At the completion of this laboratory, the student will be able to:

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The Reticulocyte Count and Erythrocyte Sedimentation Rate

Hematology II Laboratory

M
LS322



Objectives: Reticulocyte Count and ESR



At the completion of this laboratory, the student will be able to:



1. Perform reticulocyte counts matching within th
e 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 associa
ted with increased/decreased reticulocyte counts.



6. State the principle of the Erythrocyte Sedimentation Rate.



7. State the three stages of red cell settling.



8. Perform Wintrobe and Westergren ESR's.



9. Evaluate sources of error in both metho
ds of ESR's


10. Name the different ESR methods and their normal values for men and women.


11. Name the disease states that affect the ESR and the probable reason why it affects this tests.

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 oil immersion lens, count 1000 red cells (500 cells on two slides) recording the number of
reticulocytes s
een in the 500 cells. A reticulocyte is counted twice, once as a RBC and once as a re
-
ticulocyte. The number of reticulocytes seen on the two slides should match within a 1/2 range. (For
example, if 10 retics are seen on the first slide, 5
-
15 retics sho
uld be seen on the second 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 increase
s


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 R
eticulocyte 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.



Matching Criteria for Student Laboratory Results


Reported Value


Your value should match within


1. 0
-
2.0%



+

0.5%

2.
2.1
-
3.0%



+

0.8%

3. 3.1
-
6.0%



+

1.0%

4. 6.1
-
8.0%



+

1.5%

5. 8.1
-
10.0%


+

2.0%

6. > 10.0%



+

20%


Unopette Reticulocyte Count



Materials Needed:



Vital stain


Slides


Unopette pipettes (
25µl
)


Gauze squares


Applicator s
ticks


Procedure:


1.

Place drop of blood on slide. Beside it place a slightly larger drop of stain.


2.

Mix blood and stain with side of pipette.


3.

Fill pipette
-

tilt slide and hold larger end of pipette down to facilitate this; overfilling slightly i
s
recommended to ensure adequate sample for slides.


4.

Place shield over pipette and twist to secure.


5.

Allow to stand 10 minutes at room temperature.


6.

Remove shield from capillary pipette. Hold tip of pipette over slide, fit shield onto large end o
f pipette
and twist to deliver specimen onto slide.


7.

Mix specimen thoroughly with side of pipette.


8.

Prepare at least 3 good smears and allow to air dry.


9.

Using oil immersion lens, count 500 red cells (250 cells on two differ
ent slides) recording
the number
of reticulocytes seen. Calculate the reticulocyte count as follows:


# of Reticulocytes Seen

X 100 = Uncorrected Reticulocyte Count

Total Number of RBC's [500]




for example, there are 13 reticulocytes seen out of 500 total RBC's
counted. The reticulocyte count is
calculated as follows:


13

X 100 = 0.026 X 100= 2.6%

500

Erythrocyte Sedimentation Rate (ESR)


Principle



When red cells are allowed to settle from their plasma, the speed of their fall is known as the
sedimen
tation rate. It is a measure of the suspension stability of red cells. It indirectly measures an
increase in fibrinogen and serum globulins. An increase in either of these results in an increased ESR.
Red cells settle out in three stages:


1.

Initial period of aggregation: rouleaux is formed, and sedimentation is relatively slow; lasts
about 10 minutes.

2.

Fast settling stage: settling rate is constant; most of the settling occurs at this stage; lasts about
40 minutes.

3.

Packing sta
ge: continues for 10 minutes and longer.


Reagents, Equipment

Wintrobe Method



1. Wintrobe Tubes

2. 9
-
inch glass Pasteur pipettes

3. Sedimentation Tube rack

4. Patient EDTA specimen

5. Timer


Procedure


Wintrobe Method



1.

Blood

should be collected in an EDTA anticoagulant and well
-
mixed before being used.

2.

Using a 9
-
inch Pasteur pipette that will reach the bottom of the tube, slowly fill the tube with blood,
avoiding air bubbles in the column.

3.

Adjust the meniscus of the

specimen to the "0" line at the top of the tube.

4.

Place the tube in an upright position in a rack that will maintain the tube in this position.

5.

Set a timer for 1 hour.

6.

At the end of 1 hour, read the fall of erythrocytes by recording the leve
l of erythrocytes in the tube.
The ESR is read on the same side as the "0" on the tube. Reading from the top downward the ESR is
read as the fall of cells in mm per 1 hour of time.


Interpretation of Results


Normal values

Adult Males:



0 to 9 mm
/hr

Adult Females:


0 to 15 mm/hr


The ESR is not a diagnostic test for any specific disease, but it is a useful supplemental test. It is often
used to follow the course of an inflammatory process.

Disease states with increased ESR.



1. Acute

infections

2. Cancer

3. Rheumatoid arthritis

4. Multiple myeloma

5. Pregnancy after the third month


Sources of Error



A. General

1. Dirty tubes

2. Air bubbles

3. Clotted specimen

4. Dimensions of tube


B. Increa
ses ESR

1. Tilted tube

2. Increased temperature

3. Anemia

4. Increased fibrinogen

5. Macrocytic RBC's


C. Decreases ESR

1. Excessive anticoagulant

2. Old blood

3. Decreased temperature

4. Presence
of abnormally shaped RBC's

5. Polycythemia

6. Microcytic RBC's


D. Various Methods for performing ESR's

1. Wintrobe

2. Westergren

3. ZSR:

Zeta Sedimentation Ratio. This method involves spinning capillary tubes in a spe
cial
centrifuge called a zetafuge.

4. Cutler
-


micromethod

5. Landau
-


micromethod

Erythrocyte Sedimentation Rate (ESR)

Westergren Tube Method



Materials Needed:



Westergren tube and reservoir

Gauze square
s

Sedimentation rack

Clock


Procedure



1.

Thoroughly mix blood specimen.

2.

Fill reservoir to mark.

3.

Insert Westergren tube gently into blue cap.

4.

Gently work tube down to bottom of reservoir. T
he tube must be inserted all the way to the
bottom. Absorb any excess blood with a gauze square.

5.

Apply pressure to tube near its entrance to the blue cap to adjust the blood level down to zero.

6.

Place tube in stand and record patient's n
ame and position # in stand.

7.

Set timer for 1 hour

8.

At the end of 1 hour (+ 5 minutes) read how far rbc's have settled and report in mm/hr. Be sure
to read from top to bottom and not from the bottom up.


RETICESR.DOC

Saturday, February 22, 2014