Reticulocyte Counts

Reticulocyte Counts

Objectives

1. Perform reticulocyte counts matching within the required criteria.

2. Describe both procedures for performing a reticulocyte count.

3. State normal reticulocyte values for newborns and adults.

4. Perform calculations to determine reticulocyte percentages.

5. Describe disease states associated with increased/decreased reticulocyte counts.


Reticulocyte Count


Reagents, Equipment

1. New Methylene Blue - Vital Stain
2. Microscope Slides
3. 12 x 75 test tubes
4. Applicator sticks
5. Beral pipettes
6. Timers


Procedure

1. Using a Beral pipette, place 5 drops of blood in the test tube.

2. Add 5 drops of New Methylene Blue stain to the test tube.

3. Mix and allow the mixture to stand at room temperature for 10 minutes.

4. Prepare at least 3 good smears and let them air dry.

5. Using the oil immersion lens count 500 red cells on one slide. Use the hand tally counter and a differential counter to record the number of reticulocytes counted as well as the total number of red cells counted. Be sure to include the reticulocytes in the total red cell count. Record the number of retics counted. Repeat this step with the other slide.

6. Calculate as follows:

Add together the total number of reticulocytes for the two slides. This yields the num¬ber of retics per 1000 RBC's. To convert to % divide this number by 10. For example, 10 retics on slide 1 and 15 retics on slide 2 equals 25 retics total or 2.5%.

Interpretation of Results

Normal Ranges

Adult: 0.5 - 1.5%
Newborns: 0.5 - 8%


Reticulocyte count is representative of bone marrow condition

1. RBC production increased, Retic count increases
2. RBC production decreases, Retic count decreases

Disease states with increased Reticulocyte counts

1. Hemolytic Anemias
2. After acute hemorrhage
3. Pernicious Anemia or iron deficiency anemia after treatment

Disease states with decreased Reticulocyte counts

1. Aplastic Anemia
2. Iron Deficiency Anemia before treatment
3. Pernicious anemia before treatment

If no retics are seen in 500 cells, both slides should be scanned for retics. If a retic is found on scan report retic count as <0.1%. If no retics are seen, set it up again, and if no retics are seen on the new slides then report as no retics seen on smear.


Hematology Instrumentation
Flow Cytometry & Reticulocyte Counts

Reticulocytes may be counted with a flow cytometer, which is much less time consuming and analyzes far greater numbers of erythrocytes than do manual methods. Flow results correlate well with traditional methylene blue manual methods. Cells are stained with thiazole orange (UMC), acridine orange, thioflavin T, or pyronin Y. These stains are RNA specific, and since young erythrocytes contain more cytoplasmic RNA, they fluoresce more brightly than mature RBC's.


Principles of Flow Cytometry


Cells in solution are stained with a fluorescent dye. Dyes may be:
1) immunological reagent ,i.e., monoclonal antibody
2) dye that stains a specific component of the cell, or other marker with specified reactivity

Laser light is preferred because of three properties:
1) intensity
2) stability
3) monochromatism [one wavelength, ]




Brief Explanation of Principle:

The cell suspension is pressurized and passed through plastic tubing to a flow chamber, where it is injected through a needle into a stream of physiological saline called the sheath. The sheath and specimen exit the chamber through a 75m orifice. The design causes the cells to be confined to the center of the saline sheath and move in single file. The stained cells then pass through the laser beam, which causes the stained cells to fluoresce. Through a process known as the Tyndall effect, light is scattered in all directions around the fluorescing cells. Optical sensors located either in-line with the beam or at 90 degrees to the beam detect the scattered light and measure its intensity. The information is then transmitted to a computer which collates the raw data. Analysis may be performed at rates up to 50,000 cells/min.

Technical Aspects of Flow Cytometry:

With this method of blood cell counting, the sample is routed through a small passage known as a flow cell. The diameter of the flow cell is constricted so that very few cells may pass through at a time. In theory, it would be nice if only one cell could get through at a time, but in practice, this does not occur. When two or more cells pass through the flow cell counting area at once, the phenomenon is known as coincidence. As one may discern with some careful thinking, the probability of co-incidence will increase with an increase in the concentration of cells or particles in suspension. Since the concentration of, for example, red cells in whole blood is on the order of about 5.5 million per cubic millimeter, the probability that more than one cell will pass through at any one given moment will be reduced by making a dilution of the sample. If we reduce the concentration of cells by say, a factor of 400, we also reduce the chances of coincidence by a similar amount. The diluent used is an electrolyte, usually isotonic saline.

Technical Aspects of Flow Cytometry:, continued

The flow cell is arranged in such a way that a beam of laser light is directed to pass through it to a detector (photocell) on the opposite side, or at an angle to the incident beam. When a blood cell passes through the flow cell between the light source and the photocell, the cell either fluoresces (if stained with fluorescent dye), or occludes the light striking the photocell. This alters the resistance of the detection circuit and changes the current flowing through it. Since each cell produces only a momentary interruption in the intensity of light striking the photocell, there is only a momentary change in detector current. This is known as a pulse. By including in the detector circuit a device which is sensitive to these fluctuations in current, the pulses may be translated into counts, each pulse representing one cell. There is usually included on the counting instrument either a fixed or adjustable threshold. Essentially what this does is determine the amount of change in current that must be registered before the pulse is registered as a count. The higher the threshold adjustment, the larger the pulse must be before it is recognized and counted.

We mentioned coincidence before. Most automated counters also include in their circuitry a microprocessor which automatically corrects for coincidence. The correction is simply a matter of solving a probability equation, with coincidence a function of cell concentration, size, flow cell diameter, and rate of flow.

Most counters today are what is known as decade counters. This means that they register every tenth pulse instead of every pulse. Again we run into the problem of probability. If, for example, there is slight contamination of the diluent, or suspending fluid, with dust, or some other small particle, we can reduce the probability of counting this contaminant as a cell if we count only every tenth pulse. Contamination of this sort becomes a very important source of error, especially as a cell size being counted become smaller, and the threshold settings decrease.

After the required volume of fluid is counted and the pulses registered, the machine calculates the concentration of cells per cu. mm. after correcting for coincidence, dilution, and vol. counted. The results are usually presented on a digital display.



Sources of Error:

1) contamination of either glassware or diluent
2) improper dilution of sample
3) inadequate mixing of sample prior to dilution
4) contaminated flow cell
5) old or improperly collected sample
6) improper threshold settings
7) incubate in dark for 30 minutes (this step ensures that the brightness of the fluorescence will not be diminished or eliminated by bombardment of the dye with photons, the phenomenon known as "quenching".


Corrections to the Reticulocyte Count

Reticulocyte Count Correction for Anemia
Since the Reticulocyte count is expressed as a relative percentage, a change in this percentage may be due to an overall change in the number of circulating erythrocytes, and must be corrected to an absolute percentage in cases of anemia. This correction is accomplished as follows:

Patient's HCT
Corrected Retic Count = Retic % X -------------------
Normal HCT

Example: Adult male has a HCT of 30% (0.30 L/L) and a reticulocyte count of 3%. What is the corrected count?

0.30 L/L
Corrected Retic Count = 0.03 % X ------------- = 0.02 or 2 %
0.45 L/L



Reticulocyte Production Index
Used to correct the reticulocyte count for the presence of younger reticulocytes that persist in the peripheral blood longer than expected, due to large quantities being released from the bone marrow in conditions of stress (i.e., acute bleeding). The RPI gives a better indication of erythropoietic activity when these "stress" reticulocytes are present in peripheral blood. It is calculated utilizing the following table: (reference: Turgeon, Clinical Hematology, 1993. p. 61)

Maturation Time Correction Factor

HCT L/L
Maturation Time (days)

0.45
1.0

0.35
1.5

0.25
2.0

0.15
2.5




The Maturation Time may also be expressed in the form of the following equation, for values not specifically appearing on the chart:

MAT = 1 + 0.05(45 - Pt's HCT)
(y) = (b) + (m) (x) [NOTE: Linear Equation Expression]





The formula is as follows:

Corrected retic %
RPI = ---------------------------------
Maturation Time (days)


RPI, continued


Example: Corrected reticulocyte count is 2.0% and the patient's hematocrit is 0.30 L/L.

2%
RPI= ---------------- = 1.14 [NOT PERCENT!!]
1.75


> RPI Hemolytic anemia with increased peripheral blood erythrocyte destruction in the face of normal marrow response. May increase up to 7x normal.

RPI Normal Normal marrow activity

< RPI Bone marrow damage
Erythropoietin suppression
Hypoproliferative states (B12, folic acid, or Fe deficiency)

الاخ محمد الشويلي السلام عليكم
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الاخ محمد الشويلي
السلام عليكم عطاء متواصل وكبير
مواضيع اكثر من رائعة
سلمت اخي وسلمت يداك على المواضيع الطبية
ارجو ان يكون مروري على مواضيعك بقدر فائدتها لنا
لك مني كل الشكر والتقدير