[TRANSES] MODULE 4 Flashcards
sample needed
venous blood in an EDTA tube
Proper preanalytical considerations =
quality results
bases for rejection:
unlabeled specimen or a specimen containing
clots
appears as a small pipette with a short stem marked with graduations and bulb near the top
Thoma pipette
the neck of the pipette may be marked as either 101 (RBC) or 11 (WBC)
Thoma pipette
requires a sucking tube to facilitate the aspiration of the sample and diluting fluid
manual pipette
one of the most commonly used counting chambers
Levy hemocytometer with improved Neubauer ruling
other hemocytometers used in performing manual cell count
Rosenthal Hemocytometer
other hemocytometers used in performing manual cell count
Speirs – Levy Hemocytometer
Standard Brightfield Microscope
to visualize cells introduced into the counting chamber
– dictates the total magnifications you are to use
type of blood cells to count
Levy hemocytometer with improved Neubauer ruling
Counting chamber
Total area:
Divisions:
Distance between the counting chamber and the cover slip:
Total volume of 1 counting area:
Total area: 9 mm2
Divisions: 9 large squares (1 mm x 1 mm)
Distance between the counting chamber and the cover slip: 0.1 mm
Total volume of 1 counting area: 0.9 MM2
WBC – Corner squares
Divisions:
16 small squares (0.25 mm)
RBC – Central squares
Divisions:
25 small squares (0.04 mm2)
Rosenthal Hemocytometer
No. of counting chamber:
2
Rosenthal Hemocytometer
Total area per counting chamber:
16 mm2
Rosenthal Hemocytometer
Division 1:
16 large squares
Rosenthal Hemocytometer Division 1
Length:
Depth:
Length: 1 mm x 1 mm
Depth: 0.2 mm
Rosenthal Hemocytometer
Division 2:
16 small squares
Rosenthal Hemocytometer Division 2
Length:
Length: 0.25 mm x 0.25 mm
Speirs – Levy Hemocytometer
No. of counting chamber:
4
Speirs – Levy Hemocytometer
Total area per counting chamber:
10 mm2
Speirs – Levy Hemocytometer
Division 1:
10 large squares (arranged horizontally)
Speirs – Levy Hemocytometer Division 1
Length:
Depth:
Length: 1 mm x 1 mm
Depth: 0.2 mm
:
16 small squares
Speirs – Levy Hemocytometer Division 2
Length:
0.25 mm x 0.25 mm
involves the dilution of blood and counting the cells from a sample
of diluted blood
GENERAL PRINCIPLE OF HEMOCYTOMETRY
Counts are expressed as (?) because of the
linear dimensions of the hemocytometer.
cubic millimeters (mm3)
- isotonic and prevent coagulation of red blood cells.
RBC dilution fluids
– commonly weak acid solutions to lyse red blood cells to facilitate counting of white blood cells
WBC diluting fluids
RBC COUNT
Gower’s Solution
Hayem’s Solution
Toison’s Solution
Dacie’s Solution
Strong’s Solutions
Bethel’s Solution
Eagle’s Fluid (NSS)
WBC COUNT
2% Acetic Acid
Turk’s Solution
PLATELET COUNT
Rees and Ecker
Tripotassium EDTA
and Ammonium
Oxalate
CHARGING THE HEMOCYTOMETER
1. Discard the (?) of fluid in the pipetted.
2. Secure the (?) on top of the hemocytometer, making sure it will not be disturbed during charging.
3. Carefully fill the (?) with the sample as seen in the Figure 4.5A then allow cells to settle for 5 minutes.
- first few drops
- coverslip
- counting chambers
MANUAL CELL COUNT
1. (?) in each of the designated squares following the direction of the arrows as seen in Figure 4.6.
2. Use the (a) for WBC count and (b) labelled with “R.”
3. For Platelet count, use all the (?) in the central large square.
4. Count all cells within the designated squares following the (?) which implies that only cells that are touching the upper and left boundaries of the square must be counted as indicated by check marks and green circles in Figure 4.7.
- Count cells
- a] four (4) large squares labelled with “W”
b] five (5) small squares in the central large square - twenty-five (25) small squares
- Inverted L rule
States that cells settle in a random manner.
POISSON’S LAW OF DISTRIBUTION
This implies that even though there are differences in cell counts in each square, they must not go beyond the acceptable difference which is greater than or equal to 15 for white blood cell count and 20 for red blood cell count. Otherwise, the count is considered as invalid and it is recommended to charge the hemocytometer again.
POISSON’S LAW OF DISTRIBUTION
The SI unit of measurement is followed for reporting the counts which involves the number of cells found in (?).
1L of blood
contains an abundant number of cells for us to possibly count them
one-unit volume of blood
(?) the samples before proceeding with the actual counting
dilute
counting chamber has ruled areas measured in millimeters, as such, cell counts are conveniently reported in
cubic millimeters (number of cells in 1 mm3
what is being observed would be reporting (?) since it is essentially one liter
cells per one microliter (number of cells x 106 μL)
DUE TO EQUIPMENT
✓ Chipped tips
✓ Obscure markings
✓ Poor calibration
✓ Non – optically planed cover glass
✓ Unclean or wet pipettes
DUE TO TECHNICAL ERRORS
✓ Too much pressure on skin puncture site
✓ Prolonged tourniquet application
✓ Improper filling of pipette
✓ Failure to wipe off pipette tip
✓ Inadequate mixing
✓ Erroneous counting of cells
RBC Count, according to the World Health Organization (WHO), may also be referred to as the
Erythrocyte Number Concentration
not recommended to perform this method due to its tendency to have poor reproducibility and accuracy
RED BLOOD CELL COUNT
This method is usually done on bodily fluids to which a red cell count is requested.
RED BLOOD CELL COUNT
RED BLOOD CELL COUNT (RBC COUNT)
Procedure:
1. Using RBC Thoma pipette and a suction device, draw blood to the (?) (Fig. 4-8, Step 1), making sure that there are no air bubbles entering the pipette. Wipe off any excess blood on the outside of the pipette stem using a swift downward motion from the bulb to the tip.
2. Draw the diluting fluid to the (?) (Fig. 4-8, Step 2), still making sure that no air bubbles are formed.
3. Cover the tip of the pipette with your finger and remove the (?). Shake the pipette for a few minutes.
4. Discard a few drops of blood from the Thoma pipette before charging the hemocytometer. These first few drops are primarily (?). Hence the deduction of 1 unit in the computation for the dilution factor.
5. On to a clean hemocytometer with a coverslip mounted on it, dispense the mixture on both counting chambers. Allow cells to settle for at least (?) in a moist chamber.
6. Under 10x objective, locate the (?) of the counting chamber. Shift to 40x magnification and count the RBCs in the respective ruled areas. Use a tally counter to count the cells.
7. Do the same in the (?) using the same manner of counting. Take the average and compute.
- 0.5 mark
- 101 mark
- suction device
- diluting fluid
- 3 minutes
- central 1mm2 square area
- second chamber
The WBC Count, otherwise known as the
Leukocyte Number Concentration
WHITE BLOOD CELL COUNT (WBC COUNT)
1. Using a WBC Thoma pipette and a suction device, draw blood to the (?) (Fig. 4-9, Step 1), making sure that there are no air bubbles entering the pipette. Wipe off any excess blood on the outside of the pipette stem using a swift downward motion from the bulb to the tip.
2. Draw the diluent to the (?) (Fig. 4-9, Step 2), still making sure that no air bubbles are formed.
3. Cover the tip of the pipette with your finger and remove the suction device. Mix by repeated inversion or by using a pipette shaker for (?). You have now prepared a 1:20 dilution.
4. Discard a few drops of blood from the (?)before charging the hemocytometer.
5. On to a clean hemocytometer with a coverslip mounted on it, dispense the mixture on both counting chambers. To charge the mixture, hold the tip of the pipette at a (?) and dispense at the edge of the coverslip where it meets the floor of the hemocytometer. Make sure that the counting chambers are not overfilled or under filled.
6. Place the hemocytometer in the moist chamber you have prepared previously. Let it stand undisturbed for (?). This will assure that all RBCs are lysed.
7. Mount the hemocytometer on the microscope stage and use 400x total magnification. You should see the WBCs as (?). You are going to count the cells in the four corner WBC counting squares. Start on the leftmost WBC square.
8. Repeat the counting on the other side of the counting chamber. For accuracy purposes, the difference in the count for these two chambers should not exceed (?). Take the average of the two counts and use the formula to compute for the WBC count.
- 0.5 mark
- 11 mark
- 2-5 minutes
- Thoma pipette
- 45- degree angle
- 3-10 minutes
- dark dots
- 10%
There are some instances that (?) are present in the blood sample.
nucleated RBCs (NRBCs)
Unfortunately, they are not lysed by the diluting fluid and are counted during the manual WBC count since one cannot differentiate the other properly in a hemocytometer.
nucleated RBCs (NRBCs)
If you are to notice more than (?) for every 100 WBCs in a peripheral blood smear during a differential WBC count, a corrected WBC count is recommended.
5 NRBCs
CORRECTED WBC COUNT formula:
EOSINOPHIL COUNT involves utilizing a staining solution usually containing (?) to stain the eosinophils red-orange.
phyloxine B/phloxine
The staining method preserves the other types of WBCs but deem them undetectable under the microscope.
EOSINOPHIL COUNT
EOSINOPHIL COUNT is usually performed in a (?9 because it provides a greater counting area, but a Neubauer chamber will also do.
Fuchs-Rosenthal chamber
EOSINOPHIL COUNT
procedure:
1. prepare a moist chamber by applying a (?) or filter paper on the bottom of a petri dish. you may place a wooden applicator stick broken in two to serve as a stand for the hemocytometer. alternatively, the hemocytometer can be placed on a damp gauze and covered by a petri dish.
2. Using a (?), draw blood to the 1 mark, making sure that there are no air bubbles entering the pipette. Wipe off any excess blood on the outside of the pipette stem using a swift downward motion from the bulb to the tip.
3. Draw the diluent to the (?), still making sure that no air bubbles are formed.
4. Cover the tip of the pipette with your finger and remove the suction device. Mix the pipette gently for about (?). Make sure that you do not agitate the pipette harshly or too long to prevent rupturing the eosinophils.
5. Discard a few drops of sample from the pipette and introduce to (?) of a hemocytometer.
6. Let the charged hemocytometer stand in the moist chamber for (?) to allow the stain to permeate the eosinophils.
7. Count the number of eosinophils in the WBC corner squares (for a total area of (?)) under LPO. Get the average for both counting chambers.
- damp gauze
- WBC Thoma pipette and a suction device
- 11 mark
- 30 seconds
- both counting chambers
- 15 minutes
- 36 mm2
RBCs are lysed while the basophils are treated with ammonium sulfate to render their granules insoluble. Basophils are then stained with toluidine blue.
BASOPHIL COUNT
BASOPHIL COUNT
Procedure:
1. Prepare a small test tube and combine (?) of diluting fluid (EDTA-NSS solution) with (?) of blood. Mix well.
2. Add 100μL of the second solution (containing (?) cetylpyridinium chloride to lyse the RBCs, (?) ammonium sulfate, (?) toluidine blue). Mix well.
3. Introduce mixture to both sides of the (?)
4. Allow cells to settle by setting the set up aside in a moist chamber for (?)
5. Count the basophils in the (?) of both chambers.
- 80μL, 20μL
- 0.5%, 5%, 0.8%
- counting chamber
- 5 minutes
- WBC squares
WBC Differential count is also known as the (?). Based on the term itself, it involves counting a total (?) and reporting the different types in percentages.
Leukocyte Type Number Fraction
100 WBCs
WBC DIFFERENTIAL COUNT
Procedure:
1. Prepare a properly stained (?) using the following laboratory activity.
2. Under low power objective (LPO 10x), check for the adequacy of the smear. Choose the area that: (?)
3. Perform the examination under oil immersion magnification with the aid of a differential counter. The counter will sound when it reaches (?). The exaggerated (?) are often used in classifying 100 WBCs, as seen below. Take note that any (?) that can still be identified are included in the count.
- peripheral blood smear
- has no jagged edges
- has no WBC aggregation
- RBCs are barely touching each other
- 100 cells; battlement technique and lateral strip technique; distorted cells
WBC DIFFERENTIAL COUNT
Procedure:
1. Prepare a properly stained (?) using the following laboratory activity.
2. Under low power objective (LPO 10x), check for the adequacy of the smear. Choose the area that: - has no jagged edges - has no WBC aggregation - RBCs are barely touching each other
- peripheral blood smear
2.
serves as an important tool to assess the bone marrow’s ability to increase RBC production in response to an anemia
The reticulocyte count
are young RBCs that lack a nucleus but still contain residual ribonucleic acid (RNA) to complete the production of hemoglobin.
Reticulocytes
Normally, they circulate peripherally for only 1 day while completing their development.
Reticulocytes
Counting reticulocytes present in the peripheral blood allows us a glimpse of the body’s
erythropoietic activity.
The use of a supravital stain (?) helps in visualizing the leftover ribosomal ribonucleic acid (RNA) along with other organelles appearing as blue speckles all over the cell.
New Methylene Blue, Brilliant Cresyl Blue
The reticulocytes should appear like this under a microscope:
Dry Method
Procedure:
1. Mix 2-4 drops of freshly collected EDTA anticoagulated blood with 2-4 drops of prepared supravital staining solution in a small tube.
2. Incubate for 10-15 minutes at 370C .
3. Prepare a blood smear using the mixture. No need to fix and stain any further. Let it dry.
4. Examine under OIO. Count the number of reticulocytes (Fig. 4-11) per 1000 RBCs (this usually covers ten oil immersion fields).
Wet Method
Procedure:
1. Place 1-2 drops of supravital stain on a slide. Spread and let it dry.
2. Place a single drop of freshly collected EDTA anticoagulated blood on the slide.
3. Immediately place a coverslip on the drop and examine under OIO. Make sure that counting is done within 20-30 minutes.
utilizes the said reticle which is placed into the eyepiece of the microscope
Calibrated Miller Disc Method
This method provides accurate and precise results with the added benefit of reducing the tediousness of doing the manual count using the two previous methods we have discussed.
Calibrated Miller Disc Method
Calibrated Miller Disc Method
Procedure:
1. Prepare a thin blood smear, using the previously described staining methods.
2. Count both RBCs (including reticulocytes) in the smaller square (B).
3. Count only reticulocytes in the larger square (A).
According to the College of American Pathologists, a minimum of (?) should be counted in the examination of the smear.
1000 RBCs
The manner of counting where the Miller disc is involved ensures that about at least (?) are counted in the smaller square which is equivalent to (?) in the larger square.
112 cells
1008 RBCs
reflects the actual number of reticulocytes in one liter of whole blood
Absolute Reticulocyte Count (ARC)
In as much as very low RBC count due to anemia tends to decrease the (?), while increasing the (?), another computation may be done that is to eliminate this source of error.
ARC
% reticulocyte
corrects the hematocrit level by dividing the patient’s hematocrit with the normal hematocrit set at 45% prior to multiplying with the already obtained % reticulocyte
Corrected Reticulocyte Count (CRC)
is a clinically more useful tool in the evaluation of red cell production
Reticulocyte Production Index (RPI)
are produced in increased amounts when the body has increased demand for oxygenation.
red cells
Since (?) are capable of oxygen transport, the tendency in this case is the early release of reticulocytes from the bone marrow to serve that function.
reticulocytes
(?), which is also known as shift correction, is a general indicator of the capability of the bone marrow to produce blood cells in times of need
RPI
Maturation Time of Reticulocytes in Peripheral Blood Relative to Hematocrit Values
1.0
1.5
2.0
2.5
3.0
0.40-0.45
0.35-0.39
0.25-0.34
0.15-0.24
<0.15
adequate bone marrow response
RPI > 3
inadequate response
RPI < 2
