DOBLES, Hilarmina Marie P.

Score:
Hematology 1 Lab Section A 22 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Red Blood Cell Count and Red Blood Cell Indices
Task 4.1 Answer the following questions using the information from the previous video.
1. What are the parts of a hemocytometer?

The hemocytometer contains a thick piece of glass slide, the center of which contains two
counting chambers, separated by a groove. On either side of the two chambers, there are two
platforms which function to support the coverslip when loading a sample into the counting
chambers.

2. What is the dimension (length, width, and depth) of one counting chamber?

The depth of a counting chamber is 0.1 mm. The counting grid has an overall dimension
of 3x3 mm with an area of 9 sq. mm.

3. How many “smaller” and “smallest” boxes are there in one counting chamber?

Each of the four corner squares are further subdivided into 16 smaller squares measuring
0.25 by 0.25 mm in an area of 0.0625 sq. mm. The small square of the center of the grid is
divided into 25 smaller squares with each square measuring at 0.2 x 0.2 mm with an area of 0.04
sq. mm. Each of these smaller squares is further subdivided into 16 smallest squares that each
have a dimension of 0.05 x 0.05 mm with an area of 0.0025. The central small square then
comprises of a total of 400 smallest squares.

4. Differentiate the two kinds of pipettes used with the hemocytometer in counting blood cells.

The RBC pipette has a tiny, red mixing bead inside the bulb. It has three graduations: 0.5,
1 and 101. It is used for counting RBCs in a blood sample. The WBC pipette has a white bead
inside of the bulb. It has three graduations: 0.5, 1, and 11. This is used to count WBCs in blood
samples. These pipettes also have rubber tubing for drawing blood using suction force via the
mouth or any suitable suction device such as the syringe.
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 22 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Red Blood Cell Count and Red Blood Cell Indices
Task 4.2 Illustrate the procedure of Manual RBC counting based on the previous video. Accomplish
your illustrations on a short bond paper following the prescribed format for laboratory tasks provided by
your professor or instructor.

1. Take the RBC pipette pre-cleaned
with alcohol and draw blood
sample up to 0.5 mark. Avoid air
bubbles from entering pipette and
clean off excess blood from
pipette tip using cotton soaked in
alcohol
2. Carefully draw 3. Hold the pipette in a
the RBC diluting horizontal position. Rotate it
solution up to several times in a way that
101 mark makes the bead in the pipette
mix the blood and diluting
solution.

4. Take the counting chamber 6. Carefully position the tip of the

5. Discard the first
and cover slip pre-cleaned pipette on one edge of the cover
few drops from
with alcohol. Carefully slip. Let the contents to gradually
the RBC pipette.
position the cover slip on top flow in the space between the
of the support platforms. cover slip and counting chamber
through capillary force. Load the
other chamber in a similar
fashion.
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 22 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Red Blood Cell Count and Red Blood Cell Indices

7. After keeping aside the counting
chamber to let RBCs to settle, it is now
ready for microscopic observation.
8. First locate the small square at the center of the
counting grid under low power objective. Light
intensity can be adjusted to see the lines of the
counting grid more easily. Once the RBC counting
squares are located, switch to high power objective.

9. Find the 1st, 5th, 13th, 21st, and 25th smaller 10. Apply margin rule (L rule) of RBC counting.
squares. Count RBC lying inside of the square and the
ones lying on the left and lower grid lines but do
not count cells lying on the upper and right grid
lines.
11. Lastly, calculate the total RBC count using the equation:
RBC count = (Sum of no. of red blood cells in 1st, 5th, 13th, 21st, and 25th smaller squares) x 10,000
or
𝑁𝑁𝑁𝑁. 𝑜𝑜𝑜𝑜 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 × 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 × 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑ℎ 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓
𝑅𝑅𝑅𝑅𝑅𝑅 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 =
𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐
𝑁𝑁 × 200 × 10
= = 𝑁𝑁 × 10,000 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐⁄𝑐𝑐𝑐𝑐. 𝑚𝑚𝑚𝑚
1
5
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 22 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Red Blood Cell Count and Red Blood Cell Indices
Task 4.3 Given the following, calculate the RBC count.

Patient 1: Sample drawn to 0.5 mark

RBC small square 1 = 86
RBC small square 2 = 89
RBC small square 3 = 87
RBC small square 4 = 90
RBC small square 5 = 90
(86+89+ 87+90+90) × 200 ×10 442 × 200 ×10 884000
Total RBC count = 1 = =
0.2 0.2
5

= 4, 420, 000 cells/mm3

Patient 2: Sample drawn to 101 mark

RBC small square 1 = 119
RBC small square 2 = 118
RBC small square 3 = 117
RBC small square 4 = 115
RBC small square 5 = 115
(119+118+117+115+115) × 100 ×10 584 ×100 × 10 584000
Total RBC count = 1 = =
0.2 0.2
5

= 2,920,000 cells/mm3
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 22 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Red Blood Cell Count and Red Blood Cell Indices
Research task: Using your textbook and/or other reliable reference, search the answer to the following
question.
1. What is the purpose of diluting the sample in manual RBC counting?

Blood specimen is diluted using RBC diluting fluid that can help to fix and preserve the
red blood cells. A commonly used RBC diluting fluid is Hayem’s fluid which is isotonic and
does not cause damage to red blood cells. Normal saline solution can also be used but it may
cause slight RBC shrinkage and may allow rouleaux formation, leading to errors in the test
results (Batra, 2018). Venous blood in EDTA must be added to an isotonic solution like
Gower’s solution to prevent lysing and shrinkage of red blood cells. Gower’s solution also
has a fixative to preserve and prevent clotting of RBC if RBC counting could not be
accomplished in an hour (RBC count, n.d.).

2. When is a higher dilution of a sample necessary?

Whole blood contains a lot of different blood cells from thousands of white cells to
millions of red cells. That is why many cases call for dilution of the blood sample before a
manual cell count is performed. The diluent to be used along with how large the dilution to
be done is determined by the cells to be counted and the method used to perform the count.
The usual dilution for a manual RBC is 1:200 but adjustments are done to the dilution of the
sample in certain cases where cell counts could be expected to be extremely high or low
(CBC Part I: Cells Counts, n.d.).

If there is polycythemia or erythrocythemia, the number of RBC is increased significantly

and would need a higher dilution to be done. The sample of blood would have to be drawn up
to the 0.3 mark and the diluent would be brought up to the 101 mark of the RBC pipette. The
resulting dilution would be 1:333.33 and the dilution factor in the formula will change
according to this, becoming 333.33. Adjustments are also done for anemia where the sample
is drawn up to the 1 mark of the pipette and the RBC diluting fluid to 100 for a dilution of
1:100 and a dilution factor of 100 (Lecture #12a: Enumeration of Blood Cells, n.d.).
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 22 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Red Blood Cell Count and Red Blood Cell Indices

3. What are the characteristics of a good RBC diluting fluid?

According to Lecture #12a: Enumeration of Blood Cells (n.d.), the characteristics of a good RBC
diluting fluid are:
An isotonic solution
o A good preservative
o One that does not initiate the growth of molds and yeasts
o One with a high specific gravity
o One with buffer action
o Cheap and easy to prepare

4. What are the sources of error in manual RBC counting?

In Blood Cell Count (n.d.), Common sources of error in manual RBC counting are:

• Errors in dilution.
• Errors in counting.
• Errors in calculation.
• Inadequate or improper shaking of the pipette after dilution.
• Failure to wipe excess blood from the end of the pipette.
• Drying of sample during or prior to counting.
• Overflow of fluid into moot.
• Improper adjustment of light source.
• Failure to allow cells to settle into a single plane prior to counting.
• Inadequate cleaning of glass wares.
• Failure to discharge diluting fluid from the capillary prior to charging the chamber.
• Failure to focus microscope up and down during counting.

5. Why is manual RBC counting no longer performed?

It was proven that manual RBC counting methods are inaccurate and automated counters
were able to give a more accurate count of RBC. Errors in counting RBC and WBC could be
ignored when the number of erythrocytes outnumber leukocytes by a factor of 500 or higher.
However, when marked leukocytosis needs to be considered, the manual methods for
determining the counts and volumes of cells will end up with incorrect/inaccurate results unless
corrected for presence of WBC (Turgeon, 2018). In the case of no available automation, there are
other manual RBC procedures like microhematocrit and hemoglobin concentration that are more
accurate and better to use (Keohane, Otto & Walenga, 2020).
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 22 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Red Blood Cell Count and Red Blood Cell Indices
References:

Batra, S. (2018, April 6). TOTAL RED BLOOD CELL (RBC) COUNT USING
HEMOCYTOMETER / NEUBAUER’s CHAMBER (MICRO DILUTION & MACRO
DILUTION METHOD) [Web Log Post]. https://paramedicsworld.com/hematology-
practicals/total-red-blood-cell-rbc-count-using-hemocytometer-neubauer-chamber-
microdilution-macrodilution/medical-paramedical-studynotes

Blood Cell Count [Web Log Post]. (n.d.).

http://www.developmentvet.aun.edu.eg/clinical%20laboratory%20diagnosis/D.htm.

CBC Part I: Cells Counts [Web Log Post]. (n.d.).

http://ruby.fgcu.edu/courses/hogedegb/80026/hem01lab1a.htm

Keohane, E., Otto, C. & Walenga, J. (2020). Rodak’s Hematology: Clinical Principles and
Applications (6th ed.). Elsevier.

Lecture #12a: Enumeration of Blood Cells [Web Log Post]. (n.d.). http://mt-
lectures.blogspot.com/2017/08/lecture-12a-enumeration-of-blood-cells.html

RBC count [Web Log Post]. (n.d.).

https://www.medicine.mcgill.ca/physio/vlab/bloodlab/Cellindices_n.htm

ThomasTKtungnung. (2020, February 8). Haemocytometer [Video File].

https://www.youtube.com/watch?v=7IpE33WPRrc

ThomasTKtungnung. (2020, February 8). Total RBC Count [Video File].

https://www.youtube.com/watch?v=0f9p9JX4qJk

Turgeon, M. (2018). Clinical Hematology: Theory and Practice (6th ed.). Wolters Kluwer.