Understanding Clinical

Laboratory Tests
Laboratory Tests for Evaluation of Anemia

Additional Tests
Recommended Tests
(as Indicated)

• Complete blood count with • Fractionated bilirubin

indices • Stool guaiac
• Peripheral smear • Bone marrow examination
• Reticulocyte count • Coombs test
• Iron studies • Haptoglobin level
• Vitamin B12 • Hemoglobin (Hgb)
• Folate electrophoresis
• Lactate dehydrogenase • Free erythrocyte
protoporphyrin

The NCCN Clinical Practice Guidelines in Oncology: Cancer- and Treatment-Related Anemia.
Available at: www.nccn.com/professionals/physician_gls/default.asp.
Spivak JL. Semin Oncol. 1994;21:3-8.
Definitions of Laboratory Measures
Normal Laboratory Values
Laboratory measure Normal values
Hemoglobin (Hgb) 12–18 g/dL
Hematocrit (Hct) 36%–53%
Red blood cell (RBC) count 4.0 x 1012/L–5.9 x 1012/L
Serum ferritin 40–160 µg/L
Serum iron 65–165 µg/L
Total iron-binding capacity (TIBC) 300–360 µg/dL
Transferrin saturation (TSAT) 20%–50%
Reticulocytes 0.5%–2.5% of RBCs
Mean corpuscular volume (MCV) 80–98 fL
Soluble transferrin receptor (sTfR) 5.3–5.9 mg/L
RBC protoporphyrin 16–36 µg/dL RBC
Methylmalonic acid (vitamin B12) 0.05–0.26 µmol/L
Homocysteine (folic acid) < 12 µmol/L
Geaghan SM. In: Hematology Basic Principles and Practice. 4th ed. 2005:2733-2743.
Perkins S. Wintrobe’s Clinical Hematology. 10th ed. 1999:2738-2746.
Brittenham SM. In: Hematology Basic Principles and Practice. 4th ed. 2005:2733-2743.
Iron Disorders Institute. Available at: www.irondisorders.org/Forms/FActsAnemia.pdf. Accessed June 27, 2006.
Ferri FF. Laboratory tests-Protoporphyrin. Available at: www.mermedicus.com/pp/us/hcp/printpage.jsp. Accessed June 27, 2006.
Holleland G. Clin Chem. 1999;45:189-198.
Cummings KC. Physicians Reference Laboratory. Available at: www.prlnet.com/Homocysteine.htm. Accessed June 27, 2006.
Hemoglobin

• Iron-rich protein in RBCs

• Transports
– Oxygen from lungs to tissues
– Carbon dioxide from tissues to lungs
• Diagnostic value
– Direct measure of oxygen-carrying capacity of the blood
– Low levels usually indicate anemia
– Lacks specificity to accurately reflect body iron stores

Anemia Institute for Research and Education. Available at: www.anemiainstitute.org. Accessed June 27, 2006.
Cook JD, et al. Blood. 2003;101:3359-3364.
Brittenham GM. In: Hoffmann R, et al, eds. Hematology: Basic Principles and Practice. 4th ed. 2005:481-497.
Schematic Representation of Hgb

Hgb α Chain

Polypeptide chain

Iron

β Chain

Heme

Illustration created by Duarte Design.

Hematocrit

• Percentage of volume of blood occupied by RBCs

• Diagnostic value
– Less accurate than Hgb
• Time- and temperature-sensitive
• Falsely elevated in hyperglycemia
• Falsely elevated in dehydration

Amarillo Medical Specialists. Available at: www.amarillomed.com/howto.htm. Accessed June 27, 2006.
Felgar RE, et al. In: Hoffmann R, et al, eds. Hematology: Basic Principles and Practice. 4th ed. 2005:2673-2686.
RBC Count

• Number of RBCs in a volume of blood

– Also referred to as erythrocyte count
• Diagnostic value
– Subject to artifacts
• At room temperature, for example,
RBC antibodies may coagulate the
sample, falsely lowering the count

Amarillo Medical Specialists. Available at: www.amarillomed.com/howto.htm. Accessed June 27, 2006.
Felgar RE, et al. In: Hoffmann R, et al, eds. Hematology: Basic Principles and Practice. 4th ed. 2005:2673-2686.
Mean Corpuscular Volume

• Average volume of a RBC

• Diagnostic value
– The MCV varies little in a patient
– Low levels can indicate iron abnormalities
– High levels can indicate
• Deficiencies in vitamin B12 or folate
• Inefficient production of RBCs in the bone marrow
• Recent blood loss, with replacement of newer and larger cells from the bone marrow
• Medication or hemolysis
• Chemotherapy, antivirals, and alcohol

Felgar RE, et al. In: Hoffmann R, et al, eds. Hematology: Basic Principles and Practice. 4th ed. 2005:2673-2686.
Amarillo Medical Specialists. Available at: www.amarillomed.com/howto.htm. Accessed June 27, 2006.
Chen KE, et al. Available at: www.uihealthcare.com/news/currents/vol5issue4/03anemia.html. Accessed June 27, 2005.
Marks PW, et al. Hematology: Basic Principles and Practice. 4th ed. 2005:455-463.
Serum Ferritin
• Serum ferritin is an acute phase reactant protein that stores iron
in the body
• Two types of ferritin
– Intracellular
– Plasma
• Rate of synthesis of intracellular and plasma ferritin is similar
• Diagnostic value
– Is considered one of the most useful estimates of body iron storage
– Low levels usually due to iron deficiency or response to
erythropoietin therapy
– High levels can be due to malignancies, infection, and/or inflammation
Kalantar-Zadeh, et al. Am J Kidney Dis. 2001;37(3):564-572.
Brittenham GM. Hematology: Basic Principles and Practice. 4th ed. 2005:481-497.
Goodnough LT, et al. Blood. 2000;96:823-833.
Ferritin Molecule

Source: Centers for Disease Control and Prevention.

Source: Centers for Disease Control and Prevention.
Serum Iron

• Measure of the amount of iron in the blood

• Usually does not fall until iron stores (serum ferritin) have
been depleted
• Diagnostic value
– Substantial day-to-day fluctuations
– Increased levels due to iron ingestion or ineffective erythropoiesis
– Decreased levels due to infection, inflammation, and malignancy
– Needs to be taken into consideration with TIBC and/or transferrin
to calculate TSAT

Brittenham GM. In: Hoffmann R, et al, eds. Hematology: Basic Principles and Practice. 4th ed. 2005:481-497.
Total Iron-Binding Capacity

• Measure of the amount of iron that transferrin can carry

• Increases with iron deficiency, as the body tries to capture
more iron
• Diagnostic value
– High levels indicate iron deficiency, as well as normal pregnancy
– Low levels may indicate inflammation, infection, malignant tumors,
or malnutrition
– Needs to be taken into consideration with serum iron to calculate
TSAT levels

Brittenham GM. In: Hoffmann R, et al, eds. Hematology: Basic Principles and Practice. 4th ed. 2005:481-497.
Transferrin Saturation

• Transferrin—protein that binds iron

• TSAT—measurement of the iron bound to transferrin
– Indicates how much iron is immediately available to support erythropoiesis
– Diagnostic value
• Low TSAT can indicate iron deficiency, erythropoiesis, infection, or
inflammation
• TSAT values may fluctuate by 30% or more in a day2
• Elevated TSAT can indicate recent ingestion of dietary iron, ineffective
erythropoiesis, or liver disease

1Australian Iron Status Advisory Panel. Available at: www.ironpanel.org.au/AIS/AISdocs/labqlab.htm. Accessed June 27, 2006.
2Brittenham GM. In: Hoffmann R, et al, eds. Hematology: Basic Principles and Practice. 4th ed. 2005:481-497.
TSAT Calculation*

• Serum iron divided by TIBC (more accurate) or transferrin equals

the TSAT

TSAT % = Serum iron ÷ TIBC (or transferrin) x 100

*If
not performed by laboratory.
Koziol JA, et al. Clin Chem. 2001;47:1804-1810.
Reticulocytes

• Immature RBCs
• Increase in reticulocyte count indicates
– Increased RBC production, owing to
• Compensation of bone marrow
to anemia
• Healthy expansion of erythropoiesis
• Diagnostic value
– Provides a real-time assessment of
the functional state of erythropoiesis

Goodnough LT, et al. Blood. 2000;96:823-833.

Zimmerman Klima PM, et al. Available at: www.beckman-coulter.com/literature/ClinDiag/pre-rectics.pdf. Accessed June 27, 2006.
Additional Confirmatory Tests
for Evaluation of Anemia

• Iron
– sTfR
– Erythrocyte protoporphyrin
• Vitamin B12
– Methylmalonic acid (if indicated)
• Folic acid
– Homocysteine (if indicated)

Cook JD, et al. Annu Rev Med. 1993;44:63-74.

McLaren GD, et al. Clin Chem. 1975;21(8):1121-1127.
Antony AC. Hematology: Basic Principles and Practice. 4th ed. 2005:519-556.
Soluble Transferrin Receptor

• Transferrin receptors transport iron into cells

• Truncated portion of transferrin receptors coming mostly from the bone marrow
• sTfR levels increase if bone marrow lacks iron
• Diagnostic value
– Elevated sTfR levels indicate iron deficiency in the bone marrow or enhanced
erythropoiesis
– sTfR levels are not affected by inflammation
– Differentiates patients with iron deficiency from patients with anemia of
chronic disease
• sTfR levels are normal in patients with anemia of chronic disease
– Stable measurements within an individual

Cook JD, et al. Annu Rev Med. 1993;44:63-74.

Goodnough LT, et al. Blood. 2000;96:823-833.
Iron Disorders Institute. Available at: www.irondisorders.org/Forms/FActsAnemia.pdf. Accessed June 27, 2006.
Free Erythrocyte Protoporphyrin

• Protoporphyrin is involved in the incorporation of iron into Hgb

• Diagnostic value
– Slow increase at the onset of iron deficiency
– High levels
• Occurs during more advanced stage of iron-deficient erythropoiesis
• Indicator of lead poisoning and defects of Hgb synthesis

McLaren GD, et al. Clin Chem. 1975;21(8):1121-1127.

Australian Iron Status Advisory Panel. Available at: www.ironpanel.org.au/AIS/AISdocs/labqlab.htm. Accessed June 27, 2006.
Folic Acid and Vitamin B12

• Folic acid and vitamin B12 are necessary for the formation
of RBCs
– Folic acid is directly involved in the DNA synthesis of RBCs
– Vitamin B12 transports folic acid from serum to RBC

Diagnostic Products Corporation. Anemia-related laboratory tests. Available at:

www.dpcweb.com/ medical/anemia/anemia_tests.html. Accessed June 27, 2006.
Confirmatory Tests for Vitamin B12 and
Folic Acid Deficiencies

Vitamin B12 Folic acid

deficiency deficiency
Methylmalonic
Elevated Normal
acid
Normal or
Homocysteine Elevated
elevated

Antony AC. In: Hoffmann R, et al. Hematology: Basic Principles and Practice. 4th ed. 2005:519-556.
Understanding Clinical
Laboratory Tests