Work-up of Anemia

Published March 2020; Update in progress

Authors: Jeff Krimmel-Morrison, MD¹; Sioban Keel, MD²Executive Editors: Yilin Zhang, MD³; Brandon Fainstad, MD⁴

1 Clinical Instructor, Department of Medicine, University of Washington
2 Associate Professor, Department of Medicine, Division of Hematology, University of Washington
3 Assistant Professor, Department of Medicine, University of Washington – Valley Medical Center
4 Associate Professor, Department of Medicine-Internal Medicine, University of Colorado Anschutz

Objectives

Construct a framework for the evaluation of anemia.
Differentiate between types of anemia attributable to underproduction using MCV and iron studies.
Differentiate between immune-mediated and non-immune causes of hemolysis.
Interpret reticulocyte index, hemolysis labs, DAT, and peripheral blood smear results to aid in the diagnosis of anemia.

Teaching Instructions

Plan to spend at least 30-60 minutes preparing for this talk by reviewing the teaching script and clicking through the graphics animations to become familiar with the flow and content of the talk. Print out copies of the Learner’s Handout so learners can take notes as you expand on the pathophysiology and management. The first page of the handout and is a blank schematic that learners can fill in as you go through the evaluation.

The anticipate time to deliver this talk is 45-50 minutes with cases. This can also be delivered as a

Underproduction anemia + Case 1 and 4 (25 min)
Overproduction anemia + Case 2 and 3 (25 min)

Begin with reviewing the objectives for the session. We recommend progressing in order, though this gives you the flexibility of doing more focused teaching. All clickable elements are indicated by a cursor icon and shading around the button. Some elements can be clicked more than once. The cursor will disappear when all clickable elements are completed. A toolbox will appear next to key tests used in the evaluation of anemia.

Objective 1: Construct a framework for the evaluation of anemia. (Overview)

The first step in the evaluation of acute anemia is differentiating between underproduction (problem with the bone marrow) and overproduction. The reticulocyte index (click on this for how to calculate and more information) can help differentiate between these categories. In the hospital, an acute drop in hemoglobin/hematocrit may also be from large fluid shifts/hemodilution, and this will not be associated with a derangement in reticulocyte index. Of note, the reticulocyte index may be falsely low/normal if there is a concurrent underproduction process.

Objective 2: Differentiate between types of anemia attributable to underproduction using MCV and iron studies (Underproduction, Iron Labs)

Underproduction:

Within underproduction causes of anemia, the mean corpuscular volume (MCV) can be further used to differentiate between macrocytic (MCV >100), normocytic (MCV 80-100) and microcytic causes (MCV <80). Some processes may not always necessarily fall into one category. For example, primary bone marrow pathologies can be macrocytic or normocytic. Anemia of chronic disease and iron deficiency anemia (especially in early stages) can be both normocytic or microcytic.

Click on “Differential if other cell lines are affected” to help differentiate between some of the causes of macrocytic/normocytic anemia.

Bonus: Other markers of RBC morphology can help differentiate between causes.

RDW (RBC distribution width) describes the uniformity of RBC sizes. An increased RDW suggests multiple differing sizes of RBCs which may indicate a mixed process (e.g., concurrent microcytic or macrocytic process). This can also be seen in early iron deficiency.
MCHC (Mean corpuscular hemoglobin concentration) measures the mean Hgb concentration in RBCs. This is decreased in iron deficiency.

Iron studies: Click on “Iron Labs” in the left hand navigation bar learn how iron studies help differentiate between anemia of chronic disease and iron deficiency anemia.

Serum iron – this measures serum iron concentration which can fluctuate throughout the day with dietary intake. Fasting levels are the most reliable and accurate. Most circulating iron is bound to transporter protein, transferrin.
TIBC – total iron binding capacity is a measure of transferrin levels.
Transferrin saturation – this is the serum iron divided by TIBC multiplied by 100. Normal transferrin saturation is between 25-45% (some sources state 20-45%).
Ferritin – is a measure of circulating iron stores. It correlates well with iron staining in the bone marrow, once considered the gold standard for iron deficiency. It is the most sensitive measure of iron deficiency and levels <30 mg/L are diagnostic. However, ferritin is also an acute phase reactant so levels are not a reliable measure of iron stores in acute illness or diseases of chronic inflammation.

Click on “iron deficiency” and “ACD” to reveal the values in each of these conditions.

	Serum [Fe]	TIBC	% saturation	Ferritin
Iron deficiency	Low (levels may vary with recent oral intake)	Elevated (in response to low iron stores)	Low (<20% suggestive of iron deficiency; <10% is very suggestive)	Low (<30 mg/L is diagnostic)
Anemia of chronic disease (ACD)	Low (decreased dietary absorption and sequestration in macrophages)	Low – normal (inflammatory cytokines decreases transferrin levels)	Low – normal (% saturation is <20% in 80% of cases, but may be pseudo-normal if TIBC is low)	Normal-high (normal iron stores, but increased secretion by macrophages and inflammatory state elevates ferritin)
Thalassemia	Normal to elevated	Normal to elevated	Normal to elevated	Normal

What is there is concomitant iron deficiency and ACD? 20-85% of patients with ACD will also have iron deficiency. A very low transferrin saturation can be suggestive of concomitant iron deficiency. Hepcidin or soluble transferrin receptor (sTfR) can be used to distinguish between diagnoses if needed. Most patients can be trialed on iron supplementation with repeat CBC and iron studies in a few weeks to assess response.

Objective 3: Differentiate between immune-mediated and non-immune causes of hemolysis (Overproduction, Non-immune hemolysis)

Overproduction:

Causes of anemia associated with overproduction (retic index >3) can be related to blood loss, destruction (hemolysis), or sequestration. Areas of acute blood loss that should be investigated include GI loss, bleeding into long bones, intraabdominal and retroperitoneal bleeding, intrathoracic bleeding.

Identify hemolysis with hemolysis labs (click to reveal tests). These tests include: lactate dehydrogenase (LDH) which is a marker of cell damage; haptoglobin, a protein that binds free hemoglobin; total and indirect bilirubin, and a peripheral smear. LDH will be elevated in hemolysis given the presence of RBC damage and lysis. Haptoglobin will be low because it binds free hemoglobin that is released with hemolysis.

Once there is an indication of hemolysis, a direct Coombs test, or direct antibody test (DAT), can help differentiate between immune vs. nonimmune hemolysis. A DAT detects antibodies against RBCs.

Immune mediated hemolysis (click to reveal differential) or autoimmune hemolytic anemia (AIHA) can be further split into “warm” and “cold” types. Click on each of these to learn to reveal the differential. A peripheral blood smear can be helpful as they will show spherocytes. Spherocytes are formed when the spleen removes parts of the RBC membrane that are bound by antibodies.

Warm AIHA – IgG mediated. Typically associated with malignancy and autoimmune diseases. Some drugs can also cause IgG mediated hemolysis.
Cold AIHA or cold agglutin disease – IgM mediated hemolysis. Associated with malignancy (lymphoma), mycoplasma infections, and mononucleosis typically. Can also be seen with PNH and syphilis.

Click on “non-immune” to reveal a basic breakdown of non-immune causes. This is explored further on the “Non-immune” page in the left hand navigation bar.

Non-immune hemolysis: Non-immune hemolysis is broadly split into microangiopathic hemolytic anemias (fragmentation hemolysis), which are characterized by the presence of schistocytes (fragmented RBCs), and other causes.

Fragmentation hemolysis (microangiopathic hemolytic anemia, MAHA) – characterized by the presence of schistocytes. When there is concurrent thrombocytopenia, this should raise suspicion for a thrombotic microangiopathy (TMA), either primary or secondary.
- Primary TMAs – include causes like thrombotic thrombocytopeni purpura (TTP) or hemolytic uremic syndrome (HUS). Drugs can also cause TMA. DIC and HELLP also produce a TMA-like syndrome. DIC is characterized by the presence of hypofibrinogenemia.
- Other causes – Schistocytes can also be seen in other disease processes such as malignant hypertension, shear stress in prosthetic valves/ LVADs/ TIPs, or scleroderma renal crisis.
Non-fragmentation (non-MAHA) – Non-fragmentation, non-immune hemolysis can be further divided into pathologies that are intrinsic to the RBC or extrinsic to the RBC.
- Extrinsic to RBC –
  - Liver disease or hypersplenism
  - Infections can either cause hemolysis via direct destruction (malaria, Babesia, Bartonella) or via toxin-mediated destruction of RBCs (Clostridium, leishmaniasis).
  - Medications that result in oxidative stress
- Intrinsic to RBC – Causes of hemolysis intrinsic to the RBC are disorders of RBC membrane, RBC metabolism, or hemoglobinopathies. A peripheral blood smear and specialized testing can help differentiate between these causes.
  - Membrane defects – hereditary spherocytosis, elliptocytosis, stomacytosis, PNH.
  - Hemoglobin defects – thalassemia, sickle cell disease
  - Metabolism defects – G6PD deficiency, pyruvate kinase deficiency

Printouts

Learner Handout

Interactive Boards

Take Home Points

The reticulocyte index, hemolysis labs, DAT, and peripheral blood smear can help differentiate between causes of anemia.
Causes of underproduction can be further differentiated by the MCV into macrocytic, normocytic and microcytic etiologies.
Hemolysis is further differentiated by the DAT and peripheral blood smear which can help identify autoimmune hemolytic anemia and microangiopathic hemolytic anemia (fragmentation hemolysis).

References

Packman, CH. The Clinical Pictures of Autoimmune Hemolytic Anemia. Transfus Med Hemother. 2015. 42 (5):317-325.
Image from Werner EJ & Villella, AD. Sideroblastic Anemias: Diagnosis and management. In Nonmalignant Hematology. Pp 125-135.
Image fromhttps://imagebank.hematology.org/image/61458/rouleaux-in-a-peripheral-smear-multiple-myeloma
Image from Kawakami, D. Massive Hemolysis due to Clostridium perfringens infection. Intensive Care Medicine. 2020. 46, 122. https://doi.org/10.1007/s00134-019-05739-2
Weiss G, et al. Anemia of inflammation. Blood. 2019. 133 (1): 40-50.
Camaschella C. Iron deficiency. Blood. 2019. 133(1): 30-39.

Work-up of Anemia

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