on 29-Mar-2021 (Mon)

Hematocrit (HCT), also called packed cell volume (PCV), is the percentage of blood volume occupied by RBCs. It can be measured directly following centrifugation of a blood sample (picture 1 and picture 2).

When measured by an electronic cell counter, HCT is calculated from the RBC count (in millions/microL) and the mean corpuscular volume (MCV; in femtoliters [fL]): HCT = ([RBC x MCV]/10).

One set of normal ranges for hemoglobin, HCT, and RBC count is shown in the table (table 1). Other normal ranges have been proposed. World Health Organization (WHO) criteria for anemia in men and women are hemoglobin <13 and <12 g/dL, respectively [4]. However, these criteria were intended for use within the context of international nutrition studies and were not initially designed to serve as "gold standards" for diagnosing anemia [5].

The increased frequency of anemia seen with aging has led to suggestions that a different standard for the normal hemoglobin should be used in older adults [5]. Review of data from the National Health and Nutrition Examination Survey (NHANES) indicates that the mean hemoglobin values for men and women over 70 is within the usual normal range (14.5 g/dL and 13.4 g/dL, respectively), while the 5^th percentile is below the normal range (11.7 g/dL for men and 10.9 g/dL for women) [11]. This lower boundary may reflect an increased prevalence of comorbidities, especially chronic kidney disease [12].

In individuals with anemia, hemoglobin and HCT decrease in parallel, although the HCT/hemoglobin ratio (approximately 3 in most cases) may vary according to the volume (size) of the cells. The RBC count also usually parallels the hemoglobin and HCT, except in cases of extreme microcytosis such as thalassemia, in which the RBC count may be increased despite the presence of anemia. The RBC count is less commonly used to diagnose anemia for this reason. A finding of high RBC count in an individual with anemia suggests thalassemia.

Causes of lower values

• Intense physical activity – Values in endurance athletes may vary significantly from those in other healthy individuals. Various causes may contribute, including dilutional anemia from increased plasma volume, iron deficiency, and/or "march" hemolysis. (See "Exercise-related gastrointestinal disorders", section on 'Gastrointestinal bleeding' and "Non-immune hemolytic anemia due to systemic disease".)

• Pregnancy – During a healthy pregnancy, maternal red cell mass increases, but plasma volume increases to a greater degree, causing a relative decrease in hemoglobin and HCT [14]. By the criterion of RBC mass, the individual is not anemic, but hemoglobin, HCT, and RBC count frequently decrease to anemic levels (figure 1). The terms "physiologic" or "dilutional" anemia have been applied to this setting, although these individuals are not actually anemic and do not require evaluation as long as their hemoglobin remains ≥11 g/dL in the first and second trimester and ≥10 g/dL in the third trimester. (See "Anemia in pregnancy".)

• Older age – Values for hemoglobin and HCT in apparently healthy older adults are generally lower than those in younger adults, and differences between males and females that are seen in younger adults are lessened with aging [15-17]. (See 'Older adults' below.)

Causes of higher values (may occasionally mask underlying anemia)

• Smoking – Smoking causes an increase in hemoglobin, HCT, and RBC count due to increased levels of carbon monoxide, which reduces oxygen delivery. Thus, individuals who smoke or have significant exposure to secondary smoke or other sources of carbon monoxide may have HCT higher than normal [18]. A study of blood donors who smoke found a significant correlation between the patients' blood carboxyhemoglobin and hemoglobin values [19]. (See "Carbon monoxide poisoning", section on 'Pathophysiology'.)

• Hemoconcentration – Individuals with dehydration or hypovolemia related to vomiting or diarrhea will have a relative increase in hemoglobin and HCT due to hemoconcentration. Anemia will become apparent after volume replacement. This is particularly a problem in patients with severe burns, in whom substantial RBC loss may be masked by exudative loss of plasma volume until fluid resuscitation has occurred [20].

• High altitude – Persons living at high altitude have higher values than those living at sea level due to relative hypoxia [21]. (See "High altitude, air travel, and heart disease", section on 'Long-term altitude exposure'.)

Mean corpuscular volume (MCV) is the average volume (size) of the RBCs. It can be measured or calculated (MCV in femtoliters [fL] = 10 x HCT [in percent] ÷ RBC [in millions/microL]). RBCs with MCV in the normal range are roughly the same diameter as the nucleus of a normal lymphocyte on the peripheral blood smear.

Mean corpuscular hemoglobin (MCH) is the average hemoglobin content in a RBC. It is calculated (MCH in picograms [pg]/cell = hemoglobin [in g/dL] x 10 ÷ RBC [in millions/microL]). A low MCH is typically reflected in an enlarged area of central pallor in RBCs on the peripheral blood smear (greater than one-third of the RBC diameter), which defines "hypochromia" on the blood smear. This may be seen in iron deficiency and thalassemia.

Mean corpuscular hemoglobin concentration (MCHC) is the average hemoglobin concentration per RBC. It is calculated as (MCHC in grams [g]/dL = hemoglobin [in g/dL] x 100 ÷ HCT [in percent]). Very low MCHC values are typical of iron deficiency anemia, and very high MCHC values typically reflect spherocytosis or RBC agglutination. Examination of the peripheral blood smear is helpful in distinguishing these findings.

Red cell distribution width (RDW) is a measure of the variation in RBC size, which is reflected in the degree of anisocytosis on the peripheral blood smear. RDW is calculated as the coefficient of variation (CV) of the red cell volume distribution (RDW = [standard deviation/MCV] x 100).

A high RDW implies a large variation in RBC sizes, and a low RDW implies a more homogeneous population of RBCs. A high RDW can be seen in a number of anemias, including iron deficiency, vitamin B12 or folate deficiency, myelodysplastic syndrome (MDS), and hemoglobinopathies, as well as in patients with anemia who have received transfusions. Review of the peripheral blood smear often is helpful in identifying the cause.

Reticulocytes can be reported as a percentage of total RBCs or as an absolute count (table 2). Reticulocytes can be appreciated on a standard blood smear stained with Wright-Giemsa as RBCs with a blue tint (polychromatophilia) that are larger than mature RBCs, with irregular borders and a lack of central pallor (picture 3).

The appropriate count depends on the hemoglobin level. The "normal" reticulocyte count refers to the count in a non-anemic individual at steady state.

• Steady state – At steady state, approximately 1 to 2 percent of circulating RBCs are reticulocytes, corresponding to an absolute reticulocyte count of approximately 25,000 to 100,000/microL (0.25 to 1 x 10¹¹/L).

• Anemia – In anemia, the reticulocyte count rises. A normal bone marrow can increase the rate of RBC production approximately fivefold in adults (seven- to eightfold in children). Thus, under optimal conditions, reticulocyte percentages of at least 4 to 5 percent (often considerably higher) and absolute reticulocyte counts as high as 350,000/microL (3.5 x 10¹¹/L) are possible.

An increased reticulocyte count represents a normal bone marrow response to anemia. Incorporation of the reticulocyte count into the anemia evaluation can be especially helpful in diagnosing certain disorders including hemolytic anemias and multifactorial anemias

The reticulocyte stage of RBC development lasts for approximately four days. In the steady state, reticulocytes generally spend three days in the bone marrow and one day in the circulation. In severe anemia, reticulocytes can leave the bone marrow earlier and can circulate for two to three days, as illustrated in the figure (figure 2). This is the basis for the calculation of the reticulocyte production index [23]. (See "Regulation of erythropoiesis".)

If a laboratory does not report an absolute reticulocyte count or one of the corrected reticulocyte count parameters, calculators and other tools are available online (calculator 1).

While blood oxygen content is a function of hemoglobin, oxygen delivery to the tissues can also be affected by changes in the affinity of hemoglobin for oxygen, expressed as the partial pressure of oxygen at which hemoglobin is 50 percent saturated (P₅₀), as well as blood volume and tissue perfusion. (See "Oxygen delivery and consumption" and "Genetic disorders of hemoglobin oxygen affinity", section on 'Mutations that decrease the affinity of hemoglobin for 2,3-BPG'.)

Oxygen delivery is increased by:

• Decreases in pH

• Increases in RBC 2,3 bisphosphoglycerate (2,3 BPG) concentration

• Increased body temperature

Studies in animal models demonstrate that at any given HCT, systemic oxygen transport is lower with lower blood volume [25]. This is primarily a consequence of decreased tissue perfusion.

Other conditions besides hypovolemia that can impair tissue perfusion include:

• Hypotension

• Peripheral vasoconstriction

• Decreased cardiac output

• Bradycardia

• Coronary artery obstruction

Any or all of these can worsen symptoms at a given degree of anemia.

Symptoms of anemia also reflect the rate with which RBC mass declines, which determines the extent of compensatory changes. Following acute blood loss, an individual will initially have normal values for hemoglobin and HCT, but these values will decline over the ensuing 36 to 48 hours as most of the total blood volume deficit will be replaced by the movement of fluid from the extravascular into the intravascular space or with fluid resuscitation. Only then will the hemoglobin and HCT reflect blood loss. Thus, until the total blood volume deficit is fully repleted, the hemoglobin and HCT will underestimate the degree of blood loss [26]. The rapid onset of symptoms in these cases primarily reflects initial hypovolemia and hypotension and their effects on tissue oxygenation

Conversely, when anemia develops gradually over time (as with iron deficiency, vitamin B12 deficiency, or a myelodysplastic syndrome [MDS]), compensatory increases in blood volume and tissue adaptation to hypoxia may prevent symptoms from developing until the hemoglobin is very low.

Some of the more common ways to categorize anemia are based on:

● Obvious clinical features such as acute blood loss or known cause for malabsorption of nutrients needed for RBC production. (See 'Evaluation based on clinical presentation' below.)

● "Flags" on the initial CBC and chemistry panel, including other cytopenias, an abnormal differential, abnormalities of RBC shape, or evidence of kidney or liver dysfunction. (See 'Evaluation based on CBC/retic count' below.)

● Whether the RBCs are small (microcytic) or large (macrocytic). (See 'Evaluation based on MCV' below.)

● Whether the bone marrow is functioning appropriately (based on whether the reticulocyte count appropriately increased). (See 'Reticulocyte count' below.)

An experienced clinician will consider all of these frameworks simultaneously.

With some caveats, the following testing is appropriate in the initial evaluation of unexplained anemia:

● Reticulocyte count – Reflects the ability of the bone marrow to produce RBCs and can be used to categorize possible causes of anemia. (See 'Reticulocyte count' below.)

● Chemistry panel – Should include assessments of kidney and liver function, with creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

● Hemolysis labs – Lactate dehydrogenase (LDH), bilirubin, and haptoglobin (table 3), if the clinical history suggests hemolytic anemia and/or the reticulocyte count is increased. (See 'Hemolysis' below.)

● Blood smear – A review of the peripheral blood smear is always desirable in the initial evaluation of anemia. However, it is not always possible to obtain this immediately, and some workflows will direct the blood smear to an off-site reviewer who is unfamiliar with the patient. These practices may make blood smear review during the initial evaluation an unrealistic expectation.

In some settings, review of a blood smear by an experienced professional is critical to the evaluation and treatment; these settings are indicated in the following sections. Interpretation of specific findings is discussed separately.

The blood smear findings can in turn direct the subsequent laboratory tests needed to confirm or exclude a specific diagnosis.

As examples:

• Bite or blister cells (picture 5) – G6PD deficiency, evaluated by measuring the G6PD level.

• Microcytosis, target cells, teardrop cells (picture 6) – Thalassemia, evaluated by hemoglobin analysis or molecular (DNA) testing.

• Spherocytes (picture 7), elliptocytes (picture 8), or stomatocytes (picture 9) – Hereditary spherocytosis (HS), elliptocytosis (HE), or stomatocytosis (HSt).

Premenopausal women — Iron deficiency is common in premenopausal women, due to menses and/or pregnancies. RBCs are microcytic in some individuals but may be normocytic in others. Other conditions contributing to anemia can also be present.

● If the clinical history and examination are otherwise negative, we obtain iron studies (serum iron, serum transferrin or total iron binding capacity (TIBC), serum ferritin, and calculated transferrin saturation [TSAT]). (See "Iron requirements and iron deficiency in adolescents", section on 'Evaluation and presumptive diagnosis' and "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults", section on 'Diagnostic evaluation'.)

● Findings that suggest another cause of anemia should also be pursued. This may include anemia during childhood, symptoms related to hemolysis, and other findings on the complete blood count (CBC) and blood smear.

The prevalence of anemia increases substantially in patients over the age of 60 [27,28]. As noted above, we evaluate the underlying cause rather than attributing anemia to aging.

Major causes of anemia in older adults include [29]:

• Nutritional deficiencies in approximately one third.

• Kidney disease or anemia of chronic disease/inflammation (ACD/AI) in approximately one third.

• Unexplained in the remaining third.

All individuals over the age of 60 should have testing for the following:

• Kidney function – Estimation of glomerular filtration rate (GFR). eGFR <45 mL/min/1.73 m² in the absence of another diagnosis implicates chronic kidney disease (CKD) as a cause.

• Iron stores – Iron studies (serum iron, serum transferrin or TIBC, serum ferritin, and TSAT).

• Vitamin B12 – Vitamin B12 level, with methylmalonic acid level if vitamin B12 deficiency is suspected and vitamin B12 level is equivocal.

• Folate – Folate level if at risk for malnutrition.

Further testing may be appropriate in certain settings:

• Monoclonal gammopathy – Testing is indicated if the mean corpuscular volume (MCV) is increased and/or if there is reduced eGFR or hypercalcemia. Serum free light chains and serum protein electrophoresis (SPEP) with immunofixation are obtained.

• Androgen deficiency – Testing with serum testosterone level is indicated in older men with an isolated normocytic anemia in whom the testing above did not provide a diagnosis [30].

• MDS and clonal cytopenias – Testing for a clonal bone marrow disorder is indicated if the MCV is slightly elevated and/or if there are other cytopenias or morphologic abnormalities on the blood smear. Molecular testing can be performed on peripheral blood using a gene panel for myeloid disorders (myelodysplastic syndrome [MDS] panel) or clonal hematopoiesis (CH) panel. Bone marrow can be examined for signs of dysplasia for possible diagnosis of MDS.

Unexplained anemia of aging is a poorly defined syndrome often seen in older individuals. The mechanism is unclear and appears to be cytokine-mediated [31]. This is a diagnosis of exclusion in older individuals with normocytic anemia and an unrevealing workup. The diagnosis should be reassessed periodically to avoid missing a correctable disorder.

A number of specific causes of anemia diagnoses occur at increased frequency in individuals with malnutrition and/or malabsorption. These may include deficiencies of iron, vitamin B12, folate, and copper, which may occur in isolation or simultaneously. In individuals with severely reduced intake due to anorexia nervosa or starvation, the bone marrow is often affected.

● Gastric surgery – Gastric surgery, particularly bariatric surgery, is associated with malabsorption of vitamins and trace elements. This is particularly the case following Roux-en-Y procedures [32]. Rates of deficiencies with different procedures and details of routine supplementation are discussed separately. (See "Bariatric surgery: Postoperative nutritional management".)

● Zinc supplements – Zinc ingestion, as a dietary supplement or in zinc-containing denture adhesives, can cause copper deficiency by inhibiting copper absorption. (See "Causes and pathophysiology of the sideroblastic anemias", section on 'Copper deficiency'.)

● Starvation or anorexia nervosa – Anemia is seen in approximately one-third of individuals with severe malnutrition or anorexia nervosa, either alone or in combination with neutropenia or leukopenia [33]. The bone marrow may show gelatinous necrosis. Anemia will improve with restored food intake.

Iron deficiency causes microcytosis, while vitamin B12, folate, and copper deficiency cause macrocytosis. If both iron deficiency and one of the other deficiencies are present, the MCV may be in the normal range, often with an increased red cell distribution width (RDW)

Vitamin B12 and copper deficiency can cause other cytopenias; neutropenia commonly accompanies the anemia in copper deficiency. Vitamin B12 and copper deficiency both can produce posterior column neurologic abnormalities

The evaluation in all cases should include serum iron, transferrin/TIBC, ferritin, vitamin B12, and folate levels. Copper level should be measured in malnourished individuals with anemia accompanied by neutropenia and/or neuropathy, as well as those with anemia in the setting of gastric/bariatric surgery or a history of zinc ingestion. Individuals with any of these deficiencies should be evaluated for the underlying cause

Chronic anemia in patients with systemic illnesses may reflect anemia of chronic disease/inflammation (ACD/AI), particularly in disorders associated with inflammatory processes such as rheumatoid arthritis or systemic lupus erythematosus (SLE).

The reduction in hemoglobin is often mild to moderate. The red cells are typically normocytic, although there may occasionally be a moderate degree of microcytosis. Iron studies show decreased serum iron and normal or elevated ferritin concentrations. Serum erythropoietin is typically increased above the level seen in patients who are not anemic but to a lesser degree than would be observed in uncomplicated iron deficiency (figure 4). Serum hepcidin is not routinely available but would be expected to be elevated.

Underlying conditions commonly associated with ACD/AI include:

● Cancer

● Chronic kidney disease (may be associated with concomitant erythropoietin deficiency)

● Rheumatologic conditions

● Hypothyroidism

● Infections

There is debate about whether diabetes mellitus per se causes ACD/AI, or whether ACD/AI can only be caused by complications of diabetes.

Pancytopenia is the combination of anemia, thrombocytopenia, and neutropenia (or leukopenia).

Findings of particular concern that warrant hematologist involvement and bone marrow examination include:

● Severe pancytopenia.

● Blasts or immature myeloid/lymphoid forms, which suggest acute leukemia.

● Abnormal lymphocytes (hairy cells, large granular lymphocytes, prolymphocytes).

● Leukoerythroblastosis (picture 10) with or without teardrop cells (dacrocytes; (picture 11)).

● Pancytopenia with hemolysis or thrombosis.

● Pancytopenia or bicytopenia (anemia with leukopenia or anemia with thrombocytopenia) in an older individual with normal vitamin B12, folate, and copper levels.

Potential diagnoses are numerous (table 5). They include drug-induced pancytopenia (cytotoxic drugs, anti-infective drugs, anticonvulsants (table 6)), certain infections (viral [hepatitis, cytomegalovirus, Epstein Barr virus] and severe non-viral [clostridial sepsis, malaria, leishmaniasis, leptospirosis, babesiosis]), bone marrow failure (aplastic anemia), myelodysplasia, myelofibrosis, clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH), and hematologic malignancies.

Hypersplenism – Cirrhosis can cause pancytopenia due to sequestration of cells in the spleen (hypersplenism). Macrocytosis and target cells are often seen on the peripheral blood smear. The mean corpuscular volume (MCV) will typically be elevated to a moderate degree, usually no higher than 105 fL. Splenic imaging is appropriate if splenomegaly has not been previously documented.

Nutrient deficiency – Deficiency of vitamin B12, copper, and/or folate may also cause pancytopenia and should be evaluated, especially if the peripheral blood smear shows macroovalocytes (picture 12), hypersegmented neutrophils (picture 13), and/or if the MCV is >110 fL.

Autoimmune – Autoimmune cytopenias typically affect a single cell line but can affect more than one cell line simultaneously, especially if there is an underlying rheumatologic disorder such as systemic lupus erythematosus (SLE) or a lymphoid disorder such as chronic lymphocytic leukemia (CLL)

HLH – Hemophagocytic lymphohistiocytosis (HLH) may be primary (typically in children) or secondary to an infection, malignancy, or rheumatologic condition.

TMAs – Thrombotic microangiopathies (TMAs) such as thrombotic thrombocytopenic purpura (TTP) typically cause thrombocytopenia and microangiopathic hemolytic anemia, with a very low platelet count and schistocytes on the blood smear. Some types of drug-induced TMAs such as due to quinine can cause pancytopenia. Disseminated intravascular coagulation (DIC) can cause pancytopenia due to TMA plus bone marrow suppression, with coagulation abnormalities often prominent.

Reticulocyte count — Anemia can also be classified on the basis of reticulocyte production. This approach is most informative when the reticulocyte count is either very decreased or very elevated. Attention must be paid to the particular reticulocyte parameter reported (absolute count versus percentage) and is most helpful using a reticulocyte parameter that is corrected for the degree of anemia (table 2).

Reticulocytosis requires a normally functioning bone marrow replete with iron, folate, cobalamin (vitamin B12), and copper, and a normally functioning kidney that can sense a decrease in oxygen delivery and produce a compensatory increase in erythropoietin (EPO). Thus, a decreased reticulocyte count suggests underproduction of red blood cells (RBCs; bone marrow suppression), and an increased reticulocyte count usually suggests hemolysis or blood loss. If both bone marrow suppression and hemolysis or blood loss are present, the reticulocyte count will be inappropriately low

Anemia with a decreased (or inappropriately low) reticulocyte count may be due to:

- Deficiency of iron, vitamin B12, folate, or copper

- Medications that suppress the bone marrow

- Primary bone marrow disorders including myelodysplastic syndrome (MDS), myelofibrosis, or leukemia

- Very recent bleeding (within five to seven days, before bone marrow compensation has occurred)

Anemia with an increased reticulocyte count may be due to:

- Hemolysis

- Repletion of deficient iron, vitamin B12, folate, or copper (early phase of recovery)

- Recovery from bleeding

If the reticulocyte count is increased and the cause of anemia is not readily apparent, additional laboratory testing for hemolysis is appropriate (table 3).

An increase in reticulocyte count following treatment can also be very helpful in determining if the cause of anemia was determined accurately and completely. As examples:

• If anemia was attributed to a deficiency (iron, vitamin B12, folate), brisk reticulocytosis is expected to occur within one to two weeks of repletion.

• If the reticulocyte count does not increase with correction of a deficiency, this suggests an additional cause of anemia is interfering with bone marrow function. As an example, vitamin B12 or folate deficiency may occur concurrently with iron deficiency, causing a normocytic anemia (see 'Normocytic (normal MCV)' below). This combination of findings is often seen in malabsorption syndromes such as for celiac disease, autoimmune gastritis, or bariatric surgery.

Hemolytic anemia should be considered when there is a rapid fall in hemoglobin concentration with an increased reticulocyte count in the absence of blood loss (table 2). Other abnormal laboratory findings indicative of hemolysis are summarized in the table (table 3).

Schistocytes on the blood smear indicated likely hemolysis due to mechanical RBC destruction. Schistocytes plus thrombocytopenia indicate possible thrombotic microangiopathy (TMA), which may be life-threatening.

Chronic hemolysis may present with a stable hemoglobin, a high reticulocyte count, and a normal lactate dehydrogenase (LDH) and bilirubin. The combination of an increased LDH and reduced haptoglobin is 90 percent specific for acute hemolysis, while the combination of a normal LDH and a serum haptoglobin greater than 25 mg/dL is 92 percent sensitive for ruling out hemolysis [36,37].

Intramedullary hemolysis (within the bone marrow) due to ineffective erythropoiesis, as may be seen in thalassemia or vitamin B12 deficiency, may produce elevations of bilirubin and LDH without reticulocytosis

Causes of hemolysis are numerous and can be categorized in several ways, as summarized in the table (table 7) and discussed in more detail separately. (See "Diagnosis of hemolytic anemia in adults".)

Hereditary, non-immune:

• Hemoglobinopathies (sickle cell disease, thalassemias, unstable hemoglobins)

• Hereditary red cell enzyme deficiencies (glucose-6-phosphate dehydrogenase [G6PD], pyruvate kinase [PK], glucose phosphate isomerase, 5’ nucleotidase)

• Hereditary RBC membrane defects (hereditary spherocytosis [HS], elliptocytosis [HE], stomatocytosis [HSt])

Acquired, non-immune

• Membrane defects (liver disease, acquired acanthocytosis)

• Infections (malaria, babesiosis, clostridial sepsis, Bartonellosis, trypanosomiasis, visceral leishmaniasis)

• Drug-induced (oxidant stress)

• Severe burns

• Thrombotic microangiopathies (thrombotic thrombocytopenic purpura [TTP], hemolytic uremic syndrome [HUS], drug-induced TMA)

• Mechanical (intravascular devices, artificial heart valve, giant hemangioma, footstrike hemolysis)

• Hypersplenism (may have a component of phagocytosis but is not antibody mediated)

• Vasculitis

• Severe hypertension

• Heavy metals (Wilson disease, copper toxicity, arsine toxicity)

• Envenomation (snake, brown recluse spider, hobo spider)

• Hypophosphatemia

Acquired, immune-mediated

• Autoimmune (warm autoimmune hemolytic anemia [AIHA], cold agglutinin disease, paroxysmal cold hemoglobinuria)

• Hemolytic transfusion reactions

• Drug-induced

Many individuals with anemia will be otherwise well, and the clinical history and other initial findings on the complete blood count (CBC) may not be helpful for suggesting specific diagnoses leading to anemia. A diagnostic approach based on the mean corpuscular volume (MCV) is most useful in these cases, as illustrated in the flowchart (algorithm 1).

A decreased MCV (usually <80 fL) reflects a defect in cellular hemoglobin synthesis. These anemias are summarized in the table (table 8) and discussed in detail separately and briefly below.

Causes

• Iron deficiency – Restricted iron availability (iron deficiency and some cases of anemia of chronic disease/anemia of inflammation [ACD/AI], which can cause functional iron deficiency). (See "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults" and "Anemia of chronic disease/anemia of inflammation".)

• Decreased globin chains – Qualitative abnormalities in globin proteins such as thalassemia, hemoglobin (Hb) C, and HbE (but not HbS). (See "Pathophysiology of thalassemia" and "Clinical manifestations and diagnosis of the thalassemias" and "Overview of variant sickle cell syndromes".)

• Decreased heme – Abnormalities of the heme porphyrin ring, including sideroblastic anemias and lead poisoning. (See "Causes and pathophysiology of the sideroblastic anemias" and "Lead exposure and poisoning in adults".)

Very low MCV – Iron deficiency and thalassemia are the most likely causes of a very low MCV (<80 fL). The ratio of the MCV to the red blood cell (RBC) count (Mentzer index) is useful in predicting the likelihood of thalassemia trait versus iron deficiency. If the ratio of MCV (in fL) to RBC count (in millions of cells/microL) is less than 13, thalassemia is more likely than iron deficiency [38].

In practice, all individuals with a low MCV should have iron studies to evaluate iron status (and should have deficiency corrected if present), because the results of hemoglobin analysis can be altered by concomitant iron deficiency.

All patients – All patients with microcytic anemia should have serum iron, total iron binding capacity (TIBC)/transferrin, and serum ferritin concentrations measured, with calculated transferrin saturation (TSAT). Iron studies will identify iron deficiency (the most likely diagnosis for microcytic anemia) and ACD/AI in most cases. Mild microcytosis with iron studies showing low iron, low TIBC, and high-normal to high ferritin in the appropriate clinical context (eg, chronic inflammatory condition with normal MCV prior to its development) is consistent with ACD/AI.

Individuals with normal iron studies – Hemoglobin quantitation should be ordered in individuals with microcytic anemia who do not have iron deficiency or ACD/AI to identify beta thalassemia or other hemoglobinopathies. Globin gene studies may be needed to diagnose alpha thalassemia; the family history may be helpful in determining likelihood of these disorders. Minimal to mild anemia and microcytosis may indicate non-transfusion-dependent thalassemia (thalassemia trait). Basophilic stippling may also be seen with beta thalassemia [39].

Individuals with normal hemoglobin – Basophilic stippling (picture 14) suggests possible lead poisoning, and whole blood lead levels should be measured. The diagnosis of sideroblastic anemia requires a bone marrow examination.

Macrocytosis (high MCV) — An increased MCV (>100 fL) is typically attributed to asynchronous maturation of nuclear chromatin, although other causes may also be present. These anemias are summarized in the table (table 9) and discussed in detail separately and briefly below.

Megaloblastic anemia – Asynchronous nuclear maturation (megaloblastosis), in which the rate of cell division is reduced relative to cytoplasmic expansion. (See "Macrocytosis/Macrocytic anemia", section on 'Megaloblastic anemia'.)

Megaloblastic maturation can be due to:

- Vitamin B12 deficiency

- Folate deficiency

- Copper deficiency

- Myelodysplastic syndrome (MDS)

- Aplastic anemia

- Diamond Blackfan anemia

- Drugs that interfere with DNA synthesis

Membrane changes – In some cases, conditions that increase RBC membrane (such as liver disease or excess alcohol [even without liver disease]) and other underlying disorders such as hypothyroidism can cause increases in MCV. Stomatocytosis (hereditary or acquired) can also cause macrocytic anemia.

Reticulocytosis – Reticulocytes are larger than mature RBCs (picture 3), and reticulocytosis will increase the MCV. This will be associated with an increased red cell distribution width (RDW) and can often be suspected from examination of the peripheral blood smear.

Spurious – Increased concentrations of immunoglobulin or acute phase proteins such as occurs with inflammation or a polyclonal or monoclonal gammopathy may cause small rouleaux (picture 15) that are counted by electronic counters as single large cells. This is a laboratory artifact that can be assessed by viewing the peripheral blood smear.

All individuals – Serum vitamin B12 level should be measured in all patients with unevaluated macrocytosis. All individuals who are nutritionally compromised or who have had gastric surgery should also have serum folate measured. In patients without known nutritional/gastric issues who have MCV >110 fL and a normal vitamin B12 level, serum methylmalonic acid (MMA) and serum folate should be measured.

Individuals with normal vitamin B12 and folate

- Thyroid stimulating hormone (TSH) should be checked. (See "Macrocytosis/Macrocytic anemia", section on 'Hypothyroidism'.)

- Alcohol use should be assessed. The MCV typically is not >105 fL in alcohol-induced macrocytosis. (See "Hematologic complications of alcohol use", section on 'Anemia'.)

- Serum copper level should be checked, especially if neutropenia and/or neuropathy are present or if the history reveals zinc ingestion or other risk factors. (See "Copper deficiency myeloneuropathy", section on 'Causes of acquired copper deficiency'.)

- The peripheral blood smear (or report) should be reviewed. If the blood smear shows target cells, liver synthetic tests should be measured. The MCV in liver disease typically is not >105 fL. Other morphologic abnormalities such as stomatocytosis should be evaluated, if present. If the copper level is normal and the blood smear shows evidence of dysplasia such as bilobed or immature neutrophils or binucleate RBCs, or other cytopenias, refer to a hematologist for bone marrow and/or molecular (DNA) studies on bone marrow or peripheral blood.

A normal MCV (80 to 100 fL) is the most common finding in anemic men and postmenopausal women. Normocytic anemias can be more challenging to evaluate than anemias with an MCV that is obviously low or high. Causes are more numerous and may be multifactorial, an underlying condition may not be apparent, and other findings may be nonspecific

Often normocytic anemia is associated with a slightly elevated RDW, and the reticulocyte count is not substantially increased (and may be decreased). An increased RDW may indicate a population of microcytic or macrocytic RBCs that is too small to shift the MCV out of the normal range, or combined microcytic and macrocytic processes, such as iron deficiency plus vitamin B12 or folate deficiency [40]

Nutrient deficiency – Any of the causes of acquired microcytic or macrocytic anemia, especially early stages of deficiency of iron, vitamin B12, folate, or copper.

Multiple causes – Combined microcytic plus macrocytic anemia [40]. The most characteristic situation is simultaneous deficiency of vitamin B12 and iron in an individual with celiac disease or autoimmune gastritis.

• ACD/AI – Anemia of chronic disease/anemia of inflammation (ACD/AI). (See "Anemia of chronic disease/anemia of inflammation".)

• CDK – Anemia of chronic kidney disease (CKD). (See "Overview of the management of chronic kidney disease in adults", section on 'Anemia'.)

• HF – Anemia of heart failure (HF), including cardio-renal syndrome [41]. (See "Evaluation and management of anemia and iron deficiency in adults with heart failure" and "Cardiorenal syndrome: Definition, prevalence, diagnosis, and pathophysiology".)

• Endocrine – Anemia with endocrine deficiency, including hypothyroidism (most cases), androgen deficiency, or adrenal insufficiency (in adrenal insufficiency, anemia may be masked by volume contraction). (See 'Caveats for normal ranges' above and "Clinical manifestations of adrenal insufficiency in adults", section on 'Hematologic findings'.)

•

• Clonal hematopoietic stem cell disorders – Anemia due to a clonal disorder of erythropoiesis (myelodysplastic syndrome, aplastic anemia, or clonal cytopenias of uncertain significance [CCUS]) [42]. The strict definition of clonal hematopoiesis of indeterminate potential (CHIP) includes normal hemoglobin and other blood counts. (See "Clinical manifestations and diagnosis of myelodysplastic syndromes (MDS)" and "Idiopathic and clonal cytopenias of uncertain significance (ICUS and CCUS)".)

• Early blood loss – Blood loss that has not yet caused iron deficiency. (See "Evaluation of occult gastrointestinal bleeding".)

• PRCA – Pure red cell aplasia. (See "Acquired pure red cell aplasia in adults".)

• Partially treated anemia – Anemia in process of correction or following transfusion. A "dimorphic RBC population" (presence of two distinct populations of RBCs of different sizes) may be suspected when an increased RDW is present; it can be confirmed by examination of the peripheral blood smear, although the distinct populations may not always be recognized. This finding is traditionally taught as a clue to sideroblastic anemia [43]. However, it is more commonly seen during early treatment of iron deficiency or megaloblastic anemia, or following transfusion of a patient with microcytic or macrocytic anemia

Reticulocyte count and chemistry panel – All individuals with normocytic anemia of unknown cause should have a reticulocyte count and chemistry panel (or review of results of this testing) during the initial evaluation, and abnormalities of this testing should be pursued (algorithm 1).

• Iron studies and hemolysis labs – If the reticulocyte count and chemistry panel are unrevealing, determine serum iron concentration, serum TIBC/transferrin, and serum ferritin concentration measured and transferrin saturation (TSAT) calculated, in order to diagnose iron deficiency or ACD/AI. (See "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults".)

If iron stores are normal, evaluate for hemolysis.

• Additional tests – If hemolysis is absent and there are no other obvious diagnoses, consider conditions listed above including cancer, endocrine disorders, blood loss, and nutrient deficiencies.

- Normocytic anemia with estimated glomerular filtration rate (eGFR) <45 mL/min/1.73 sq m and no other identified cause is most probably the anemia of chronic kidney disease. (See "Overview of the management of chronic kidney disease in adults", section on 'Anemia'.)

- Evaluation for disorders common in older adults is generally reasonable, including testing for monoclonal gammopathies, clonal cytopenias, androgen deficiency (in men), and bone marrow evaluation for myelodysplasia and pure red cell aplasia.

Acquired copper deficiency has been recognized to cause a myelopathy in humans relatively recently [1,2]; however, cases of myelopathy described among zinc-smelter workers in the 19^th century are now felt likely to be due to copper deficiency myelopathy [3].

The neurologic manifestations of copper deficiency are usually, but not always, accompanied by the typical hematologic derangements of anemia and leukopenia

The age range of reported cases of copper deficiency myeloneuropathy is 30 to 82 years [4,5]. More cases in women than in men are reported.

There are limited pathologic studies in individuals with acquired copper deficiency. A few reports of nerve biopsies in patients confirm the presence of axonal degeneration [1,13-16]. A single postmortem examination in one patient revealed significant degeneration in the dorsal and lateral columns of the spinal cord [17]. In animals, neuronal loss in the cerebellum and spinal tracts with extensive demyelination is reported [18].

The mechanism underlying neurologic damage in individuals with copper deficiency is uncertain. Copper is a component of enzymes that have a critical role in the structure and function of the nervous system [6-12]. It permits electron transfer in key enzymatic pathways

Although rare, acquired copper deficiency has been well documented in humans [6,18]. The most common causes are malabsorption and zinc ingestion

Gastric surgery is the most common cause of acquired copper deficiency, underlying approximately half of reported cases [1,4,13,19-33]. Typically, neurologic manifestations are delayed by years following gastric surgery.

Excessive zinc ingestion is another cause of copper deficiency [3,30,31,34-38]. Copper and zinc are competitively absorbed from the gastrointestinal tract. In addition to overuse of zinc supplements [39], ingestion of denture cream [17,35-37,40] and parenteral zinc overloading during chronic hemodialysis [41] have also been linked to copper deficiency myelopathy.

Dietary copper deficiency is rare but has been described in premature infants receiving milk formulas without adequate copper supplementation [6,42]. Copper deficiency may also be a complication of prolonged total parenteral nutrition, in particular when copper supplementation is withheld due to cholestasis [8,31,43].

Enteropathies associated with malabsorption, such as cystic fibrosis, celiac disease, and inflammatory bowel disease, may be associated with copper deficiency [1,6,18,31,44,45]. In some cases the neurologic manifestations have occurred in the absence of gastrointestinal symptoms [44,46,47]. Bacterial overgrowth has also been implicated as a cause of copper deficiency in a patient with a prior history of gastric surgery [23].

Prolonged use of proton pump inhibitors was speculated to contribute to impaired intestinal absorption of copper and subsequent copper deficiency and myeloneuropathy in a series of patients without another known cause for copper deficiency [48].

Over-treatment of Wilson disease with zinc and chelators has only rarely been reported to cause copper deficiency-related neurologic manifestations [49-53]. The antiparasitic agent, clioquinol, is a copper chelator and was linked to many cases of myelo-optico-neuropathy (a syndrome similar to copper deficiency myeloneuropathy) in Japan in the 1960s [54-56]. Another copper chelator, tetrathiomolybdate, has been associated with copper deficiency [57].

The coexistence of multiple causes of copper deficiency increases the risk of a clinically significant deficiency state [58]. In some cases, the cause of copper deficiency is not evident even after extensive investigation [1,4,5,15,28,59].

The most common neurologic manifestation of acquired copper deficiency is that of a myelopathy or myeloneuropathy [1,2,5,19,20,34,37,44,46,60]. Patients typically present with a subacute gait disorder with prominent sensory ataxia and/or spasticity. Examination reveals long tract signs with spasticity in the legs and Babinski response, along with impaired vibration and position sense and a positive Romberg sign. Bladder dysfunction is relatively uncommon, but can occur [2,5,29]. This pattern of neurologic deficits reflects involvement of the dorsal columns of the spinal cord and is similar to that seen in patients with subacute combined degeneration secondary to vitamin B₁₂ deficiency.

Symptoms and signs suggesting an associated peripheral neuropathy are common [37]. The neuropathy may take different forms. A complaint of lower limb paresthesias is present in nearly all patients, and most demonstrate a stocking distribution of impaired sensation to pain and temperature [1,2,20]. Superimposed neuropathy may lead to suppressed reflexes in the legs, although many patients have brisk reflexes instead, reflecting the myelopathy [1,2]. A common finding is brisk knee jerks with depressed or absent ankle jerks.

Less common peripheral nerve manifestations include a wrist or foot drop [ 1]; asymmetric sensory symptoms of the face and trunk as well as limbs, suggesting a sensory ganglionopathy [15]; and a progressive, asymmetric weakness with electrodiagnostic evidence of denervation suggesting lower motor neuron disease [14,36]

In most cases, the associated neuropathy develops along with the myelopathy; however, in some cases, these develop before the myelopathy [ 13] or in the absence of a myelopathic symptoms [27,28,35,49,61].

Optic nerve involvement of varying severity has been reported as part of this syndrome in a number of patients [23,27,29,62], although not in most. Affected patients typically have subacute onset of bilateral vision loss, without optic disc edema.

Rare neurologic symptoms associated with copper deficiency in isolated case reports include myopathy (along with myelopathy) [47], myelo-optico-neuropathy with hyposmia and hypogeusia [23], and cognitive impairment [36]. However, the precise significance of these reported associations is unclear and needs further confirmation.

Hematologic — The hematologic hallmark of copper deficiency is anemia and leukopenia; these are present in most, but not all, patients with associated neurologic deficits [1,2,4,16,28,30,31,34,37,63-65].

The anemia may be microcytic, macrocytic, or normocytic. Thrombocytopenia and pancytopenia are relatively rare.

In some cases, patients with copper deficiency were initially given a diagnosis of sideroblastic anemia, myelodysplastic syndrome, or aplastic anemia.

One case series and a case report describe hepatic iron overload and/or cirrhosis in five patients with copper deficiency myeloneuropathy [30,67]. The authors speculated that this was caused by the secondary deficiency of ceruloplasmin, which is responsible for oxidizing iron for binding to transferrin, which facilitates iron mobilization

In particular, the subacute combined degeneration resulting from vitamin B₁₂ deficiency can have a very similar neurologic presentation. Some diagnoses of copper deficiency myeloneuropathy were made only after vitamin B₁₂ supplementation failed to halt neurologic progression [1,4,20,26,35,62]. Copper and vitamin B₁₂ deficiency may also coexist, particularly in patients with a history of gastric surgery [1,2,4,35,68].

Nitrous oxide abuse can also lead to subacute combined degeneration, by inactivation of vitamin B₁₂ [69-71].

Other causes of a dorsal cord syndrome include multiple sclerosis (more typically the primary progressive form), tabes dorsalis, Friedreich ataxia, vascular malformations, epidural and intradural extramedullary tumors, cervical spondylotic myelopathy, and atlantoaxial subluxation [72]. Adrenomyeloneuropathy and hereditary spastic paraplegia should also be considered in some cases.

Laboratory indicators of copper deficiency include a decrease in serum copper or ceruloplasmin levels (more than 90 percent of circulating copper is bound to ceruloplasmin). Measurement of 24-hour urinary copper excretion is a less sensitive marker for copper deficiency [31,73].

All described cases of copper deficiency myeloneuropathy have had low serum copper levels [2,4,16,60]. However, in marginal copper deficiency, serum copper levels may be normal [8,74]. Ceruloplasmin is an acute phase reactant and will be elevated in the presence of inflammatory conditions and also at times in pregnancy, oral contraceptive use, liver disease, malignancy, hematologic disease, myocardial infarction, infections, smoking, diabetes, and uremia. It is possible that in these settings, levels of serum copper and ceruloplasmin may be normal, even while stores of copper are low, and a copper deficiency could be masked. Copper levels may also be low in the absence of true copper deficiency in patients with Wilson disease or in heterozygous carriers of the Wilson disease gene [75].

When excessive zinc ingestion is suspected or when the cause of copper deficiency is unknown, serum zinc and 24-hour urinary zinc excretion levels should be obtained. A significant elevation in these should prompt an investigation for an exogenous source of zinc

Vitamin B₁₂ levels should be checked, as vitamin B₁₂ deficiency may coexist with copper deficiency, particularly in cases of prior gastric surgery [4]. Measurement of methylmalonic acid and homocysteine, which are elevated in vitamin B₁₂ deficiency, increases the sensitivity of laboratory testing and should be performed in high-risk patients.

Neuroimaging — Imaging of the spinal cord, usually with MRI, should be undertaken as part of the initial evaluation in all patients with a myelopathy.

MRI of the spine may be normal [1,2,4,13,16,23,37,46,47,76,77], but many patients have increased T2 signal involving the dorsal column in either the cervical or thoracic cord, or both (image 1) [1,2,4,15,16,19,37,60,64,78,79]. The signal change may also involve the central cord or lateral column [78], and also may extend into the brainstem [80]. Contrast enhancement is typically absent. These findings are similar to those reported in patients with vitamin B₁₂ deficiency [4]. After treatment, the signal abnormality may resolve [4,79].

Evoked potential studies may provide electrophysiologic evidence of posterior column dysfunction [2,16]. Somatosensory evoked potentials, in particular, appear to be abnormal in many patients with copper deficiency myeloneuropathy [1,2,4,15,16,23,46]. Visual evoked potentials may also be prolonged [23].

The standard dose is 2 mg of elemental copper per day. This dose of elemental copper may be given intravenously; a commonly used regimen is 2 mg of elemental copper administered intravenously (over two hours) daily for five days and periodically thereafter. Some patients require higher doses, particularly if administered orally and in the setting of malabsorption. In our practice, we give 8 mg of elemental copper each day orally for a week, 6 mg for the second week, 4 mg for the third week, and 2 mg thereafter. When copper salts are used, the dosing should be adjusted to make sure that the elemental copper is at least 2 mg. Replacement doses are empiric; periodic assessment of serum copper is essential to determine adequacy of replacement and to decide on the most appropriate long-term administration strategy.

Discontinue zinc – In those patients in whom excess zinc ingestion is the likely cause, stopping zinc supplementation may suffice and no additional copper supplementation may be required. However, copper supplementation is generally started at least initially in addition to stopping zinc supplementation [34].

Treat other deficiencies – Copper deficiency in gastrointestinal disease may be accompanied by deficiencies of zinc, vitamin B₁₂, vitamin D, vitamin E, and iron [45]; these should be supplemented as well when appropriate. However, iron therapy in a patient with anemia of indeterminate cause may decrease copper absorption.

Copper supplementation generally prevents further neurologic deterioration, but improvement of neurologic signs and symptoms is variable, often subjective, and limited to sensory symptoms [1,2,4,5,23,24,26,33-35,37,39,44,60,64,79]. Most patients have residual deficits [2,13,14,19,29].

The duration and severity of symptoms prior to supplementation may influence prognosis [82]

By contrast, response of the hematologic parameters (including bone marrow findings) is prompt and often complete [1,2,5,16,28,35,37,63]. Hematologic recovery may be accompanied by reticulocytosis.

The natural history of this disorder is not well known, but it is likely that symptoms will continue to progress in the absence of treatment. In one exceptional case of copper deficiency myeloneuropathy related to excessive zinc ingestion that was not diagnosed for several months, neurologic disability progressed to include diaphragmatic weakness and, ultimately, fatal aspiration [17].

Gastric surgery is the most common cause of acquired copper deficiency.

The most common neurologic manifestation of acquired copper deficiency is that of a myeloneuropathy. Patients typically present with a subacute onset of a gait disturbance.

The hematologic hallmark of copper deficiency is anemia and leukopenia; these are present in most, but not all, patients with associated neurologic deficits.