Reticulocyte Count

 

 

MLT 130: Hematology I

Lab Week #3: Reticulocyte

Objectives:

1.      To perform, within 20% accuracy of instructor’s values, reticulocyte counts on whole blood specimens.

2.      To perform reticulocyte counts with and without a Miller ocular.

3.      List reference ranges for reticulocytes.

4.      Correlate the reticulocyte count results with normal and disease states.

5.      List sources of error in the reticulocyte count.

6.      Calculate the reticulocyte count and corrected reticulocyte count.

7.      Calculate the Reticulocyte Production Index (RPI).

8.      List 2 common supravital stains.

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 supravital stains (methylene blue or brilliant cresyl blue) that precipitate the RNA and organelles.  These stains cause 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).  The reticulocyte count is a means of assessing the erythropoietic activity of the bone marrow.  The number of reticulocytes is expressed as a percentage of the total number of erythrocytes counted.

The reticulocyte count is elevated in patients with hemolytic anemia, hemorrhage, and following the treatment of iron deficiency anemia.  The reticulocyte count is decreased in cases of aplastic anemia and disorders resulting in ineffective erythropoiesis.

 

 

 

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.   RBCs 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 amount of time.

Quality Control:       

Three slides should be made for each retic count performed.  The final calculated retic% from the first two slides should agree within 15% of each other.  If this criterion is not met, count the third slide.

Reagents, Supplies, and Equipment:

1.      Methylene Blue

2.      Microscope slides

3.      Microscope

4.      Disposable pipettes

5.      Timer

6.      Miller ocular (if available)

Procedure:

            Preparation of Smears

1.       Label a tube with patient’s last name, ID number, and your initials.

2.      Add 3 drops of a well mixed EDTA anticoagulated blood to the labeled test tube.

3.      Add 3 drops of the methylene blue to the labeled test tube.

4.      Mix the contents by gently shaking.

5.      Incubate at room temperature (20-24°C) for 10 minutes.

6.      After the 10 minute incubation, gently mix the stain/blood solution.

7.      Using the wedge smear technique, makethree smears (not to thick or thin.

8.      Allow to AIR dry. (Do not blow to hasten drying)

9.      Label the slides with initals and date.

 

 

 

 

Manual Method of Counting (WITHOUT Miller disc)

1.       Place the first slide on the microscope stage and, using the 10x (low power) objective, find an area in the thin portion of the smear in which the red cells are evenly distributed and NOT touching each other. 

2.      Without moving the slide, change to 100x (oil immersion objective), add oil, and find an area of the smear in which there are approximately 100 red cells visible per field.  A method for finding a field with around 100 RBCs is to mentally divide the field into 4 quadrants and count one of those 4 quadrants.  If you count 22-28 RBCs in one quadrant, then the area is acceptable.  In other words, there should be around 25 RBCS in ¼ of the oil immersion field visible.

3.      Begin counting both the RBCs and the retics.  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 as retics and RBCs. (See procedure notes for inclusions that may be confused with reticulum.) 

4.      Continue counting consecutive oil immersion fields until you have reached 1000 total red cells (RBCs + Retics).  You may count 500 cells on two slides, as an alternate method.

5.      Record the number or Retics counted on each smear on the data sheet.

6.      If the two slides do not agree within ±15% roughly two units), perform a retic count on the third slide.

7.      Calculate the percent of reticulocytes as follows:

 

% Retics=   # of reticulocytes counted in 1000 total red cells

        10

                                    Example:

Slide 1= 46 in 1000 erythrocytes

Slide 2= 48 in 1000 erythrocyte

Average= 47

                                    Retics counted = 47 in 1000 erythrocytes

                                    % Retics = 47/10 =4.7%

 

 

 

 

 

                                                                                                               Field of View

            Method using the Miller disk:                                                 Miller disk

                                                                                                        Retic counting sq.

                                                                                                    RBC counting sq.

1.      Use a 100x objective and a 10x ocular secured with a Miller disc.  The Miller disc imposes two squares onto the field of view of the oil immersion objective. The retic counting square is 9x the area of the RBC counting square. 

2.      Find a suitable area of the smear that shows 3-10 RBCs in the Retic counting square.

3.      Count the retics within theretic counting square including the cells touching the top and right sides of the small square.

4.      Count all cells in the RBC counting square.  Count retics in this square as both a retic and an RBC.Cells touching the lines should only be counted on the bottom and left sides.

5.      Continue counting in other acceptable fields.

6.      Stop counting when you have reached 111 total RBCs (RBCs +Retics) from the RBC counting square.  This number of cells should be reached in 15-20 fields and corresponds to 999 RBCs counted with the standard manual procedure.

7.      Calculate the % Reticulocytes  using the Miller disc method by the following formula:

                        % Retics = Total Retics counted in retic counting square

                        10

** The large retic counting square ENCOMPASSES the small RBC counting square. See picture above.

 

                        Example:

                        Retics counted in retic counting sq=20

                        RBCs counted in RBC counting sq=111 (Constant #. Not part of the calculation)

                                          %retics= 20/10 = 2%

 

            Calculation of corrected reticulocyte for Low Hematocrit

The reticulocyte count is most often expressed as a percentage of total red cells.  In an anemic state with a low hematocrit, the reticulocyte % is not a true reflection of the reticulocyte production because the whole blood in these patients has fewer RBCs resulting in an elevated percentage. 

                        Corrected retic count=Retic% x patient’s Hct%

                                                                          Avg. normal Hct %

           

Gender (adult)	Average normal Hct %
Male	45
Female	42

           

                        Example:

                        Uncorrected retic% = 5.0%

                        Patient Hct= 25.0% (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.  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	Hematocrit %
1 day	45
1.5 days	35
2 days	25
3 days	15

 

The value obtained is called the reticulocyte production index (RPI).  An RPI ≥2 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.

            Calculation of the RPI:

                        RPI=Corrected retic count in %

                                 Maturation time in days

                                                Example:

                                                Patient’s corrected retic = 12%

                                                Patient’s Hct = 25%

                                                RPI= 12%/2= 6

Procedural 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.      Incubation >15 minutes falsely increases the retic count due to dye adhering to mature RBCs.

3.      Filtration of the stain is necessary when precipitated material is present which can resemble a reticulocyte.

4.      Erythrocyte inclusions should not be mistaken for reticulocytes. 

Inclusion	Appearance
Howell-Jolly bodies	one or sometime two, deep-purple dense structures.
Heinz bodies	stain a lightblue-green and are usually present at the edge of the erythrocytes.
Pappenheimer bodies	Most of confused due to difficulty distinguishing.  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	multiple small dots in every cell.

                       

5.      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.

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

7.      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.

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

Reporting Results:

                        Reference values:

                        Newborn (0-2 weeks):  2.5-6.0%

                        Adult:                           0.5-2.5%

 

 

 

                       

 

Reticulocyte Data Sheet

 

Student name: ___________________________Date:_________________________

ReticSet lot #: __________________________         Expiration date:________________

Instructions:  Perform TWO reticulocyte counts on TWO different patients.  Show your calculations. Remember to use correct units when reporting the data.

	Retics counted	Patient Hct	Calculation	Retic Result	Normal?	Corrected Retic	Reticulocyte Production Index
1.	Slide 1 _____   Slide 2 _____   (Slide 3 ____)   Average: _____					Calculation:             Result:_____	Calculation:             Result:_____
2.	Slide 1 _____   Slide 2 _____   (Slide 3 ____)   Average: _____					Calculation:         Result:_____	Calculation:         Result:_____

 

 

 

 

Reticulocyte Lab

Study Questions

 

1. What is the reticulocyte used to assess?

 

 

2. What constitutes reticulum?

 

 

3. What is the ration of blood to stain in the manual retic procedure?

 

 

 

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

 

 

5. What is a “stress reticulocyte”?

 

 

6. List 3 conditions in which the reticulocyte count is increased.

a.       _________________________________________

b.      _________________________________________

c.       _________________________________________

 

7.      List 3 conditions in which the reticulocyte count is decreased.

a.        _________________________________________

b.      _________________________________________

c.       _________________________________________

 

 

8.      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.________________________________________________________________________________________________________________________________________

 

9. How can you distinguish reticulum from artifact?

 

 

10. State the reference range for the following patient populations:

                        Adult:_____________________________

                        1 week old baby:____________________

11. Calculate the following retic count which was done using the MANUAL method.  Show calculations:

            1^st slide= 15 retics counted in 500 cells

            2^nd slide= 17 retics counted in 500 cells

 

 

 

 

 

12. Correct the retic count obtained in question 11 because the patient is an adult female with a hematocrit of 25%.  Show calculation:

 

 

13.  Calculate the following retic count which is done using the Miller disc method. The patient is an adult male. Show your calculation.

            # retics counted in larger square: 26

            # RBCs (all cells) in smaller square: 111

 

 

 

 

14.  Why would a doctor request and RPI?

 

 

 

 

 

 https://laboratorytests.org/reticulocyte-count/

https://wps.prenhall.com/wps/media/objects/684/700987/ch07RET.pdf 

 https://www.healthline.com/health/reticulocyte-count#risks

 https://www.verywellhealth.com/reticulocyte-count-uses-4583797

 https://www.kau.edu.sa/files/0007058/files/60658_reticulocytes%20count.pdf

 https://diagnolab.com.na/images/SOPs/SOP--321-RETIC.pdf

 https://neit.instructure.com/courses/45412/files/5512131/download?wrap=1

 http://biolabdiagnostics.com/image/catalog/pdf/RETICULOCYTES%20Literature%20New%20Final.pdf

 

.

.