LABORATORY TESTING

• Disc is composed of two squares, with the area of the

RETICULOCYTE COUNT smaller square measuring 1/9 the area of the larger
• Assess the erythropoietic activity of the bone marrow square.
• Principle: • Disc is inserted into the eyepiece of the microscope
o Supravital stains, such as new methylene blue N or • RBCs are counted in the smaller square, and
brilliant cresyl blue, bind, neutralize, and cross-link reticulocytes are counted in the larger square.
RNA. These stains cause the ribosomal and residual • A minimum of 112 cells should be counted in the small
RNA to coprecipitate with the few remaining square, because this is equivalent to 1008 red cells in the
mitochondria and ferritin masses in living young large square
erythrocytes to form microscopically visible dark blue Formula:
clusters and filaments (reticulum).
• Procedure Reticulocyte (%) = no. of reticulocytes in square A x 100
o Mix equal amounts of blood and new methylene blue no. RBCs in square B (small square) x 9
stain (2 to 3 drops, or approximately 50 mL each)
o Allow to incubate at room temperature for 3 to 10 Example:
minutes Reticulocytes =15
o Remix preparation Red Blood Cells = 112
o In an area in which cells are close together but not
touching, count 1000 RBCs under the oil immersion Reticulocyte (%) = 15 x 100
objective lens (1000x total magnification). 112 x 9 = 1.5 %
Reticulocytes are included in the total RBC count • Sources of Error/Comments
(i.e., a reticulocyte counts as both an RBC and a o Very anemic → adjust the proportion of dye
reticulocyte). o Not mixed = error (not stained well)
o To improve accuracy, have another laboratorian o Refractile bodies may resemble reticulocytes.
count the other film; counts should agree within 20%. o Other RBC inclusions
o Calculate the % reticulocyte count: ▪ Heinz = appear round or oval, and tend to
Formula: adhere to the cell membrane
▪ Howell-Jolly bodies = round nuclear fragments
Reticulocyte (%) = number of reticulocytes x 100 and are usually singular
1000 (RBCs counted) ▪ Pappenheimer bodies = confirmed with an iron
OR stain, such as Prussian blue.
Reticulocyte (%) = number of reticulocyte x 0.1
Example:
Reticulocyte =15

Reticulocyte (%) = 15 x 100

1000 = 1.5 %

Reference Values
• Adult = 0.5% to 1.5 %
• Neonates = 2.5% to 6.5 %

ABSOLUTE RETICULOCYTE COUNT

• Actual number of reticulocytes in 1 liter (L) or 1 microliter
(mL) of blood
Formula:

ARC = reticulocytes (%) x RBC count (x10^12L)

100
Example:
Reticulocytes =2%
RETICULOCYTE COUNT (MILLER DISC)
RBC count = 2.20x10^12/L

ARC = 2 x (2.20x10^12/L)
100 = 44x10^9/L
Reference Interval: 20 to 115x10^9/L
CORRECTED RETICULOCYTE COUNT
• Few RBCs → low hematocrit → falsely elevated
reticulocyte
• Used of correction factor as 45%
Formula:

CRC (%) = Reticulocytes (%) x Patient HCT (%)

45
Example:
• Reduce the labor-intensive process. Reticulocytes (%) = 3%

MONTEMOR, DJ. 1
 HCT = 35 %

CRC (%) = 3 x 35 %
45 % = 2.3%

Reference Interval:
HCT of 35% = 2 to 3% reticulocyte
HCT of <25% = 3 to 5% reticulocyte

RETICULOCYTE PRODUCTION INDEX

• Anemia → shift reticulocytes → falsely increased.
• Principle
o Reticulocytes that are released from the marrow
prematurely are called shift reticulocytes. These
reticulocytes are “shifted” from the bone marrow to
the peripheral blood earlier than usual to
compensate for anemia. Instead of losing their
reticulum in 1 day, as do most normal circulating
reticulocytes, these cells take 2 to 3 days to lose their
reticula. When erythropoiesis is evaluated, a
correction should be made for the presence of shift
reticulocytes if polychromasia is reported in the red
blood cell morphology. Most normal (nonshift)
reticulocytes become mature red blood cells within 1
day after entering the bloodstream and thus
represent 1 day’s production of red blood cells in the
bone marrow. Cells shifted to the peripheral blood
prematurely stay longer as reticulocytes and
contribute to the reticulocyte count for more than 1
day. For this reason, the reticulocyte count is falsely
increased when polychromasia is present, because
the count no longer represents the cells maturing in
just 1 day.
With Shift Reticulocyte
Mon Tues Wed
SR1 3 3 3
SR2 4 4
SR3 5
R1 2
R2 2
R3 2
Total 5 9 14

• Patient’s hematocrit is used to determine the appropriate

correction factor (reticulocyte maturation time in days):
Patient’s Hematocrit Correction Factor
Value (%) (Maturation Time, Days)
40–45 1
35–39 1.5
25–34 2
15–24 2.5
<15 3

Formula:
RPI = reticulocyte (%) x (%HCT/45)
maturation time
OR

RPI = CRC
maturation time

Example:
Reticulocyte count = 7.8%
HCT = 30%

RPI = 7.8 x (30%/45)

2 = 2.6

Reference Interval:
RPI >3 = inadequate bone marrow response
RPI <2 = inadequate bone marrow response

MONTEMOR, DJ. 2
 LEUKOCYTES
• Neutrophil – bacteria (fine lilac granules)
• Eosinophil – parasitic (red- orange)
• Basophil – Hypersensitivity reaction (dark purple/blue
black)
• Monocyte – Phagocytic (glass appearance)
• Lymphocyte -production antibodies (pale blue cytoplasm)
[Differential count only 100 cells]
5. Mix the contents of the pipette for 3-5 minutes to ensure
WBC COUNTING AREA
even distribution of cells. Expel unmixed and relatively
• WBC DILUTING FLUIDS:
cell-free fluid from the capillary portion of the pipette
o 1-3% Acetic acid with Gentian violet [commonly
(usually 4 drops).
used]
o 1% Hydrochloric acid
o Turk’s [preferred]
• WBC COUNTING
o The four large squares placed at the comers are
used for white blood cell count. Since their
concentration is lower than red blood cells a larger
area is required to perform the cell count

6. Place the forefinger over the top (short end) of the pipet,
hold the pipet at a 45 angle, and touch the pipet tip to
the junction of the cover glass and the counting
chamber. Similarly, fill the opposite chamber of the
hemacytometer

• WHITE BLOOD CELL PIPETTE

o The stem of the WBC pipette, is the portion from 0.0
to 1.0 and the mixing chamber is the portion from 10
to 11. The volume of the stem is exactly 10 times the
volume of the mixing chamber. Thus the bulb bolds
10 units of volume
7. Allow the cells to settle for about 3 minutes, Under LPO
and reduced light, focus on the ruled area and observe
for even distribution of cells.
8. Count the white cells in the 4 large corners in each of
• PROCEDURE: two chambers.
1. Swab the tip of a little-used finger with 70% ethanol 9. Count all the white cells lying within the square and
those touching the upper and right-hand center lines.
The white cells that touch the left-hand and bottom lines
are not to be counted. In each of the four areas, conduct
the count as indicated by the "snake-like" line.

2. Lance with quick, fil jab to the side of the pad of the
finger, wipe away first blood.

[Inverted L, do not count over the 3rd line; if touch w/first &
second line still include]
3. Using dilution pipit with the WHITE mixer, draw up to the 10. Compute:
0.5 mark. Do not allow blood to congeal in pipette. Formula:

No. of cells/mm³ = total no. of cells counted

Area x depth x dilution

= total no. of cells counted

1/4 x 1/10 x 1/10 or100
4. Immediately draw diluting fluid to the 11" mark while
rotating the pipet between the thumb and forefinger to =cell counted x 10.000
mix the specimen and diluent. Hold the pipet upright to
prevent air bubbles in the bulb. • Presence of Nucleated RBC

MONTEMOR, DJ. 3
 o Not lysed by WBC diluting fluid
o Falsely counted as WBCs
• Formula for Correction if:
o ADULTS->5NRBC/1000WBC Differential
o NEWBORNS->10NRBC/1000

Corrected WBC Count: Uncorrected WBC Count X 100

100+NRBCs

MONTEMOR, DJ. 4