The Reticulocyte Count

Anaemia induces production of erythropoietin which promotes differentiation of the hemopoietic stem cell along the erythroid lineage. With maturation, the erythroid precursors shrink in size, loose nucleus and haemoglobinize (see Morphology of Erythroid Precurssors). A newly released erythrocyte contains ribosomal RNA that it looses over 24-36 hours. These young erythrocytes are called reticulocytes because the ribosomal RNA gives a reticular appearance when stained by certain supravital stains like new methylene blue.

Anaemia may result from an increased destruction or impaired production of erythrocytes. The former is characterised by an increased erythrocyte production and the latter by an inappropriately low erythrocyte production. The normal life span of the erythrocyte is about 120 days. About 0.8% of the erythrocytes are destroyed everyday. If erythrocyte production ceases completely, a 10% fall in erythrocyte count (also in haemoglobin) would take about two weeks. Though a greater fall would indicate a loss of erythrocytes in the form of haemolysis or acute blood loss, the erythrocyte count is of little in diagnostic value in assessing bone marrow activity accompanying anaemia. The number of the reticulocytes indicates the bone marrow activity in a short period preceding assessment (a day or two) and allows classification anaemia into those resulting from decreased production and increased destruction.

The reticulocyte

On Romanowsky staining, other than polychromasia and a slightly larger size, there are no morphological features to differentiate reticulocytes from other erythrocytes. Differentiation on the basis of size is difficult and all reticulocytes do not show polychromasia. When reticulocytes are stained with new methylene blue or brilliant cresyl blue the ribosomes precipitate as a blue staining reticulum (hence the name reticulocytes). As the staining is done on cells that are not fixed (are live) it is knows as supravital staining. New methylene blue stains the cytoplasm blue-green obviating the need for a cytoplasmic counterstain. The density of the reticulum falls with age of the reticulocyte. The most mature reticulocytes may show only one or two dots. The majority of reticulocytes have a few dots and the precise definition of a reticulocyte has a bearing on the reticulocyte count. The definition of a reticulocyte is any non-nucleated erythroid cell containing two or more bluish–stained material corresponding to ribosomal RNA. The reticulocyte needs to be differentiated from other intracellular inclusions viz. Pappenheimer bodies, Heinz bodies, Howell-Jolly Bodies and Hb H inclusions.

Performing the Reticulocyte Count

The count is performed by mixing 2-3 drops of new methylene blue (or brilliant cresyl blue) with 2-4 volumes of EDTA-anticoagulated blood, allowing the mixture to stand for 15-20mins, making films on a glass slide and examining these when dry under 100X oil immersion. The number of erythrocytes and reticulocytes is counted till at least 100 reticulocytes and a total of 10 oil immersion fields are counted. The reticulocyte count is expressed as follows:

Reticulocyte count = [number of reticulocytes counted]/[number of erythrocytes counted]

The reticulocyte count may also be expressed as absolute reticulocyte count as follows:

Absolute reticulocyte count = [RBC count X Reticulocyte count]/100

The normal reticulocyte count is 0.5-1.5% and the normal absolute reticulocyte count is 50-100X10⁹/L

Correcting the reticulocyte count for anaemia

Anaemia decreases the amount of time the reticulocyte spends in the marrow. The reticulocytes of patients with haematocrits in the range of 45% are estimmated to spend 3.5 days in the marrow and about 1 day in the peripheral blood. At a haematocrit 15% these times are 1.5 and 2.5 days respectively. Decreases RBC count gives a false elevation in reticulocyte count. The haematological parameters of four patients given in the table below highlight the limitations of a reticulocyte count.

	Patient A	Patient B	Patient C	Patients D
Haemoglobin	13g/dL	5 g/dL	7.5 g/dL	7.2 g/dL
Erythrocyte Count	4.4 Million/mm3	1.2 Million/mm3	2.5 Million/mm3	3.5 Million/mm3
Haematocrit	40.48%	16.2%	23.25%	25.2%
MCH	29.55 pg	41.67 pg	30 pg	20.57 pg
MCV	92 fl	135 fl	93 fl	72 fl
MCHC	32 g/dL	31 g/dL	32 g/dL	29 g/dL
Reticulocyte Count	1.3%	3%	15%	2%
Absolute Reticulocyte count	57200 /mm3	36000/mm3	375000/mm3	70000/mm3
Corrected Reticulocyte Count	1.17	1.08	7.75	1.12
Reticulocyte Production Index	0.78	0.43	3.1	0.56

• Low erythrocyte count can cause an false impression of reticulocytosis: The patient B has an erythrocyte count of 1.2 million/mm³, a hematocrit of 16.2% and a reticulocyte count of 3%. The absolute reticulocyte count is 36000/mm³. Patient A has a erythrocyte count of 4.4 million/mm³, a haematocrit of 40.48% and a reticulocyte count of 1.3%. The absolute reticulocyte count is 57200/mm³. Despite having a higher reticulocyte count patient B actually has a lower absolute reticulocyte count and a less active marrow than patient A. Corrected reticulocyte count corrects reticulocyte count for low erythrocyte count and is calculated as follows:

Corrected Reticulocyte Count = Reticulocyte count X (Patinets Haemotocrit/Normal Haemotocrit)

(Haemoglobin or erythrocyte count may be used for the correction instead of haematocrit)

The corrected reticulocyte count in both patients is almost identical.

• In addition to correcting for erythrocyte count one must correct for a premature release of erythrocytes: Patinet A and patient B have almost identical corrected reticulocyte count. Does this mean they are producing the same number of erythrocytes per day? The reticulocyte spends about 2.5 days in peripheral blood in the patient A and about 1.5 days in the patient B. Only 14,400/mm³ (36,000/number of days reticulocyte spends in peripheral blood) reticulocytes are produced every day in patient B and about 38,000/mm³ produced in patient A. Despite having an almost identical corrected reticulocyte count as patient B, patients A is producing twice as many reticulocytes as patient B. The corrected reticulocyte needs to be further corrected for an early release of erythrocyte in anaemia. This gives the reticulocyte production index as follows

  Reticulocyte Production Index = Corrected Reticulocyte Count X Correction Factor

  The correction factor is 1 for haemotocrit of 40-45%, 1.5 for haemotocrit of 35-40%, 2 for haematocrit of 25-35% and 2.5 for haematocrit of 15-25%.

  A reticulocyte production index of <2 in the presence of anaemia indicates a bone marrow pathology. Patient C, a patient of haemolytic anaemia, and has a RPI of 3.1 indicating a normally responding bone marrow. Patient D is a patients with a hypochromic n=microcytic anaemia with a low RPI. This indicates a impaired erythropoiesis which in the case is most likely to be an iron deficiency anaemia.

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