Lecture 1 Flashcards
(45 cards)
1
Q
What is EDTA
A
(ethylene –diamine tetra acetic acid) is the anticoagulant of choice
• It maintains the cell’s shape and size.
• Prevents blood from clotting by binding with calcium
2
Q
What is CBC
A
evaluation of the formed elements of blood: red cells, white cells and platelets. A CBC includes counting these elements and then inspecting them on a blood film microscopically.
3
Q
What does heparin do
A
Heparin anticoagulant
• cause clumping of cells
• gives blue background staining
4
Q
What does citrate do
A
anticoagulant
• Dilutes blood due to volume of liquid
• Exception-can be used for platelet count only
5
Q
Handling and storage of CBC samples
A
- Refrigerate at 4ºC until testing
- Referrals –ice pack
- Mix Samples well before use x 6
- Test Samples the same day
- Blood Film/ Smears must be made the same day
- Samples >24 hrs old: No good for testing
6
Q
Effects of old samples:
A
- Platelets clump
- Nucleus of WBCs deteriorate
- Cytoplasm of WBC has vacuoles and dark granules
- RBCs swell or lyse
7
Q
Three important steps Blood Film Preparation
A
- Making
- Fixing
- Staining
8
Q
Three methods of Blood Film Preparation
A
- Wedge-manual
- Spun-automated slide spinner
- Automated-Hematology analyzer
9
Q
Thickness of the film
A
- If the hemoglobin/hematocrit is increased the angle of the spreader slide should be decreased to 30 degrees
- If the hemoglobin/hematocrit is decreased the angle of the spreader slide should be increased to 40 degrees
10
Q
Area used for microscopic evaluation
A
- Cell morphology and differential are done in the mono layer area
- Cells overlap at the start (thick area)
- Cells are distorted at the end of the blood film
11
Q
Features of a well-made peripheral blood film
A
- Correct size of drop
- The film is 2/3 to 3/4 the length of the slide
- The film is finger shaped with slightly rounded or feathered edge
- The film is smooth without irregularities, holes or streaks
- The whole drop of blood is picked up and spread
- The blood film should not touch the ends of the slide
- The angle of the spreader should be about 35º ( the greater the angle the thicker and shorter the blood film. The lower the angle the longer and thinner the blood film)
12
Q
Common causes of unacceptable blood film
A
- Chipped or rough end spreader slide
- Drop of blood too big or too small
- Dirt or grease on the slide
- Holes in the film – Lipemic
- Too long or too short
- Too thick or too thin
- Dried up drop of blood
- Ripples
- Jazzed edge
13
Q
Staining and fixing
A
- Allow blood film to dry completely before staining. Do not blow to dry as moisture from breath will cause RBC artifact
- Methanol is the fixative of choice
- Stain has methanol
- Timing and pH are critical
14
Q
Romanowsky Staining
A
- Wrights
* Giemsa or Wright/Giemsa mixture
15
Q
Stain composition
A
• A Cationic or basic dye (methylene blue), stains the cell nucleus and granules of basophils blue –grey.
• An anionic or acidic dye (eosin), stains haemoglobin and eosinophil granules red –orange
Azure B
16
Q
Effects of poorly stained blood films - RBC’s too pink or red
A
- Staining time /buffering time too short
- Over –rinsing
- Stain or buffer pH too acidic
17
Q
Effects of poorly stained blood films - RBC’s grey-blue, WBC’s too dark or Eosinophil granules are grey
A
- Stain or buffer pH too alkaline
- Inadequate rinsing
- Prolonged staining
- Heparinized blood
18
Q
Effects of poorly stained blood films - Stain Deposit
A
- Dirty slide
- Stain solution not filtered
- Inadequate rinsing
- Stain dried up on slide ( excess timing)
- Staining rack not levelled
19
Q
Corrective actions: if slides are too blue
A
- decrease stain time
- increase wash time
- decrease buffer pH
20
Q
Corrective actions: if slides are too pink
A
- increase stain time
- decrease wash time
- increase buffer pH
21
Q
- COAGULATION TESTS
Type of specimen
A
- Plasma is required
- Citrate is the anticoagulant for PT and PTT
- Sodium citrate binds calcium, preventing coagulation
- Tubes must be filled to it’s draw capacity
- Record if patient is on anticoagulant therapy or aspirin
22
Q
Handling and storage coagulation samples
A
- Centrifuge sample immediately and separate plasma
- Some clotting factors are highly labile and deteriorate significantly at room temperature
- Freeze plasma if not tested within 4 hrs
23
Q
Haematolgy QC
A
Activities that ensure that specific steps in the procedure meet acceptable standards or parameters
24
Q
Controls and Calibrators
A
- Commercial control ( Low, High or Normal ) -Used to check precision for verifying known manufacturer’s values. Must fall within 2SD
- Analyze controls daily or in batch. Plot Levey- Jennings Chart
- Commercial controls or calibrators-Have a short shelf life and must be refrigerated between uses.
- Calibrators measure accuracy, verifying reference centre values.
- If 2 results are out of 2SD, run samples again, then get new controls to see if it’s the samples or the controls. IF neither works then you know its not the control, sample. Then change the reagent, do it again . If not then it’s the instrument .
25
For accuracy
controls and calibrators of known value are used
26
For precision:
An in lab prepared control with known value is used
27
For linearity:
A Calibrator is used to ensure that the instrument is sensitive to very low or high values. Patient results are never reported below or above the linearity values.
28
Cell Counters Manual
The manual procedure, using a diluting chamber/ hemocytometer, is no longer used except for white cell counts in body fluids with extremely few cells, e.g. cerebrospinal fluid
29
Disadvantage of Manual Counts
* Time consuming
| * Cell identification error
30
Advantage of Automated Counts
• More efficient and cost effective
31
Coulter Principle
Particles suspended in a conductive electrolyte solution are drawn through a small aperture by a vacuum . The volume of blood is determined by a vacuum.A background current (DC) runs between two electrodes (one internal and one external)
As a particle passes through the aperture, a volume of electrolyte equivalent to the immersed volume of the particle is displaced from the sensing zone. This causes a short-term change (increased) in the impedance (resistance) across the aperture. The voltage increase and can be measured as a voltage pulse or a current pulse
32
What does the coulter graph look like
Change in electrical resistance produced by cells as they go through a small aperture.
Gives only total count it ..ALL WBC but you have to differeniate between the enosophil, neutrophil, etc
• when a cell passes into the aperture spike in voltage
• number of spikes (impulses) = number of cells
height of spike = volume (size) of cell neutrophil would have a smaller height than monocyte
33
HOW DO WE GET CELL COUNTS
• Blood is diluted by isotonic solution
• RBC’s, platelets and MCV are measured.
• Lyse is added. WBC and Hb are measured
Hct is calculated: Hct = RBC count x MCV
34
OPTICAL LIGHT SCATTER
used to differentiate white blood cells ( differential).
• Lyse destroys RBCs and causes WBC membrane to shrink around the nucleus and cytoplasmic granules
• Each WBC flows in a single line in a flow cell
• A lazer light focuses on the cells
• Light scatter occurs in different direction.
• Scattered light is converted by a photo-detector and converted to electrical impulse
• Scattered light tells about the cells structure , shape and reflectivity
Enosphils would scatter because they have lots of granules , Moncytes would not because they don’t
35
What does the index of refraction measure in opitcal light scatter
• Index of refraction (Df) of each cell is measured.
o Forward angle light scatter=cell size and volume
o Side scatter = granularity and nuclear size
• Cell volume is also determined by impedance.
• Differential is determined by grouping the cells by size, nuclear and internal structure (granulation)
36
Radio Frequency Probe (VCS technology)
* Volume: measured by electrical impedance.
* Conductivity: radio frequencies penetrate the cell and measure internal structure and size
* Scatter: laser beam gives information about cell surface and granularity
37
Automated Intelligent Morphology (AIM),
enhanced multi-dimensional high-definition cellular analysis solution utilizes flow cytometric technology to deliver results to physicians and patients faster.
38
Peroxidase method:
stain with peroxidase WBC’s which are analyzed for size and stain intensity
White blood cells are stained with peroxidase and the cells are counted based on size and staining characteristics.
The analyzer also provides an automated differential cell count by separating the cells into clusters, using the peroxidase channel
39
Basophil method:
The basophil method involves stripping the cells of cytoplasm and counting nuclei.
40
- Interfering agents that can cause erroneous results when trying to measure WBC
* RBC that resist lysis
* Nucleated RBCs
* Fragmented WBCs
* Platelet clumps of large platelet
* Specimens containing fibrin, cell fragments
41
- Interfering agents that can cause erroneous results when trying to measure RBC
* Very high WBC (greater than 99.9)
* Large platelets
* Clumped RBC’s
* Specimens containing fibrin, cell fragments
42
- Interfering agents that can cause erroneous results when trying to measure Hgb
• Very high WBC count
• Severe lipemia or increased triglycerides
• High bilirubin
-gross hemolysis effect hemoglobin
43
- Interfering agents that can cause erroneous results when trying to measure Plt
* Very small red cells
* Cell fragments
* Clumped platelets
44
What are the indexes
MCV, MCH LOOK at slides and figure it out
45
What is reticulyte
young RBC
