The condition needs to be differentiated from pure red cell aplasia. In aplastic anemia, the patient has pancytopenia (i.e., leukopenia and thrombocytopenia) resulting in decrease of all formed elements. In contrast, pure red cell aplasia is characterized by reduction in red cells only. The diagnosis can only be confirmed on bone marrow examination. Before this procedure is undertaken, a patient will generally have had other blood tests to find diagnostic clues, including a complete blood count, renal function and electrolytes, liver enzymes, thyroid function tests, vitamin B and folic acid levels.

The following tests aid in determining differential diagnosis for aplastic anemia:

1. Bone marrow aspirate and biospy: to rule out other causes of pancytopenia (i.e. neoplastic infiltration or significant myelofibrosis).

2. History of iatrogenic exposure to cytotoxic chemotherapy: can cause transient bone marrow suppression

3. X-rays, computed tomography (CT) scans, or ultrasound imaging tests: enlarged lymph nodes (sign of lymphoma), kidneys and bones in arms and hands (abnormal in Fanconi anemia)

4. Chest X-ray: infections

5. Liver tests: liver diseases

6. Viral studies: viral infections

7. Vitamin B and folate levels: vitamin deficiency

8. Blood tests for paroxysmal nocturnal hemoglobinuria

9. Test for antibodies: immune competency

The diagnosis of hemolytic anemia can be suspected on the basis of a constellation of symptoms and is largely based on the presence of anemia, an increased proportion of immature red cells (reticulocytes) and a decrease in the level of haptoglobin, a protein that binds free hemoglobin. Examination of a peripheral blood smear and some other laboratory studies can contribute to the diagnosis. Symptoms of hemolytic anemia include those that can occur in all anemias as well as the specific consequences of hemolysis. All anemias can cause fatigue, shortness of breath, decreased ability to exercise when severe. Symptoms specifically related to hemolysis include jaundice and dark colored urine due to the presence of hemoglobin (hemaglobinuria). When restricted to the morning hemaglobinuria may suggest paroxysmal nocturnal haemoglobinuria. Direct examination of blood under a microscope in a peripheral blood smear may demonstrate red blood cell fragments called schistocytes, red blood cells that look like spheres (spherocytes), and/or red blood cells missing small pieces (bite cells). An increased number of newly made red blood cells (reticulocytes) may also be a sign of bone marrow compensation for anemia. Laboratory studies commonly used to investigate hemolytic anemia include blood tests for breakdown products of red blood cells, bilirubin and lactate dehydrogenase, a test for the free hemoglobin binding protein haptoglobin, and the direct Coombs test to evaluate antibody binding to red blood cells suggesting autoimmune hemolytic anemia.

Regular full blood counts are required on a regular basis to determine whether the patient is still in a state of remission.

Many patients with aplastic anemia also have clones of cells characteristic of the rare disease paroxysmal nocturnal hemoglobinuria (PNH, anemia with thrombopenia and/or thrombosis), sometimes referred to as AA/PNH. Occasionally PNH dominates over time, with the major manifestation intravascular hemolysis. The overlap of AA and PNH has been speculated to be an escape mechanism by the bone marrow against destruction by the immune system. Flow cytometry testing is performed regularly in people with previous aplastic anemia to monitor for the development of PNH.

Anemia is typically diagnosed on a complete blood count. Apart from reporting the number of red blood cells and the hemoglobin level, the automatic counters also measure the size of the red blood cells by flow cytometry, which is an important tool in distinguishing between the causes of anemia. Examination of a stained blood smear using a microscope can also be helpful, and it is sometimes a necessity in regions of the world where automated analysis is less accessible.

In modern counters, four parameters (RBC count, hemoglobin concentration, MCV and RDW) are measured, allowing others (hematocrit, MCH and MCHC) to be calculated, and compared to values adjusted for age and sex. Some counters estimate hematocrit from direct measurements.

Reticulocyte counts, and the "kinetic" approach to anemia, have become more common than in the past in the large medical centers of the United States and some other wealthy nations, in part because some automatic counters now have the capacity to include reticulocyte counts. A reticulocyte count is a quantitative measure of the bone marrow's production of new red blood cells. The reticulocyte production index is a calculation of the ratio between the level of anemia and the extent to which the reticulocyte count has risen in response. If the degree of anemia is significant, even a "normal" reticulocyte count actually may reflect an inadequate response.

If an automated count is not available, a reticulocyte count can be done manually following special staining of the blood film. In manual examination, activity of the bone marrow can also be gauged qualitatively by subtle changes in the numbers and the morphology of young RBCs by examination under a microscope. Newly formed RBCs are usually slightly larger than older RBCs and show polychromasia. Even where the source of blood loss is obvious, evaluation of erythropoiesis can help assess whether the bone marrow will be able to compensate for the loss, and at what rate.

When the cause is not obvious, clinicians use other tests, such as: ESR, ferritin, serum iron, transferrin, RBC folate level, serum vitamin B, hemoglobin electrophoresis, renal function tests (e.g. serum creatinine) although the tests will depend on the clinical hypothesis that is being investigated.

When the diagnosis remains difficult, a bone marrow examination allows direct examination of the precursors to red cells, although is rarely used as is painful, invasive and is hence reserved for cases where severe pathology needs to be determined or excluded.

Ringed sideroblasts are seen in the bone marrow.

The anemia is moderate to severe and dimorphic. Microscopic viewing of the red blood cells will reveal marked unequal cell size and abnormal cell shape. Basophilic stippling is marked and target cells are common. Pappenheimer bodies are present in the red blood cells. The mean cell volume is commonly decreased (i.e., a microcytic anemia), but MCV may also be normal or even high. The RDW is increased with the red blood cell histogram shifted to the left. Leukocytes and platelets are normal. Bone marrow shows erythroid hyperplasia with a maturation arrest.

In excess of 40% of the developing erythrocytes are ringed sideroblasts. Serum iron, percentage saturation and ferritin are increased. The total iron-binding capacity of the cells is normal to decreased. Stainable marrow hemosiderin is increased.

In the morphological approach, anemia is classified by the size of red blood cells; this is either done automatically or on microscopic examination of a peripheral blood smear. The size is reflected in the mean corpuscular volume (MCV). If the cells are smaller than normal (under 80 fl), the anemia is said to be microcytic; if they are normal size (80–100 fl), normocytic; and if they are larger than normal (over 100 fl), the anemia is classified as macrocytic. This scheme quickly exposes some of the most common causes of anemia; for instance, a microcytic anemia is often the result of iron deficiency. In clinical workup, the MCV will be one of the first pieces of information available, so even among clinicians who consider the "kinetic" approach more useful philosophically, morphology will remain an important element of classification and diagnosis.

Limitations of MCV include cases where the underlying cause is due to a combination of factors – such as iron deficiency (a cause of microcytosis) and vitamin B12 deficiency (a cause of macrocytosis) where the net result can be normocytic cells.

All beta thalassemias may exhibit abnormal red blood cells, a family history is followed by DNA analysis. This test is used to investigate deletions and mutations in the alpha- and beta-globin-producing genes. Family studies can be done to evaluate carrier status and the types of mutations present in other family members. DNA testing is not routine, but can help diagnose thalassemia and determine carrier status. In most cases the treating physician uses a clinical prediagnosis assessing anemia symptoms: fatigue, breathlessness and poor exercise tolerance. Further genetic analysis may include HPLC should routine electrophoresis prove difficult.

Sideroblastic anemias are often described as responsive or non-responsive in terms of increased hemoglobin levels to pharmacological doses of vitamin B.

1- Congenital: 80% are responsive, though the anemia does not completely resolve.

2- Acquired clonal: 40% are responsive, but the response may be minimal.

3- Acquired reversible: 60% are responsive, but course depends on treatment of the underlying cause.

Severe refractory sideroblastic anemias requiring regular transfusions and/or that undergo leukemic transformation (5-10%) significantly reduce life expectancy.

Anemia is often discovered by routine blood tests, which generally include a complete blood count (CBC). A sufficiently low hemoglobin (Hb) by definition makes the diagnosis of anemia, and a low hematocrit value is also characteristic of anemia. Further studies will be undertaken to determine the anemia's cause. If the anemia is due to iron deficiency, one of the first abnormal values to be noted on a CBC, as the body's iron stores begin to be depleted, will be a high red blood cell distribution width (RDW), reflecting an increased variability in the size of red blood cells (RBCs).

A low mean corpuscular volume (MCV) also appears during the course of body iron depletion. It indicates a high number of abnormally small red blood cells. A low MCV, a low mean corpuscular hemoglobin or mean corpuscular hemoglobin concentration, and the corresponding appearance of RBCs on visual examination of a peripheral blood smear narrows the problem to a microcytic anemia (literally, a "small red blood cell" anemia).

The blood smear of a person with iron-deficiency anemia shows many hypochromic (pale, relatively colorless) and small RBCs, and may also show poikilocytosis (variation in shape) and anisocytosis (variation in size). With more severe iron-deficiency anemia, the peripheral blood smear may show hypochromic, pencil-shaped cells and, occasionally, small numbers of nucleated red blood cells. The platelet count may be slightly above the high limit of normal in iron-deficiency anemia (termed a mild thrombocytosis), but severe cases can present with thrombocytopenia (low platelet count).

Iron-deficiency anemia is confirmed by tests that include serum ferritin, serum iron level, serum transferrin, and total iron binding capacity (TIBC). A low serum ferritin is most commonly found. However, serum ferritin can be elevated by any type of chronic inflammation and thus is not consistently decreased in iron-deficiency anemia. Serum iron levels may be measured, but serum iron concentration is not as reliable as the measurement of both serum iron and serum iron-binding protein levels (TIBC). The ratio of serum iron to TIBC (called iron saturation or transferrin saturation index or percent) is a value with defined parameters that can help to confirm the diagnosis of iron-deficiency anemia; however, other conditions must also be considered, including other types of anemia.

Further testing may be necessary to differentiate iron-deficiency anemia from other disorders, such as thalassemia minor. It is very important not to treat people with thalassemia with an iron supplement, as this can lead to hemochromatosis. A hemoglobin electrophoresis provides useful evidence for distinguishing these two conditions, along with iron studies.

While no single test is reliable to distinguish iron deficiency anemia from the anemia of chronic inflammation, there are sometimes some suggestive data:

- In anemia of chronic inflammation without iron deficiency, ferritin is normal or high, reflecting the fact that iron is sequestered within cells, and ferritin is being produced as an acute phase reactant. In iron deficiency anemia ferritin is low.

- Total iron-binding capacity (TIBC) is high in iron deficiency, reflecting production of more transferrin to increase iron binding; TIBC is low or normal in anemia of chronic inflammation.

Abdominal pain due to hypersplenism and splenic infarction and right-upper quadrant pain caused by gallstones are major clinical manifestations. However, diagnosing thalassemiæ from symptoms alone is inadequate. Physicians note these signs as associative due to this disease's complexity. The following associative signs can attest to the severity of the phenotype: pallor, poor growth, inadequate food intake, splenomegaly, jaundice, maxillary hyperplasia, dental malocclusion, cholelithiasis, systolic ejection murmur in the presence of severe anemia and pathologic fractures. Based on symptoms, tests are ordered for a differential diagnosis. These tests include complete blood count; hemoglobin electrophoresis; serum transferrin, ferritin, total iron-binding capacity; urine urobilin and urobilogen; peripheral blood smear, which may show codocytes, or target cells; hematocrit; and serum bilirubin.

Anemia of chronic disease is usually mild but can be severe. It is usually normocytic, but can be microcytic. The presence of both anemia of chronic disease and dietary iron deficiency in the same patient results in a more severe anemia.

Conventionally, a definitive diagnosis requires a demonstration of depleted body iron stores obtained by bone marrow aspiration, with the marrow stained for iron. However, with the availability of reliable blood tests that can be more readily collected for iron-deficiency anemia diagnosis, a bone marrow aspiration is usually not obtained. Furthermore, a study published April 2009 questions the value of stainable bone marrow iron following parenteral iron therapy.

Experimental gene therapy exists to treat hereditary spherocytosis in lab mice; however, this treatment has not yet been tried on humans due to all of the risks involved in human gene therapy.

In a peripheral blood smear, the red blood cells will "appear" abnormally small and lack the central pale area that is present in normal red blood cells. These changes are also seen in non-hereditary spherocytosis, but they are typically more pronounced in hereditary spherocytosis. The number of immature red blood cells (reticulocyte count) will be elevated. An increase in the mean corpuscular hemoglobin concentration is also consistent with hereditary spherocytosis.

Other protein deficiencies cause hereditary elliptocytosis, pyropoikilocytosis or stomatocytosis.

In longstanding cases and in patients who have taken iron supplementation or received numerous blood transfusions, iron overload may be a significant problem. This is a potential cause of heart muscle damage and liver disease. Measuring iron stores is therefore considered part of the diagnostic approach to hereditary spherocytosis.

An osmotic fragility test can aid in the diagnosis. In this test, the spherocytes will rupture in liquid solutions less concentrated than the inside of the red blood cell. This is due to increased permeability of the spherocyte membrane to salt and water, which enters the concentrated inner environment of the RBC and leads to its rupture. Although the osmotic fragility test is widely considered the gold standard for diagnosing hereditary spherocytosis, it misses as many as 25% of cases. Flow cytometric analysis of eosin-5′-maleimide-labeled intact red blood cells and the acidified glycerol lysis test are two additional options to aid diagnosis.

Definitive therapy depends on the cause:

- Symptomatic treatment can be given by blood transfusion, if there is marked anemia. A positive Coombs test is a relative contraindication to transfuse the patient. In cold hemolytic anemia there is advantage in transfuse warmed blood

- In severe immune-related hemolytic anemia, steroid therapy is sometimes necessary.

- In steroid resistant cases, consideration can be given to rituximab or addition of an immunosuppressant ( azathioprine, cyclophosphamide)

- Association of methylprednisolone and intravenous immunoglobulin can control hemolysis in acute severe cases

- Sometimes splenectomy can be helpful where extravascular hemolysis, or hereditary spherocytosis, is predominant (i.e., most of the red blood cells are being removed by the spleen).

PA may be suspected when a patient's blood smear shows large, fragile, immature erythrocytes, known as megaloblasts. A diagnosis of PA first requires demonstration of megaloblastic anemia by conducting a full blood count and blood smear, which evaluates the mean corpuscular volume (MCV), as well the mean corpuscular hemoglobin concentration (MCHC). PA is identified with a high MCV (macrocytic anemia) and a normal MCHC (normochromic anemia). Ovalocytes are also typically seen on the blood smear, and a pathognomonic feature of megaloblastic anemias (which include PA and others) is hypersegmented neutrophils.

Serum vitamin B levels are used to detect its deficiency, but they do not distinguish its causes. Vitamin B levels can be falsely high or low and data for sensitivity and specificity vary widely. Normal serum levels may be found in cases of deficiency where myeloproliferative disorders, liver disease, transcobalamin II deficiency, or intestinal bacterial overgrowth are present. Low levels of serum vitamin B may be caused by other factors than B deficiency, such as folate deficiency, pregnancy, oral contraceptive use, haptocorrin deficiency, and myeloma.

The presence of antibodies to gastric parietal cells and intrinsic factor is common in PA. Parietal cell antibodies are found in other autoimmune disorders and also in up to 10% of healthy individuals, making the test nonspecific. However, around 85% of PA patients have parietal cell antibodies, which means they are a sensitive marker for the disease. Intrinsic factor antibodies are much less sensitive than parietal cell antibodies, but they are much more specific. They are found in about half of PA patients and are very rarely found in other disorders. These antibody tests can distinguish between PA and food-B malabsorption. The combination of both tests of intrinsic factor antibodies and parietal cell antibodies may improve overall sensitivity and specificity of the diagnostic results.

A buildup of certain metabolites occurs in B deficiency due to its role in cellular physiology. Methylmalonic acid (MMA) can be measured in both the blood and urine, whereas homocysteine is only measured in the blood. An increase in both MMA and homocysteine can distinguish between B deficiency and folate deficiency because only homocysteine increases in the latter.

Elevated gastrin levels can be found in around 80-90% of PA cases, but they may also be found in other forms of gastritis. Decreased pepsinogen I levels or a decreased pepsinogen I to pepsinogen II ratio may also be found, although these findings are less specific to PA and can be found in food-B malabsorption and other forms of gastritis.

The diagnosis of atrophic gastritis type A should be confirmed by gastroscopy and stepwise biopsy. About 90% of individuals with PA have antibodies for parietal cells; however, only 50% of all individuals in the general population with these antibodies have pernicious anemia.

Forms of vitamin B deficiency other than PA must be considered in the differential diagnosis of megaloblastic anemia. For example, a B-deficient state which causes megaloblastic anemia and which may be mistaken for classical PA may be caused by infection with the tapeworm "Diphyllobothrium latum", possibly due to the parasite's competition with host for vitamin B.

The classic test for PA, the Schilling test, is no longer widely used, as more efficient methods are available. This historic test consisted, in its first step, of taking an oral dose of radiolabelled vitamin B, followed by quantitation of the vitamin in the patient's urine over a 24-hour period via measurement of the radioactivity. A second step of the test repeats the regimen and procedure of the first step, with the addition of oral intrinsic factor. A patient with PA presents lower than normal amounts of intrinsic factor; hence, addition of intrinsic factor in the second step results in an increase in vitamin B absorption (over the baseline established in the first). The Schilling test distinguished PA from other forms of B deficiency, specifically, from Imerslund-Grasbeck Syndrome (IGS), a vitamin B12-deficiency caused by mutations in the cobalamin receptor.

The treatment of PA varies by country and area. Opinions vary over the efficacy of administration (parenteral/oral), the amount and time interval of the doses, or the forms of vitamin B (e.g. cyanocobalamin/hydroxocobalamin). More comprehensive studies are still needed in order to validate the feasibility of a particular therapeutic method for PA in clinical practices. A permanent cure for PA is lacking, although repletion of B should be expected to result in cessation of anemia-related symptoms, a halt in neurological deterioration, and in cases where neurological problems are not advanced, neurological recovery and a complete and permanent remission of all symptoms, so long as B is supplemented. Repletion of B can be accomplished in a variety of ways.

Those with hereditary elliptocytosis have a good prognosis, only those with very severe disease have a shortened life expectancy.

Genetic testing for the presence of mutations in protein molecules is considered to be a confirmatory testing technique. It is important to know the risks regarding the transmission and dangers of HPP.

The serum iron and total iron-binding capacity (transferrin) are helpful but not diagnostic; it is quiet possible to have co-existing ineffective iron utilisation and iron deficiency, as determined by bone marrow iron status, e.g. in rheumatoid arthritis.

Typical causes of microcytic anemia include:

- Childhood

- Iron deficiency anemia, by far the most common cause of anemia in general and of microcytic anemia in particular

- Thalassemia

- Adulthood

- Iron deficiency anemia

- Sideroblastic anemia, In congenital sideroblastic anemia the MCV (mean corpuscular volume) is either low or normal. In contrast, the MCV is usually high in the much more common acquired sideroblastic anemia.

- Anemia of chronic disease, although this more typically causes normochromic, normocytic anemia. Microcytic anemia has been discussed by Weng et al.

- Lead poisoning

- Vitamin B (pyridoxine) deficiency

Other causes that are typically thought of as causing normocytic anemia or macrocytic anemia must also be considered, and the presence of two or more causes of anemia can distort the typical picture.

There are five main causes of microcytic anemia forming the acronym TAILS. Thalassemia, Anemia of chronic disease, Iron deficiency, Lead poisoning and Congenital sideroblastic anemia. Only the first three are common in most parts of the world. In theory, these three can be differentiated by their red blood cell (RBC) morphologies. Anemia of chronic disease shows unremarkable RBCs, iron deficiency shows anisocytosis, anisochromia and elliptocytosis, and thalessemias demonstrate target cells and coarse basophilic stippling. In practice though elliptocytes and anisocytosis are often seen in thalessemia and target cells occasionally in iron deficiency. All three may show unremarkable RBC morphology. Coarse basophlic stippling is one reliable morphologic finding of thalessemia which does not appear in iron deficiency or anemia of chronic disease. The patient should be in an ethnically at risk group and the diagnosis is not confirmed without a confirmatory method such as hemoglobin HPLC, H body staining, molecular testing or another reliable method. Course basophlic stippling occurs in other cases as seen in Table 1

Basically classified by causative mechanism, types of congenital hemolytic anemia include:

- Genetic conditions of RBC Membrane

- Hereditary spherocytosis

- Hereditary elliptocytosis

- Genetic conditions of RBC metabolism (enzyme defects). This group is sometimes called "congenital nonspherocytic (hemolytic) anemia", which is a term for a congenital hemolytic anemia without spherocytosis, and usually excluding hemoglobin abnormalities as well, but rather encompassing defects of glycolysis in the erythrocyte.

- Glucose-6-phosphate dehydrogenase deficiency (G6PD or favism)

- Pyruvate kinase deficiency

- Aldolase A deficiency

- Hemoglobinopathies/genetic conditions of hemoglobin

- Sickle cell anemia

- Congenital dyserythropoietic anemia

- Thalassemia

Congenital hemolytic anemia (or hereditary hemolytic anemia) refers to hemolytic anemia which is primarily due to congenital disorders.

The incidence of hereditary elliptocytosis is hard to determine, as many sufferers of the milder forms of the disorder are asymptomatic and their condition never comes to medical attention. Around 90% of those with this disorder are thought to fall into the asymptomatic population. It is estimated that its incidence is between 3 and 5 per 10,000 in the United States, and that those of African and Mediterranean descent are of higher risk. Because it can confer resistance to malaria, some subtypes of hereditary elliptocytosis are significantly more prevalent in regions where malaria is endemic. For example, in equatorial Africa its incidence is estimated at 60-160 per 10,000, and in Malayan natives its incidence is 1500-2000 per 10,000. Almost all forms of hereditary elliptocytosis are autosomal dominant, and both sexes are therefore at equal risk of having the condition. The most important exception to this rule of autosomal dominance is for a subtype of hereditary elliptocytosis called hereditary pyropoikilocytosis (HPP), which is autosomal recessive.

There are three major forms of hereditary elliptocytosis: common hereditary elliptocytosis, spherocytic elliptocytosis and southeast Asian ovalocytosis.

Common hereditary elliptocytosis is the most common form of elliptocytosis, and the form most extensively researched. Even when looking only at this form of elliptocytosis, there is a high degree of variability in the clinical severity of its subtypes. A clinically significant haemolytic anaemia occurs only in 5-10% of sufferers, with a strong bias towards those with more severe subtypes of the disorder.

Southeast Asian ovalocytosis and spherocytic elliptocytosis are less common subtypes predominantly affecting those of south-east Asian and European ethnic groups, respectively.

The following categorisation of the disorder demonstrates its heterogeneity:

- Common hereditary elliptocytosis (in approximate order from least severe to most severe)

- With asymptomatic carrier status - "individuals have no symptoms of disease and diagnosis is only able to be made on blood film"

- With mild disease - "individuals have no symptoms, with a mild and compensated haemolytic anaemia"

- With sporadic haemolysis - "individuals are at risk of haemolysis in the presence of particular comorbidities, including infections, and vitamin B deficiency"

- With neonatal poikilocytosis - "individuals have a symptomatic haemolytic anaemia with poikilocytosis that resolves in the first year of life"

- With chronic haemolysis - " individual has a moderate to severe symptomatic haemolytic anaemia (this subtype has variable penetrance in some pedigrees)"

- With homozygosity or compound heterozygosity - "depending on the exact mutations involved, individuals may lie anywhere in the spectrum between having a mild haemolytic anaemia and having a life-threatening haemolytic anaemia with symptoms mimicking those of HPP (see below)"

- With pyropoikilocytosis (HPP) - "individuals are typically of African descent and have a life-threateningly severe haemolytic anaemia with micropoikilocytosis (small and misshapen erythrocytes) that is compounded by a marked instability of erythrocytes in even mildly elevated temperatures (pyropoikilocytosis is often found in burns victims and is the term is commonly used in reference to such people)

- South-east Asian ovalocytosis (SAO) (also called stomatocytic elliptocytosis) - "individuals are of South-East Asian descent (typically Malaysian, Indonesian, Melanesian, New Guinean or Filipino, have a mild haemolytic anaemia, and has increased resistance to malaria"

- Spherocytic elliptocytosis (also called hereditary haemolytic ovalocytosis) - "individuals are of European descent and elliptocytes and spherocytes are simultaneously present in their blood"