ANEMIA

Microcytic Anemia
Normocytic Anemia
Macrocytic Anemia
Classification of Anemia

Schilling's Test

Peripheral Brain Directory

References

 

Basic Lab Workup: CBC with indices, examine smear, platelet count, ferritin level, TSH, BUN/Cr, AST, LDH, indirect bilirubin. Check reticulocyte count and haptoglobin if hemolysis is suspected strongly.

First, use the MCV (mean corpuscular volume) to determine whether the anemia is microcytic, normocytic, or macrocytic.

Microcytic Anemia (MCV<80 fL)                   top of page

Causes: The most common cause is iron deficiency anemia.

Diagnosis: 

Step 1: Check for a Low Ferritin::  The definitive test for iron deficiency anemia is measurement of the serum ferritin level.  An iron deficiency anemia in the presence of a clearly normal or elevated ferritin is very unlikely.  The serum iron, total iron binding capacity and transferrin saturation cannot distinguish iron deficiency from anemia of chronic disease.  Bone marrow aspirate when negative for iron may be helpful, but an empiric trial of iron replacement can be a more useful way to deal with equivocal cases.  Increased RDW (red cell distribution width) favors iron deficiency.  Microcytic anemia with increased red blood cell count is most often Thalassemia trait (MCV/RBC < 13 usually indicates thalassemia,  MCV/RBC >13 is usually iron deficiency).  Microcytosis without anemia is possible in thalassemia trait and in polycythemia associated with iron deficiency. Anisocytosis and poikilocytosis are usually present  in iron deficiency anemia. In severe cases cigar shaped cells or elliptocytes may be present.  Polychromasia, basophilic stippling, and target cells are not typical of iron deficiency.  Reactive thrombocytosis is common in iron deficiency anemia.

Step 2: Evaluation of Microcytic Anemia with Normal Ferritin:  Ask yourself whether the microcytosis is old or new ?  If it is old, then it may be a congenital disorder such as thalassemia.  If it is new, then it may be an acquired non-thalassemic microcytic anemia.

Thalassemic Syndromes:  97% of normal adult hemoglobin (hemoglobin A) is made up of equal quantities of alpha and beta globin chains.  Thalassemia is defined as a decreased production of either of the these two globin chains (e.g., alpha-thalassemia and beta-thalassemia) or a structurally abnormal globin chain (e.g., hemoglobin E).  The abnormal globin causes microcytosis and an abnormal electrophoresis pattern.  The test of choice for investigating thalassemia is hemoglobin electrophoresis, but remember it does not always detect thalassemia (see below).

Alpha-Thalassemia: alpha-globin is controlled by 4 genes (2/haploid chromosome). Consider family history and ethnic origin.

Mutation in 4/4 genes causes hydrops fetalis and death.
Mutation in 3/4 genes causes beta chains to form tetramers of hemoglobin H with severe microcytic anemia. Electrophoresis is abnormal.
Mutation in 2/4 genes causes microcytosis and mild anemia, called alpha-thalassemia trait. Electrophoresis is normal. PCR and southern blot tests can show thalassemia trait.
Mutation in 1/4 genes creates a silent carrier with no microcytosis or anemia.

Beta- Thalassemia: beta-globin is controlled by 2 genes (1/haploid chromosome).

Mutation of 1/2 genes causes beta-thalassemia trait. Note that in this condition with normal ferritin levels Hemoglobin A2 will generally go from a normal value of 2% up to 3-6%. However, if the patient has low iron, then this may not occur. A normal Hemoglobin A2 level must be accompanied by a normal ferritin level to be sure you are not accidentally missing a beta-thalassemia trait condition in which iron is lacking causing a low Hemoglobin A2.
Mutation of 2/2 genes causes symptomatic beta thalassemia. Electrophoresis will show mostly Hemoglobin F (alpha2,gamma2). A slight increase in Hemoglobin F may be seen in beta thalassemia trait and compound heterozygotes. Therefore, electrophoresis will generally be adequate for evaluating beta-thalassemia with genetic testing only needed on occasion.

Structurally Abnormal Globin Chain Thalassemia: some structural hemoglobinopathies can cause a microcytic phenotype due to decreased globin production. The three examples below can be identified by electrophoresis often.

Ex:  Hemoglobin E, structural hemoglobinopathy common in SE Asia due to RNA splice mutation.
Ex: Hemoglobin Lepore, due to fusion of a delta and beta-globin genes.
Ex:  Hemoglobin Constant Spring, due to a stop codon mutation resulting in a longer unstable globin messenger RNA. 

Non-Thalassemic Conditions Associated with Microcytosis other than Iron Deficiency: the differential diagnosis of this includes: Anemia of Chronic Disease and Hereditary or Acquired Sideroblastic Anemia.

Anemia of Chronic Disease: this anemia is usually normocytic. However, some systemic diseases can result in a microcytic process, e.g., rheumatoid arthritis, polymyalgia rheumatica, diabetes mellitus, connective tissue diseases, chronic infection, Hodgkin's Lymphoma (HL), Castleman disease (CD), renal cell carcinoma (RCC), and myelofibrosis with myeloid metaplasia (MMM). The first five cause mild microcytosis, with normal RDW, and normal smear.  Diagnosis is made clinically. Microcytic anemia in the last three is often associated with systemic symptoms: fever, constitutional symptoms, pruritus (HL), lymphadenopathy (HL,CD), monoclonal gammopathy (CD), hematuria (RCC),  splenomegaly (MMM, HL, CD), or leukoerythroblastosis (nucleated red cells on peripheral smear (MMM).  

Normocytic Anemia (MCV 80-100fL)             top of page

Causes:  Many.

Diagnosis:

Step 1: Rule out readily treatable causes.

Nutritional Anemia: may be related to both iron deficiency and/or B12/folate deficiency.  First, determine a ferritin and B12/folate levels.

Anemia of Renal Insufficiency: there will be a normal blood smear with an inappropriately elevated erythropoietin level. Can be severe when Cr>3 mg/dL, mild to moderate when Cr 1.5-3.0 mg/dL. Consider hemolysis.

 Hemolytic Anemia: look for evidence of red cell destruction with elevated serum LDH;  increased red cell catabolism with increased indirect bilirubin, decreased level of haptoglobin (serum protein that clears free hemoglobin); and increased reticulocytes demonstrating increased bone marrow regenerative action. Therefore, when hemolysis is suspected check: reticulocyte count, indirect bilirubin, serum LDH, and haptoglobin.  None of these tests are specific. Urinary hemosiderin is absent in extravascular hemolysis, but present in intravascular hemolysis  

Extravascular hemolytic processes: red cell intrinsic and immune mediated hemolytic anemia. 
Intravascular hemolytic processes: microangiopathic processes, infection associated, and chemical-induced hemolytic anemia.

Differential Diagnosis of Hemolytic Anemia: 

Red Cell Intrinsic causes:

Membranopathies (e.g., hereditary spherocytosis- the MCHC>36)
Enzymopathies (e.g., G6PD deficiency- interacts with sulfa)
Hemoglobinopathy (e.g., sickle cell disease)

Red Cell Extrinsic causes:

Immune Related

Autoimmune

Drug
Viral
: parvovirus
Lymphoid
 
Idiopathic

Alloimmune

Immediate Transfusion
Delayed  Transfusion
Neonatal Hemolysis

Microangiopathic (e.g., TTP/HUS)

Infection Related (e.g., falciparum malaria)

Chemical Agent (e.g., spider venom)

Step 2: Normocytic Anemia not associated with Nutritional deficiency, Renal Insufficiency, or Hemolysis will include: Anemia of Chronic Disease or Primary Bone Marrow disorder or other causes.

Anemia of Chronic Disease: usually normocytic, but can be microcytic. Current theory is that it is caused by a cytokine-mediated inhibition of red cell production or erythropoietin production or function. Often associated with diabetes mellitus, connective tissue disease, chronic infections, and malignancy. Serum iron and transferrin saturation may be low  in these conditions, therefore always check a ferritin level, this is the best test to differentiate iron deficiency from anemia of chronic disease.

Anemia due to a Primary Bone Marrow Disorder: check the peripheral smear in these conditions. Consider a bone marrow biopsy, especially in younger patients with histories of chemotherapy or abnormal blood smears.

Myelodysplastic Syndrome: look for an increased RDW, oval macrocytes, hyposegmented neutrophils, or monocytosis.
Bone Marrow Infiltration: think about metastatic cancer and myeloid metaplasia.  Look for nucleated RBC's, immature myeloid cells. Rouleaux may form in multiple myeloma. Consider lymphoma, leukemia.
Aplastic Anemia: look for severe anemia with low reticulocyte count. Look for platelet and white cell abnormalities or low counts which are common in many bone marrow effecting conditions. Consider  pure red cell aplasia, congenital, idiopathic, paroxysmal nocturnal hemoglobinuria, Fanconi anemia.

Other Causes: 

Drug Effects
Alcoholism
Radiation Therapy
Chemical Exposure
Acute Blood Loss
Recent Trauma
Recent Surgery

Macrocytic Anemia (MCV>100 fL)               top of page

Diagnosis:

Step 1: Rule out drugs that cause macrocytosis.

Consider: alcohol, hydroxyurea, methotrexate, trimethoprim, zidovidine, 5-fluorouracil.  Hydroxyurea can cause MCV>110 fL.

Step 2: Rule out Nutritional Causes of Macrocytic Anemia.

Check for vitamin B12 and folate deficiencies with a B12 level, methylmalonic acid level, folate level, red cell folate level, and homocysteine level.  Folate levels can be altered by recent dietary intake.  Red cell folate levels can be imprecise. Homocysteine levels will be high in folate deficiency due to impaired folate dependent conversion of homocysteine to methionine.  Therefore, a normal homocysteine level is good evidence against folate deficiency.  B12 levels may be falsely low during pregnancy, in the elderly, and when white cell counts are low. The methylmalonic acid level is a more sensitive and specific test for B12 deficiency. If the methylmalonic acid level is normal then B12 deficiency is very unlikely.  B12 is a cofactor for converting methylmalonyl coenzyme A to succinyl coenzyme A, so if methylmalonic acid levels are high then B12 may be low.  In this setting, renal insufficiency and certain inborn errors of metabolism can cause an increased methylmalonic acid level.

Once a diagnosis of B12 deficiency is made, then you must search for a cause for it.  The first test is a screen for intrinsic factor antibodies.  If it is positive you have diagnosed pernicious anemia and begin replacement therapy.  If it is negative then a Schilling's test can be performed to help differentiate pernicious anemia from intestinal malabsorptive disorders. Malabsorptive disorders may include: tropical sprue, celiac sprue, inflammatory bowel disease, amyloidosis, intestinal lymphoma, pancreatic insufficiency (creates a lack of pepsin needed to release food bound B12), bacterial overgrowth, and gastric atrophy (fail to release acid to allow release of food bound B12).

Step 3: Evaluate for Non-Drug Induced, Non-Nutritional Macrocytic Anemia.

First, classify whether the macrocytosis is mild (MCV 100-110 fL) or marked (>110).

Marked Macrocytosis: when this is not due to nutritional deficiency it is nearly always due to a primary bone marrow disease such as: Myelodysplastic syndrome, Aplastic anemia, pure red cell aplasia, or large granular lymphocyte disorder.  A bone marrow biopsy is indicated if it will make a difference in care.

Mild Macrocytosis: First, study the peripheral smear.  Polychromasia (indicates reticulocytosis) suggests hemolysis. Red cells appearing round or as target cells suggests liver disease or hypothyroidism.

Schilling's Test Procedure                       top of page

Performed in two stages, but stage two is performed only if stage 1 is abnormal.

In stage 1, patients receives, 1 mg B12 IM to saturate B12 binding proteins and 1 mcg of radiolabeled crystalline B12 orally. A 24 hour urine collection is started. This urine is checked for radioactivity. If >7% of the ingested load of B12 is found in the urine then the patient has no problem absorbing crystalline B12.

A normal stage 1 rules out pernicious anemia.  Malabsorption may still be a problem.  Elderly people may lack gastric acid due to gastric atrophy or pepsin needed to release food bound B12. Abnormal stage 1 results suggest either pernicious anemia or malabsorption. Rarely, bacterial overgrowth or pancreatic insufficiency may cause abnormal stage 1 results.

In stage 2, patients with abnormal stage 1 results are given intrinsic factor (60 mg) along with oral B12.  If the B12 is absorbed adequately then pernicious anemia is present.  If it does not correct, then you cannot absolutely rule out pernicious anemia because the gut epithelium may be abnormal preventing absorption and mimicking malabsorption. Therefore the best time to do a Schilling's test is after two weeks of treatment with B12 to assure the absorptive surfaces are healed adequately. 

Classification of Anemia              top of  page

Microcytic Anemia

Iron Deficiency anemia

Inadequate Absorption

Poor Bioavailability
Antacid Rx or hi gastric pH
Excess Dietary Bran, Tannins, Phytates or Starch
Competition for other metals (e.g. copper or lead)
Loss or dysfunction of eneterocytes
Short Bowel Syndrome or bowel resection
Celiac Disease
Inflammatory Bowel Disease
Intrinsic Enterocyte decrease

Increased Loss

Gastrointestinal Blood Loss

Epistaxis
Varices
Gastritis
Peptic Ulcer Disease
Colon Cancer
Stomach Cancer
Esophageal Cancer
Meckel's Diverticulum
Parasites
Milk enteropathy of early childhood
AV malformations
Inflammatory Bowel Disease
Diverticulosis
Hemorrhoids

Genitourinary Blood Loss

Menstruation/Menorrhagia
Cancer
Chronic Infection

Pulmonary Blood Loss

Pulmonary Hemosiderosis
Chronic Infection

Other Blood Loss

Trauma
Excessive Phlebotomy


Dietary Deficiency

Vegetarian Diets
Poor dietary intake



Thalassemia

Alpha Thalassemia
Beta Thalassemia

Non-thalassemic Conditions associated with microcytosis 

Anemia of Chronic Disease

Rheumatoid Arthritis
Hodgkin Lymphoma
Polymyalgia Rheumatica
Diabetes Mellitus
Connective Tissue Diseases
Chronic Infection
Castleman Disease
Renal Cell Carcinoma
Myelofibrosis with Myeloid Metaplasia

Sideroblastic Anemia

Hereditary
Acquired
Lead Poisoning

Normocytic Anemia

 Blood Loss, Acute

Nutritional Anemia

Iron Deficiency Anemia
B12 Deficiency

Anemia of Renal Insufficiency

Hemolytic Anemia

Red Cell Intrinsic causes

Membranopathies

Hereditary Spherocytosis

Enzymopathies (Annals Int Med, 1985; 103:245-57)

Glucose-6-phosphate Dehydrogenase deficiency
Hexose Monophosphate Shunt deficiencies
Pyruvate Kinase Deficiency
Hexokinase Deficiency
Glucose Isomerase Deficiency
Phosphofructokinase Deficiency
Aldolase Deficiency
Phosphoglycerate Kinase Deficiency
Lactate Dehydrogenase Deficiency
2,3-DiPhosphoglycerate mutase and Phosphatase Deficiency
Adenylate Cyclase Enzymopathy
Adenosine Deaminase Enzymopathy
Pyrimidine Nucleotidase Deficiency

Hemoglobinopathy

Sickle Cell Disease

Red Cell Extrinsic causes

Immune Mediated

Autoimmune

Drug
Virus
Lymphoid Disorder
Idiopathic

Alloimmune

Immediate Transfusion Rx
Delayed Transfusion Rx
Neonatal Hemolytic

Microangiopathic

HUS
TTP

Infection Related

Falciparum Malaria
Septicemia

Chemical Agent

Spider Venom

Anemia of Chronic Disease

Primary Bone Marrow Disorder

Intrinsic to hematopoietic stem cells

Aplastic Anemia

Idiopathic
Paroxysmal Nocturnal         Hemoglobinuria  
Fanconi Anemia

Pure Red Cell Aplasia

Acquired
Congenital

Ineffective Erythropoiesis

Myelodysplastic Syndrome
Other Myeloid Process

Extrinsic Causes

Drugs
Toxins
Virus 

Parvovirus B19

Immune Mediated
Bone Marrow Infiltrating Process

Lymphoma
Metastatic Cancer

Macrocytic Anemia

Drug Induced

Hydroxyurea, methotrexate, zidovidine, etc.

Nutritional

B12
Folate

Non-Drug, Non-Nutritional

Marked Macrocytosis

Clonal Disorder

Myelodysplastic disorder
Aplastic Anemia
Large Granular Lymphocyte Disorder

Mild Macrocytosis

Clonal Disorder

Oval Macrocytes

Alcohol or Liver Disease

Round Macrocytes

Hemolytic Anemia- reticulocytosis

Spurious

Cold agglutinins present
Hyperglycemia

References:

Teferri,A, Anemia in Adults: A contemporary Approach to Diagnosis.  Mayo Clinic Proceedings. 2003;78:1274-80.       Plagiarized heavily for this page !!  Thanks.
Andrews,NC, Disorders of Iron Metabolism. NEJM. 1999;341:1986-94.
Valentine,WN,et al, Hemolytic Anemias and Erythrocyte Enzymopathies. Annals Int Med. 1985;103:245-57.

 

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