Reticulocyte count is performed by microscopy or by electronic instruments. In both cases, the distinctive marker is polyribosomal RNA, which is well represented in reticulocytes. Preparation for microscopic counting is performed by supravital staining of a part of incoagulable blood suspended in an equal part of an isotonic solution of brilliant cresyl blue. Other supravital stainings are the new methylene blue and Azur B. Also, dyes for fluorescence microscopy, such as acridine orange and auramine, are commonly used. The observation is done by immersion (1000×) on a freshly swiped slide (Fig. 1). The reticulocytes observed are compared to 1000 erythrocytes, and their number is expressed as a percentage; a CV of 10% is acceptable, but this value must consider the variability among operators. Therefore, in practice, even higher values are reported to the point of invalidating their clinical-diagnostic significance. Although the advent of automatic instruments has displaced microscopic counting, it is still used in less-developed healthcare facilities.

Fig1. Reticulocytes after supravital staining

Automated instruments allow not only obtaining a more accurate and precise reticulocyte count than microscopy- based methods but also measuring reticulocyte characteristics that cannot be captured by microscopic observation. In the automatic procedure, the blood is incubated with an iso tonic solution of a dye, which crosses the cytoplasmic mem brane and binds to RNA; the detection of reticulocytes is carried out by colorimetry or fluorescence through the use of supravital dyes, such as the new methylene blue, or fluorescent dyes, such as thiazole orange, osazine 750, and others.

The reticulocyte count allows us to evaluate the pro duction rate and, indirectly, the replacement rate of the erythrocyte population. It can be reported as an absolute count or as a percentage of reticulocytes relative to erythrocytes. In this way, it is possible to distinguish between anemias of different origins, such as those resulting from hemolysis or other destructive processes, and those due to deficient production or deficiency of essential factors for erythropoiesis. The reticulocyte count allows deter mining not only if their production is qualitatively increased during anemia but also if the entity of increased production is adequately sustained and leads to the recovery of the state of normality. On the other hand, a decrease in the reticulocyte count can be a consequence of conditions such as deficiency states, bone marrow insufficiency, and cancer (Table 1).

Table1. Conditions accompanying changes in the reticulocyte count

The reference range for the adult percentile count is 0.5 1.5%. The absolute reticulocyte count is obtained by calculating its value over the total number of red blood cells; for example, in a subject with 4,800,000/fL erythrocytes and 1.5% reticulocytes: 4,800,000/(100 × 1.5) × 72,000/fL.

Hematological instruments automatically provide this calculation. If there is a state of anemia, the percentage of reticulocytes does not accurately reflect the true responsiveness of the bone marrow to the anemic state. It is necessary to correct the percentage value according to the hematocrit value. While it appears evident that the absolute value does not need any correction as it expresses the actual number of reticulocytes, the percentage value needs correction for two main reasons: one intrinsic to the data itself and the other due to the period of maturation of the reticulocytes.

Reticulocyte Production Index

 The first correction is performed by the reticulocyte production index (RPI) or corrected reticulocyte count using the following calculation:

RPI =  Reticulocyte Count × patient hematocrit / Hematocrit 45%.

A hematocrit of 45% is taken as a reference. The RPI is normally between 0.5% and 2.5%. Alternatively, there are formulas based on hemoglobin, such as the following:

 RPI =  Reticulocyte Count × patient hematocrit / normal hemoglobin

A normal hemoglobin value is 150 g/L.

The second condition is a little more complex and depends on the period of maturation of reticulocytes from the loss of the nucleus (erythroblast stage) to maturation into circulating erythrocytes. Usually, this maturation takes place over a period of 4.0–4.5 days, of which 3.0–3.5 days are spent in the marrow and one in the circulation. However, under conditions of erythropoietic stress, such as hypoxia due to anemia, premature release of reticulocytes occurs from the marrow, and the days of maturation in the circulation increase up to 2–3. This leads to increased reticulocyte counts that do not accurately reflect normal production and release kinetics. Graphs such as the one shown in Fig. 2 have been proposed to correct this effect.

Fig2. The correction factor is the divisor to be applied to the per cent count to modify the effect due to the lengthening of circulating maturation in case of anemia. (Copyright EDISES 2021. Reproduced with permission)

Immature Reticulocyte Fraction

In the evaluation of anemia, the state of maturation of reticulocytes can introduce elements of complexity when a correct meaning must be attributed to their count. However, it pro vides further diagnostic opportunities that have been progressively perfected with automation. The microscopic observation gives the opportunity to detect different entities of staining in relation to the progressive reduction of RNA until its disappearance when the reticulocyte becomes an erythrocyte. To define this phenomenon, as evidenced very well by automatic instruments, an index called immature reticulocyte fraction (IRF) has been proposed and adopted internationally. IRF considers the reticulocytes subdivided into populations of different maturation degrees. Generally, instruments divide reticulocytes into three populations of different immaturities: low, medium, and high. The IRF index groups the fractions of medium and high immaturity. The reference range is 0.11–0.38% (percentage of medium and high immature reticulocytes in the total erythrocyte count), with possible variations related to the instrumentation used. The parameter represents an early and sensitive index of bone marrow erythropoietic activity, with the following main diagnostic features:

• After marrow transplantation, IRF may demonstrate successful engraftment earlier than other laboratory parameters, including absolute neutrophil granulocyte count. An increase in IRF of more than 20% over the post-transplant value is indicative of successful erythroid engraftment.

 • IRF is a sensitive and early index of the recovery of bone marrow activity after chemotherapy treatment.

 • IRF, measured in patients with anemia associated with chronic renal failure, neoplasms, infections, and chronic conditions, is an early and reliable index of response to erythropoietin therapy.

• In surgical procedures with preservation of the patient’s blood, it may be useful to monitor the response to erythropoietin.

 • It can be used to monitor the effectiveness of treatments for deficient anemias, such as those caused by iron, vita min B12, and folic acid deficiency.

Evaluation of IRF along with reticulocyte counts may provide additional insight, as shown in Table 2.

Table2. Clinical conditions using the combination of immature reticulocyte fraction (IRF) and reticulocyte count

Reticulocyte Hemoglobin Content Hemoglobin content

in reticulocytes (reticulocyte hemoglobin content [CHr]) provides a measure of the efficiency of hemoglobin synthesis that the reticulocyte has implemented in the previous few (3–4) days. The reference range is approximately 24–32 pg. Values <24  pg are indicative of iron deficiency. Since hemoglobin synthesis depends on iron availability, CHr provides a measure of functional iron and, therefore, is an index for early assessment of iron deficiency. Accordingly, it could be useful in the following conditions:

• Pediatric age.

 • Pregnancy.

• Complex clinical pictures, such as chronic inflammation and chronic kidney disease, as the ferritin level in these conditions, is elevated as an acute-phase reactant, despite the reduced iron stores.

 • Nonmacrocytic anemias, which may be a better predictor of iron stores than traditional indices.

 Reticulocyte Hemoglobin Equivalent

 The hemoglobin equivalent in reticulocytes (Reticulocyte Hemoglobin equivalent [Ret-He]) is a parameter with a very similar meaning to the previous one. The reference range is approximately 28–35 pg. Values <28 pg are indicative of iron deficiency.

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مواضيع ذات صلة

Sideropenic and Assimilated Anemia

Automated Differential Count

Differential Leukocyte Count (Leukocyte Formula)

lactose tolerance test (Hydrogen breath test)

Lactoferrin

lactic dehydrogenase (LDH, Lactate dehydrogenase)

lactic acid (Lactate)

ischemia-modified albumin (IMA)

iron level and total iron-binding capacity (Fe and TIBC, Transferrin saturation, Transferrin)

Platelet Indices and Platelet Count

Red Cell Distribution

Flow Cytometry