Unit 3

RED BLOOD CELL STUDIES

Introduction

The erythrocyte was one of the first microscopic structures

recognized and described after the development of the microscope. It
was not until 1865, however that the ability to carry oxygen in the red
pigment (the heme portion of the hemoglobin molecule) was
understood.

Mature red blood cells have a limited life span of about 120
days (+20 days) and are removed from circulation by the liver and
spleen. The red blood cell is about 7µ in diameter, approximately the
same size as a lymphocyte nucleus. A number of disease states are
recognized as being exclusively erythrocytic in nature.

A Medical Technologist / Medical laboratory scientist could

determine the rate of production of red blood cells in the peripheral
circulation by performing reticulocyte count.

This unit includes (1) Normal Maturation Series; (2)

Reticulocytes; (3) Erythrocytes; (4) Hemoglobin; (5) Hematocrit; (6)
RBC Indices; (7) Erythrocyte Sedimentation Rate; (8) Osmotic
Fragility Test; and (9) Variations of RBC Morphology. This units is
designed to provide students with theoretical background about the
laboratory methods of RBC studies and their clinical significance.

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Learning Outcomes

At the end of this unit, students will be able to:

1. Discuss the importance of the different metabolic pathways in the

life of the erythrocyte.
2. State the diagnostic importance of RBC inclusion bodies,
poikilocytes, and anisocytes.
3. Discuss the importance of red cell distribution width in the
diagnosis of certain hematological diseases.
4. State the clinical significance of the reticulocyte count, corrected
reticulocyte count, and the reticulocyte production index.
5. State the clinical significance of the erythrocyte count and the
reason for the difference in the RBC counts of children, male adults
and female adults.
6. Trace the formation of hemoglobin synthesis.
7. Explain the different types of hemoglobin and explain the formation
of such types.
8. Discuss the different types of porphyrias, hemoglobinopathies, and
thalassemias.
9. Enumerate and explain the principles behind the different tests
which may be used to diagnose porphyrias, hemoglobinopathies,
and thalassemias.
10. Discuss the importance of hematocrit, red blood cell indices, and
erythrocyte sedimentation rate in the clinical diagnosis of certain
diseases and the various factors that affect the results of these
tests.
11. Compute and interpret for the Red Blood Cell Indices (Mean
Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean
Corpuscular Hemoglobin Concentration)
12. Discuss the Red Cell Distribution Width (RDW)
13. Correlate the results of Hct , ESR & blood indices to diseases
affecting man.
14. Discuss the proper procedures , sources of error in the
determination, and clinical uses.
15. Differentiate the methods of ESR, Hct, and OFT.
16. Explain the variations of red blood cells.
Topic 1: Normal Maturation Series

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Learning Objectives

At the end of this topic, students will be able to:

1. List the maturational times for the various erythrocyte developmental phases.
2. Describe the major morphological features of each of the erythrocyte maturational
stages.
3. Explain the events that occur during reticulocyte maturation.
4. Define the terms shift or stress reticulocytes.

Activating Prior Learning

Answer/do the following:

1. List the normal RBC maturation series

a. _____________
b. _____________
c. _____________
d. _____________
e. _____________
f. _____________
g. _____________
2. __________ is the youngest cell not anymore capable of mitosis
3. __________ and __________ are the most commonly used supravital stains for
reticulocytes.

Presentation of Contents

RBC Normal Maturation Series

1. Rubriblast / Pronormoblast / Erythroblast

2. Prorubricyte / Basophilic normoblast
3. Rubricyte or polychromatophilic normoblast
4. Metarubricyte / Orthrochromic normoblast
5. Reticulocyte/Diffusely Basophilic Erythrocyte/
6. Polychromatophilic Erythrocyte
7. Mature Erythrocyte

Erythrocyte Normal Maturation Series

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1. Rubriblast / Pronormoblast / Erythroblast
} Size: 12-25 um in diameter
} Cytoplasm:
Deeply basophilic
Relatively small amount
Perinuclear halo
} Nucleus
Large
Round to sl. Oval
Reddish purple
1-3 nucleoli
} N/C ratio: 8:1
} 1% of nucleated cells in bone marrow
} A single pronormoblast gives rise to *16 RBCs (Henry, Harmening,
Steininger) *8-32 (Wintrobe) *8 (Rodak)

2. Prorubricyte / Basophilic Normoblast

} Size: 12-17 um in diameter
} Cytoplasm: intensely
basophilic
} Nucleus:
Relatively large
Round to slightly oval
Coarser chromatin
Not visible nucleoli
Occupies 75% of the cell
N/C Ratio: 6:1
} 1-3% of nucleated cells of bone marrow

3. Rubricyte or Polychromatophilic Normoblast or polychromatophilic

Erythroblast
} Size: 12-15 um in diameter
} Cytoplasm: blue-gray to pink gray
} Polychromasia (opaque, violet-blue color
cytoplasm due to blue RNA mixed with red
Hb)
} Hemoglobinization becomes evident
(start of hemoglobin synthesis)
} Nucleus:
Round, eccentric
Smaller
More condensed chromatin

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 Stains deeper blue-purple
N/C Ratio: 4:1
} 13-30% of nucleated cells in bone marrow
} This is the last cell division during maturation (last stage capable of mitosis
} Characterized by having a checkerboard appearance, its parachromatin
remains unstained and the mingling of blue (RNA) & pink (Hb) gives a
muddy/gray appearance

4. Orthochromic Normoblast (Metarubricyte/ Orthochromic Erythroblast)

} Size: 8-12 um in diameter
} Cytoplasm: pinker, increased amount of Hb
} Nucleus:
Pyknotic (dense mass of
degenerated chromatin)
Eccentric
N/C Ratio: 1:2
} 1-4% of nucleated cell in bone marrow
} Nucleus is extruded at this stage (last
nucleated stage)
} Youngest cell not anymore capable of mitosis

5. Reticulocyte
Diffusely Basophilic Erythrocyte*
Polychromatophilic Erythrocyte*
} Size: 7-10 um in diameter
} Cytoplasm: Pink to pinkish gray
Still contains small
amounts of RNA
(polychromasia)
} Nucleus: none
} Remain in the bone marrow for 2 to 3 days
before being released in the circulation
} Within 24-48 hrs, the cell loses the organelles
& assumes a biconcave shape end of
hemoglobin synthesis
} Immature erythrocyte stage w/ reticulum
network
a. RNA cytoplasmic remnants

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 b. Protoporphyrin remnants (responsible for the fluorescence of some
erythrocytes – fluoresces)
} Stained SUPRAVITALLY
a. New Methylene Blue (NMB)
b. Brilliant Cresyl Blue (BCB)
} Precipitates the ribosomal RNA of the immature RBC to form a deep – blue,
meshlike network
} If stained w/Wright’s – will demonstrate a slight blue tint in some
erythrocytes w/c is described as POLYCHROMATOPHILIA or
POLYCHROMASIA
} When the large reticulocytes normally found in the bone marrow are present
in the peripheral blood, they are referred to as shift or stress reticulocytes.

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