Anemia Evaluation and Microcytic and Macrocytic Anemias

Anemia is established from a hemoglobin concentration below the appropriate reference interval. Age, sex, pregnancy, altitude, smoking, plasma volume, and the laboratory method affect that interval. The RBC count, indices, reticulocyte response, and blood film then show how the anemia developed and which tests can narrow its cause.^1,2

Establishing the anemia pattern

Hemoglobin is the main operational measure of anemia. Hematocrit supports the interpretation but also changes with cell size and plasma volume. The first laboratory review should place the complete blood count (CBC) in the patient’s age and physiologic setting, compare current results with prior values, and look for internal inconsistencies or analyzer flags. The red-cell indices are mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Red-cell distribution width (RDW) describes variation in cell volume.

For adult pattern recognition, an MCV below approximately 80 fL is microcytic, 80 to 100 fL is normocytic, and above 100 fL is macrocytic. These are teaching intervals. The performing laboratory’s validated interval controls patient interpretation. A normal MCV can conceal two populations, such as microcytes and macrocytes whose average falls within range. An increased RDW or a broad, bimodal RBC histogram can reveal that mixture. With a low MCV, increased RDW commonly favors iron deficiency over the usual thalassemia-trait or inflammation pattern, with enough overlap that RDW carries no diagnosis by itself.²

The absolute reticulocyte count measures current marrow output more directly than the reticulocyte percentage. In anemia, a percentage can appear high because mature RBCs are fewer. A corrected reticulocyte percentage and reticulocyte production index (RPI) can adjust for anemia and early release of larger reticulocytes. The normal hematocrit denominator, maturation factor, and RPI decision limits are conventional and vary among references. An RPI below about 2 supports an inadequate production response; a value above about 3 supports strong regeneration when the marrow has had time to respond.²

Worked interpretation. A patient has hemoglobin 8.2 g/dL, hematocrit 25%, RBC count 4.0 × 10¹²/L, MCV 62.5 fL, and reticulocytes 2.2%. The absolute reticulocyte count is 88 × 10⁹/L. Using a 45% normal hematocrit and a 2-day maturation factor gives a corrected reticulocyte count of 1.22% and an RPI of about 0.61. The absolute count may fall within a general adult interval, but the RPI shows an inadequate response for this degree of anemia. The result is a hypoproliferative microcytic pattern. Iron studies, inflammatory context, and hemoglobin analysis determine the cause.

The immature reticulocyte fraction can rise early when marrow releases less mature reticulocytes. Reticulocyte hemoglobin reflects iron available to the newest cells. Both parameters depend on the analyzer and should be interpreted with method-specific intervals.³

The film should answer a focused question raised by the CBC. Relevant findings include:

Morphology terms and grading should follow the laboratory’s procedure. The College of American Pathologists glossary and International Council for Standardization in Haematology recommendations provide common terminology.^4,5

Production, destruction, and loss

An inadequate reticulocyte response points toward insufficient or ineffective erythropoiesis. Causes include unavailable iron, impaired DNA or globin synthesis, reduced erythropoietin signaling, marrow failure, or marrow replacement. An increased response points toward blood loss or shortened RBC survival once enough time has passed for marrow output to rise. Bilirubin, lactate dehydrogenase, haptoglobin, urinalysis, and the direct antiglobulin test help separate hemorrhage from hemolysis. Detailed immune-hemolysis testing is covered in the Blood Banking topics.²

The hematopoiesis and erythrocyte physiology topic explains erythropoietin signaling, reticulocyte release, red-cell clearance, and iron recycling.

Acute hemorrhage

Acute hemorrhage removes plasma and red cells together. Hemoglobin and hematocrit may therefore remain near baseline immediately after the loss. Interstitial fluid movement and intravenous fluids often dilute the remaining blood over approximately 24 to 72 hours, and the measured hemoglobin and hematocrit fall. The timing varies with bleeding, fluid replacement, and physiologic compensation. The early cells are usually normocytic and normochromic.

A catecholamine stress response can produce an early neutrophilic leukocytosis through demargination. A marrow left shift and reactive thrombocytosis are later and variable. If erythropoiesis is intact, polychromasia and an absolute reticulocytosis usually become evident after about 3 to 5 days and peak around 7 to 10 days. Nucleated RBCs can appear after severe loss. A single acute bleed initially leaves iron studies intact; repeated or chronic loss can deplete iron stores.²

Uncomplicated hemorrhage lacks the biochemical pattern of hemolysis. Concurrent disease can change bilirubin, lactate dehydrogenase, haptoglobin, and direct antiglobulin test results, so those results must be interpreted together.

Microcytic anemia patterns

Microcytosis reflects inadequate hemoglobin assembly. The defect may involve available iron, heme synthesis, or globin-chain production. Several processes can coexist.

These typical patterns come from current guidance and references for iron deficiency, inflammation, myeloid neoplasms, lead exposure, and thalassemia.^{6,7,8,9,10,11,12}

Serum iron varies during the day and with recent intake. Ferritin rises with inflammation, liver disease, malignancy, infection, and other conditions. Soluble transferrin receptor, reticulocyte hemoglobin, zinc protoporphyrin, and marrow iron can add evidence, but their methods and decision limits are less uniform. A pattern should be supported by more than one result.^6,7

Iron deficiency

Iron deficiency progresses from reduced stores to restricted iron delivery and then iron-deficient erythropoiesis. The sequence can overlap, especially when inflammation restricts iron release while storage iron remains present.

• Storage depletion: ferritin falls as macrophage and hepatocyte iron is used.
• Iron-restricted erythropoiesis: transferrin saturation and reticulocyte hemoglobin fall as less iron reaches developing cells.
• Iron-deficiency anemia: hemoglobin falls; microcytosis, hypochromia, anisocytosis, and an inadequate reticulocyte response become more apparent.

A low ferritin strongly supports depleted stores. The World Health Organization uses less than 15 µg/L to indicate deficiency in otherwise healthy adults. In adults with infection or inflammation, WHO conditionally permits a value below 70 µg/L to indicate iron deficiency; the certainty of evidence is low. Clinical laboratories and specialty guidelines may use different limits for the population and setting. A normal or increased ferritin during inflammation can coexist with iron deficiency, so C-reactive protein or another inflammation marker, transferrin saturation, CBC findings, and the clinical source of loss remain part of the interpretation.^6,7

Common causes include chronic blood loss, increased need during growth or pregnancy, reduced intake, and impaired absorption. Ongoing hemoglobinuria and repeated phlebotomy or blood donation can also remove iron. The laboratory finding establishes iron deficiency; the source of the deficiency requires clinical investigation.

Transferrin saturation is calculated from serum iron and total iron-binding capacity. The core chemistry calculations topic gives the formula and unit checks.

Anemia of inflammation

Inflammation increases hepatic hepcidin. Hepcidin binds ferroportin and reduces iron export from intestinal cells and iron-recycling macrophages. Plasma iron falls while storage iron remains inside cells. Cytokines also reduce erythropoietin production or response, suppress erythroid progenitors, and can shorten RBC survival. The result is iron-restricted erythropoiesis with an inadequate reticulocyte response.⁸

The usual profile combines low serum iron, low or normal transferrin or TIBC, transferrin saturation that is low or sometimes within the local interval, and normal or increased ferritin. The anemia is often mild to moderate and normocytic, with microcytosis appearing in some cases. Coexisting true iron deficiency can lower ferritin and increase TIBC, but inflammation can blur both changes. Soluble transferrin receptor or an assay-specific reticulocyte hemoglobin result may help in selected cases. Increased erythropoietic drive and thalassemia can also raise soluble transferrin receptor, which limits its specificity.⁸

Sideroblastic and lead-associated patterns

Sideroblastic anemia results from impaired mitochondrial heme production or iron use. Iron accumulates in erythroblast mitochondria around the nucleus. Perls Prussian blue staining of marrow shows ring sideroblasts. Each cell has at least five iron granules around at least one-third of the nuclear circumference. A reported percentage must name the erythroblast denominator used by the laboratory. Ring sideroblasts occur in inherited disorders, clonal myeloid neoplasms, and acquired secondary conditions. Current myeloid classifications use genetic and morphologic criteria for the clonal categories.⁹

Secondary causes include alcohol, lead, copper deficiency, zinc excess, and drugs such as isoniazid, linezolid, and chloramphenicol. The CBC may be microcytic, normocytic, macrocytic, or dimorphic, depending on the cause. Serum iron, ferritin, and transferrin saturation are often increased. Film findings can include basophilic stippling and Pappenheimer bodies. A marrow ring-sideroblast result must be correlated with exposure history, medications, nutritional studies, cytogenetic or molecular findings, and other evidence of a myeloid neoplasm.

Lead inhibits aminolevulinate dehydratase and ferrochelatase in heme synthesis. It also interferes with degradation of erythrocyte RNA, contributing to coarse basophilic stippling. Zinc protoporphyrin (ZPP), free erythrocyte protoporphyrin (FEP), and urinary aminolevulinic acid can rise, but these are adjuncts. Diagnosis uses a blood lead concentration. For children, a capillary result at or above the Centers for Disease Control and Prevention blood lead reference value of 3.5 µg/dL should be confirmed with a venous specimen. Lead exposure can harm health below this population-based action threshold.¹⁰

Thalassemia syndromes

Thalassemias reduce production of a structurally normal globin chain. The unpaired partner chains damage developing or circulating erythrocytes, causing ineffective erythropoiesis, hemolysis, or both. Reduced hemoglobin synthesis produces microcytosis that can be striking relative to the degree of anemia. A β⁰ allele produces no β chain, a β⁺ allele reduces production by a mutation-specific amount, and a β^silent allele has a mild effect.

Age, genotype, transfusion history, iron status, assay method, and coinherited variants change the hemoglobin fractions. Iron deficiency and δ-globin variants can lower Hb A₂. Hb Constant Spring may be present at a concentration below routine detection. Exact percentages should therefore be read against the method’s validated interpretation and the patient’s setting.^11,12

Laboratory sequence

1. Confirm persistent microcytosis and review hemoglobin, RBC count, RDW, reticulocytes, and the film.
2. Assess iron status. Iron deficiency can coexist with thalassemia and can alter Hb A₂.
3. Perform hemoglobin analysis by a validated fractionation method when a thalassemia or hemoglobin variant pattern remains likely. The result identifies and quantifies fractions; method-specific coelution and comigration can create ambiguity.
4. Use molecular testing to identify HBB variants or HBA1/HBA2 deletions and sequence variants when the hemoglobin pattern is inconclusive, α-thalassemia is suspected, or a precise genotype is required. The assay must cover the relevant variant types.

Hemoglobin fractionation supports the phenotype. Molecular testing can establish the genotype when it identifies pathogenic or likely pathogenic variants within the assay’s coverage. A variant of uncertain significance leaves the diagnosis unresolved. Laboratories combine these methods when either one leaves uncertainty.¹³

The Mentzer ratio, MCV divided by RBC count, can support initial pattern recognition. A value below about 13 favors thalassemia trait and a higher value favors iron deficiency in the populations where the rule was studied. Mixed deficiency, age, pregnancy, and other disorders reduce its accuracy. Iron studies and hemoglobin analysis carry the diagnostic weight.²

Macrocytic anemia patterns

Macrocytosis separates into megaloblastic and nonmegaloblastic patterns. The blood film, reticulocyte count, vitamin studies, liver and thyroid tests, medication history, and other cytopenias guide the next step. Megaloblastic anemia often produces an MCV from about 100 to 150 fL and can exceed 120 fL. Nonmegaloblastic causes often produce an MCV near 100 to 110 fL, although greater values occur. RDW is often normal or only mildly increased in a uniform nonmegaloblastic population.²

The International Council for Standardization in Haematology defines neutrophil hypersegmentation as at least one neutrophil with six or more lobes or more than 3% of neutrophils with five lobes in a 100-cell count. This standard helps separate a reproducible finding from occasional five-lobed cells.⁵

Megaloblastic change can lower leukocytes and platelets as well as RBCs. A film with macro-ovalocytes, hypersegmented neutrophils, and pancytopenia should prompt vitamin B12 and folate evaluation. Similar marrow morphology can occur in myelodysplastic and other dyserythropoietic disorders, so biochemical confirmation matters.^2,5

Vitamin B12 and folate

Folate supplies one-carbon units used to make thymidylate for DNA. Vitamin B12 supports methionine synthase, which regenerates tetrahydrofolate from 5-methyltetrahydrofolate, and methylmalonyl-CoA mutase, which converts methylmalonyl-CoA to succinyl-CoA. During B12 deficiency, folate becomes trapped as 5-methyltetrahydrofolate and methylmalonic acid accumulates. Impaired DNA synthesis produces nuclear-cytoplasmic asynchrony and ineffective erythropoiesis.^14,15

The testing relationships and limitations follow current NIH and NICE guidance.^14,15,16

Many laboratories consider total B12 below approximately 200 to 250 pg/mL subnormal and use methylmalonic acid when total B12 is indeterminate. These values remain assay dependent. Neurologic B12 deficiency can occur with normal hemoglobin and MCV. Clinical suspicion therefore remains relevant when the CBC is normal.^14,16

Autoimmune gastritis damages gastric parietal cells and reduces intrinsic factor, impairing B12 absorption. Pernicious anemia is the hematologic expression of this process. Anti-intrinsic-factor antibody is relatively specific but insensitive. If suspicion remains after a negative result, the applicable diagnostic pathway may include parietal-cell antibody, gastrin, or additional clinical evaluation. Gastrin can rise with acid-suppressing medication and other conditions, so it is an adjunct.¹⁶

Other causes of B12 deficiency include inadequate intake, food-cobalamin malabsorption, gastric or ileal surgery, ileal disease, pancreatic insufficiency, bacterial overgrowth, and inherited transport defects. Folate deficiency can follow inadequate intake, alcohol use disorder, increased need, malabsorption, dialysis, or medications that block folate metabolism. The medication and clinical history should be interpreted alongside the laboratory pattern.

Nonmegaloblastic macrocytosis and spurious results

Reticulocytes are larger than mature RBCs, so hemolysis, recovery after hemorrhage, or response to effective therapy can raise MCV. Liver disease and alcohol-related changes can produce round macrocytes, sometimes with target cells or stomatocytes. Hypothyroidism, marrow disease, and drugs that affect DNA synthesis or cell turnover are additional causes. Persistent macrocytosis with other cytopenias or dysplastic cells warrants evaluation for a marrow disorder.²

An implausible CBC should be checked for analytic interference before a disease interpretation is released:

• Cold agglutinins clump RBCs, producing a falsely low RBC count and falsely high MCV and MCHC. Warming the specimen to 37 °C and repeating the count usually resolves the pattern.
• Marked hyperglycemia can cause analyzer-dependent in-vitro RBC swelling after dilution and a spuriously increased MCV.
• Lipemia, icterus, marked leukocytosis, hemolysis, clots, specimen age, and IV-fluid contamination can disturb selected CBC measurements.

The correction follows the analyzer’s validated procedure and may require warming, dilution, plasma replacement, repeat analysis, or a fresh uncontaminated specimen.¹⁷

References

1. World Health Organization. Guideline on Haemoglobin Cutoffs to Define Anaemia in Individuals and Populations. World Health Organization; 2024. Accessed August 29, 2026.
2. Keohane EM, Preston MM, Mirza KM, Walenga JM, eds. Rodak's Hematology: Clinical Principles and Applications. 7th ed. Elsevier; 2025. Accessed August 29, 2026.
3. Obstfeld AE, Davis BH, Han JY, Urrechaga E. Report of the International Council for Standardization in Haematology working group for standardization of reticulocyte parameters. Int J Lab Hematol. 2024;46(2):266-274. doi:10.1111/ijlh.14209.
4. College of American Pathologists. Hematology, Clinical Microscopy, and Body Fluids Glossary. 2025. Accessed August 29, 2026.
5. Palmer L, Briggs C, McFadden S, et al. ICSH recommendations for the standardization of nomenclature and grading of peripheral blood cell morphological features. Int J Lab Hematol. 2015;37(3):287-303. doi:10.1111/ijlh.12327.
6. World Health Organization. WHO Guideline on Use of Ferritin Concentrations to Assess Iron Status in Individuals and Populations. World Health Organization; 2020. Accessed August 29, 2026.
7. Fletcher A, Forbes A, Svenson N, Thomas DW. Guideline for the laboratory diagnosis of iron deficiency in adults (excluding pregnancy) and children. Br J Haematol. 2022;196(3):523-529. doi:10.1111/bjh.17900.
8. Weiss G, Ganz T, Goodnough LT. Anemia of inflammation. Blood. 2019;133(1):40-50. doi:10.1182/blood-2018-06-856500.
9. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022;36(7):1703-1719. doi:10.1038/s41375-022-01613-1.
10. Centers for Disease Control and Prevention. Recommended actions based on blood lead level. Updated August 21, 2025. Accessed August 29, 2026.
11. Langer AL. Beta-thalassemia. In: Adam MP, Bick S, Mirzaa GM, et al, eds. GeneReviews. University of Washington, Seattle; 2000. Revised February 12, 2026. Accessed August 29, 2026.
12. Tamary H, Greenberg-Kushnir N, Dgany O. Alpha-thalassemia. In: Adam MP, Bick S, Mirzaa GM, et al, eds. GeneReviews. University of Washington, Seattle; 2005. Updated April 23, 2026. Accessed August 29, 2026.
13. Agarwal AM, Rets AV. Advances in hemoglobinopathies and thalassemia evaluation. Clin Lab Med. 2024;44(3):441-453. doi:10.1016/j.cll.2024.04.006.
14. National Institutes of Health Office of Dietary Supplements. Vitamin B12: fact sheet for health professionals. Accessed August 29, 2026.
15. National Institutes of Health Office of Dietary Supplements. Folate: fact sheet for health professionals. Accessed August 29, 2026.
16. National Institute for Health and Care Excellence. Vitamin B12 Deficiency in Over 16s: Diagnosis and Management. NICE guideline NG239. Published March 6, 2024. Accessed August 29, 2026.
17. Gulati G, Song J, Florea AD, Gong J. Unreliable automated complete blood count results: causes, recognition, and resolution. Ann Lab Med. 2022;42(5):515-530. doi:10.3343/alm.2022.42.5.515.