**2. Principles of erythrocyte morphology**

Circulating red cells are formed from bone marrow stem cells. Stem cells are pluripotent; they self-replicate and differentiate to specialized cells in circulation through different lineages. Red cells are formed from the myeloid stem cell lineage (colony forming unit—granulocytes, erythroid, myeloid and megakaryocytes). The earliest recognizable red cell precursor in the bone marrow is the pronormoblast. The pronormoblast undergoes series of maturation to become the orthochromatic normoblast. Upon extrusion of its nucleus, the late normoblast becomes the shift reticulocytes, which is released into the circulation. Finally, DNA remnants and other chromatin materials in the reticulocytes is removed by the pitting action of the spleen, hence the mature red cells.

Erythrocytes cannot be seen with the naked eyes. Typically, morphology of red cells is performed on peripheral blood smears, once there is an indication. Erythrocyte morphology is either indicated by a clinical request or laboratory flags. Examples of clinical indications for peripheral blood film/erythrocyte morphology are listed in **Table 1**.

Erythrocyte morphology may also be indicated when significant deviations from the normal are seen in the laboratory during blood work (full blood count) irrespective of a clinical request. For instance, a significantly reduced haemoglobin level with low MCV and raised RDW may suggest iron deficiency anaemia. This is an indication for red cell morphology and other ancillary investigation for iron deficiency.

Blood for peripheral blood film is collected through venipuncture. Anticoagulant of choice is the potassium EDTA. Specimens should be analysed as


**9**

*Erythrocyte Morphology and Its Disorders DOI: http://dx.doi.org/10.5772/intechopen.86112*

Cytoplasm with RNA and nuclear Remnants (e.g. polychromasia, basophilic stippling's)

**Table 2.**

**Cell component Colour** Chromatin (including H-J bodies) Purple

Mature red cells Pink

*Romanowsky staining characteristics of red cell components.*

tion, preferably in the first hour.

**3. Red cell morphologic disorders**

soon as possible, preferably within 2 hours of blood collection. Samples not analysed immediately should be stored at 2–6°C in a refrigerator, or the blood smear should be made, dried and fixed, for subsequent staining with Romanowsky dyes. Asides automated slide makers, the commonest method for preparation of peripheral blood film is the slide 'wedge' or push technique. This technique typically requires microscope slides, pipette/blood dropper, spreader slide and the blood specimen to be analysed. Standard precautions must be observed to prevent transmission of infectious pathogens such as human immunodeficiency virus and hepatitis viruses. Quality control measures will include ensuring proper anticoagulant: blood ratio, sample processing/analysis within sample viability period and adequate mixing of the blood before smearing. Each slide must be labelled with at least two patient identifiers such as name and laboratory, and date of procedure. Once the smear is air-dried in about 5–10 minutes, fixation of the blood tissue is another very important step. Fixation helps to preserve the architecture of the cells, which ensures good morphology. A dried slide should be fixed within 4 hours of prepara-

In polychromatic red cells, RNA produces a blue colour, which offsets the pink colour imparting a purple tinge Basophilic stippling: appears as blue granules dispersed within the cytoplasm in a punctate appearance

For routine morphology, the glass slides are stained with Romanowsky dyes. Romanowsky dyes are differential stains composed of both acidic and basic components. The acidic component is eosin and the basic part is azure B or polychrome methylene blue. Examples of Romanowsky stains include Leishman stain, Jenner, Wright stain, May-Grunwald-Giemsa stain and Giemsa stain. Generally, the eosin part of the dye binds to the basic component of the cell such as the haemoglobin molecules in the red cell and stains it pink. The basophilic part of the dye binds to the acidic component of the cells such as the nucleus and stains it blue. Other components of the cells appear in different colour shades that contrasts and compares with the dye. The term, azurophilic is used to describe a neutral to sky-blue colour shade. For instance, the cytoplasm of a neutrophil is described as azurophilic in colour. Furthermore, the characteristic staining quality of different red cell components is presented in **Table 2**. Staining procedure and the stain contact time depends on the type of dye in use. Staining protocols are contained in standard laboratory texts and reagent manuals. Red cell morphology should be examined at the monolayer region of the film which is 2–4 × 10 fields from the feathered edge. In this place red cells are randomly distributed with most lying singly and only a few overlapping. If area is too thin, the RBCs will appear flat with no central pallor. If too thick, false rouleaux may be reported and morphology may be difficult to evaluate because red cells are packed.

The haemato-morphologist reviews the red cell morphology under the compound microscope and notes any significant abnormalities for reporting/diagnosis

#### **Table 1.**

*Clinical indications for peripheral blood film.*


#### **Table 2.**

*Erythrocyte*

picture with paucity of red cells, numerous red cell fragments, increased polychro-

Circulating red cells are formed from bone marrow stem cells. Stem cells are pluripotent; they self-replicate and differentiate to specialized cells in circulation through different lineages. Red cells are formed from the myeloid stem cell lineage (colony forming unit—granulocytes, erythroid, myeloid and megakaryocytes). The earliest recognizable red cell precursor in the bone marrow is the pronormoblast. The pronormoblast undergoes series of maturation to become the orthochromatic normoblast. Upon extrusion of its nucleus, the late normoblast becomes the shift reticulocytes, which is released into the circulation. Finally, DNA remnants and other chromatin materials in the reticulocytes is removed by the pitting action of the

Erythrocytes cannot be seen with the naked eyes. Typically, morphology of red cells is performed on peripheral blood smears, once there is an indication. Erythrocyte morphology is either indicated by a clinical request or laboratory flags. Examples of clinical indications for peripheral blood film/erythrocyte morphology

Erythrocyte morphology may also be indicated when significant deviations from the normal are seen in the laboratory during blood work (full blood count) irrespective of a clinical request. For instance, a significantly reduced haemoglobin level with low MCV and raised RDW may suggest iron deficiency anaemia. This is an indication for red cell morphology and other ancillary investigation for iron deficiency. Blood for peripheral blood film is collected through venipuncture.

Anticoagulant of choice is the potassium EDTA. Specimens should be analysed as

• Features of congenital haemolytic anaemias such as splenomegaly, jaundice or bone pains • Suspected chronic or acute myeloproliferative disease, e.g. Chronic myeloid leukaemia

• Features of hyperviscosity syndrome as in paraproteinaemias, leukaemic hyperleucocytosis,

• Evaluation of disseminated intravascular coagulopathy and other red cell fragmentation syndromes

A clinical request for a PBF may be prompted by the following indications:

• Unexplained anaemia, leucopenia or thrombocytopenia • Unexplained leukocytosis, lymphocytosis or monocytosis

• Suspected organ failure such as renal disease, liver failure

• Advanced malignancies with possible bone marrow involvement

• Suspected chronic lymphoproliferative such as chronic lymphocytic leukaemia

• Severe bacterial sepsis and parasitic infections

• Suspected cases of nutritional anaemia

*Clinical indications for peripheral blood film.*

• Advanced lymphoma with leukaemic spills

• Unexplained jaundice or haemolysis

polycythaemia

matic red cells suggests a micro-angiopathy or fragmentation syndrome. This chapter aims to discuss principles of red cell morphology, as well as describe red cells in terms of morphology, identify morphologic abnormalities

associated with different disease conditions.

**2. Principles of erythrocyte morphology**

spleen, hence the mature red cells.

are listed in **Table 1**.

**8**

**Table 1.**

*Romanowsky staining characteristics of red cell components.*

soon as possible, preferably within 2 hours of blood collection. Samples not analysed immediately should be stored at 2–6°C in a refrigerator, or the blood smear should be made, dried and fixed, for subsequent staining with Romanowsky dyes.

Asides automated slide makers, the commonest method for preparation of peripheral blood film is the slide 'wedge' or push technique. This technique typically requires microscope slides, pipette/blood dropper, spreader slide and the blood specimen to be analysed. Standard precautions must be observed to prevent transmission of infectious pathogens such as human immunodeficiency virus and hepatitis viruses.

Quality control measures will include ensuring proper anticoagulant: blood ratio, sample processing/analysis within sample viability period and adequate mixing of the blood before smearing. Each slide must be labelled with at least two patient identifiers such as name and laboratory, and date of procedure. Once the smear is air-dried in about 5–10 minutes, fixation of the blood tissue is another very important step. Fixation helps to preserve the architecture of the cells, which ensures good morphology. A dried slide should be fixed within 4 hours of preparation, preferably in the first hour.

For routine morphology, the glass slides are stained with Romanowsky dyes. Romanowsky dyes are differential stains composed of both acidic and basic components. The acidic component is eosin and the basic part is azure B or polychrome methylene blue. Examples of Romanowsky stains include Leishman stain, Jenner, Wright stain, May-Grunwald-Giemsa stain and Giemsa stain. Generally, the eosin part of the dye binds to the basic component of the cell such as the haemoglobin molecules in the red cell and stains it pink. The basophilic part of the dye binds to the acidic component of the cells such as the nucleus and stains it blue. Other components of the cells appear in different colour shades that contrasts and compares with the dye. The term, azurophilic is used to describe a neutral to sky-blue colour shade. For instance, the cytoplasm of a neutrophil is described as azurophilic in colour. Furthermore, the characteristic staining quality of different red cell components is presented in **Table 2**.

Staining procedure and the stain contact time depends on the type of dye in use. Staining protocols are contained in standard laboratory texts and reagent manuals. Red cell morphology should be examined at the monolayer region of the film which is 2–4 × 10 fields from the feathered edge. In this place red cells are randomly distributed with most lying singly and only a few overlapping. If area is too thin, the RBCs will appear flat with no central pallor. If too thick, false rouleaux may be reported and morphology may be difficult to evaluate because red cells are packed.

#### **3. Red cell morphologic disorders**

The haemato-morphologist reviews the red cell morphology under the compound microscope and notes any significant abnormalities for reporting/diagnosis in light of patient clinical context. Red cell morphology is evaluated in terms of size, shape, colour, distribution and intra cytoplasmic inclusions. In general, red cells have a fairly uniform variation in size, with a red cell distribution width of 11–15% in normal individuals. Abnormal variations in sizes and shape are termed anisocytosis and poikilocytosis, respectively [1].
