Searching for clues beyond iron deficiency
by Pierre Laneuville, MD and Chantal Cassis, MD
Vol.15, No.05, May 2007

What are the causes of anemia?
Anemia can result from deficient hemoglobin production, increased red blood cell (RBC) destruction, or from blood loss. Impaired hemoglobin production leads to hypoproliferative anemias with low or inappropriately normal reticulocyte counts. Increased RBC destruction or hemolysis and/or blood loss cause hyperproliferative anemias with elevated reticulocyte counts. In adults, the most common variants are iron deficiency anemia, megaloblastic anemias due to folate or vitamin B12 deficiency, anemia of chronic disease, anemia due to renal failure, and myelodysplastic syndrome (MDS). Since iron deficiency anemia has been discussed in depth in the April 2007 issue of Parkhurst Exchange, we will focus this discourse on anemias with other causes.

How are anemias classified?
Mechanistically, anemias are hypoproliferative, i.e. reticulocyte counts are low due to an inability to synthesize proper amounts of RBCs, or hyper-proliferative. In the hyperproliferative case, reticulocyte counts are high but can't compensate for increased blood loss or destruction of RBCs. The reticulocyte count is used to differentiate between these two variants.

Anemias can be further subdivided according to the mean corpuscular volume (MCV) of the RBCs. The cells can be microcytic (MCV < 80 fL), macrocytic (MCV > 100 fL) or normocytic (80 fL < MCV < 100 fL). This classification aids in identifying the source of the anemia (see Table). It's important to rule out sources of pseudo-macrocytosis when interpreting the MCV. RBC agglutination and very elevated reticulocyte numbers can give a falsely high MCV, as the machine may count them as large RBCs.

How does classification help pinpoint the cause of anemia?
Hypoproliferative anemias often manifest as microcytic and megaloblastic macrocytic anemias. Iron deficiency, acquired and congenital sidero-blastic anemias, vitamin B12 or folate deficiencies, MDS or other primary marrow failure processes, and drugs such as antiretrovirals, methotrexate and alcohol all give rise to hypoproliferative anemias. Other important causes that present with normal MCVs include anemia of chronic disease, renal failure with decreased erythropoietin production, multiple myeloma and endocrinopathies such as hypothyroidism.

Hyperproliferative anemias include all causes of normocytic anemia secondary to RBC destruction. Hemolysis can be immune-mediated -- as is the case in auto-immune hemolytic anemia -- or nonimmune-mediated. Nonimmune hemolytic anemias may be caused by factors intrinsic to the RBCs such as cell membrane disorders (hereditary spherocy-tosis), hemoglobinopathies (sickle cell anemia) or enzymopathies (glucose-6-phosphate dehydrogenase deficiency [G6PD]). They can also be the result of factors extrinsic to the RBC such as infectious agents (malaria), prosthetic valves or fibrin clots like those formed in disseminated intravascular coagulation. Nonmegaloblastic macrocytic anemias due to alcohol abuse, liver disease or hypothyroidism also present with elevated reticulocyte counts. Finally, thalassemia syndromes are the sole microcytic anemias that can present with increased reticulocyte counts. Usually, these counts are normal or slightly elevated in thalassemia.

What information should we get from the history?
Often, you can clarify the etiology of anemia solely based on laboratory investigations, but the history and physical exam focus the laboratory workup and avoid unnecessary tests which may be costly, invasive and time-consuming to the patient.

You first need to assess the impact of anemia on the person's health. Is the individual so fatigued that he or she is unable to participate in the activities of daily life? Is the anemia worsening cardiopulmonary function in a patient at risk? If so, you should treat more aggressively and consider temporary measures such as blood transfusions.

The history should also provide clues as to the etiology of the anemia. Based on the mechanisms mentioned above, look out for a history of overt or occult blood loss. In men, this includes asking about bloody stools, melena, bloody vomit and blood in the urine. The same questions apply to women, but also take a detailed history of menses including regularity, length of cycle in days, number of days with heavy bleeding, number of pads or tampons used per day or hour, waking at night to change, and the presence of clots. There are no official abnormal values for these parameters, so use clinical judgement in interpreting the numbers. Menses should usually last no longer than 7 days -- with heavy bleeding for 3 days or less.

What risk factors should we look out for?
For hypoproliferative anemias, inquire about the ethnic background, longstanding anemia and family history, as they're common factors in thalassemia. Gastrointestinal (GI) symptoms, known GI pathology or GI surgeries, as well as symptoms of autoimmune disease -- e.g. joint pain, synovitis, vitiligo, skin rash, photosensitivity and aphthous ulcers -- suggest deficiencies in folate, vitamin B12 or iron. Prolonged use of proton pump inhibitors or histamine receptor blockers may also lead to vitamin B12 malabsorption. Excessive alcohol consumption can be directly toxic to the bone marrow, resulting in macrocytic anemia. The presence of inflammatory illnesses, malignancy or chronic renal failure would support a diagnosis of anemia of chronic disease.

Patients with hyperproliferative anemias secondary to cell membrane disorders, hemoglobinopathies or enzymopathies often have family histories of anemia or are from geographic regions at high risk. G6PD deficiency occurs worldwide, but it's more prevalent in the tropical and subtropical zones of the Eastern Hemisphere. Sickle cell anemia is particularly common among people whose ancestors come from sub-Saharan Africa, Spanish-speaking regions such as South America, Cuba and Central America, Saudi Arabia, India and Mediterranean countries. Other risk factors for hyperproliferative anemias include the presence of prosthetic cardiac valves and recent travel to areas with a high prevalence of malaria. Recent RBC transfusions -- i.e. within the past 120 days -- are a risk factor for immediate or delayed immune-mediated hemolysis. Finally, ask about a history compatible with acute hemolytic crisis or a microangiopathic hemolytic process.

What's the focus of the physical exam?
Lymph nodes, spleen and liver are important to examine. Any enlarged lymph nodes or splenomegaly or exquisite bony tenderness may indicate a primary marrow problem. Jaundice and splenomegaly with a history of gallstones suggest a chronic hemolytic process. A thorough examination of the skin, mucous membranes and nails can also give clues.

A neurologic exam may be warranted if you suspect vitamin B12 deficiency; this should include an assessment of muscle tone and strength, vibration sense and proprioception as well as gait. You are looking for the classic features of subacute combined degeneration of the dorsal and lateral spinal columns. Anemia or macrocytosis don't necessarily precede the neuropsychiatric manifestations of vitamin B12 deficiency, especially in older patients.

What baseline blood work should we order?
A complete blood count (CBC) with blood smear, reticulocyte count, international normalized ratio, partial thromboplastin time and prothrombin time will be helpful in all patients. With the reticulocyte count you can differentiate between hypoproliferative and hyperproliferative anemia. The CBC provides the MCV and red cell distribution width (RDW) -- the variation in RBC size. The combination of reticulocyte count with the MCV and RDW narrows the differential diagnosis of anemia tremendously (see Table). Pancytopenia -- i.e. a reduction in red and white blood cells (WBC) as well as platelets -- or bicytopenia indicate a primary bone marrow failure. Abnormal platelet counts and coagulation studies suggest occult bleeding as a cause of anemia.

The morphology of RBCs, platelets and the WBC on a smear can also be useful. The presence of schistocytes -- RBC fragments resulting from membrane damage -- in combination with a reduced platelet count indicates that a microangiopathic hemolytic process is occurring. Spherocytes -- round rather than donut-shaped RBCs -- occur in the presence of an autoimmune hemolytic process or can be inherited. Rouleaux, i.e. clumps of blood cells that take the shape of stacks, are seen in multiple myeloma and Waldenström's macroglobulinemia. Intra-erythrocyte inclusion bodies, such as Heinz bodies, basophilic stippling and malaria, may also be useful for making a diagnosis. Neutrophils can demonstrate characteristic features of vitamin B12 deficiency or myelodysplastic syndrome, e.g. hyper--segmentations (> 4 lobes in > 1 neutrophil or > 5 in 1 neutrophil) and pseudo Pelger-Huët cells (bilobed neutrophils), respectively.

How do we interpret the reticulocyte count?
The reticulocyte count is often reported as a percentage. It needs to be converted either to an absolute count or to be adjusted for the total number of RBCs present. To calculate the absolute reticulocyte count, multiply the percentage with the RBC count in units of cells/µL. To adjust the reticulocyte percentage, multiply it by the patient's hematocrit divided by a normal hematocrit (preferably age and gender appropriate). For practical purposes, an absolute reticulocyte count < 100,000 cells/µL or a corrected count < 2% suggests a hypoproliferative anemia.

How do we proceed after the baseline workup?
Results of the baseline investigations will direct your subsequent workup. This second step should usually yield the diagnosis.

If you find a hypoproliferative anemia with low MCV, you should suspect iron deficiency anemia, thalassemia or sideroblastic anemias. In this case, you order a ferritin level, fasting iron profile and hemoglobin electrophoresis. Also make sure to rule out the presence of risk factors for acquired sideroblastic anemias such as isoniazid use, alcoholism or lead exposure. If a patient is found to be iron-deficient, the search for a cause is very important.

In the case of a hypoproliferative anemia with high MCV, you should look for causes of megaloblastic anemia and order vitamin B12 and folate levels. Medications such as antiretrovirals or immunosuppressants for auto-immune diseases are risk factors. If vitamin B12 levels are equivocal (> ~74 pmol/L -- the exact range is assay-dependent), measure-ment of the serum concentrations of homocysteine (HC) and methylmalonic acid (MMA) are helpful in clarifying the diagnosis. HC and MMA metabolism is dependant on cobalamin, and a deficiency in the latter leads to increased concentrations of HC and MMA. Elevated HC and MMA levels have been shown to precede the decrease in serum vitamin B12. In fact, they're more sensitive than vitamin B12 levels in diagnosing cobalamin deficiency. Combined elevated levels of HC and MMA have a sensitivity of 99.8%.

Many studies have demonstrated that if we rely solely on low levels of serum vitamin B12 to diagnose cobalamin deficiency, the rate of missed diagnoses can range from 10-50%. If normalization of elevated HC and MMA levels in response to vitamin B12 replace-ment therapy is used as diagnostic criteria, 50% more cases of cobalamin deficiency can be detected. When using HC and MMA levels, however, a few caveats need to be kept in mind. Increased HC levels can also be caused by folate or vitamin B6 deficiencies, while elevated MMA levels may occur with renal failure. These conditions must be ruled out before a diagnosis of cobalamin deficiency is made. If you diagnose vitamin B12 or folate deficiencies, you must pursue further testing to identify their etiology. Upper GI endoscopy with biopsies can reveal H. pylori or parasitic (Giardia) infections, as well as celiac disease as the cause of the deficiencies. Lower GI endoscopy is used to rule out inflammatory bowel disease or other causes of terminal ileum disease that can lead to nutritional deficiencies. A Schilling test and intrinsic factor antibodies are helpful in diag-nosing pernicious anemia.

Hypoproliferative anemias with normal MCV occur in anemia of chronic disease including chronic renal failure, endocrinopathies and primary marrow pathologies such as aplastic anemia and multiple myeloma. A workup should include a serum protein electrophoresis and possibly urine protein electropho-resis, renal function tests, calcium and a search for inflammation. Make sure to exclude vitamin B12 and folate deficiency in normocytic anemias since morphologic features can lag behind the development of the anemia. A bone marrow biopsy may also be useful here.

Hyperproliferative anemias with normal or large MCVs should be investigated with liver and thyroid function tests as well as a hemolysis profile including total and direct bilirubin, lactate dehydrogenase and haptoglobin. If the history or baseline blood work suggests a particular hemolytic syndrome, order the appropriate special test. Hereditary spherocytosis is diagnosed with an osmotic fragility test, G6PD deficiency with a G6PD assay and sickle cell anemia with hemoglobin electrophoresis. If you suspect autoimmune hemolysis, a direct antiglobulin test will confirm the presence of auto-antibodies.

If the history is strongly suggestive of a specific cause of anemia, you may add the specific test at the time of the baseline workup. Also, since iron, vitamin B12 and folate deficiencies are common causes of anemia, you may want to order the appropriate tests at the same time.

When should we perform a bone marrow biopsy?
Bone marrow aspiration and biopsy permit the evaluation of cellular morphology and marrow architecture. Special studies can be performed on both the aspirate and biopsy. Flow cytometry on the aspirate may help clarify the origin of abnormal marrow elements by identifying cell surface proteins. Cytogenetics is performed on the aspirate to identify genetic abnormalities, which may be useful in diagnosing primary marrow problems such as lymphomas or leukemias. The study can be performed with standard techniques (Giemsa banding) or by fluorescent in situ hybridization (FISH). While standard cytogenetics look at all chromosomes, FISH can only search for specific genetic abnormalities. Immunohistochemistry and molecular studies may also be performed on the biopsy.

Bone marrow aspiration and biopsy are invasive procedures, so be careful to consider your options before deciding to pursue these investigations. You should determine beforehand what special studies will be helpful and what samples are needed to perform them. You also need to know how to collect, preserve and send the samples to the proper centre. If uncertain, we recommend communicating with the laboratory that performs the special studies. If MDS, leukemia or lymphoma are suspected, aspirate samples should be sent for flow cytometry and cytogenetics in heparinized tubes. Also consider a bone marrow aspirate and biopsy in cases of unclear hypoproliferative anemias.

Why do some patients not perfectly "fit" a diagnosis?
Anemia may have more than one cause, which can lead to a mixed picture. Patients with chronic hemolysis from hereditary disorders such as sickle cell anemia may develop folate or vitamin B12 deficiency from the high RBC turnover rate and therefore not have the expected elevated reticulocyte count. Young women with thalassemia can have superimposed iron deficiency anemia from heavy menses, yielding a false negative hemoglobin electrophoresis due to insufficient quantities of abnormal hemoglobin synthesis. So if investigations don't "fit" a single diagnosis, consider multiple etiologies.

Anemias are also not an "all or none" phenomenon. Those secondary to nutritional deficiencies progress slowly over time, so patients may present to your office before fully developing the classic clinical and biochemical features of a particular anemia. These incomplete profiles explain the diagnostic difficulties encountered in some cases. Early iron deficiency anemia can also present with normal MCV, and early vitamin B12 and folate deficiency can lack the classic high MCV.

When should we refer to a hematologist?
Cases where diagnostic uncertainty remains after all is said and done should be referred to a hematologist. Patients with diagnoses of cell membrane disorders, hemoglobinopathies or enzymopathies may need to be followed in a specialized hematology clinic. MDS, leukemia, lymphoma or multiple myeloma are condi-tions that need urgent evaluation by a hematologist. But general practitioners can diagnose and treat iron deficiency anemia, vitamin B12 or folate deficiency and anemias of chronic disease.

Pierre Laneuville, MD, FRCPC is an associate professor in the Department of Medicine and Oncology at McGill University and the former Head of Hematology for the McGill University Health Centre in Montreal, Quebec.

Chantal Cassis, MD, is a Hematology Fellow at the McGill University Health Centre.

feature image
Classification and causes of anemia

HYPOPROLIFERATIVE ANEMIA (corrected reticulocyte count < 2%)

elevated RDW

variable RDW

elevated RDW

normal RDW

normal to elevated RDW

elevated RDW

  • lipid abnormalities


  • Hoffman R (ed). Hematology: Basic Principles and Practice, 4th Ed. Churchill Livingstone, 2004.
  • Young NS et al. Clinical Hematology. Philadelphia, PA. Mosby Elsevier, 2006.
  • Oh R, Brown DL. Vitamin B12 deficiency. Am Fam Physician 2003;67:979-86.
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