PRACTICAL CLINICAL HEMATOLOGY

Reticulocyte Count

Dr. Mayank AgarwalMa.gsvm@gmail.com

RETICULOCYTES Reticulocytes are juvenile, non nucleated red blood

cells containing remnants of reticular material (RNA).

RETICULOCYTES STAINS

On Romanowsky stain (Leishman, Geimsa, Wright) reticulocyte appears polychromatic cell.

Vital stain : stains living cells in vivo Supravital stain : stains living cell in vitro

Commonly used supravital stains :1. New Methylene blue (C18H22N3S:SCl ZnCl2, methylene blue : C16H18N3SCl)

2. Azure B3. Brilliant cresyl blue

New methylene blue stains the reticulo-filamentous material in reticulocytes more deeply and uniformly. Azure B is a satisfactory substitute for new methylene blue; it has the advantage that the dye does not precipitate and is available in pure form

PRINCIPLE The supravital stain causes the ribosomal and

residual cytoplasmic RNA to co-precipitate with the few remaining mitochondria and ferritin masses in living reticulocytes to form dark-blue clusters and filaments (reticulum).

The most immature reticulocytes are those with the largest amount of precipitable material; in the least immature, only a few dots or short strands are seen. Reticulocytes can be classified into four groups, ranging from the most immature reticulocytes, with a large clump of reticulin (group I), to the most mature, with a few granules of reticulin (group IV)

REQUIREMENTS 1. Disposable syringe2. Microscope slides3. Microscope4. EDTA vial5. Staining solution : prepared by dissolving 1 gm. of

supravital stain in 100ml of 3% trisodium citrate saline solution (30 gm sodium citrate in 1L saline).

6. Capillary tube7. Miller ocular or Ehrlich ocular (if available)

PROCEDURE 1. Draw blood in EDTA vial.2. Add equal amount of supravital stain3. Mix the contents by gently shaking sideways, and

allow to incubate at body temperature (37°C) for a minimum of 15 minutes.

4. At the end of 15 minutes, gently mix the blood/stain solution.

5. Using a capillary tube, place a drop of the mixture on three slides for making a peripheral smear using the wedge smear technique.

6. When dry, examine the films without fixing or counterstaining

The exact volume of blood to be added to the dye solution for optimal staining depends on the RBC. A larger proportion of anaemic blood, and a smaller proportion of polycythaemic blood, should be added than of normal blood.

In a successful preparation, the reticulofilamentous material should be stained deep blue and the non reticulated cells should be stained diffuse shades of pale greenish blue.

Films should not be counterstained. The reticulofilamentous material is not better defined after counterstaining and precipitated stain overlying cells may cause confusion. Moreover, Heinz bodies will not be visible in fixed and counterstained preparations.

COUNTING THE RETICULOCYTES CELLS:An area of film should be chosen for the count where

the cells are undistorted and where the staining is good

To count the cells, use the 100X oil-immersion objective and, if possible, eyepieces provided with an adjustable diaphragm (Ehrlich ocular).

If eyepieces with an adjustable diaphragm are not available, a paper or cardboard diaphragm, in the centre of which has been cut a small square with sides about 4 mm in length, can be inserted into an eyepiece and used as a less convenient substitute.

Count 1000 red cells (satisfies the college of American pathologists hematology standard for manual reticulocyte count) in consecutive oil immersion fields. Record the number reticulocytes seen.

You may count 500 cells on two slides each. They should agree within ± 20% of each other. If they do not, repeat the reticulocyte count on the third smear.

CALCULATION Number of reticulocytes in n fields = xTotal number of red cells in n fields = yReticulocyte percentage = x/y * 100 %Absolute reticulocyte count = RBC count *

reticulocyte %

METHOD USING THE MILLER DISC

1. Use a 100x objective and a 10x ocular secured with a Miller disc. The Miller disc imposes two squares (one 9 times the

area of the other) onto the field of view. Find a suitable area of the smear. A good area will show

3-10 RBCs in the smaller square of the Miller disc.2. Count the reticulocytes within the entire large

square including those that are touching the lines on the left and bottom of the ruled area. Count RBCs in the smaller square whether they contain stained RNA or not. A retic in the smaller square should be counted as an RBC.

3. Continue counting until a minimum of 111 RBCs have been observed. This would correspond to 999 RBCs counted with the standard procedure.

MILLER DISC METHOD OF COUNTING

The factor 9 corrects for the difference in area between the smaller and the larger square.

REPORTING RESULTS Reticulocyte count Percent : Normal value : Adult: 0.5 to 1.5%Newborn (0-2 weeks): 2.0 - 6.0%

Absolute Reticulocyte Count (ARC): is the actual number of reticulocytes in 1L of whole blood. Normal Absolute count in adults: 25 to 75 X 109/L

Corrected Reticulocyte Count is calculated based on a normal hematocrit of 45%.

Reticulocyte Production Index (RPI) = Corrected retic count (%) / Days (Maturation time)

CALCULATION OF CORRECTED RETICULOCYTE

In specimen with low hematocrit, the reticulocyte percentage may be falsely elevated because whole blood contains fewer RBCs.

The corrected reticulocyte count my be calculated by the following formula:

Example: Uncorrected retic% = 5.0%

Patient Hct= 25.0% (adult male) Corrected retic% = 5.0% x 25% / 45% = 2.8%

CALCULATION OF THE RETICULOCYTE PRODUCTION INDEX (RPI)

Estimating RBC production by using the corrected reticulocyte count may yield erroneously high values in patients when there is a premature release of younger reticulocytes from the marrow (owing to increased erythropoietin stimulation).

The premature reticulocytes are called “stress or shift” reticulocytes. These result when the reticulocytes of the bone marrow pool are shifted to the circulation pool to compensate for anemia. The younger stress reticulocytes present with more filamentous reticulum.

Normally, reticulocytes lose their reticulum within 24 hours after entering the peripheral circulation. The premature stress retics have increased reticulum and require 2 to 2.5 days to lose their reticulum, resulting in a longer peripheral blood maturation time

Cells shifted to the peripheral blood prematurely stay longer as reticulocytes and contribute to the reticulocyte count for more than 1 day- causing a false increase. Maturation Time Hematocrit %1 day 40-451.5 days 35-392 days 25-342.5 days 15-243 days <15

An RPI >3 represents an adequate response to anemia by the bone marrow, whereas an RPI <2 is considered an inadequate response of erythopoiesis by the bone marrow to a state of anemia.

INTERPRETATION The Reticulocyte count is an important diagnostic tool:

The number of Reticulocytes is a good indicator of bone marrow activity, because it represents recent production. It is used to differentiate anemia's caused by bone marrow failure from those caused by hemorrhage or hemolysis.

It used also to check the effectiveness of treatment in pernicious anemia and folate and iron deficiency.

To assess the recovery of bone marrow function in aplastic anemia and to determine the effects of radioactive substance on exposed workers.

A low reticulocyte count may mean a need for a bone marrow biopsy. This can tell if is a problem with how new reticulocytes are made by the bone marrow.

Reticulocytosis (Increased RBC Production) Reticulocyte Index >3%, Reticulocyte Count >1.5%

I. PHYSIOLOGICAL:1. Newborns and infants2. High altitude

II. PATHOLOGICAL 3. Acute blood loss or hemorrhage4. Hemolytic Anemia 5. Proliferation of bone marrow 6. Post-Anemia Treatment

Folate Supplementation Iron Supplementation Vitamin B12 Supplementation

Reticulocytopenia (Decreased RBC Production) Reticulocyte Index <1%, Reticulocyte Count <0.5%1. Aplastic Anemia2. Bone Marrow suppression or failure

1. Sepsis2. Chemotherapy or radiotherapy

3. Ineffective erythropoiesis1. Iron Deficiency Anemia2. Vitamin B12 Deficiency3. Folate Deficiency4. Sideroblastic Anemia5. Anemia of Chronic Disease6. Hypothyroidism

4. Blood transfusion5. Liver disease6. Hypopituitarism

SOURCES OF ERROR1. A refractile appearance of erythrocytes should not be

confused with reticulocytes. 2. Filtration of the stain is necessary when precipitated

material is present which can resemble a reticulocyte.3. Falsely decreased reticulocyte counts can result from

under staining the blood with new methylene blue. Be sure the stain/blood mixture incubates atleast 20 minutes.

4. High glucose levels can cause reticulocytes to stain poorly.

5. There is high degree of inaccuracy in the manual reticulocyte count owing to error (± 2% in low counts and ± 7% in high counts) and a lack of reproducibility because of the inaccuracy of the blood film. This inaccuracy has been overcome by the use of automated instruments using flow cytometry.

6. If no reticulocytes are observed after scanning at least two slides, report “none seen”.

7. Erythrocyte inclusions should not be mistaken for Reticulocytes.

Howell-Jolly bodies appear are round, solid staining, dark-blue to purple inclusions, 1 to 2 µm in size. They are nuclear remnants predominantly composed of DNA. Its presence is associated with hemolytic anemias, pernicious anemia, and particularly post splenectomy.

Heinz bodies are inclusions, 0.2 to 2.0 µm in size. They represent precipitated, denatured hemoglobin and are clinically associated with congenital hemolytic anemia, G6PD deficiency, and some hemoglobinopathies.

Pappenheimer bodies are more often confused with reticulocytes and are the most difficult to distinguish. If Pappenheimer bodies are suspected, stain with Wright-Giemsa to verify their presence. These siderotic granules are they are associated with iron-loading anemias, hyposplenism, and hemolytic anemias

Hemoglobin H inclusions will appear as multiple small dots in every cell. These precipitated bodies represent polymers of the beta chains of HbA

Basophilic stippling (fine) and punctate stippling (coarse).

REFERENCES

Clinical Hematology Theory and Procedures 5ed, Mary L. Turgeon

Dacie and Lewis Practical Haematology 11ed, Barbara J Bain

Hematolgy clinical principles and application 4ed, B.E. Rodak

Clinical Hematology and Fundamentals of Hemostasis, 3ed, Denise M. Harmening

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