The diagnosis of pyruvate kinase deficiency can be done by full blood counts (differential blood counts) and reticulocyte counts. Other methods include direct enzyme assays, which can determine pyruvate kinase levels in erythrocytes separated by density centrifugation, as well as direct DNA sequencing. For the most part when dealing with pyruvate kinase deficiency, these two diagnostic techniques are complementary to each other as they both contain their own flaws. Direct enzyme assays can diagnose the disorder and molecular testing confirms the diagnosis or vice versa. Furthermore, tests to determine bile salts (bilirubin) can be used to see whether the gall bladder has been compromised.

The diagnosis is generally suspected when patients from certain ethnic groups (see epidemiology) develop anemia, jaundice and symptoms of hemolysis after challenges from any of the above causes, especially when there is a positive family history.

Generally, tests will include:

- Complete blood count and reticulocyte count; in active G6PD deficiency, Heinz bodies can be seen in red blood cells on a blood film;

- Liver enzymes (to exclude other causes of jaundice);

- Lactate dehydrogenase (elevated in hemolysis and a marker of hemolytic severity)

- Haptoglobin (decreased in hemolysis);

- A "direct antiglobulin test" (Coombs' test) – this should be negative, as hemolysis in G6PD is not immune-mediated;

When there are sufficient grounds to suspect G6PD, a direct test for G6PD is the "Beutler fluorescent spot test", which has largely replaced an older test (the Motulsky dye-decolouration test). Other possibilities are direct DNA testing and/or sequencing of the G6PD gene.

The "Beutler fluorescent spot test" is a rapid and inexpensive test that visually identifies NADPH produced by G6PD under ultraviolet light. When the blood spot does not fluoresce, the test is positive; it can be falsely negative in patients who are actively hemolysing. It can therefore only be done 2–3 weeks after a hemolytic episode.

When a macrophage in the spleen identifies a RBC with a Heinz body, it removes the precipitate and a small piece of the membrane, leading to characteristic "bite cells". However, if a large number of Heinz bodies are produced, as in the case of G6PD deficiency, some Heinz bodies will nonetheless be visible when viewing RBCs that have been stained with crystal violet. This easy and inexpensive test can lead to an initial presumption of G6PD deficiency, which can be confirmed with the other tests.

The gold standard for the diagnosis of Vitamin B deficiency is a low blood level of Vitamin B. A low level of blood Vitamin B is a finding that normally can and should be treated by injections, supplementation, or dietary or lifestyle advice, but it is not a diagnosis. Hypovitaminosis B can result from a number of mechanisms, including those listed above. For determination of cause, further patient history, testing, and empirical therapy may be clinically indicated.

A measurement of methylmalonic acid (methylmalonate) can provide an indirect method for partially differentiating Vitamin B and folate deficiencies. The level of methylmalonic acid is not elevated in folic acid deficiency. Direct measurement of blood cobalamin remains the gold standard because the test for elevated methylmalonic acid is not specific enough. Vitamin B is one necessary prosthetic group to the enzyme methylmalonyl-coenzyme A mutase. Vitamin B deficiency is but one among the conditions that can lead to dysfunction of this enzyme and a buildup of its substrate, methylmalonic acid, the elevated level of which can be detected in the urine and blood.

Due to the lack of available radioactive Vitamin B, the Schilling test is now largely a historical artifact. The Schilling test was performed in the past to help determine the nature of the vitamin B deficiency. An advantage of the Schilling test was that it often included Vitamin B with intrinsic factor.

6-phosphogluconate dehydrogenase (6PGD) deficiency has similar symptoms and is often mistaken for G6PD deficiency, as the affected enzyme is within the same pathway, however these diseases are not linked and can be found within the same person.

Most affected individuals with pyruvate kinase deficiency do not require treatment. Those individuals who are more severely affected may die in utero of anemia or may require intensive treatment. With these severe cases of pyruvate kinase deficiency in red blood cells, treatment is the only option, there is no cure. However, treatment is usually effective in reducing the severity of the symptoms.

The most common treatment is blood transfusions, especially in infants and young children. This is done if the red blood cell count has fallen to a critical level. The transplantation of bone marrow has also been conducted as a treatment option.

There is a natural way the body tries to treat this disease. It increases the erythrocyte production (reticulocytosis) because reticulocytes are immature red blood cells that still contain mitochondria and so can produce ATP via oxidative phosphorylation. Therefore, a treatment option in extremely severe cases is to perform a splenectomy. This does not stop the destruction of erythrocytes but it does help increase the amount of reticulocytes in the body since most of the hemolysis occurs when the reticulocytes are trapped in the hypoxic environment of the spleen. This reduces severe anemia and the need for blood transfusions.

Physical examination may show an enlarged spleen. Tests that may be done include: Complete Blood Count (CBC), Hemoglobin electrophoresis, Peripheral blood smear, and Blood hemoglobin.

Genetic counseling may be appropriate for high-risk couples who wish to have a baby.

The blood film can point towards vitamin deficiency:

- Decreased red blood cell (RBC) count and hemoglobin levels

- Increased mean corpuscular volume (MCV, >100 fL) and mean corpuscular hemoglobin (MCH)

- Normal mean corpuscular hemoglobin concentration (MCHC, 32–36 g/dL)

- The reticulocyte count is decreased due to destruction of fragile and abnormal megaloblastic erythroid precursor.

- The platelet count may be reduced.

- Neutrophil granulocytes may show multisegmented nuclei ("senile neutrophil"). This is thought to be due to decreased production and a compensatory prolonged lifespan for circulating neutrophils, which increase numbers of nuclear segments with age.

- Anisocytosis (increased variation in RBC size) and poikilocytosis (abnormally shaped RBCs).

- Macrocytes (larger than normal RBCs) are present.

- Ovalocytes (oval-shaped RBCs) are present.

- Howell-Jolly bodies (chromosomal remnant) also present.

Blood chemistries will also show:

- An increased lactic acid dehydrogenase (LDH) level. The isozyme is LDH-2 which is typical of the serum and hematopoetic cells.

- Increased homocysteine and methylmalonic acid in Vitamin B deficiency

- Increased homocysteine in folate deficiency

Normal levels of both methylmalonic acid and total homocysteine rule out clinically significant cobalamin deficiency with virtual certainty.

Bone marrow (not normally checked in a patient suspected of megaloblastic anemia) shows megaloblastic hyperplasia.

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.

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.

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.

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.

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.

There are 3 possible ways to test the fetal antigen status. Free Cell DNA, Amniocentesis, and Chorionic Villus Sampling. Of the three, CVS is no longer used due to risk of worsening the maternal antibody response. Once antigen status has been determined, assessment may be done with MCA scans.

- Free Cell DNA can be run on certain antigens. Blood is taken from the mother, and using PCR, can detect the K, C, c, D, and E alleles of fetal DNA. This blood test is non-invasive to the fetus and is an easy way of checking antigen status and risk of HDN. Testing has proven very accurate and is routinely done in the UK at the International Blood Group Reference Laboratory in Bristol. Sanequin laboratory in Amsterdam, Netherlands also performs this test. For US patients, blood may be sent to either of the labs. In the US, Sensigene is done by Sequenome to determine fetal D status. Sequenome does not accept insurance in the US, but US and Canadian patients have had insurance cover the testing done overseas.

- Amniocentesis is another recommended method for testing antigen status and risk for HDN. Fetal antigen status can be tested as early as 15 weeks by PCR of fetal cells.

- CVS is possible as well to test fetal antigen status but is not recommended. CVS carries a higher risk of fetal maternal hemorrhage and can raise antibody titers, potentially worsening the antibody effect.

There are several intervention options available in early, mid and late pregnancies.

There are several intervention options available in early, mid and late pregnancies.

There are 3 possible ways to test the fetal antigen status. Free Cell DNA, Amniocentesis, and Chorionic Villus Sampling. Of the three, CVS is no longer used due to risk of worsening the maternal antibody response. Once antigen status has been determined, assessment may be done with MCA scans.

- Free Cell DNA can be run on certain antigens. Blood is taken from the mother, and using PCR, can detect the K, C, c, D, and E alleles of fetal DNA. This blood test is non-invasive to the fetus and is an easy way of checking antigen status and risk of HDN. Testing has proven very accurate and is routinely done in the UK at the International Blood Group Reference Laboratory in Bristol. Sanequin laboratory in Amsterdam, Netherlands also performs this test. For US patients, blood may be sent to either of the labs. In the US, Sensigene is done by Sequenome to determine fetal D status. Sequenome does not accept insurance in the US, but US and Canadian patients have had insurance cover the testing done overseas.

- Amniocentesis is another recommended method for testing antigen status and risk for HDN. Fetal antigen status can be tested as early as 15 weeks by PCR of fetal cells.

- CVS is possible as well to test fetal antigen status but is not recommended. CVS carries a higher risk of fetal maternal hemorrhage and can raise antibody titers, potentially worsening the antibody effect.

MCA scans Middle cerebral artery - peak systolic velocity is changing the way sensitized pregnancies are managed. This test is done noninvasively with ultrasound. By measuring the peak velocity of blood flow in the middle cerebral artery, a MoM (multiple of the median) score can be calculated. MoM of 1.5 or greater indicates severe anemia and should be treated with IUT.

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

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.

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.

There are 3 possible ways to test the fetal antigen status. Free Cell DNA, Amniocentesis, and Chorionic Villus Sampling. Of the three, CVS is no longer used due to risk of worsening the maternal antibody response. Once antigen status has been determined, assessment may be done with MCA scans.

- Free Cell DNA can be run on certain antigens. Blood is taken from the mother, and using PCR, can detect the K, C, c, D, and E alleles of fetal DNA. This blood test is non-invasive to the fetus and is an easy way of checking antigen status and risk of HDN. Testing has proven very accurate and is routinely done in the UK at the International Blood Group Reference Laboratory in Bristol. Sanequin laboratory in Amsterdam, Netherlands also performs this test. For US patients, blood may be sent to either of the labs. In the US, Sensigene is done by Sequenome to determine fetal D status. Sequenome does not accept insurance in the US, but US and Canadian patients have had insurance cover the testing done overseas.

- Amniocentesis is another recommended method for testing antigen status and risk for HDN. Fetal antigen status can be tested as early as 15 weeks by PCR of fetal cells.

- CVS is possible as well to test fetal antigen status but is not recommended. CVS carries a higher risk of fetal maternal hemorrhage and can raise antibody titers, potentially worsening the antibody effect.

There are several intervention options available in early, mid and late pregnancies.

It is unclear if screening pregnant women for iron-deficiency anemia during pregnancy improves outcomes in the United States. The same holds true for screening children who are "6 to 24 months" old.

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.

Diagnosis of canine phosphofructokinase deficiency is similar to the blood tests used in diagnosis of humans. Blood tests measuring the total erythrocyte PFK activity are used for definitive diagnosis in most cases. DNA testing for presence of the condition is also available.

Treatment mostly takes the form of supportive care. Owners are advised to keep their dogs out of stressful or exciting situations, avoid high temperature environments and strenuous exercise. It is also important for the owner to be alert for any signs of a hemolytic episode. Dogs carrying the mutated form of the gene should be removed from the breeding population, in order to reduce incidence of the condition.