MANUAL WBC COUNT Flashcards
1
Q
REASONS FOR MANUAL COUNTING
A
- Every automated methods require a backup method
- Not all samples can be evaluated by automated methods
- Specimen Quality
2
Q
The capability of instruments to count cells accurately
A
Instrument Linearity
3
Q
When WBC values are at the extreme ends of the spectrum (i.e. extremely high or extremely low WBC count) there will be _______
A
Loss of instrument linearity
4
Q
Those that have ________ that may interfere with the instruments ability to
count leukocytes accurately
A
abnormal proteins or any other elements
5
Q
is charged (filled) with the well-mixed dilution and placed under a microscope and the number of
cells in the 4 large corner squares (4 mm2) is counted
A
Hemocytometer
6
Q
the number of WBCs in 1 liter (L) or 1 microliter (µL) of blood
A
WBC count
7
Q
Typical dilution
A
1:20
8
Q
Diluent for leukocytes must be?
A
must be capable of lysing (non nucleated) RBCs without
destroying leukocytes
9
Q
Specimen
A
WB with EDTA (Heparin or Ammonium Potassium Oxalate may be used)
Capillary blood
10
Q
Diluting fluids for Leukocyte Count
A
3% Glacial Acetic Acid
1% HCl
1% Buffered Ammonium Oxalate
1% Turk’s sol’n
11
Q
Diluent of choice for manual leukocyte count
A
3% Glacial acetic acid
12
Q
Enhances leukocyte nuclear definition (details of the nucleus of leukocytes)
A
1% Turk’s sol’n
13
Q
Components of Turk’s solution
A
3 mL glacial acetic acid
1 mL aqueous gentian violet
96 mL distilled water
1 pinch of thymol
14
Q
Lyses (non nucleated) RBCs
A
3 mL glacial acetic acid
15
Q
Enhances staining of WBC nuclei
A
1 mL aqueous gentian violet
16
Q
Solvent
A
96 mL distilled water
17
Q
1 pinch of thymol
A
Prevents fungal growth
18
Q
Most Common Hemocytometer/Counting chamber
A
Levy Chamber with improved Neubauer ruling
19
Q
Hemocytometer is composed of 2 raised surfaces
A
each with a 3 mm × 3 mm square counting area or grid separated by H
shaped moat
20
Q
Total area of hemocytometer
A
9 mm2
21
Q
Hemocytometer is composed of
A
Nine (9) 1 mm × 1 mm squares
4 corner squares → subdivided into 16 squares
1 center square → subdivided into 25 smaller squares
Each smaller square → 0.2 mm × 0.2 mm = 0.04 mm
22
Q
Distance between the counting surface and the coverslip (depth)
A
0.1 mm
23
Q
Total volume of one entire grid or counting area (one side)
A
0.9mm3
24
Q
Total volume of one entire grid or counting area both sides
A
1.8 mm3
25
Difference bet. the counted cells on both side:
Less than 10%
26
Thoma diluting pipette/pipet
WBC Thoma Pipette
RBC Thoma Pipette
27
used to make 1:10 or 1:20 dilutions
WBC Thoma Pipette
28
used to make 1:100 or 1:200 dilutions
RBC Thoma Pipette
29
procedure
1. Mix the blood sample.
2. Aspirate blood to exactly 0.5 mark on pipet stem
3. Wipe excess blood.
4. Draw WBC diluting fluid up to 11 mark.
5. Immediately cover the tip of the pipette with your middle finger and carefully remove the suction device. Place your
thumb over the open tip and vigorously rotate the pipette back and forth, moving only the wrist, for 30-45 seconds.
○ After diluting the blood specimen, allow the dilution to sit for 10 minutes to ensure lysis of the RBCs (Dilution
becomes clear)
○ NOTE: WBC count should be performed 3 hours of specimen dilution
6. After thorough mixing, discard the first few drops and then gently fill the chamber until the platform is filled (charging).
○ Drops: Ensures that the sample placed in the hemocytometer is really the diluted blood sample
7. After charging the hemacytometer, place it in a moist chamber for 10 minutes before counting.
○ Moist Chamber:
■ Allows the cells to settle
■ Prevents the evaporation of the sample from the hemocytometer
8. Under the microscope, count all of the cells (WBCs) in the four corner squares
○ NOTE:
■ Cells that touch the top and left lines should be counted
■ Cells that touch the bottom and right lines should NOT be counted
30
How to Make a Moist Chamber
1. Place a piece of damp filter paper in the bottom of a Petri
dish
2. Break the applicator stick into half and place each piece on
top of the damp filter paper
3. Place the hemacytometer above the applicator sticks
31
Anticipated WBC Count (× 109 /L)
0.1 - 3.0
WHAT IS THE RECOMMENDED DILUTION?
1:10
32
Anticipated WBC Count (× 109 /L)
0.1 - 3.0
What Type of Thoma Pipette is appropriate to use?
WBC
33
Anticipated WBC Count (× 109 /L)
3.1 - 30.0
WHAT IS THE RECOMMENDED DILUTION?
1:20
34
Anticipated WBC Count (× 109 /L)
3.1 - 30.0
What Type of Thoma Pipette is appropriate to use?
WBC
35
Anticipated WBC Count (× 109 /L)
>30.0
WHAT IS THE RECOMMENDED DILUTION?
1:100
36
Anticipated WBC Count (× 109 /L)
>30.0
What Type of Thoma Pipette is appropriate to use?
RBC
37
Anticipated WBC Count (× 109 /L)
≥100.0
WHAT IS THE RECOMMENDED DILUTION?
1:200
38
109 /L)
≥100.0
What Type of Thoma Pipette is appropriate to use?
RBC
39
ALTERNATIVE PROCEDURE (W/O WBC THOMA PIPETTE)
1. Mix 25 µL blood sample and 475 µL of WBC diluting fluid in a small test tube
2. Allow the dilution to sit for 10 minutes to ensure lysis of RBCs
3. Charge the hemocytometer using microhematocrit tube
4. Moist chamber for another 10 minutes
40
WBC COUNT (ADULT)
Conventional Units
(cells/mm3 or cells/µL)
3,600 – 10,600
41
WBC COUNT (ADULT)
Standard International
(SI) Units (×109/L)
3.6 – 10.6
42
SOURCES OF ERRORS AND COMMENTS
1. Hemocytometer and coverslip should be cleaned properly before they are used.
2. The diluting fluid should be free of contaminants.
3. If the count is low, a greater area may be counted (e.g., 9 mm2) to improve accuracy.
4. The chamber must be charged properly to ensure an accurate count.
5. After the chamber is filled, allow the cells to settle for 10 minutes before counting.
6. Correct the WBC count in the presence of 5 or more (adult) or 10 (newborn) nucleated RBCs using the formula
7. The accuracy of the manual WBC count can be assessed by performing a WBC estimate on a Wright-stained
peripheral blood film made from the same specimen.
43
Leukocytosis (Increased WBC Count)
BALPUU
○ Bacterial infections
○ Appendicitis
○ Leukemia
○ Pregnancy
○ Uremia
○ Ulcers
44
Leukopenia (Decreased WBC Count)
○ Viral infection
○ Brucellosis
○ Typhoid fever
○ Rheumatoid arthritis
○ Cirrhosis
45
Provide reservoir containing a pre-measured diluent and calibrated pipettes that automatically measure the
appropriate amount of sample required for diluting blood specimens in preparation for manual counting
DISPOSABLE BLOOD CELL COUNT DILUTION SYSTEMS
46
Older; replaced by the LeukoChek system
Unopette System
47
It is an example of capillary pipette and diluent reservoir system which is commercially available for both WBC
and platelet counts
LeukoChek System
48
LeukoChek
Diluting Fluid
1% buffered ammonium oxalate
49
Unopette
Diluting Fluid
3% glacial acetic acid
50
LeukoChek
Volume of Diluting Fluid
1.98 mL
51
Unopette
Volume of Diluting Fluid
0.475 mL/475 µL
52
LeukoChek
Volume of Blood
20 µL
53
Unopette
Volume of Blood
25 µL
54
LeukoChek
Dilution
1:100
55
Unopette
Dilution
1:20
56
DISPOSABLE BLOOD CELL COUNT DILUTION SYSTEMS
SPECIMEN
WB EDTA or Capillary Blood
57
DISPOSABLE BLOOD CELL COUNT DILUTION SYSTEMS
REAGENTS AND MATERIALS
● Plastic reservoir
● Capillary pipette
● Hemocytometer and coverslip
58
Contains 1.98 mL of 1% buffered ammonium oxalate
Plastic reservoir
59
Calibrated to accept 20 µL of blood
Capillary pipette
60
DISPOSABLE BLOOD CELL COUNT DILUTION SYSTEMS
PROCEDURE
1. Blood from a well-mixed EDTA-anticoagulated specimen or from a skin puncture is allowed to enter the pipette by
capillary action to the fill volume
2. Blood is added to the reservoir making a 1:100 dilution
3. Mix
4. Stand for 10 minutes to allow the lysis of the red blood cells
5. The reverse end of the capillary pipette is placed in the reservoir cap making a dropper
6. Discard the first 3 or 4 drops of the diluted sample and the capillary pipette is used to charge the hemocytometer
7. WBCs are counted in all 9 large squares (9 mm2) using low power.
8. Platelets are counted in the 25 small squares in the center square (1 mm2) using high power
61
Number of eosinophil per liter of blood
ABSOLUTE EOSINOPHIL COUNT
62
Still occasionally requested for diagnostic purposes because it's more appropriate and clinically useful to determine the
absolute number of eosinophil in a certain volume of blood compared or done to determine the relative number from the
differential count
ABSOLUTE EOSINOPHIL COUNT
63
MANUAL ABSOLUTE EOSINOPHIL COUNT
PRINCIPLE
Using a suitably diluted specimen and appropriate stain, eosinophils can be identified and counted microscopically,
after the specimen is loaded on a hemocytometer. The count is reported in terms of cells per liter
64
SPECIMEN
● Venous blood with EDTA/Heparin
● Capillary blood
65
REAGENTS AND MATERIALS
● Thoma WBC diluting pipette or the Unopette system for absolute eosinophil count
● Diluting fluid
1. Phloxine B solution
2. Pilot’s Solution
3. Randolph’s Stain
● Hemocytometer
1. Fuchs-Rosenthal Hemocytometer
2. Speirs-Levy Hemocytometer
66
Phloxine B solution
● Phloxine → Stains eosinophil red
● Sodium carbonate → Enhances eosinophil granule staining
● Propylene glycol → Lyses RBCs
● DIstilled water → Solvent
67
Stains eosinophil red
Phloxine
68
Enhances eosinophil granule staining
Sodium carbonate
69
Lyses RBCs
Propylene glycol
70
Solvent
Distilled water
71
72
inhibits leukocyte clumping
Heparin
73
contains Phloxine B solution in propylene
glycol & distilled water
Reservoir
74
used to aspirate 25 μL of blood
sample
Capillary pipet:
75
Unopette System for Eosinophil Count
Dilution
1:32
76
It has 2 ruled areas each consisting of one 4×4-mm square
Fuchs-Rosenthal Hemocytometer
77
Hemocytometer’s depth = 0.2 mm
Fuchs-Rosenthal Hemocytometer
Speirs-Levy Hemocytometer
78
Fuchs-Rosenthal Hemocytometer
Total volume in each ruled area (16 mm2 × 0.2 mm) =
3.2 mm3
79
Speirs-Levy Hemocytometer
Total volume in each counting area (10 × 1 mm2 × 0.2 mm) =
2.0 mm3
80
Fuchs-Rosenthal Hemocytometer
Total volume that may be counted on the chamber (2 × 3.2 mm3) =
6.4 mm3
81
Speirs-Levy Hemocytometer
Total volume that may be counted on the chamber (4 × 2.0 mm3) =
8.0 mm3
82
MANUAL ABSOLUTE EOSINOPHIL COUNT
PROCEDURE
1. Make a 1:10 dilution by drawing well mixed blood to the 1.0 mark of WBC Thoma pipette and adding eosinophil diluting
fluid up to 11 mark.
● Alternatively, use a 25-μL pipet to aspirate specimen and dilute in eosinophil Unopette reservoir (1:32 dilution)
2. Mix blood and diluting fluid (approximately 2 minutes)
3. Discard 4-5 drops
4. Fill the counting chamber
5. Place the filled counting chamber into a moist Petri dish
6. Stand for 15-20 minutes for cells to settle, red cells to lyse, and eosinophils to stain
7. Count eosinophils under low power (10×) objective
83
REFERENCE RANGE
Eosinophil Count (Adult)
Conventional Units
(cells/µL)
0 - 300
84
REFERENCE RANGE
Eosinophil Count (Adult)
Standard International
(SI) Uits (× 109/L)
0.0 - 0.3
85
Eosinopenia (Decreased Eosinophil Count)
○ Hyperadrenalism (Cushing’s disease)
86
Eosinophilia (Increased Eosinophil Count)
○ Allergic reactions
○ Parasitic infections
○ Brucellosis
○ Certain Leukemias
87
It was historically used to test adrenocorticotropic function
THORN TEST (EOSINOPHIL DEPRESSION TEST)
88
THORN TEST (EOSINOPHIL DEPRESSION TEST)
Procedure
1. 1st sample is collected for fasting absolute eosinophil count
2. The patient is given an injection of ACTH (stimulator).
3. After 4 hours, 2nd sample is collected for absolute eosinophil count
89
2nd absolute count should be at least 50% less than the fasting count
Normal adrenocortical function
90
fasting and 4-hour counts are approximately of equal value
Hypoadrenalism
91
MANUAL ABSOLUTE BASOPHIL COUNT
REAGENT AND MATERIAL
● Cooper and Cruickshank stain
● Fuchs-Rosenthal or Speirs-Levy Hemocytometers
92
MANUAL ABSOLUTE BASOPHIL COUNT
PRINCIPLE
● Most methods for counting basophils are based on the metachromatic staining of mucopolysaccharide in the basophil
granules by aqueous solutions of toluidine blue. Saponin is used to lyse erythrocytes and potassium or ammonium
sulphate mixtures are sometimes added to keep the diluted blood in a fluid state
● Counts may be completed in approx 10 minutes
● Blood dilution is considered to be stable in that counts may be performed up to 1 week after the mixture is prepared
● Staining process is fast
93
REFERENCE RANGE
Conventional Units
(cells/µL)
Basophil Count (Adult)
0 - 200
94
REFERENCE RANGE
Standard International
(SI) Units (× 109/L)
Basophil Count (Adult)
0.0 - 0.2
95
Basopenia (Decreased Basophil Count)
○ Allergic reactions
96
Basophilia (Increased Basophil Count)
○ Chronic myelogenous leukemia
○ Polycythemia vera
○ Myelofibrosis
○ Myxedema
○ Colitis
○ Chronic hemolytic anemia
97
(Manual RBC Count)
● Diluting Fluids Used:
○ Isotonic saline (most common)
○ Sodium citrate (3.2%)
○ Dacie’s/Formol citrate
○ Hayem’s solution
○ Toisson’s solution
○ Bethel’s solution
○ Gower’s solution
