DOBLES, Hilarmina Marie P.

Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination
Task 3.1 After watching the video, explain the principle of the method, list the reagents and equipment
used, identify specimen requirements, narrate the procedure, and identify the sources of error.

Principle:

In the Cyanmethemoglobin method or Drabkin’s method, a reagent solution of Potassium

cyanide and Potassium ferricyanide is used to dilute blood. The Potassium ferricyanide would act
on the blood to turn hemoglobin into methemoglobin. The Potassium cyanide would then turn
the methemoglobin into cyanmethemoglobin. The resulting solution ’s absorbance will be
measured at a wavelength of 540 nm through a spectrophotometer or colorimeter that uses a
yellow-green filter.

Equipment:

• Micropipette
• Spectrophotometer/Colorimeter
o Same principle applies to both spectrophotometer and colorimeter but
wavelengths differ.

Reagents:

• Drabkin’s solution
o The pH of solution must be checked every month and kept at 7.0 – 7.4 and the
solution should be clear and pale yellow in color; the solution should be
discarded if turbid and pH is outside 7.0 – 7.4 range.
o The solution should not be exposed to sunlight to prevent it from becoming
unstable and should be stored at room temperature in brown borosilicate
bottles
o When measured against water as a blank, its absorbance at the wavelength of
540 nm in a spectrophotometer must be adjusted to 0
o It must be kept in a refrigerator if the room temperature is above 30°C but
brought to room temperature before use; it must never be frozen
o It should not be pipetted by mouth

• Hb standard solution
o It is available commercially at a specific concentration
o Due to its photosensitivity, it must be kept in a brown bottle; exposure to light
deteriorates the standard’s strength.

• Venous blood sample in EDTA (whole blood) or capillary blood by finger prick
sample
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination
Procedure:

Take 5 mL of Drabkin’s solution and put it in a test tube. Take the blood sample tube and
mix it through gentle inversion. Draw 0.2 mL of the blood sample into the pipette and wipe off
excess blood on outside of the pipette tip with a tissue paper. Place the pipette into the tube with
Drabkin’s solution and slowly expel the blood into the solution. Mix well and let it stand
undisturbed for 15 minutes. Measure the absorbance of this solution at 540 nm in a
spectrophotometer after adjusting the optical density at 0 by using Drabkin’s solution as blank.

Possible sources of error:

• Abnormal plasma proteins can cause turbidity

• High leukocyte count can cause turbidity on dilution of blood
• The rate of conversion of blood containing carboxyhemoglobin is slowed considerably
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination
Task 3.2 Explain the principle and procedure of the following methods of hemoglobin determination.

1. Acid hematin method

Principle:

Hemoglobin is converted into brown-colored acid hematin when blood is mixed

with an acid solution. Acid hematin is diluted with distilled water until the color of the
acid matches the brown glass standard. Estimation of hemoglobin is then done by reading
the value directly from the scale.

Materials and Reagents:

1. 0.1N Hydrochloric acid

2. Distilled water
3. Sahli’s hemoglobinometer – equipment consists of a comparator with a brown glass
standard and Sahli’s graduated hemoglobin tube which is marked in percent and
gram.
4. Hemoglobin pipette/Sahli’s pipette (0.02 ml or 20 μl)
5. Glass stirring rod
6. Dropper

Method:

1. 0.1 N Hydrochloric acid is put into Sahli’s graduated tube up to 2 grams.

2. Take a blood sample up to 20 μl using Sahli’s pipette and wipe away blood on the
outer part of the pipette with absorbent paper or gauze piece.
3. Add the blood sample to the 0.1 N Hydrochloric acid solution which is put into
Sahli’s graduated tube. Mix together with the use of a glass stirring rod and allow the
tube to stand for 10 minutes.
4. Drop by drop, add distilled water to the mixture in Sahli’s graduated tube until the
color of the solution matches the brown glass standard.
5. Take the reading using the lower meniscus from the graduated tube in grams.

2. Copper sulfate method

Principle:

Copper Sulphate Method: It is based on specific gravity of blood. A blood droplet

is allowed to fall into copper sulphate solution of a specific gravity 1.053 and the
movement of droplet is observed for at least 15 seconds.
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination

Method:

1. Produce one drop of blood (approximately 0.05 mL) from swabbed finger of blood
donor.
2. Put the working copper sulfate solution with a specific gravity of 1.053 into a clear
beaker. The depth should at least be 3 inches. The drop of blood is then allowed to
fall into the 30 mL solution from a height of 1 cm.
3. The result is declared after 15 seconds of careful observation. If hemoglobin content
is more than 12.5 g/dL, the blood droplet will sink. If it is less than 12.5 g/dL, it will
float.
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination
Task 3.3 After watching the video, explain the principle of the method, list the reagents and equipment
used, identify the specimen requirements, narrate the procedure, and identify the sources of error.

Principle:

A volume of anticoagulated blood is placed in a glass tube which is centrifuged so the blood
is separated into three layers. The three layers are RBC, buffy coat of WBCs and platelets,
plasma. There should be complete separation of the layers. Hematocrit is the ratio of the height
of the red blood cell column to whole blood in the tube. The Wintrobe tube has a length of 110
mm, an internal diameter of 2.5 mm, and total diameter of 3 mm. It is graduated from 0 to 10 cm
in ascending and descending order on both sides of the tube. The scale with the markings for 0 to
10 cm scale from top to bottom is used to determine ESR; from bottom to top, the 0 to 10 cm
scale is used to measure hematocrit or PCV.

Reagents and equipment:

• Wintrobe tube
• Centrifuge machine
• Pasteur pipette

Procedure:

Under aseptic conditions, obtain a venous blood sample. Carefully mix the blood sample
in EDTA through repeated inversion. Fill the Wintrobe tube with the help of Pasteur pipette to
the 10 cm mark and avoid air bubbles. Place the Wintrobe tube into the centrifuge machine, and
balance with other Wintrobe tubes with water. Turn the centrifuge on a slow speed and gradually
increase the speed. Centrifuge for 30 minutes at 3000 rpm; after 30 minutes, turn off the
centrifuge machine and allow it to stop rotating by itself. Take out the Wintrobe tube and note
the readings directly off the graduation. The red blood cells are seen packed at the bottom and
straw-colored plasma is seen above that. In between, there is a buffy coat, a grayish white layer
consisting of white blood cells and platelets, that should be 0.5 – 1 mm in thickness. Read the
level at which the erythrocytes meet the leukocytes.
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination

Sources of error:

• Hemolyzed sample may give falsely decreased results and should not be used.
• Blood must be well mixed and should be at room temperature before testing
• Time and speed of centrifugation are important in obtaining maximum packing of red
blood cells; inadequate centrifugation will result in falsely elevated results due to
excessive trapped plasma.
• Blood to anticoagulant ratio is important in using EDTA; excessive EDTA additive can
cause a falsely decreased hematocrit due to RBC shrinkage.
• Buffy coat might be accidentally included in reading. It may cause falsely increased
hematocrits especially in people with diseases which causes significant error.
• Failure to read hematocrit within 10 minutes after centrifugation will result in pre-
dispersion incompatibility of RBC in plasma and a slanting of the red blood cell/plasma
interface. This can cause a falsely elevated reading.
• Even when there is proper centrifugation of hematocrit, there may be trapped plasma in
the red cell column. Manual hematocrit results are 1-2% higher than automated
instruments that do not have trapped plasma.
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination
Task 3.4 Using your textbook and other reliable references, search the answer for the following
questions.

1. What is the principle and procedure of the copper sulfate method of hemoglobin
determination?

Principle:

Copper Sulphate Method: It is based on specific gravity of blood. A blood droplet is

allowed to fall into copper sulphate solution of a specific gravity 1.053 and the movement
of droplet is observed for at least 15 seconds.

Method:

1. Produce one drop of blood (approximately 0.05 mL) from swabbed finger of blood
donor.
2. Put the working copper sulfate solution with a specific gravity of 1.053 into a clear
beaker. The depth should at least be 3 inches. The drop of blood is then allowed to
fall into the 30 mL solution from a height of 1 cm.
3. The result is declared after 15 seconds of careful observation. If hemoglobin content
is more than 12.5 g/dL, the blood droplet will sink. If it is less than 12.5 g/dL, it will
float.

2. How does dehydration, altitude and age affect hematocrit levels?

Hematocrit can be affected by many factors. Dehydration and being in high

altitude can cause higher hematocrit levels. There is less oxygen in the air of places of
high altitude, and living in such places can elevate your hematocrit levels. Dehydration
can cause high hematocrit levels because the less water there is in the blood, the more red
blood cells per volume of fluid goes up artificially. However, this can be remedied by
having adequate fluid intake.

Hematocrit levels are also affected by age. Hematocrit levels, along with
erythrocyte count and hemoglobin levels, are seen at birth at their highest normal value
but gradually decreases for 2 months after birth. This decrease will be followed by a
steady increase until normal adult values are reached at 14 years of age. It is good to note
that by the age of 12, males are found to have higher erythrocyte, hemoglobin, and
hematocrit levels than females. These hematologic values change in a very little amount
in normal older adults and increase slightly after menopause despite anemia being
prevalent in older people.
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination

3. Does the source of sample (venous, capillary, arterial) affect hematocrit levels?

Although very little, there is still a difference in the normal values of blood
samples in regards to the different sites the blood could be drawn from. For example,
between venous and capillary blood, there is hardly any difference however there are
some blood values like hemoglobin, hematocrit, and platelet counts that differ. Capillary
blood is said to be known for having higher hemoglobin and hematocrit values than
veous blood.

According to Oikonen (2008) in Hematocrit, Smaller vessels in tissue usually

have blood with lower hematocrit levels than in large veins or arteries where blood
samples are taken with the exception of splenic blood that has hematocrit that is nearly
double the hematocrit level in arterial blood. Splenic blood aside, microvessels may have
less than half of the systemic hematocrit. This is said to be attributed to the Fahraeus
effect. However, short capillary transit time can limit the diffusion of oxygen because
oxygen in erythrocytes does not equilibrate with tissue or at least in working muscle.
Conditions that elevate skeletal muscle blood flow can increase capillary HCT and causes
a higher red blood cell surface area to capillary luminal surface area that augments the
oxygen diffusing capacity.
DOBLES, Hilarmina Marie P. Score:
Hematology 1 Lab Section A 3 Oct 2020
TOPIC: Laboratory Evaluation of Erythrocytes: Hemoglobin and Hematocrit Determination
References:

Capillary blood - Can it replace venous blood? (n.d.). Retrieved from

https://www.human.de/lab-professionals/trends-topics/capillary-blood-can-it-replace-venous-
blood/#:~:text=The%20differences%20between%20capillary%20and,values%20and%20in%20p
latelet%20counts.&text=It%20is%20well%20known%20that,hematocrit%20values%20than%20
venous%20blood.

Dayyal Dg. (2016, July 29). Sahli’s Acid Hematin Method for the Estimation of Hemoglobin
[Web Log Post]. Retrieved from https://www.bioscience.com.pk/topics/cell-biology/item/165-
sahlis-acid-hematin-method#:~:text=Principle,value%20directly%20from%20the%20scale.

Hematocrit [Web Log Post]. (n.d.). Retrieved from https://labtestsonline.org/tests/hematocrit

Hematocrit Test [Web Log Post]. (n.d.). Retrieved from

https://www.healthline.com/health/hematocrit

LabsforLifeProject. (2018, June 7). Haemoglobin by Cyanomethemoglobin Method [Video file].

https://www.youtube.com/watch?v=deydnUQdhzA&feature=youtu.be

LabsforLifeProject. (2018, June 7). Haematocrit or PCV [Video file].

https://www.youtube.com/watch?v=RoS3w_Sng_Q&feature=youtu.be

Oikonen, V. (2008, March 27). Hematocrit [Web Log Post]. Retrieved from
http://www.turkupetcentre.net/petanalysis/hematocrit.html.

Shiraz, A. , Debroy, A. (2019). Haemoglobin Screening Methods in BloodDonors- Where Do

We Stand Now? National Journal of Laboratory Medicine, 8(2), PO01-PO04.