Peripheral blood smear (PBS)examination, also known as peripheral blood film or blood cell morphology examination, is a diagnostic Pathology laboratory test used to evaluate blood cells and their morphology, size, and number. The main objective of performing this test is to identify and diagnose a wide range of hematological disorders and
use to diagnose blood disorders, blood cancers and infections. These conditions can happen when bone marrow cells mutate and become abnormal cancerous cells known as blasts.
Examples include:
Leukemia
anemia
heart failure
lymphoma
autoimmune diseases
malaria.
when patient is already done CBC and CBC reports shows abnormal blood cell count, then they will order a PBS. For example, your CBC results may indicate your white blood cells, red blood cells and/or platelets appear abnormal means low or High , abnormal number of any given type of cell. A microscopic view of your cells may help your doctors find out how and/or why your blood cells look abnormal or you have an abnormal number of cells.
Results from a peripheral blood smear test is not diagnosis test. doctors make diagnoses based on your medical history, physical examination and results from laboratory tests, like a PBS,
PBS tests may help to have more information about the role your blood cells and platelets.
(RBC )Red blood cells: Your red blood cells carry oxygen from your lungs to your tissues. Your tissues use oxygen to create energy. When your tissues create energy, they release carbon dioxide. Your red blood cells carry carbon dioxide back to your lungs. When you exhale, you’re getting rid of the carbon dioxide.
(Normal RBC )
(WBC)White blood cells: Your white blood cells are part of your immune system that helps protect your body from infection. There are five different white blood cell types. Each type fights infection in different ways.(Neutrophil ,Basophil ,eosinophil ,monocyte, lymphocyte )
Platelets: Platelets help your blood to clot. Too few platelets may be a sign of cancer, infections or other health problems.
Importance Of The Test:
The importance of peripheral blood smear examination lies in its ability to provide valuable information about the patient's overall blood health in a relatively simple and cost-effective way. By examining the morphology of various blood cells, such as red blood cells, white blood cells, and platelets, doctors can get an insight into the patient's underlying medical condition and recommend further diagnostic investigations and treatments accordingly.
The procedure for performing a peripheral blood smear examination in a clinical laboratory involves the following steps:
Specimen type:
1. Collect a blood sample using a standard venipuncture technique and anticoagulant.
2. EDTA Whole Blood .
3. No any prepration require before test.
Test Procedure :
Preparation of thin blood film :
1. Take a clean, dry grease free slide.
2. Mix the whole blood sample well by inverting it 4-5 times placing a small drop of venous blood on a glass microscope slide, using a glass capillary pipette, micro pipette, used for this purpose.
3. Transfer a small drop (approximately 5 ul) of blood about 1 cm from one end of the slide.
4. Place the spreader at an angle of 30 degrees; Pull back the spreader until it touches the drop of blood. Let the blood run along the edge of the spreader.
5. Push the spreader forward to the end of the slide with a smooth movement.
6. Air dry the blood smear.
7. Label the slide with patients SID, name of the patient.
8. The ideal smear is tongue shaped (3 zones well identified) with smooth edges.
a) Head
b) Body
c) Tail.
Steps For Blood Film
How To Prepare A Blood Smear:
Identification marking :
All slides are uniquely identified with patient’s na me,age,sex and Identification number. With a pencil identification can be written on the frosted area of the slide. Also labeling area is used permnant maker pen.
• Air Fixing the Smear:
The slide should be fixed after marking the smear. Air-dry the smear. Methanol present in the stain fixes the smear. If the staining is to be done later, the blood smear must be fixed with methanol for 2 to 3 minutes to prevent distortion of cells.
Manual staining by Lieshman Stain.
1) Make thin film of blood & allow it to dry at room temperature.
2) Cover the smear with Lieshman satin by adding 10-15 drops on smear & wait for 2 minutes.
3) After 2 minutes add. equal drop of Lieshman buffer.Provided with stain.
4) Mix the reaction mixture by gently blowingon it by pipette & wait for 6 minutes.
5) Look for greenish scum formationon the reaction mixture which means reacting is adequate.
6) Pour tap water from beaker/jar & wash the slides.
7) Hold the slide under slow running tap water& wash the back side of slide.
8) Allow the slide to dry by placing vertically on stand.
9) Examine under Microscope.
Manual Method – Field Staining
• Fill up two coplin jar bottle
• Field stain A (Blue stain)
• Field stain B (Red stain)
• Make a Blood film on clean glass slide, and it is dried in the air
• Fix in ethanol for 1 min
• Dry in the air
• Dip fixed smear to field stain B for 5-6 Seconds
• Wash in running tap water
• Dip smear in to field stain A for 10- 30 seconds
• Wash in running tap water
• Dry at air and see under oil immersion objective.
• Examination of Film
1. First examine the stained smear under the low power ie, 10 X. In an ideal smear three zones will appear (i) Thick area (Head) (ii) Body and (iii) Thin end of the smear (Tail).
2. Choose the portion slightly before the tail end where the red cells are beginning to overlap.
3. Place a drop of immersion oil on the smear. Switch to the oil immersion objective and increase the light.
4. Examine the film by moving from one field to the next, in vertical direction from top to bottom or (bottom to top). Systematically. Record the type of leukocyte seen in each field.
5. Count at least a total of 100 leucocytes. Counting 500 leucocytes gives high degree of accuracy.
6. Study Red Blood Cells morphology, abnormalities.
7. Check if platelets are adequate or inadequate. Also observe for macro platelets if present.
WBC examination:
Scanning technique for Total leukocyte count and Differential Count
1. First examine the smear at low power10 X, Overview for TLC .
2. Focus the smear on 40 X for differential count , use manual cell counter (which is specified with BASO,EOSIN,SEG,LYMPH,MONO, and total of 100 cells are counted.)
3. For TLC count ,observe for number of leukocytes per field, observe atleast 100 fields. Take average of this fields multiply the average with 2000. That will be the final TLC count.
4. For ideal morphology review examine the smear under 100 X (oil immersion lense.)
5. Ten microscopic fields are examined in a vertical direction from bottom to top (or top to bottom).
6. The slide is horizontally moved to the next field
7. Ten microscopic fields are counted vertically.
8. The procedure is repeated until 100 leukocytes have been counted (for a 100-cell count).
The different types of leucocytes seen in a normal peripheral blood film may be divided into three groups
a) Granulocytes
b) Monocytes
c) Lymphocytes
a) Granulocytes: There are three types of granulocytes which derive their names from the staining
1. Neutrophils: Diameter 12 – 16 µm. The nucleus is divided in to one to four lobes. Segmented polymorph with purple granules.
Abnormalities to be observed : 1. Shift to left, Hyper segmentedpolymorphs, Toxic granules.
2. Eosinophil: Diameter 10 – 12 µm. Cytoplasm contains large, oval or round, red-orange granules. Nucleus shows fewer lobes, probably two. Polymorph with large orange granules.
3. Basophil: Diameter 8 – 10 µm. The nucleus is not easily seen due to the presence of large, round, deep blue or back granules. Polymorph with large blue granules.
b) Monocytes: Diameter 16 – 22 µm. The nucleus is kidney shaped or horse shoe shaped. Sometimes it may be round or oval its stain pale violet and has fine chromatic arrangement. The cytoplasm is plentiful and stains pale grayish blue and contains a number of very fine pinkish blue granules.
Abnormality to be observed : Mono blasts.
c) Lymphocyte: Dark blue nuclei with clear blue cytoplasm.
Two forms observed 1) Large lymphocyte 2) Small Lymphocyte
1. Large Lymphocyte is about 12 –15 µm in diameter. This has abundant clear pale blue cytoplasm and a large round or slightly indented nucleus with dense chromatin.
2. Small Lymphocyte is about 10 – 12 µm in diameter. It has vary little blue cytoplasm and often little more than just a rim around the nucleus. The nucleus is dark round and some times intended.
Abnormalities to be observed : Reactive lyphocytes.
Platelet morphology: Platelet size and morphology is mentioned in the remark if following variations observed in peripheral blood smear examination.
• Giant platelets, Large platelets
• Platelet aggregates
Scanning technique for Total Platelet count
1. Observe the smear under 10 X low poer , overview for platelet clumps, platelet aggregates, and giant platelets at tail end and body of smear.
2. For platelet count use oil immersion lenses (100 X) , count no. of platelets per field . Count atleast 10 fields , take average count of those fields multiply with 10000. That will be the total platelet count.
This test reports is provide by pathologist.
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