Belinda Solanna Rodriguez

8th September, 2016

Lab#4
Title: Hemoglobin Solubility and Erythrocyte Sedimentation Rate (ESR)
Objectives:
To determine the solubility of hemoglobin using the sodium dithionate procedure
To determine the erythrocyte sedimentation rate using the Wintrobe method
To determine the RBC morphology of a blood sample.
Principles:
Hemoglobin S will sickle and become rigid under low oxygen tension. This is what
causes the RBC to lose their deformability in decreased oxygen conditions for
people who have the Sickle Cell trait (HbAS) and Sickle Cell Anemia (HbSS). The
solubility test is based on the fact that sickle hemoglobin precipitates when it is
reduced therefore producing a turbid solution while normal hemoglobin remains in
solution and produces a clear liquid. (Student Health Centre Manuals, 2013)
The erythrocyte sedimentation rate (ESR) is a nonspecific measurement used to
detect and monitor an inflammatory response to tissue injury. lt represents the
presence of disease but not its severity. Ln normal blood, the red blood cells remain
more or less separated by being negatively charged. Ln certain diseases, this
negative change is reduced causing the consequent formation of rouleaux. These
larger masses of red blood cells have a faster rate of fall (increased ESR). (Student
Health Centre Manuals, 2013)
Introduction:
Materials & Equipment:
PPE (lab coat, gloves)

Slide

Bleach for decontamination

Test Tubes 12 x 75 mm

Kim-wipes/paper towels

Test Tube rack

EDTA whole blood

Pipetts, 2 ml and 10uL

Lined reader scale

10g Saponin

500 ml distilled water

Microscope

Sharps Container

Timer

Volumetric flask

Wintrobe sed rack and Sedrate tube

Monobasic potassium phosphate , crystals (KH2PO4)

50 mg sodium hydrosulphite (dithionite) Na2S2O4

Pasteur pipette

Wrights stain

216g Dibasic potassium phosphate anhydrous (K2HPO4)

Immersion Oil

Procedures of Section I

Preparation of buffer solution

1. Five hundred mL of distilled water was placed in a 1Lvolumetric flask.
2. the dibasic potassium phosphate was added and mix until dissolved.
3. the monobasic potassium phosphate was added and mix until dissolved.
4. the saponin was added and mix until dissolved.
Working solution
5. Ten mL of buffer solution was poured in a conical flask. the rest (stock solution) were
refrigerated at 40C for up to one month.
6. Fifty mg of sodium dithionite was added to the 10mLbuffer solution.
Solubility Test
1. one ml of whole blood was centrifuged at 1500-2000 g for 5 minutes.
2. the plasma and buffy coat layers were removed using a pipette.
3. Two mL of working solution (room temp) to a test tube for each patient and controls
to be tested
4. Ten uL of centrifuged cells was added to each appropriately labeled tube.
5. the tubes were Mixed well and allowed to stand at room temperature for 5-6 minutes.
6. each tube was placed in front of the lined reader.
7. If lines were visible through the test solution it was reported as negative for HbS.
8. If lines were not visible through the test solution it was reported as positive for HbS.
Procedure for section II: Turgeon, M.L. (2005), pages 444-445.
Erythrocyte Sedimentation Rate
1. anticoagulated whole blood was Thoroughly mixed.
2. Blood was drawn into the Pasteur pipette with attached pipette bulb.
3. the tip of the pipette was placed into the Wintrobe tube until the tip touches the bottom of
the tube.
4. the bulb was Gently pressed and the tip was moved slowly up the bottom of the tube in a
continuous motion to avoid introducing air bubbles into the column of blood.
5. the Wintrobe tube was filled up to the 0 mark.
6. the filled tube was placed into the leveled Wintrobe tube rack.
7. the tube was allowed to stand for 1 hour at room temperature in a draft-free room.
8. the tube was read from the bottom of the plasma meniscus to the top of the sedimented
RBC in millimeters.
Evaluating RBC Morphology
1. EDTA whole blood sample was Thoroughly mixed.
2. a suitable smear was made using the wedge method.

3.
4.
5.
6.
7.
8.

smear was allowed to air dry for 15 minutes.

Smear was placed in methanol solution for 1 minute.
smear was placed in Wrights stain for 5 minutes.
stained smear was rinsed with cold water.
smear was allowed to air dry for 10-15 minutes.
smear on a light microscope was mounted and RBCs were examined under oil immersion
lens.
9. the color, shape and size of the RBCs was evaluated.
10. The poikilocytosis, anisocytosis and palor of the RBCs were reported.
Results:

Patients
ID
Johan
Alonso

Sex
M

Ag
e
25

ESR
7mm/h
r

ESR
reference
Westergren
0-15mm/hr

Interpretation
No inflammation, as the Sedimentation rate
fell within normal range.

Table 1 shows the patient information and results for ESR and the reference range
which is Westergren since that was the method use. The interpretation was
formulated using the results and ESR reference.
Field of
view 1

Field of
view 2

Field of
view 3

Field of
view 4

Poikilocytos
is
Anisocytosi 1
1
s
Palor of
RBC
Normal
TMTC
TMTC
TMTC
TMTC
Table 2 Morphology of RBC shows the table made the EDTA slide

Field of
view 5

TMTC

Discussion:
Sickle Cell test is used to detect the presence of hemoglobin S, to monitor the
amount of Red blood cell and hemoglobin level. The reduced amount of oxygen will
cause the abnormal sickle-shaped cells to form. Saponin is used to lyse the red
blood cells. When Sodium Hydrosulfite is added it reduces the released hemoglobin.
Reduced Hb-S is insoluble when the concentrated phosphate buffer is added and
forms a cloudy, turbid suspension. Some hemoglobin S will be present in those who
carry one sickle cell gene (sickle cell trait) and much more will be present in those
who have sickle cell disease. This test detects the presence of hemoglobin S and
does not differentiate between sickle cell disease and trait. The test is done when
Pain due to sickle cell crises. This is the most common symptoms of sickle cell
disease. Episodes of pain that can last for extended periods of time. The pain can
occur throughout the body and often involves the bones, joints, lungs, and stomach.
The results of sample B would means that there is presence of HbS. A person with
sickle cell trait will produce mostly normal hemoglobin A and some Hb S, while
those with sickle cell disease (anemia) will produce mostly Hb S with no Hb A.

Sample A was negative for Sickle cell as the lines showed. This test is not run on
infants because it would lead to false positive due to the presence of Hemoglobin F.
Another Reason for False positive would be people who are heterozygous for two
different hemoglobin variants will usually produce varying amounts of both types.
(Lab Test Online, 2013)

The erythrocyte sedimentation rate is a simple nonspecific test that has been used
for the dection of inflammation which can be associated with infections, cancers
and autoimmune diseases. It is said to be non specific as it only indicates the
presence of inflammation but does not give specifics such as Where the
inflammation is and what is causing it. Therefore, it should be done with other tests.
An ESR may be ordered when a condition or disease is suspected of causing
inflammation somewhere in the body. There are numerous inflammatory conditions
that may be detected using this test. Test is done in cases where arthritis, Systemic
Lupus Erythematosus as well as anemia is suspected. (Lab Test Online, 2014)
Sample A was normal as it fell to 7 mm/hr, since normal range for westerngren is 015 mm/hr for males. If the concentration of EDTA is greater than recommended, the
ESR will be falsely decreased, (blood volume is less than 2.5 ml in a 5 ml tube). If
the ESR stands for more than 30 minutes, the ESR will be falsely elevated. Tilting of
the ESR tube increases the sedimentation rate. Bubbles in the blood cause invalid
results. Fibrin clots present in the blood validate test results. The ESR should be set
up within 2 hours of blood collection, or within 6 hours, if specimen drawn into EDTA
tube and refrigerated. (Student Health Centre Manuals, 2013)
Normal, mature red blood cells are uniform in size and do not have a nucleus as
most other cells do. They are round and flattened with a depression in the middle.
Giving the biconcave shape. Due to the hemoglobin inside the RBCs, they appear
pink to red in color with a pale center after staining the blood smear. When the
appearance of RBCs (RBC morphology) is normal, it is often reported as
normochromic and normocytic. Using this description the Cells from Sample A was
normochromic and normocytic. However, not all RBC will be perfect if there is a
significant amount of different sizes, shapes and color is indication of a disease,
such as anemia and bone marrow disorders. (Lab Tests Online, 2014) Anisocytosis:
this is a variation in size of RBCs; it may be an indication of anemia. Poikilocytosis is
a variation in the shape of an RBC and may include several different abnormalities
at the same time. Acanthrocytes (spur, thorn or spiculated cells) are irregular
shaped cells with 5-10 spicules; may be present in the blood of people who have
had their spleen removed (splenectomy) and with, for example, chronic alcoholism
(cirrhosis), hemolytic anemia, or thalassemia. They are also present in an inherited
disorder called abetalipoproteineimia. Sickle cells are crescent-shaped RBCs that are
characteristic of sickle cell anemia. Pallor of RBC may be due to Hypochromasia
may be seen in a variety of disorders including thalassemia and iron deficiency. The
RBC is pale in color due to insufficient hemoglobin and contains a large, hollow
middle (central pallor) of the cell. Hyperchromasia is when the RBC is darker in color
than normal; this may be due to dehydration or presence of spherocytes.

Polychromasia is the blue-staining RBCs, indicating that they are immature due to
early release from the bone marrow. (Lab Tests online, 2007)
Conclusion:
The lab was successful as the hemoglobin solubility was observed where sample B
was positive for sickle cell and sample A was not. The erythrocyte sedimentation
rate was conducted using westergren method, the sample tested fell within normal
range. The RBC morphology was determined to be normochromic and normocytic,
since there were only a 3 over all acanthrocytes seen in all 5 fields viewed.

Bibliography
Lab Test Online. (2013). Sickle Cell Tests. Retrieved from
https://labtestsonline.org/understanding/analytes/sickle/tab/test/
Lab Test Online. (2014). ESR. Retrieved from
https://labtestsonline.org/understanding/analytes/esr/tab/test/
Lab Tests online. (2007). Blood Smear: Details on RBCs, WBCs. Retrieved from
https://labtestsonline.org/understanding/analytes/blood-smear/details
Lab Tests Online. (2014). Blood Smear. Retrieved from
https://labtestsonline.org/understanding/analytes/blood-smear/tab/test/
Mayo Clinic. (2014). Disease and conditions: Sickle Cell Anemia . Retrieved from
http://www.mayoclinic.org/diseases-conditions/sickle-cell-anemia/basics/testsdiagnosis/con-20019348
Student Health Centre Manuals. (2013). Erythrocyte Sedimentation Rate. Retrieved
from http://shs-manual.ucsc.edu/policy/erythrocyte-sedimentation-rate
Student Health Centre Manuals. (2013). Erythrocyte Sedimentation Rate. Retrieved
from http://shs-manual.ucsc.edu/policy/erythrocyte-sedimentation-rate