*5.1.3 Light microscopic method*

Equal volumes of anticoagulated (preferably EDTA) peripheral whole blood and supravital stains are mixed and incubated for at least 20 minutes. A thin smear of the stained blood preparation is made on a microscope slide, a counterstain (usually Wright's) is applied, and the slide is examined by light microscopy. An adequate number of RBCs (minimum 1000 for optimal analytical precision) in a well-stained area are examined, and the proportion of reticulocytes is determined. Reticulocytes possess a blue granular precipitate, which can vary from individual small blue granules to a network of blue reticular material. Reticulocytes have a faint, diffuse basophilic hue termed as polychromasia (**Figure 2**).

The reticulocyte count is usually reported as a percentage of total RBC count. The normal mean percentage reticulocyte count by NMB light microscopy is 1.0% to 3.0%. In cases of anemia, this relative reticulocyte count is misleading because reduced RBC count causes erroneous elevation in reticulocyte count. Under these circumstances, reticulocyte count is corrected with respect to patient's packed cell volume (PCV) as Reticulocyte index to compensate the decrease in mature RBCs. The PCV corrects the percentage reticulocyte count to the baseline PCV i.e. 0.45 using the following formula:

$$\text{Reticulocyte Index} \left(RI \right) = \text{Reticulocyte count} \left( \% \right) \times \frac{\text{Patient'sPCV}}{0.45}$$

Absolute count of reticulocytes is more accurate term to correct the effect of anemia. This is calculated as follows:

( ) ( ) ( ) <sup>3</sup> Absolute Reticulocyte count Reticulocyte *ARC* = × Count % count *RBC per mm*

The normal ARC is between 50,000 and 150,000 reticulocytes/mL (5× 1010 and 1.5 × 1012 reticulocytes/L).

Manual microscopic RC is highly subjective and tedious, which results in high level of imprecision, with a coefficient of variations of up to 50% [30]. This imprecision in manual counting of reticulocytes can be attributed to:

#### **Figure 2.**

*Photomicrograph of peripheral blood smears stained with new methylene blue, 400x. Reticulocytes (arrowhead) are differentiated from mature RBCs (arrow) by the presence of an intracellular granular precipitate [29].*

*Reticulocytes-Mother of Erythrocytes DOI: http://dx.doi.org/10.5772/intechopen.107125*


The College of American Pathologists and National Committee for Clinical Laboratory Standards (NCCLS) has defined reticulocytes as the cell containing two or more precipitate granules not attached to cytoplasmic membrane following staining by new methylene blue and the use of Miller ocular disks, to standardize the microscopic counting and reduce the imprecision.

Hence, clinical serial assessment of erythropoietic activity of bone marrow in patients receiving myelotoxic or hematinic therapies by this manual counting of reticulocytes is impractical due to poor precision and methodologic limitations.

### **5.2 Immunofluorescence method**

First attempt of reticulocyte enumeration by using immunofluorescence microscope was done in 1950s by Kozenow and Mai. They used RNA specific fluorochrome dye i.e., acridine orange. Subsequently, different fluorochromes used by others for reticulocyte enumeration later on like pyronin Y, thioflavin T, DiOCl, proflavin. Reticulocyte enumeration by this method gives spuriously high count sometimes due to some interference present in the blood sample such as platelet clumps, nucleated RBCs, Howell-Jolly bodies, Heinz bodies, Pappenheimer bodies, Basophilic stippling and Malarial parasites.

## **5.3 Fluorescence microscopy**

The prefixed whole blood in formalin is mixed with a dilute, buffered solution of the dye and counted as a wet preparation. Ultraviolet to blue light is required for the excitation of these dyes, and a green long-pass filter must be placed for detection of the fluorescence emission.

## **5.4 Conventional flow cytometry**

Thioflavin T analogue has been widely used as specific fluorochrome for reticulocyte enumeration to improve counting accuracy and made flow cytometric reticulocyte enumeration practical as TO excitation occurs in the visible region of the spectrum. These flow cytometers rely on the enumeration of reticulocytes using nucleic acid binding fluorescent dyes and measurement of fluorescence emission. Reticulocytes are identified and enumerated within the gated RBC population on the basis of fluorescence intensity. The quantitation of the intensity of green fluorescence reveals young, immature reticulocytes as brightly fluorescent (high RNA content),

while maturing reticulocytes show an intermediate fluorescence intensity (intermediate RNA content), and older reticulocytes show dim fluorescence (low RNA content).

#### **5.5 Automated reticulocyte instrumentation**

All automated instruments for reticulocyte enumeration are capable of rapid analysis of whole blood sample in flow through systems, with the red cell population interrogated on a cell-by-cell basis by laser light. Now-a-days the reticulocyte counting is fully integrated into the automated complete blood count (CBC) in high-throughput hematology analyzers. The reticulocyte RNA content is detected by fluorochrome Auramine O, while the cell size is determined by forward light scattering using an argon ion laser as the light source. Automated flow cytometric analysis has replaced traditional microscopic quantitation of reticulocytes.

#### **5.6 Reticulocyte-specific monoclonal antibodies**

Reticulocyte analysis with fluorochrome-labeled monoclonal antibodies against reticulocyte surface receptors is available only for research studies of the reticulocyte and diseases of the erythron. Fluorochrome-labeled monoclonal antibodies specific for different reticulocyte antigens have been evaluated as reagents for reticulocyte enumeration. The antigens presently evaluated include the CD71 molecule (transferrin receptor, TfR, and T9 antigen), CD36, and other antigens. CD71 receptors are abundant on the surface of early reticulocytes, but gradually decrease during reticulocyte maturation, and are absent from mature RBCs. CD36 and the integrin α4-β1 complex are expressed on reticulocytes but not mature erythrocytes. CD36-positive reticulocytes correlate with the stress reticulocyte fraction in patients with chronic hemolytic diseases.

### **6. Indices of reticulocytes**

The recent development in automation of reticulocyte counts provide accurate RC with enhanced precision along with reliable and accurate measurements of RNA content and cellular indices such as mean reticulocyte volume, mean reticulocyte Hb concentration, and its content. These novel parameters have been studied with prompt interest for their clinical usefulness and the utility of reporting these analytes with their appropriate interpretation.

## **7. Reticulocyte production index (RPI)**

During intense erythropoietic stimulation, immature, large and basophilic precursor macroreticulocytes ("shift cells") from the bone marrow are released prematurely into the peripheral blood. This causes a shortened reticulocyte maturation time in the bone marrow (depends on the severity of stress erythropoieisis), and the longer reticulocyte maturation time in the peripheral blood. Since these shift cells have a cell volume about 25% larger than that of normal cells, a correction for RBC maturation time and the PCV must be done when they comprise more than 5% of the total reticulocytes [31]. The correction is referred as "shift correction" or "reticulocyte production index", and calculated by the following formula (**Figure 3**).

#### **Figure 3.**

*The relationship between PCV (hematocrit) and blood maturation time. The total RBC maturation time is approximately 4.5 days. During normal erythropoieisis 3.5 days of maturation occurs in bone marrow and last one day in peripheral blood. However, in anemia the marrow maturation time progressively shortens, and reticulocytes circulate for a correspondingly longer period of time in the peripheral blood to compensate the degree of anemia [32].*


RPI was proposed to correct the reticulocyte percentage for peripheral blood maturation time based on the hematocrit value. It helps to alleviate the effect of the premature release of reticulocytes by taking into account maturation time of reticulocytes. Thus RC is corrected or adjusted for both premature release of reticulocytes and the degree of anemia. Stressed erythropoiesis is accomplished by increased production and shortening of the fraction of time that reticulocytes mature in marrow and proportionally prolongs their maturation time in circulation, thus increasing reticulocyte circulation time. RPI is used for evaluating erythropoiesis and classifying anemias. RPI is normally between 2 and 3.

RPI of less than 2 suggests for hypo-proliferative erythropoiesis and more than 3 is applied for hypo-proliferative state of erythropoiesis.

#### **Clinical interpretation of RPI**

	- Hemolytic anemias (Autoimmune)
	- Recent hemorrhage
	- Marrow response to therapy in nutritional anemia (EPO)
	- Chemotherapy induced anemia
	- Hypoproliferative disorder (i.e., aplastic anemia)
	- Ineffective erythropoiesis
	- Megaloblastic anemia
