AUTOMATION Flashcards
Most common cinical application of flow-cy
Diagnosis of Leukemias and lymphomas
most significant discovery that improved flowcy and its widespread application
monoclonal antibodies
what are other particles detected in flow cy aside from cells
chromosomes, microorganisms, and proteins.
composition of flow cytometry
fluidics, a light source (laser), multiple detectors, and a compute
advantage of flow cytometry from other techniques
its ability to quickly and simultaneously analyze multiple parameters
in a large number of cells
most commonly analyzed specimens in flow cytometry
bone marrow, peripheral blood, lymphoid tissues, body cavity fluids and solid tissues
spx. collection for Peripheral blood and BM spx
must be processed within 24 to 48 hours from time of collection
temp. and anticogaulant for PB and BM spx. for flow cytometry
Room temp, Heparin
injecting a cell suspension into a stream of sheath fluid.
hydrodynamic focusing
forward scatter detects
particle volume / size
Side scatter detects
Surface complexity and internal structures(granules & vacuoles)
hematopoietic cell marker
CD34
B lymphocyte markers
CD 19, 20, 22
Kappa
Lambda
T lymphocyte markers
CD 2, 3, 4, 5, 7, 8
Megakaryocytic markers
CD 41, 42, 61
Granulocytic/Monocytic markers
CD 33, 13, 15, 14
Erythroid markers
CD 71, Glycophorin A
A method useful for Acute Leukemias
Cytochemistry
Specimens in cytochemistry
BM
Lymph nodes
Peripheral blood
for enzymatic techniques
Fresh smears
nonenzymatic techniques (PAS/SBB)
remain stable for months if stored at room temp
cytochem stain for differentiation Acute granulocytic leukemias from monocytic leukemias
Esterase
Periodic Acid Scchiff is stain for
Glycogen
PAS is useful in identifying
FAB M6 Leukemia
Terminal deoxynucleotidyl transferase (TdT)
for primitive lymphoid cells
basic components of most hema analyzers
- Hydraulics
- Pneumatics
- Electrical systems
(vacuums and pressures for operating valves and moving the sample through the system)
pneumatics
electronic analyzers and computing circuitry for processing data
Electrical systems
aspirating unit, dispensers, dilutors, mixing chambers, aperture baths and/or flow cells, and hemoglobinomete
hydraulics
Coulter principle / Electrical impedance
-a.k.a: Electronic Resistance or low-voltage direct current (DC) resistance
most common methodolgy used in analyzers
Coulter principle / Electrical impedance
a.k.a. alternating current (AC) resistance
-otherwise known as RF resistance or high-voltage electromagnetic current;
measures conductivity
Radiofrequency (RF)
displays clusters of cells number of dots represents the concentration of a specific cell type
Scatterplot
instrumental errors
- aperture plugs
- bubblesin sample
- extraneous electrical pulses
- excessive lysing of RBCs
- improper setting of aperture current/threshold will cause either positive or negative error
most common problem in cell counting
aperture plugs
produce negative error
excessive lysing of RBCs
nature of the specimen (errors)
- giant platelets
- fragments of WBC cytoplasm
- Some abnormal RBCs
Blood cell histograms provide
size distribution of the different cell populations
These types of histograms will provide an approximate
number of cells on the y-axis and the cell size on the x-axis.
RBC histogram:
shift to the right
RBCs LARGER than normal
RBC histogram:
shift to the left
RBCS smaller than normal
histogram curve is bimodal,
2 cell population
-blood transfusion
Other conditions that will cause a bimodal distribution curve
- cold agglutinin disease, 2. hemolytic anemia with
schistocytes present, or 3. anemias with different size cell populations
actual ounting of platelets is in
RBC aperture
Platelet histograms obtained from
2-20 fL
flags in histograms
H
L
H flag
when a parameter value is higher than the set normal limit
L flag
when a parameter value is lower than the set normal limit
– warns of increased interference in the area left of the lymphocyte peak (approx. 35 fL)
- typically caused by sickled RBCs, nucleated RBCs, or clumped and giant platelets being counted
in the WBC aperture bath.
R1
warning is caused by excessive overlap of cell populations at the mononuclear/granulocyte
boundary (approximately 160 fL), which is due to the increased presence of immature granulocytes
(i.e., bands, metamyelocytes)
R3
warns of excessive overlap of cell populations at the lymphocyte/mononuclear cell boundary
(approximately 90 fL) caused by the presence of abnormal cell types, such as atypical lymphocytes,
blast, or plasma cells
R2
warning is caused by the extension of the cell distribution past the upper end of the WBC threshold
(approximately 450 fL). This most commonly occurs when the granulocyte population is very high.
R4
is the error code for multiple region overlap
RM
3 Distribution peaks of WBC histogram
First peak
Second peak
third peak
45 - 90 fL
First peak
90 - 160 fL
Second peak
160 - 450 fL
Third peak
at what distribution peak is normal mature types of granulocytes
Third peak
distribution peak of where large mononuclear cells appear
second peak
