7 Red Blood Cell Count
OVERVIEW OF RBC
MORPHOLOGY
Red blood cell or erythrocyte is a non-nucleated
cell, meaning it doesn’t have any nucleus inside.
DIMENSIONS
● Diameter: 7-8 um
● Thickness: 1.5-2.5 um
SHAPE
● Biconcave disk
COLOR
● Appears salmon pink to reddish pink under
a stained smear with a central pale color in
the middle
● The central pale color or the concavity in
the middle almost occupies already 1/3 of
the dimension of the red cell
● Immature erythrocytes or RBC stays in the
circulation for approximately 120 days
LOCATION AND TIMELINE
LOCATION
● Peripheral circulation
TIMELINE
● Approx. 120 days
○ A mature erythrocyte or RBC stays in the
circulation for about, approximately
120 days. So, as the red cell ages. Ages
between 100 to 120th day, the red cells
start to lose its cell membrane. As it goes
through or enter into the tiniest or
narrowest capillaries, it enters into the
red pulp of the spleen it loses its cell
membrane and eventually it is
degraded and ruptured, which means
will be the end of red blood cells.
TRANSFORMERS
Hematology 1 (Lec)
FUNCTION
● To deliver or carry oxygen from Lungs to
other Tissues
○ Accomplished by the attachment of
the oxygen to the hemoglobin present
inside the red blood cell
○ As it circulates in the body, it supplies
and deliver oxygen to the different
tissues of the body
RBC MEASUREMENT
METHOD
● Why is it first necessary to do RBC Counting?
○ It is necessary to have a quantitative
count of red cells since this helps the
physicians or the diagnosticians to
have a clear assessment of the
different erythrocyte indices.
○ So, the basic information that they
need in order to be able to assess
erythrocyte indices is of course the
quantitative number of a red cell and
the qualitative picture under the
peripheral blood smear. So, this
erythrocyte disorder assessment, is
accomplished with the quantitative,
qualitative measurement of RBCs. Also,
it includes with the RBC indices the
hematocrit and the hemoglobin.
1. ELECTRONIC CELL COUNTING
● Most commonly used nowadays because it
involves automation.
● It deals with automated analyzers for cell
counting
● Commonly used in different laboratories
● Faster, easier and less tedious than the
manual cell counting
2. MANUAL CELL COUNTING
● As Medical Technologists, it is still important
to know manual cell counting despite
having the automated methods. This is to
troubleshoot in case of machine
breakdown and if there is an unavailability
of electronic or automated analyzers.
● Manual cell counting involves Dilution. This is
to:
○ Lessen the number of blood cells
■ Remember that an adult has millions
of RBCs so it is impossible for the MT
to quantitatively count RBCs.
Dilution is done to have an estimate
or to have a clear picture of the
number of red cells
○ Lyse cells not needed in the count
■ If you are only going to count, it is
important to use a diluting fluid that
will lyse or eliminate other cells that
are not included in the counting
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7 Red Blood Cell Count Hematology 1 (Lec)

○ Stain particular cell type NEUBAUER COUNTING CHAMBER

■ Staining is important for us to easily
count the specific cells that we want
to count
● Units of Measurement used in Manual cell
counting
○ Traditional unit are expressed by per
cubic mm (mm3) or per microliter (uL)
○ S.I unit are expressed by per Liter (L)
● Another material that is necessary for
MATERIALS AND EQUIPMENT counting is the Neubauer counting
RBC DILUTING FLUID chamber or the Hemocytometer
● For RBC, the diluting fluid should be an ● This Neubauer counting chamber has two
isotonic solution. Isotonic solution, will lessen moats with each having one counting
the damage or at least no effect for the chamber. And each of this counting
RBC where it will not cause shrinking or chamber has a calibrated grits that is used
swelling. In cases of placing RBCs in a for the counting of cells
Hypertonic solution, the RBC would shrink.
And if an RBC is placed in a Hypotonic
solution, it would cause swelling and
eventually, hemolysis. So, if you are using
diluting fluids that are hyper or hypotonic,
then you would not see/count RBC then.
Hence, we must use an ISOTONIC
SOLUTION.
● RBC diluting fluids used:
○ Hayem’s solution ● So, this is the Ruled Areas of counting
○ Gower’s solution chamber
○ Toisson’s solution ● A Neubauer counting chamber has:
○ Formol-Citrate or Dacie’s fluid ● 9 primary large squares (9 mm^2)
○ NSS ○ Each square= 1 mm^2
THOMA DILUTING PIPETTE ○ each of these squares has an area
● 2 types of diluting pipette: of 1 mm^2 with a total of 9 mm^2
○ RBC Pipette ● There are only specific squares that are
■ Has a larger bulb than that used for specific cells
of the WBC ○ First, are the WBC squares
■ Bead color: Red ■ Used are the 4 Primary corner
■ Has large dilution range than squares (WBC)
WBC because they are ● Each of these primary
many compared to WBC squares for the WBC
○ WBC Pipette contains 16 secondary
■ Bead color: White squares
○ For RBC
■ Used is 1 Central square
(RBC)
● Inside this primary central
square contains 25
secondary squares
○ Inside of those 25
● The 2 types differ on the size of the bulb, the secondary squares, it is
bead inside further divided into 16
● Has a bulb and a stem tertiary squares
○ Calibrated stem has different ○ But we will not be using
markings: 0.5, 1, 101 markings all of the 25 secondary
● Range of dilution: 1:100 – 1:1000 squares for counting
● Stem: can hold 1 unit volume blood RBC, we will only be
● Bulb: 100-unit volume of blood using 5 of them which
● In total, stem + bulb: 101 unit are the 4 corner
secondary squares
and the central most
square (A, B, C, D, E).
16(5) = 80 Tertiary square would be counted

TRANSFORMERS 2
7 Red Blood Cell Count
INVERTED L- RULE IN COUNTING
How does it work?
● So, let's say for example this one square. The
inverted L says that all of those cells that will
touch the left and the upper corner of the
square will be included in the count. As well
as the cells that are inside the square will
be counted
● We will be disregarding the cells that will
touch the right and the lower corner of the
square
● Vary on the cells that are on the outside
lines
● The manner of counting should be in the
Battlement method
● This is same with the peripheral blood
smear. So, we have to have a method in
counting so para hindi ulit-ulit or mag-iisip
ka kung nabilang mo na ba ‘to or not.
● So, you will start on the upper left corner
area of the square, pababa.
● Let us try to count red cells, so we will count
the square B
● So, these are the 5 secondary squares in a
central square (most lower right corner in
the pic). So, we have here the A, B, C, D, E.
● Let us only count the cells that will touch
the left and the upper corner of the square.
All of these will be counted on except those
who are touching the right and the lower
area of the square. (Watch it @ 15:13 para
mas maintindihan).
TRANSFORMERS
Hematology 1 (Lec)
○ A: 80
○ B: 89
○ C: 88
○ D: 87
○ E: 83
● Note: NO more than 10 cell difference for
each square
○ So, let's say for example you have
counted only 60 on square A and
then 50 on square E. So, there is
more than 10 cell difference. What
you have to do if you encounter
that problem, you have to repeat
the entire process of charging your
hemocytometer. Because that
means that the RBC are not equally
distributed. In the first place, you
have to check first if the RBCs are
well distributed in the
hemocytometer before you start
counting.
● So, after you counted the 5 squares, you
get the total count and adding them and
we will get 427 counts (count for 1st
chamber)
Acceptable Difference
● It is necessary that the difference of each
chamber should not be more than 10%
○ Note: The difference between the
total cells counted on each side
should be <10%
● Let’s say we have already counted already
counted the chamber 1 and the chamber
2. The formula for that is:
𝐶1 − 𝐶2
× 100
𝐶1 + 𝐶2
2
○ C1: Chamber that has the higher
count of cells (total count: 407)
○ C2: Chamber that has the lower
count of cells (total count 399)
● Solution:
𝐶1 − 𝐶2
× 100
𝐶1 + 𝐶2
2
407 − 399
× 100
407 + 399
2
= 1.99%
○ This is acceptable because it is lower than 10%
● If you have encountered a difference more
than 10 %, repeat counting and do the
charging in the hemacytometer because
red cells are not well distributed. As it results
to inaccurate result.
3
 7 Red Blood Cell Count
CALCULATIONS
Now, that we all know how to count, what are the
rules in counting and which squares to count for the
red blood cells. Let us now go forward to
calculations. It doesn’t end on the counting of the
cells under hemocytometer, we use formula for the
total count of red blood cells.
GENERAL FORMULA
Total count (cells/mm3) =
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝐴𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 (𝑚𝑚2 ) × 𝐷𝑒𝑝𝑡ℎ (𝑚𝑚)
or
Total count (cells/mm3) =
No. of cells counted × DCF × VCF
DCF = Dilution Correction Factor
VCF = Volume Correction Factor
NO. OF CELLS COUNTED
● It is the total number or average number of
RBC counted on 2 chambers
● Average of chamber 1 and 2 to get the
total number of cells factor
● The difference between the total cells
counted on each side should be <10%
FORMULA:
𝐶ℎ𝑎𝑚𝑏𝑒𝑟 1 + 𝐶ℎ𝑎𝑚𝑏𝑒𝑟 2
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 =
2
EXAMPLE:
● Chamber 1: 407
● Chamber 2: 399
COMPUTATION:
407 + 399
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 =
2
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 = 403
DILUTION CORRECTION FACTOR
Total count (cells/mm3) =
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝐴𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 (𝑚𝑚2 ) × 𝐷𝑒𝑝𝑡ℎ (𝑚𝑚)
Total count (cells/mm3) =
No. of cells counted × DCF × VCF
● FORMULA:
!"#$%& "( )#""* $+&*
DCF =
,"-.# "( )#""* / 01#$-123 (#$1* 12 %14123 )$#)
×4
● GIVEN:
○ x = DCF or dilution correction factor
○ Volume of blood used = Volume of blood
aspirated. It is usually up to 0.5 mark
[standard]. This changes if the dilution or
aspiration of the blood would be
■ 0.3 [polycythemia which has a high
count, you have to increase the
dilution by decreasing the volume of
blood]
TRANSFORMERS
Hematology 1 (Lec)
0.8 or up to 1 mark [anemia which has
■
a low count, decrease the dilution by
increasing the volume of blood]
■ Volume of blood is the one that is
changing
○ Total of blood + Diluting fluid in mixing
bulb = A bulb can hold 100 units. It is
constant, 100 in the denominator.
● SOLUTION:
0.5 1
○ DCF =
100
× 4
0.5 (4) 1 (100)
○ DCF =
0.5
× 0.5
1 (100)
○ DCF =
0.5
○ DCF = 200
AREA COUNTED (mm2)
Total count (cells/mm3) =
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝐴𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 (𝑚𝑚2 ) × 𝐷𝑒𝑝𝑡ℎ (𝑚𝑚)
Total count (cells/mm3) =
No. of cells counted × DCF × VCF
1 central square = 1 mm3
1 central square has 25 intermediate squares.
Area of 1 secondary square = 0.04 mm2
● For you to get the area counted, you have
to know the number of squares counted
multiplied by the area of one (1) square.
● How many squares did we use in counting
RBCs?
○ We used five (5) secondary squares.
● Saan galing yung 0.04?
○ This is the area of 1 square. Why? This
is because 1 central square yung
ginamit natin sa RBC and that
central square measures 1 mm3.
● Inside each central square, there are 25
intermediate squares, so you’re going to
divide the area into 25 — you will get 0.04
1
mm2 (this is the area of 1 secondary square).
If you multiply 5 by the area of 1 square, which is
0.04, you will get an area of 0.2.
Again, this is for the standard RBC count. This
changes if you add more number of squares
counted (used in cases such as anemia or
polycythemia vera), there will be adjustments.
However, for the standard, we only use secondary
squares.
4
7 Red Blood Cell Count Hematology 1 (Lec)

(Based on the Q & A portion)

Total no. of square counted 5
Ex. 15 squares are added Total area counted 0.2 𝑚𝑚2
Simply replace 5 with 15 Depth 0.1 mm

15 x 0.04 = 0.6 (area) Total volume counted 0.02 𝑚𝑚2

---hindi ko masyado naiintindihan yung ibang Volume Factor 50

sinabi ni maam dito :< ---
DEPTH (mm)
● Easiest because it is constant
● Depth is the distance between coverslip
and mount of the counting chamber.
● Depth: 0.1mm (constant for the formula)
VOLUME CORRECTION FACTOR
● This is the desired volume over the number
of cells counted multiplied by the area of
one square multiplied by the depth
0&+18&* !"#$%&
VCF =
9"."( +:$.8&+ ;"$2-&* × =8&. "( 1 +:$.8& × 0&>-?
● The denominator is also known as the total
volume.
● The desired volume is the volume of a 1
primary square. Because when you use one
primary central square, that is the desired
volume, which is the 1 cubic millimeter.
● The no. of squares counted in the RBC is the
secondary squares. Each of the secondary
square measures as an area of 0.04 mm2,
and the depth would be 0.1 mm.
1993
VCF =
5 × 0.04992 × 0.199
● We will get a volume correction factor of
50. This is for standard RBC count.
● Kailan naman nag-iiba ung volume
correction factor? We adjust this when we
add more squares in case of anemia. But
for in normal cases, our Volume Correction
factor would be 50.
SUMMARY
Total count (cells/mm3) =
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝐴𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 (𝑚𝑚2 ) × 𝐷𝑒𝑝𝑡ℎ (𝑚𝑚)
Total count (cells/mm3) =
No. of cells counted × DCF × VCF
Standard RBC Count
Dilution Factor 200
Area of 1 square 0.04 𝑚𝑚
Practice Exercise
What is the RBC count of a patient with 451 cell
count of on the first chamber and 445 on the
second chamber?
a. Is the difference of two chambers acceptable?
● Yes, the acceptable difference of 2
chambers should be less than 10%
𝐶1 − 𝐶2
× 100
𝐶1 + 𝐶2
2
451 − 445
= × 100
451 + 445
2
= 1.34%
b. Should you continue to the calculations?
● Yes
c. Total number of cells counted?
451 + 445
= 448
2
d. What is total RBC Count?
● 4.48 mil/𝑚𝑚3
Total count (cells/mm3) =
𝑁𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝐴𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 (𝑚𝑚2 ) × 𝐷𝑒𝑝𝑡ℎ (𝑚𝑚)
Total count (cells/mm3) =
448 × 200
0.2𝑚𝑚2 × 0.1𝑚𝑚
Total count (cells/mm3) = 4,480,000/ mm3 or 4.48
106 /uL
OTHER METHODS
UNOPETTE SYSTEM
● It is an alternative device used for dilution
● Dilution: 1:200
● Dilution Factor: 200
● The difference is that there is no diluting
pipette
● Unopette has already have calibrated
capillary where we can aspirate blood
directly from the puncture site or from an
EDTA tube
● After aspiration from the capillary tube of
unopette, insert it to the reservoir
● Inside the reservoir, there is already a pre-
2 filled diluent or diluting fluid
● Mix for a couple of minutes, then let it settle
● Charge it then to the counting chamber

TRANSFORMERS 5
7 Red Blood Cell Count Hematology 1 (Lec)

TEST TUBE DILUTION METHOD HIGH COUNT/VALUES

● If no unopette Here are some cases wherein we have to change
● Use of Serological pipet the dilution, for high count we have polycythemia
○ Aspirate 0.2 mL of blood POLYCYTHEMIA
○ Mix it with 4 mL of diluting fluid ● Also known as erythrocytosis
● Shake the tube to mix, then let it stand. ● Term used to refer in the increase of red
● Use capillary tube to sample mixture blood cell
○ After the red cells have settled, you ● Polycythemia – increase in red cells
use a capillary tube to get a sample ○ “poly” – many
from the mixture o “cyte” – cells
● Use capillary tube in charging the counting o “emia” – heme/ blood cells
chamber ● Erythrocytosis
● The manner of counting, the squares you are o “erythro” – red cells
counting on the hemocytometer would still o “cytosis” – increase in red cells
be 5 secondary squares for red blood cells.
● INCREASE the dilution
3 Methods: o What we do when there is an
● Thoma Diluting Pipette increase red cells is that we have to
● Unopette system increase the dilution
● Test tube dilution method
Aspirate blood up to 0.3 mark (instead
of 0.5), and increase dilution up to 101
CLINICAL SIGNIFICANCE mark. The dilution then will be 1:333.33
NORMAL COUNT/VALUES DILUTION OF BLOOD Blood: 0.3 mark
FEMALE 3.80 – 5.20 mil/cumm Diluting fluid: 101 mark
MALE 4.20 – 6.00 mil/cumm DILUTION 1:333.33
NEWBORN 4.10 – 6.10 mil/cumm *if magiiba yung dilution (like in this case
mil/cumm = million per cubic millimeter magiincrease), sa formula natin magiiba din lahat,
Reference: Rodak’s like the dilution correction factor (DCF), volume
correction factor (VCF)
FACTORS THAT AFFECT RBC COUNT
1 Altitude LOW COUNT/VALUES
2 Body Fluids ● Decrease or count of red blood cell is call
3 Age and Sex anemia
ALTITUDE ANEMIA
When higher in higher altitude, it triggers or causes ● Low RBC count
hypoxia. When a patient is experiencing hypoxia, ● Decrease the dilution
the erythropoietin (EPO) is stimulated to produce DILUTION OF BLOOD Blood: 0.8 or 1 mark
more red blood cells (rbcs). Therefore, attaining an (depends)
increased rbc when in a higher altitude area. Diluting fluid: 101 mark
BODY FLUIDS DILUTION 1:100
● Liquid portion of blood is plasma
● If plasma is decreased, RBCs look like it is Note: basta mas mataas sa standard (0.5) ng
increased because RBCs become more blood
concentrated due to loss of plasma ● Dilution depends on the severity of anemia
● Decreased plasma = Increased Red cells (example only yung sa table)
AGE AND SEX VARIATION TECHNIQUE
● Same dilution of 1:200
● Gender
● No. of squares counted are increased by 5
○ Males generally they have larger
MULTIPLYING FACTOR
built. So, the demand for the oxygen
● 10 sq= 5, 000
is higher. So, mas maraming
● 15 sq= 3, 340
medyong napoproduce na red
cells than of the females. ● 25 sq = 2, 000
● Only by 5, since red blood cells is already
● Age
less and increasing by 1 is not enough or will
○ Newborns are generally has higher
just make you tired.
red cell count because they have
more active bone marrow to
produce red blood cells or general
blood cells than of the adult.

TRANSFORMERS 6