• Ferritin < 15 µg/L has 99% specificity for absolute iron deficiency and confirms the diagnosis without further testing. 2, 3

• Ferritin < 30 µg/L indicates low body iron stores in the absence of inflammation. 2, 3, 4

• Ferritin 30–100 µg/L with elevated CRP/ESR suggests a mixed picture of true iron deficiency plus anemia of chronic disease; confirm with TSAT < 20%. 1, 2

• Ferritin > 100 µg/L with TSAT < 20% and elevated CRP defines anemia of chronic disease with functional iron deficiency, not true iron deficiency. 1, 2

• Ferritin > 150 µg/L makes absolute iron deficiency unlikely even in the presence of inflammation. 2

Transferrin Saturation Interpretation

• TSAT < 16–20% confirms iron-deficient erythropoiesis and is the decisive marker when ferritin is equivocal or falsely elevated by inflammation, malignancy, or liver disease. 1, 2, 5

• TSAT is more sensitive than hemoglobin alone for detecting iron deficiency. 3

Extended Work-up When Initial Tests Are Inconclusive

Additional Iron Studies

• Measure soluble transferrin receptor (sTfR) when ferritin and TSAT provide conflicting information; elevated sTfR confirms true iron deficiency because it is not affected by inflammation. 1, 2

• Assess percentage of hypochromic red cells and reticulocyte hemoglobin content for more precise iron-status evaluation when standard tests are borderline. 1, 2

Evaluation for Other Deficiencies

• Obtain vitamin B12 and folate levels to exclude megaloblastic anemia, which can coexist with iron deficiency and produce a mixed micro- and macrocytic picture with elevated RDW. 1, 2

• Macrocytosis (MCV > 100 fL) raises suspicion for vitamin B12 or folate deficiency, medication effects (azathioprine), or reticulocytosis. 1

Hemolysis Evaluation

• When reticulocytes are elevated, order haptoglobin, lactate dehydrogenase, and bilirubin to evaluate for hemolysis, which excludes deficiency states. 1, 2

Renal Function

• Measure serum creatinine and urea when anemia etiology remains unclear, as chronic kidney disease contributes to anemia via reduced erythropoietin and functional iron deficiency. 1, 2

Investigation of Underlying Cause

Gastrointestinal Evaluation

• In adult men and postmenopausal women with confirmed iron deficiency, gastrointestinal bleeding is the presumptive source until proven otherwise; occult malignancy must be excluded. 2, 6

• Perform bidirectional endoscopy (upper endoscopy with duodenal biopsies plus colonoscopy) promptly because colorectal and gastric cancers are the most common serious causes in this age group. 2, 6

• Upper endoscopy with duodenal biopsies screens for celiac disease, which accounts for 2–3% of iron deficiency anemia cases and may present with isolated anemia. 2

• Colonoscopy detects colonic carcinoma, adenomatous polyps, and angiodysplasia, which are high-yield findings in older adults. 2

• Do not delay endoscopic evaluation even if hemoglobin is above fast-track referral thresholds; investigation is indicated at any anemia level when iron deficiency is present. 2

Menstrual and Obstetric Assessment

• In premenopausal women, assess menstrual blood loss as the most common cause, but do not attribute iron deficiency solely to menstruation without excluding gastrointestinal pathology. 2, 4

• Screen all pregnant women in the first trimester and again at 24–28 weeks of gestation, as iron deficiency affects up to 84% of pregnant women in the third trimester. 7, 4

Malabsorption Disorders

• Screen for celiac disease with serologic testing before endoscopy if malabsorption is suspected. 2

• Evaluate for atrophic gastritis, Helicobacter pylori infection, and bariatric surgical history, which impair iron absorption. 2, 4

Special Population Considerations

Inflammatory Bowel Disease

• In IBD patients in remission, ferritin < 30 µg/L reliably indicates iron deficiency. 1, 2

• During active IBD inflammation, use ferritin < 100 µg/L as a screening threshold and confirm iron deficiency with TSAT < 20%. 1, 2

• Monitor every 6–12 months in remission or mild disease, and at least every 3 months in active disease. 2

Chronic Kidney Disease and Heart Failure

• In CKD and chronic heart failure, use ferritin < 100 µg/L (instead of < 30 µg/L) as the screening threshold for iron deficiency. 2

• Iron-deficiency criteria in heart failure are ferritin < 100 ng/mL or ferritin 100–300 ng/mL together with TSAT < 20%. 2

Algorithmic Approach

1. Confirm anemia using WHO hemoglobin thresholds: men < 13 g/dL, non-pregnant women < 12 g/dL, pregnant women < 11 g/dL. 1, 2

2. Order initial panel: CBC with MCV/MCH/RDW, reticulocyte count, ferritin, TSAT, and CRP. 1, 2

3. Interpret ferritin in context of inflammation:

   • No inflammation (normal CRP): ferritin < 30 µg/L confirms iron deficiency. 2
   • Inflammation present (elevated CRP): ferritin 30–100 µg/L with TSAT < 20% indicates iron deficiency; ferritin > 100 µg/L with TSAT < 20% indicates anemia of chronic disease. 1, 2

4. If ferritin is equivocal or TSAT is borderline, add sTfR to confirm true iron deficiency. 1, 2

5. Evaluate for hemolysis if reticulocytes are elevated (order haptoglobin, LDH, bilirubin). 1, 2

6. Screen for vitamin B12 and folate deficiency if MCV > 100 fL or RDW is markedly elevated. 1, 2

7. Investigate the source of iron loss:

   • Adult men and postmenopausal women: bidirectional endoscopy. 2, 6
   • Premenopausal women: assess menstrual loss and consider endoscopy if severe or refractory. 2, 4
   • All patients: screen for celiac disease and malabsorption. 2

8. If microcytosis persists after iron repletion, order hemoglobin electrophoresis to exclude β-thalassemia trait. 2, 3

Critical Pitfalls to Avoid

• Do not rely on ferritin alone when inflammation is present; always calculate TSAT because functional iron deficiency can exist with high ferritin but low TSAT. 1, 2

• Do not attribute severe iron deficiency in adults solely to dietary insufficiency or menstrual loss; gastrointestinal pathology must be investigated. 2

• Do not postpone endoscopic investigation while awaiting response to iron therapy; malignancy work-up should proceed in parallel. 2

• Do not use hemoglobin electrophoresis as a first-line test; reserve it for cases with normal or borderline iron parameters or when MCV is disproportionately low. 2, 3

• Do not overlook combined deficiencies; iron deficiency can coexist with vitamin B12 or folate deficiency, recognizable by elevated RDW. 1, 2

• Do not assume mild anemia is less indicative of serious underlying disease; no evidence supports down-ranking its clinical significance. 2