CHINESE GENERAL HOSPITAL COLLEGES

1ST SEMESTER, A.Y. 2017-2018

HEMATOLOGY 1
Laboratory Activity # 9
ERYTHROCYTE SEDIMENTATION RATE

I. INTRODUCTION

Erythrocyte Sedimentation Rate (ESR) refers to the speed at which the erythrocytes settle or fall. This test is used
as a general indicator of inflammation but NOT specific for a particular disease. Erythrocytes in anticoagulated whole blood
will gradually separate from the plasma and settle at the bottom of the tube after standing for 60 minutes (1 hour). The most
important factor that influences the rate is the action of plasma proteins.

II. OBJECTIVES
1. To perform the test that measures the ESR.
2. To know the principle involved in ESR determination.
3. To list the different factors that may affect the result.
4. To learn the clinical significance of the test.

III. PRINCIPLE
If the anticoagulated blood is allowed to stand undisturbed, the red cells will normally settle down at the bottom of the
tubes since the erythrocytes are heavier than the plasma in which they are suspended. Thus, in ESR, one measures the time
it takes for the upper level of the red cell column to reach a specified point and the distance covered by the upper level in a
specified period of time.

IV. STAGES OF ESR

1. Rouleaux formation : 10 minutes
2. Period of fast settling : 40 minutes
3. Final period of packing : 10 minutes

V. IMPORTANCE OF ESR
1. It is used as an indicator or index of the presence of an active disease.
2. It measures the suspension stability of erythrocytes.
3. It indicates the abnormal concentration of plasma proteins.

VI. MATERIALS
WINTROBE METHOD WESTERGREN METHOD
a. Anticoagulated blood (Oxalated blood) a. Citrated Blood
b. Wintrobe Tube b. Westergren Tube
c. Wintrobe Rack c. Westergren Rack
d. Pasteur pipette / syringe with canula d. Pasteur pipette / syringe with canula
e. Timer e. Timer

VII. PROCEDURE
A. WINTROBE AND LANDSBERG METHOD
1. With the use of Pasteur pipette or syringe with canula, fill the Wintrobe tube with anticoagulated blood up to mark
0.
2. Raise the canula as you gradually expel the blood sample to avoid air space or bubbles.
3. Place the tube in a Wintrobe rack vertically.
4. Allow it to stand, undisturbed, for 60 minutes (1hour).
5. Record the ESR value (in mm/hour) by reading directly on the LEFT portion of the Wintrobe tube.

NOTE: The Wintrobe tube is graduated from 0 to 100 millilitres and marked in number centimetres.

NORMAL VALUES:
MALES : 0 to 10 mm/hr. FEMALES : 0 to 20 mm/hr. CHILDREN: 0 to 13 mm/hr.

B. WESTERGREN AND LANDAU METHOD – most sensitive and most specific method of ESR determination for serial
study of chronic diseases. This is based on the same principle of the Wintrobe method. They just differ in the amount
of blood sample needed, anticoagulant used, and the size and design of the tube used for the test.
1. Fill the Westergren tube with blood with the use of an aspirator.
2. Tube must be held firmly by the clip at the top of the Wetergren rack in an exact vertical position.
3. Reading is done after an hour and after 2 hours.

NOTE: Westergren tube is a straight pipette that measures 30 cm. Long with 2.5 mm. Internal diameter.
NORMAL VALUES:
MALES: 3 - 5 mm/hour and 7 - 15 mm/ 2 hours FEMALES: 4 - 7 mm/hour and 12 mm/ 2 hours

CHINESE GENERAL HOSPITAL COLLEGES

1ST SEMESTER, A.Y. 2014 - 2015
HEMATOLOGY 1
Laboratory Activity # 9
ERYTHROCYTE SEDIMENTATION RATE

Name:__________________________________________________________ Section:_________ Date:_____________

QUESTIONS:

1. Write your observations of the group’s result.

2. Enumerate the factors that affect the test.
a. the tube used for blood collection
b. The temperature
c. Disease
d. The procedure
3. Differentiate the Wintrobe from Westergren method.
a. Tube- citrated for westerngreen , but for wintrobe it uses oxalated or anticoagulated blood.
b. Description and function-westerngreen is the most specific and most sensitive for determination of chronic
disease
4. Why do men have lower ESR values than women?
a. Currently. High esr occurs more in women than men. But due to the question itself, I think the reason is due
to the lifestyle that some men did and they are very prone to disease like CHF and Myeloma.
5. List down the common sources of errors in ESR determination.
a. Temperature
b. Clerical error-due to the medtech that types beside the esr setup
6. Illustrate the Wintrobe tube and Westergren tube.

CHINESE GENERAL HOSPITAL COLLEGES

1ST SEMESTER, A.Y. 2014 - 2015
HEMATOLOGY 1
Laboratory Activity # 10
BLOOD SMEAR STAINING

I. INTRODUCTION
Stains are applied to blood smears in order to better evaluate cellular and other formed elements. The stained
smear is evaluated in a procedure called differential count, in which the relative numbers of leukocytes could be
determined. Erythrocytes and platelets can also be evaluated. Parasites such as malaria and microfilariae can
also be identified. Bone marrow aspirate may be examined to evaluate the blood cell production. Polychromatic
stains are the most commonly used for routine microscopic examination. These stains contain methylene blue
and methyl alcohol, a fixative. The two most commonly used differential blood stains are Wright’s and Giemsa’s.

II. OBJECTIVES
1. To know the purpose of staining.
2. To discuss the information that may be obtained from the stained blood smear.
3. To stain a blood smear.
4. To identify some precautions for proper blood smear staining.

III. MATERIALS
A. Blood smears D. Buffer solution
B. Wright’s / Giemsa’s Stain E. Distilled water
C. Droppers F. Staining pan

IV. PROCEDURE
1. Prepare a blood smear.
2. Place the slides on a level staining pan.
3. Add stain to the blood smear, making sure that the entire smear is covered.
4. Add an equal amount of buffer solution over the whole slide.
 5. Mix the buffer and stain thoroughly by blowing gently on the mixture until a metallic sheen forms on the surface. Total
staining time is 3 to 5 minutes.
6. Wash of the stain off by running water directly onto the center of slide to prevent precipitation of stain on the blood
film. Continue washing for 30 seconds.
7. Air dry. Remove any film of stain at the backside of the slide by wiping with a clean gauze or piece of cloth.
8. Examine under the OIO of the microscope.

METHODS OF STAINING:
1. Staining Dish Method – involves placing the blood smear on a rack placed in a dish.
2. Staining Jar or “DIP” Method – involves immersing or “dipping” the blood smear into a jar containing the stain.
3. Automated Method
a. Hemastainer Automatic Slide Stainer
Freshly prepared staining solutions are used daily or every 4-8 hours during operation
STATION 1 – Methanol
STATION 2 – Wright’s or Wright’s-Giemsa stain (500 mL)
STATION 3 – Stain-buffer mixture
Wright’s or Wright’s-Giemsa (80mL)
Phosphate buffer (420 mL)
STATION 4 – Deionized water (1L)
STATION 5 – Phosphate buffer (500mL)
STAION 6 – Warm air

b. Hema-tek 1000 Slide Stainer

The bottles of the stain-pak are first opened by making a small hole and a canula is inserted into each
solution. A pump tube set is installed to transport each solution.
TUBING 1 – stain
TUBING 2 – buffer
TUBING 3 – rinse solution

c. Hema-tek 2000 Slide Stainer

This stainer employs the same principle as Hema-tek 1000. The innovation is the improved staining system
through the use of new pumps and volume controls. The operator can electronically adjust the stain, buffer,
and rinse solutions.

d. Sysmex SP-100 and Beckman Coulter

These automated machines can both perform blood smear preparation as well as automatic staining.

TYPES OF STAIINS USED IN DIFFERENTIAL COUNT:

1. Romanowsky Stains – contains methylene blue or its oxidation product such as azure B and eosin B or eosin Y. The
dyes produce multiple colors when used on cells and cellular components. This is the reason why they are
considered polychromatic stain.

A. Wright’s Stain – Most satisfactory in general routine studies.

COMPOSITION: Oxidized Methylene Blue and Eosin azures

B. Giemsa Stain – excellent stain for the demonstration of inclusion bodies and intracellular parasites aside from
staining white blood cells.
COMPOSITION: Eosin Y with azure B and Methylene Blue in methanol with glycerin

C. Leishman, Jenner, and May-Grunwald – similar to Wright’s stain except for the method used to oxidize methylene
blue.

2. Panopic Stains – consists of Romanowsky stain and another dye.

A. Wright’s-Giemsa
B. Jenner-Giemsa Stain
C. May-Grunwald-Giemsa Stain