Reticulocyte Parameters: A Window Into Erythropoiesis & Anemia Monitoring

Introduction

Reticulocytes, precursors to red blood cells, play a vital role in maintaining the body's homeostasis. Formed in the bone marrow, red cells lose their nucleus when they enter the blood stream. These early blood cells still contain RNA and continue to synthesize hemoglobin for a few days before becoming mature erythrocytes.

The reticulocyte count serves as a measure of erythropoiesis activity in the bone marrow and predicts the status of future red blood cells in the peripheral blood. A high reticulocyte count indicates a compensatory response to anemia whereas a low value may indicate an impairment in red cell production.

Methods for Reticulocyte Measurement

Traditionally, reticulocyte counts were performed using manual/visual methods – microscopic counting after staining with supra-vital stains such as New Methylene Blue or Brilliant Cresyl Blue, which stain the RNA content.

Automated flow cytometric methods have been developed to improve measurement precision and efficiency. Counts are automated through the simultaneous measurement and analysis of multiple physical characteristics of single particles, including relative size, granularity, and fluorescence intensity.

HORIBA Technology

HORIBA uses flow cytometry and impedance measures with a reagent containing a fluorescent stain: thiazole orange (TO). This nucleic-acid-specific dye binds to RNA, resulting in a significant increase in fluorescence emission. The degree of fluorescence emission is proportional to the amount of RNA in the erythrocyte.

Fluorescent Staining + Flow Cytometry + Impedance
Thiazole Orange: an RNA specific nucleic acid (recommended by the CLSI) that enters the cells by permeability
Advantages of Thiazole Orange
- Precision
- Speed
- Measurement of reticulocyte maturity
- No user manipulation
Incubation 25 seconds
The degree of fluorescence emission is directly proportional to the amount of RNA in the reticulocyte

Reticulocyte Matrix and Parameters

The reticulocyte matrix is generated from measurements of resistivity volume and orthogonal fluorescence of cells. Mature red blood cells with little or no RNA show minimal fluorescence, while reticulocytes are separated by their fluorescence, which is proportional to RNA content and immaturity.

Reticulocyte parameters

Reticulocyte Count: RET (%) and RET (#)
Corrected Reticulocyte Count: CRC (%)
Immature Reticulocyte Fraction: IRF (%)
Maturation Classes: RETL, RETM, RETH (%)
Mean Fluorescence Index: MFI (%)
Mean Reticulocyte Volume: MRV (fL)
Reticulocyte Hemoglobin Content calculated: RHCc (pg)

Reticulocyte parameters, including reticulocyte count (RET%; RET), RETL%, RETM%, RETH%, and CRC, are used to evaluate normal or pathologic conditions affecting erythropoiesis. These parameters provide valuable information for diagnosing anemia, assessing regenerative activity after anemia treatment, monitoring patients receiving recombinant human erythropoietin, and detecting sport-induced anemia and doping.

Immature Reticulocyte Fraction (IRF)

The Immature Reticulocyte Fraction (IRF) is the ratio of immature reticulocytes to the total number of reticulocytes. IRF is useful for evaluating the degree of erythropoiesis, predicting RBC fluctuations, and diagnosing anemia types.

Reticulocyte Hemoglobin Content calculated (RHCC)

Reticulocyte Hemoglobin Content calculated reflects the amount of hemoglobin production in the bone marrow and is a useful parameter for diagnosing iron deficiency anemia.

This parameter gives an indication of the functional iron available for incorporation into hemoglobin within RBCs over the previous 3-4 days.

Calculating the reticulocyte hemoglobin concentration content (RHCc) is useful in the following situations:

Diagnosis of iron-deficient erythropoiesis (absolute or functional) in complex clinical settings.
Better predictor of marrow iron stores than traditional serum iron parameters in non-macrocytic subjects.
A stronger predictor of iron deficiency than hemoglobin when screening infants and adolescents for iron deficiency prior to the development of anemia.
Monitoring early response to intravenous iron therapy in patients on rHuEPO (recombinant Human Erythropoietin) because it increases significantly after only 48 hours.

Mean Reticulocyte Volume (MRV)

Mean reticulocyte volume refers to the general volume of reticulocytes, which are naturally larger than mature RBCs. This parameter is useful in assessing iron storage disorders and response to therapy, with smaller reticulocytes being produced in patients with iron-deficiency.

Conclusion

Reticulocyte parameters provide valuable insights into erythropoietic activity and are essential tools for diagnosing and monitoring various clinical conditions, including anemia, bone marrow disorders, and iron deficiency. The development of flow cytometric methods has significantly improved the accuracy and efficiency of reticulocyte measurement, making these parameters indispensable in modern clinical laboratories.

References:

1. Reticulocyte count and its parameters: comparison of automated analyzers, flow cytometry, and manual method. Varun Uppal, Shano Naseem, Ishwar Bihana, Man Updesh, Singh Sachdeva, Neelam Varma

2. CLSI_H44-A2. Methods for Reticulocyte Counting (Automated Blood Cell Counters, Flow Cytometry, and Supravital Dyes)

3. Rodrigo Proto-Siqueira. Reticulocyte parameters: why should clinical laboratories evaluate and report them? Rev bras hematol hemoter. 2014; 36(1):3-4.

4. Lee LG, Chen CH, Chiu LA. Thiazole orange: a new dye for reticulocyte analysis. Cytometry. 1986 Nov; 7(6):508-17.

5. Figuerres E, Olszewski M, Kletzel M. A flow cytometric technique using thiazole orange to detect platelet engraftment following pediatric stem-cell transplants. Cytotherapy. 2001; 3(4):277-83.