RBC Inclusions and Reticulocyte Procedure HEMO

What are RBC inclusions?

Discreet structures within the cytoplasm of erythrocytes

Why is it important to recognize, identify and report RBC inclusions?

Their presence may indicate a specific disease

What are the characteristics of basophilic stippling?

Numerous, irregular granules distributed evenly throughout the cytoplasm; fine to coarse, blue to black

What does basophilic stippling consist of?

Precipitated RNA

What does coarse basophilic stippling suggest?

Impaired hemoglobin production

What conditions are associated with coarse basophilic stippling?

Lead poisoning, thalassemias, hemoglobinopathies, megaloblastic anemia

How does basophilic stippling form?

RNA breakdown is impaired in pathologic states, causing large amounts of RNA to remain in RBCs in circulation

Why is a supravital stain normally used to see RNA in reticulocytes?

Wright-Giemsa stain is insensitive and there is too little RNA in normal reticulocytes to see

Is coarse basophilic stippling ever normal?

No, it always indicates abnormal hemoglobin production

What are the characteristics of Howell-Jolly bodies?

Round, densely stained pyknotic granules 1 µm in size, eccentric, dark purple, usually only 1 per cell

What do Howell-Jolly bodies consist of?

Fragment of DNA

When do Howell-Jolly bodies develop?

During periods of abnormal or accelerated erythropoiesis

What conditions are associated with Howell-Jolly bodies?

Megaloblastic anemias, hemolytic anemia, thalassemia, hyposplenism, post-splenectomy

What are the two ways Howell-Jolly bodies form?

A fragment of chromosome detaches during cell division, or a portion of the nucleus is left behind during extrusion

Why are numerous Howell-Jolly bodies seen in hyposplenism or post-splenectomy?

The spleen usually pits these inclusions from the cytoplasm, but cannot keep up when the spleen is underactive or absent

What are Pappenheimer bodies also known as?

Siderotic granules

What are the characteristics of Pappenheimer bodies?

Small, irregular (angular), deposits of iron-containing granules (iron-protein matrix), usually in clusters along the periphery, light to dark blue

What is the presumptive evidence for iron on a Wright's stained smear?

Pappenheimer bodies

What is the confirmatory test for iron in Pappenheimer bodies?

Prussian blue stain

What conditions are associated with Pappenheimer bodies?

Sideroblastic anemia (vitamin B6 deficiency), hemoglobinopathies, severe anemias (NOT IDA), post-splenectomy

How do Pappenheimer bodies form?

Organelles damaged by iron form a digestive vacuole, lysosomes digest contents, autophagosome condenses creating a compact inclusion of protein and iron

What are Cabot rings?

Remnants of mitotic spindle left over after cell division

What do Cabot rings look like?

Thin reddish-purple loops or figure eights

What conditions are associated with Cabot rings?

Severe anemias, megaloblastic anemia, thalassemia, post-splenectomy, myelodysplastic syndrome

Why is it important not to confuse Cabot rings with Malaria?

Correct diagnosis is essential to treatment and survival of patient

What are Heinz bodies?

Precipitates of denatured hemoglobin

How do Heinz bodies vary?

Greatly in size, may be single or multiple

In what stain are Heinz bodies visible?

Only in supravital stains (NOT Wright's stain)

What conditions are associated with Heinz bodies?

G6PD deficiency, unstable hemoglobinopathy, post-splenectomy

What evidence on a Wright's stain indicates a Heinz body has been removed by the spleen?

Bite cells

What are the two ways erythropoiesis may be monitored?

Bone marrow examination and reticulocyte count

What are reticulocytes?

Immature red blood cells released from the bone marrow into the peripheral blood

What do reticulocytes contain that allows them to be visualized?

RNA remnants

How do reticulocytes appear on a Romanowsky stain?

As polychromatic red cells

What stain is used to visualize and count reticulocytes?

Supravital stain

What does the retic count indicate?

The amount of effective red cell production taking place in the bone marrow (erythropoiesis)

What are the three commonly used supravital stains for retics?

New methylene blue, brilliant cresyl blue, Nile blue

How do supravital stains allow retics to be detected?

They precipitate the RNA

How many red cells are counted for a % retic count?

1000

How is the % retic calculated?

retics / 10

What is the reference range for % retic in adults?

0.5-2.0%

What is the reference range for % retic in newborns?

2.5-6.0%

What is the reference range for % retic in infants?

2.0-6.0%

What percentage of circulating red blood cells are replaced daily in an adult?

Approximately 1%

What is the definition of a supravital stain?

A stain applied to living, unfixed cells

Why can a supravital stain demonstrate structures that may be lost after fixation?

Because the cells are not fixed, the stain can enter intact RBCs

What is the absolute retic count?

The actual number of reticulocytes in 1 L of blood

What is the formula for absolute retic count?

Absolute retics = # retics × RBC (10^12/L) = abs retic (10^9/L)

What is the reference range for absolute retic count?

24-84 × 10^9/L

What is reticulocytopenia?

A decreased retic count

What conditions cause reticulocytopenia?

Aplastic anemia, untreated pernicious anemia

What is reticulocytosis?

An increased retic count

What conditions cause reticulocytosis?

Hemolytic anemia, following treatment for IDA or megaloblastic anemia, thalassemia, chronic or acute hemorrhage

Why may the reticulocyte percentage appear elevated in patients with low hematocrit?

Reticulocytes are expressed as a percentage of total circulating RBCs, and there are fewer mature RBCs present

What does the corrected reticulocyte percentage adjust for?

The patient's degree of anemia

What is the reference hematocrit used for corrected retic count?

0.450 L/L (45%)

What is the formula for corrected retic %?

Corrected Retic % = Retic % × (patient hct (L/L) ÷ 0.450 L/L)

When should correction for hematocrit be done for a male?

If patient hct is below 0.420 L/L

When should correction for hematocrit be done for a female?

If patient hct is below 0.370 L/L

When calculating the absolute reticulocyte count, which reticulocyte percentage should be used?

The original (uncorrected) reticulocyte percentage

What is the degree of inaccuracy in the manual retic count?

+/- 2-7%

How can the inaccuracy of manual retic counts be overcome?

By performing automated counts

What is the retic count detected by flow cytometry based on?

The presence of RNA

What dyes are commonly used in flow cytometry to detect RNA in retics?

Coriphosphine-O and thiazole orange

What does the Coulter retic count reagent contain?

Two solutions that precipitate basophilic RNA and remove hemoglobin

What concept is used to measure the total cell population in the Coulter retic count?

Volume, conductivity, and light scatter

How long do retics spend in the bone marrow?

2 days

How long do retics spend in peripheral blood before becoming mature RBCs?

1 day