EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

RETICULOCYTE COUNT
LAB OBJECTIVE The student will perform, within 20% accuracy of instructors values, two reticulocyte counts on whole blood specimens. The student will calculate the reticulocyte value, corrected reticulocyte and the reticulocyte production index. PRINCIPLE Reticulocytes are immature RBCs that contain remnant cytoplasmic ribonucleic acid (RNA) and organelles such as mitochondria and ribosomes. Reticulocytes are visualized by staining with supra-vital dyes (stain living cells) that precipitate the RBC and organelles. This stain causes the ribosomal and residual RNA to coprecipitate with the few remaining mitochondria and ferritin masses in living young erythrocytes to form microscopically visible dark-blue clusters and filaments (reticulum). An erythrocyte still possessing RNA is referred to as a reticulocyte. The reticulocyte count is a means of assessing the erythropoietic activity of the bone marrow. SPECIMEN Whole blood that is anticoagulated with either EDTA or heparin is suitable. Capillary blood drawn into heparinized tubes or immediately mixed with stain may also be used. Red blood cells must still be living when the test is performed therefore it is best to perform it promptly after blood collection. Blood may be used up to 8 hours after collection. Stained smears retain their color for a prolonged period of time. QUALITY CONTROL Three slides should be made for each retic performed. Final calculated reticulocyte precentage from two slides counts should agree within 15%. If this agreement is not reached, count the third slide. REAGENTS, SUPPLIES AND EQUIPMENT 1. Commercially prepared liquid new methylene blue solution. It should be stored in a brown bottle. If precipitate is a problem on the smear, the stain should be filtered prior to use. 2. Microscope slides 3. Microscope 4. 10 x 75 mm test tubes 5. Pasteur pipets (with bulb if pipets are glass) 6. Capillary tubes 7. Miller ocular (if available)

PROCEDURE

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LAB EXERCISES MLAB 1315

EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

Preparation of smears 1. Add 5 drops of new methylene blue solution to 5 drops of thoroughly mixed EDTA anticoagulated blood to a glass 10 x 75 mm test tube. 2. Mix the contents by gently shaking and allow to incubate at room temperature for a minimum of 10 minutes. 3. At the end of 10 minutes, gently mix the blood/stain solution. 4. Using a capillary tube, place a drop of the mixture on each of three slides near the frosted edge as you would when making a peripheral smear. 5. Using the wedge smear technique, make acceptable smears not too thick or thin. 6. Label the slides with patient name, ID# and date. 7. Allow to air dry. (Do not blow to hasten to drying.) Manual method of counting without the Miller disc: 1. Place the first slide on the microscope stage and, using the low power objective (10x), find an area in the thin portion of the smear in which the red cells are evenly distributed and are not touching each other. Carefully change to the oil immersion objective (100x) and further located an area in which there are approximately 100 red cells per oil immersion field. Do this by finding a field where the cells are evenly distributed and mentally divide the field into 4 quadrants. Count the cells in 1 quadrant. If there are about 25, you are in a good area. There will be a lot of open space between the red cells. 2. When you have selected a proper area for counting, the retics may be counted. Be sure to count all cells that contain a blue-staining filament or at least 2 or more discrete blue aggregates of reticulum in the erythrocyte. (See procedure notes for inclusions that may be confused with reticulum.) 3. Count 1000 red cells in consecutive oil immersion fields. Record the number reticulocytes seen. You may count 500 cells on two slides each. They should agree within 15% of each other. If they do not, repeat the reticulocyte count on the third smear.

Calculation Using the Manual Method Calculate the percent of reticulocytes as follows: Page 2 LAB EXERCISES MLAB 1315

EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

FUNC {%~Reticulocytes~=~{\#~of~reticulocytes~counted ~\in~1,000~red~cells} over {10}} EXAMPLE: Retics counted = 47 in 953 erythrocytes % Retics = 47 = 4.7% 10 Method using the Miller disc 1. Use a 100x objective and a 10x ocular secured with a Miller disc. See figure 31-15 on page 599 of textbook. 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 310 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 and a retic. Record RBC # counted and retic # counted separately. 3. Continue counting until a minimum of 111 RBCs have been observed (usually 15-20 fields). This would correspond to 999 RBCs counted with the standard procedure. Calculation Using the Miller Disc Method % Reticulocytes = Total retics in larger square X 100 Total RBC in smaller square X 9 EXAMPLE: Retics counted in large square = 20 RBCs counted in small square = 111 % retics = 20 X 100 = 2.02% 111 x 9

Calculation of corrected reticulocyte The reticulocyte count is most often expressed as a percentage of total red cells. In states of anemia, the reticulocyte percentage is not a true reflection of reticulocyte production. A correction factor must be used so as not to overestimate marrow production, because each reticulocyte is released into whole blood containing few RBCs - a low hematocrit (Hct) - thus relatively increasing the percentage. The corrected reticulocyte count my be calculated by the following formula: Corrected reticulocyte count = Retic % x patients Hct % Average normal hct* Page 3 LAB EXERCISES MLAB 1315

EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

*Average normal hct is 45 for men and 42 for women. EXAMPLE: Uncorrected retic% = 5.0% Patient hct = 25.0% (male) Average normal hct for male = 45% Corrected retic% = 5.0% x 25% = 2.8% 45% 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. The mature reticulocyte may present with granular dots representing reticulum. Normally, reticulocytes lose their reticulum within 24 to 27 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.
Maturation Time 1 day 1.5 days 2 days 3 days Hematocrit% 45 35 25 15

The peripheral blood smear should be reviewed carefully for the presence of many polychromatophilic macrocytes, thus indicating stress reticulocytes and the need for correction for both the RBC count and the presence of stress reticulocytes. The value obtained is called the reticulocyte production index (RPI). An RPI equal to or greater than 3 represents an adequate response to anemia by the bone marrow, whereas an RPI of less than 2 is considered an inadequate response of erythopoiesis by the bone marrow to a state of anemia. Calculation of the RPI: RPI = Corrected retic count in % Page 4 LAB EXERCISES MLAB 1315

EXERCISE 3: Reticulocyte Count Maturation time in days EXAMPLE: Patients corrected retic = 12% Patients hct = 25% RPI =

MLAB 1315 Hematology

12% = 6 2 PROCEDURE NOTES Interpretation of results 1. The reticulocyte count is elevated: 1) in patients with hemolytic anemia, 2) in those with hemorrhage (acute and chronic), 3) following treatment of iron-deficiency anemia and the megaloblastic anemias, and 4) in patients with uremia. 2. The reticulocyte count is decreased in cases of: 1) aplastic anemia, 2) aplastic crises of hemolytic anemias, 3) ineffective erythropoiesis as seen in thalassemia, pernicious anemia and sideroblastic anemia. 3. Reticulocytopenia in the presence of a suggested hemolytic anemia may often make diagnosis difficult. The diagnosis of a hemolytic anemia can be made because the combination of both hemolysis and reticulocytopenia results in a rapidly falling hemoglobin and hematocrit. Sources of error 1. A refractile appearance of erythrocytes should not be confused with reticulocytes. Refractile bodies are due to poor drying owing to moisture in the air. To rule out refractile material, fine focus up and down. The refractile material will get shiny and reticulum will remain blue. 2. Filtration of the stain is necessary when precipitated material is present which can resemble a reticulocyte. 3. Erythrocyte inclusions should not be mistaken for reticulocytes. Howell-Jolly bodies appear as one or sometime two, deep-purple dense structures. Heinz bodies stain a light blue-green and are usually present at the edge of the erythrocyte. Pappenheimer bodies are more often confused with reticulocytes and are the most difficult to distinguish. These purple-staining iron deposits generally appear as several granules in a small cluster. If Pappenheimer bodies are suspected, stain with Wright-Giemsa to verify their presence. Hemoglobin H inclusions will appear as multiple small dots in every cell. 4. Falsely decreased reticulocyte counts can result from understaining the blood with new methylene blue. Be sure the stain/blood mixture incubates the full 10 minutes. 5. High glucose levels can cause reticulocytes to stain poorly. 6. 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 Page 5 LAB EXERCISES MLAB 1315

EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

7.

automated instruments using flow cytometry. If no reticulocytes are observed after scanning at least two slides, report none seen.

REPORTING RESULTS Normal values Newborn (0-2 weeks):2.5-6.0% Adult: 0.5-2.0% REFERENCES Harmening., Denise, Clinical Hematology and Fundamentals of Hemostasis, 3rd edition, pp. 599-601. Turgeon, Mary Louise, Clinical Hematology - Theories and Procedures, 3rd edition, pp324-326.

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LAB EXERCISES

MLAB 1315

EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

Students name:____________________________________________Date:________________ Retic stain lot #:_____________________________ Expiration Date:______________

Grading criteria: Slide quality, slide labeling, accuracy of result. Adult normal range: _____________________ Newborn normal range: _________________ Show your calculations Patient name/ID # / gender/age Cells counted Slide 1_____ Slide 2_____ (Slide 3_____) Average:_____ Slide 1_____ Slide 2_____ (Slide 3_____) Average:_____ Hct Calculation Result Normal?

Show calculations
Patient ID 1st patient Retic Corrected Retic Calculation: Reticulocyte Production Index Calculation:

Result:______________

Result:____________

2nd patient

Calculation:

Calculation:

Result:______________

Result:____________

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LAB EXERCISES

MLAB 1315

EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

STUDY QUESTIONS
Name_ _______________________________ Date_________________________________ 1 pt 1. What is the reticulocyte used to assess?

2 pt

2.

What criterion is used to identify reticulocytes?

1 pt

3.

What is reticulum composed of?

1 pt

4.

What is the ratio of blood to stain in the usual retic procedure?

5. 1 pt

How long must the blood incubate with the new methylene blue before making the retic smear?

1 pt

6.

What is a stress reticulocyte?

3 pt

7.

List 3 conditions in which the reticulocyte count is increased. A. Page 8

LAB EXERCISES

MLAB 1315

EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

B. C. List 3 conditions in which the reticulocyte count is decreased. A. B. C.

3 pt

8.

6 pt 6 pts. 9. List 3 RBC inclusions that could be confused with reticulocytes on a retic smear and state how you could differentiate them from retics. A. B. C. 1 pt 10 How can you distinguish between reticulum and artifact?

2 pt

11.State the normal range for the following patient populations: Adult:_______________ Newborn:____________

2 pt

12. Calculate the following retic count which was done using the manual method. Show your calculations. 1st slide = 15 retics counted in 500 RBCs 2nd slide = 17 retics counted in 500 RBCs

1 pt

13. Correct the retic count obtained in question # 12. The patient is an adult female with a hematocrit of 25%. Show your calculation. Page 9

LAB EXERCISES

MLAB 1315

EXERCISE 3: Reticulocyte Count

MLAB 1315 Hematology

2 pts

14. Calculate the following retic count which was done using the Miller disc method. The patient is an adult male. Show your calculation. # retics counted in larger square: 26 # RBCs in smaller square: 111

2 pts

15. Why would a doctor request an RPI?

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