**4. Classification of reticulocytes**

A number of classification systems have been attempted based on the maturation of reticulocytes along with their morphology. Maturity classification is based on the quantity of the granular/reticular filamentous substance and its distribution in their cytoplasm.

The first attempt was made by Heilmeyer and Westharer, who divided the cells into 4 groups (Groups I-IV), designated by Roman numerals and characterized by a progressive reduction in the compactness of the reticulum. Therefore, reticulocyte maturation predominantly assesses the relative proportion of mRNA (see **Figure 1**).

**Group 0** – nucleated RBCs with a dense perinuclear reticulum.

**Group I** – most immature reticulocytes with extruded nuclei having a dense, coherent mass of RNA.

**Group II** – reticulocytes with extensive but loose reticular network.

**Group III** – reticulocytes with scattered reticular network.

**Group IV** – most mature reticulocytes with scattered remnants of RNA.

According to Lowenstein, in steady-state erythropoiesis the circulating reticulocytes are more than 60% in Group IV, 30% in Group III, and less than 1% in Groups I or II.

#### **4.1 Stress Reticulocytosis**

Some specific hematological disturbances (*e.g.* severe anemia or hemolysis) are compensated with accelerated erythropoiesis (called stress erythropoiesis), which causes release of the immature, larger and more stained form of reticulocytes into the peripheral blood, which are called "stress reticulocytes" and appear as polychromatophilic cells on Romanowsky staining. All polychromatophils are reticulocytes on a Wright-stained blood smear. Reticulocyte production can increase up to 20 folds above the base line values of 1 to 2 million reticulocytes per second when stressed with intense hematopoiesis. This increased production is accomplished by increased production and shortening of marrow maturation time [26, 27]. Stress reticulocytosis is seen in bone marrow regeneration following autoimmune hemolysis, chemotherapy induced anemia, administration of therapy in nutritional anemia, and use of erythropoiesis-stimulating agents (ESAs). Stress reticulocytes are multilobular and motile, in contrast to the cup-shaped non-motile mature reticulocytes.

### **5. Reticulocyte counting**

Till the end of last century, the standard method of counting reticulocytes was based on the visual detection of RNA ribosomal networks (reticulum), by microscopic examination of supravitally stained peripheral blood smears. But, this method has several sources of imprecision in the manual microscopic counting of reticulocytes, including different staining with variation, distributional variability for quality of blood film, intra and inter-observer variations, and inadequate number of cells counted. As per NCCLS-ICSH guidelines, supravital staining with New Methylene Blue (NMB) still remains the recommended method for optimal agreement/correlation assessments. NMB staining procedures have been standardized to improve the accuracy and reliability of reticulocyte manual counting [28].

Reticulocyte counting should be done within 6 hours if the sample is kept at room temperature, or up to 72 hours if the blood sample is refrigerated at 2-6°C.

#### **5.1 Manual methods**

#### *5.1.1 Photographic methods*

In 1960s photographs used to be obtained on wet preparation from fresh oxalated blood mixed with brilliant cresyl blue in isotonic saline. 30–35 fields containing reticulocytes used to be printed on glossy paper with final magnification of approximately 4000x.

#### *5.1.2 Planimetric method*

Reticulocytes stained with brilliant cresyl blue wet preparations are photographed and the resultant Kodachromes are projected onto butcher paper. Then, pairs of reticulocytes and adjacent RBCs are circled and traced with a planimeter.
