SCYM ASCP ACTUAL FINALS QUESTIONS AND
ANSWERS SURE A+
✔✔BONE MARROW BIOPSY OR FINE NEEDLE ASPIRATE (FNA) - ✔✔Submerge in
RPMI tissue culture medium for optimal cell viability (sterile saline is acceptable). Send
at room temperature
within 12 hours of collection.
✔✔FRESH TISSUE - ✔✔Mince tissue and submerge in RPMI tissue culture medium for
optimal cell viability (sterile saline is acceptable). Send at room
temperature within 12 hours of collection.
✔✔BONE MARROW ASPIRATE - ✔✔Add 1 - 2 mL of first pull bone marrow aspirate to
a green top (sodium heparin) tube. Send intact specimen stored at room
temperature within 12 hours of collection. Include 4 unstained aspirate smears, an
unstained peripheral blood smear and a
copy of the patient's most recent complete blood count (CBC) and white cell differential.
✔✔study sample preparation - ✔✔staining, disaggreation, lysing agents, aggregates,
filtering, fixation etc (study protocols)
✔✔density gradient centrifugation - ✔✔Density gradient centrifugation is commonly
used for the isolation of specific cell populations from whole blood. This technique takes
advantage of the differences in density between the various leukocytes and the density
gradient medium. As a result, the particular cell populations isolated will depend on the
density of the medium that is used.
✔✔Signal Cell Sorting - ✔✔Fluorescence-activated cell sorting (FACS) is a specialized
type of flow cytometry. It provides a method for sorting a heterogeneous mixture of
biological cells into two or more containers, one cell at a time, based upon the specific
light scattering and fluorescent characteristics of each cell.
,✔✔CSF OR BODY FLUID - ✔✔Add body fluid (e.g., CSF, pleural, peritoneal) to a
sterile, leak-proof container. Include a copy of the patient's most recent
body fluid cell count and differential. If possible, an original cytospin preparation
(preferably unstained) should be included
with CSF specimens for correlative morphological evaluation. Send at room
temperature within 12 hours of collection. Note
that the volume required for CSF and Body Fluid is dependent on the cellularity of the
specimen. Immunophenotypic analysis
may not be successful if cell count is below 5 cells/cmm with ≤ 1 mL of sample.
✔✔rare events - ✔✔In flow cytometry, an "event" is defined as a single particle detected
by the instrument. The term "rare" generally refers to a frequency of 0.01% and below.
Accurate detection of rare-cell events using flow cytometry requires the ability to detect
single cells with specific characteristics in a heterogeneous population of cells. This
detection can be additionally complicated by the challenge of detecting the cells of
interest in a limited sample or in the presence of cell debris or other artifacts of sample
preparation. Minimal sample preparation is recommended, to avoid these artifacts and
minimize cell loss. "No-lyse/no-wash" or "lyse/no-wash" procedures can help maintain
the cells' native characteristics while maximizing recovery of the rare population. When
investigating a rare population of cells, it may be necessary to acquire millions of events
to obtain a sufficient number of cells for statistically significant detection. The number of
events needed for analysis depends on three main factors: the ratio of cells to debris in
the sample; the signal-to-noise ratio of the detected cells compared to background
fluorescence; and the frequency of the cell population of interest in the sample.
✔✔Microvesicles/Microparticle - ✔✔Microvesicles originate from cells and have the
same analysis requirements as cells. For these and other reasons, flow cytometry is a
popular choice for microvesicle analysis. However, there are pitfalls with small particle
flow cytometry that have led to many conflicting publications. The only way to avoid
these mistakes is to first identify them and then take measures to prevent them. 4
important things to account:
1. take into account pre-analytical variations. When the particles are this small, even the
slightest protocol variations can affect your experiment.
2. use beads to determine micovesicle size
3. Microvesicles are between 0.1 - 1 micron diameters in size. Though most fall in the
smaller end of that range.
4. Accounting for coincidence events is important to every flow cytometry experiment,
including experiments measuring and reporting microvesicles.
✔✔Magnetic Beads - ✔✔Gentle, tube-based magnetic separation with Invitrogen
Dynabeads or MagniSort beads are technologies of choice when you want to isolate
high yields of pure, viable and functional cells. Magnetic separation helps ensure that
the isolated cells are not affected by passage through a dense column and that the
cells' native characteristics are preserved.
, ✔✔fetal hemoglobin assay - ✔✔Murine monoclonal antibodies directed against fetal
hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used
in a multiparametric flow cytometric assay developed for the quantitation of fetal red
cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton
X-100 permeabilization prior to monoclonal antibody incubation was developed, along
with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells.
The performance of the assay can be correlated with the Kleihauer-Betke acid elution
method.
✔✔Isolation of Mononuclear Cells from Peripheral or Cord Blood - ✔✔To isolate
mononuclear cells (MNCs) from peripheral blood, cord blood and bone marrow, it is
recommended to use media with a density of 1.077 g/mL. For this particular application,
diluted blood is layered on top of the density gradient medium and the samples are
centrifuged. Granulocytes and erythrocytes have a higher density than mononuclear
cells and during centrifugation they sediment through the density medium layer. The
less dense mononuclear cells remain at the plasma-density medium interface. This
process is greatly facilitated by performing the density gradient centrifugation in a
SepMate™ tube.
✔✔Multiplex Assay - ✔✔In the biological sciences, a multiplex assay is a type of
immunoassay that uses magnetic beads to simultaneously measure multiple analytes in
a single experiment. A multiplex assay is a derivative of an ELISA using beads for
binding the capture antibody.
✔✔Cell Cycle - ✔✔Cell Cycle Analysis. Cell cycle analysis is a very common flow
cytometry application. By using a DNA-specific stain, one can determine a DNA profile
e.g. find percentage of the population in G0/G1, S, and G2/M. This information can be
used to, for example, monitor the effect of an anticancer treatment.
✔✔Stem Cell - ✔✔Flow cytometry and cell sorting are absolutely indispensable
techniques for both the identification and isolation of embryonic and adult stem cells in
both bone marrow and other tissues. As with most things in biology, there is no single
characteristic that adequately identifies stem cells by itself.
✔✔Immunophenotyping of Live Human Pluripotent Stem Cells - ✔✔Human induced
pluripotent stem cells (hiPSC), which are derived from somatic cells through the ectopic
expression of a specific set of transcription factors and share the ESC properties of self-
renewal and pluripotency, have overcome many of the limitations associated with
hESC. Despite the advantages, there remain many obstacles to overcome prior to using
hiPSC in the treatment of disease.
✔✔Panel Design Uses - ✔✔selection of fluorochromes and antigenic targets
✔✔cell adhesion molecules (CAMs) - ✔✔Proteins on plasma membrane of cells that bin
similar proteins on other cells, thereby meiating cell-cell adhesion. There are 4 classes:
ANSWERS SURE A+
✔✔BONE MARROW BIOPSY OR FINE NEEDLE ASPIRATE (FNA) - ✔✔Submerge in
RPMI tissue culture medium for optimal cell viability (sterile saline is acceptable). Send
at room temperature
within 12 hours of collection.
✔✔FRESH TISSUE - ✔✔Mince tissue and submerge in RPMI tissue culture medium for
optimal cell viability (sterile saline is acceptable). Send at room
temperature within 12 hours of collection.
✔✔BONE MARROW ASPIRATE - ✔✔Add 1 - 2 mL of first pull bone marrow aspirate to
a green top (sodium heparin) tube. Send intact specimen stored at room
temperature within 12 hours of collection. Include 4 unstained aspirate smears, an
unstained peripheral blood smear and a
copy of the patient's most recent complete blood count (CBC) and white cell differential.
✔✔study sample preparation - ✔✔staining, disaggreation, lysing agents, aggregates,
filtering, fixation etc (study protocols)
✔✔density gradient centrifugation - ✔✔Density gradient centrifugation is commonly
used for the isolation of specific cell populations from whole blood. This technique takes
advantage of the differences in density between the various leukocytes and the density
gradient medium. As a result, the particular cell populations isolated will depend on the
density of the medium that is used.
✔✔Signal Cell Sorting - ✔✔Fluorescence-activated cell sorting (FACS) is a specialized
type of flow cytometry. It provides a method for sorting a heterogeneous mixture of
biological cells into two or more containers, one cell at a time, based upon the specific
light scattering and fluorescent characteristics of each cell.
,✔✔CSF OR BODY FLUID - ✔✔Add body fluid (e.g., CSF, pleural, peritoneal) to a
sterile, leak-proof container. Include a copy of the patient's most recent
body fluid cell count and differential. If possible, an original cytospin preparation
(preferably unstained) should be included
with CSF specimens for correlative morphological evaluation. Send at room
temperature within 12 hours of collection. Note
that the volume required for CSF and Body Fluid is dependent on the cellularity of the
specimen. Immunophenotypic analysis
may not be successful if cell count is below 5 cells/cmm with ≤ 1 mL of sample.
✔✔rare events - ✔✔In flow cytometry, an "event" is defined as a single particle detected
by the instrument. The term "rare" generally refers to a frequency of 0.01% and below.
Accurate detection of rare-cell events using flow cytometry requires the ability to detect
single cells with specific characteristics in a heterogeneous population of cells. This
detection can be additionally complicated by the challenge of detecting the cells of
interest in a limited sample or in the presence of cell debris or other artifacts of sample
preparation. Minimal sample preparation is recommended, to avoid these artifacts and
minimize cell loss. "No-lyse/no-wash" or "lyse/no-wash" procedures can help maintain
the cells' native characteristics while maximizing recovery of the rare population. When
investigating a rare population of cells, it may be necessary to acquire millions of events
to obtain a sufficient number of cells for statistically significant detection. The number of
events needed for analysis depends on three main factors: the ratio of cells to debris in
the sample; the signal-to-noise ratio of the detected cells compared to background
fluorescence; and the frequency of the cell population of interest in the sample.
✔✔Microvesicles/Microparticle - ✔✔Microvesicles originate from cells and have the
same analysis requirements as cells. For these and other reasons, flow cytometry is a
popular choice for microvesicle analysis. However, there are pitfalls with small particle
flow cytometry that have led to many conflicting publications. The only way to avoid
these mistakes is to first identify them and then take measures to prevent them. 4
important things to account:
1. take into account pre-analytical variations. When the particles are this small, even the
slightest protocol variations can affect your experiment.
2. use beads to determine micovesicle size
3. Microvesicles are between 0.1 - 1 micron diameters in size. Though most fall in the
smaller end of that range.
4. Accounting for coincidence events is important to every flow cytometry experiment,
including experiments measuring and reporting microvesicles.
✔✔Magnetic Beads - ✔✔Gentle, tube-based magnetic separation with Invitrogen
Dynabeads or MagniSort beads are technologies of choice when you want to isolate
high yields of pure, viable and functional cells. Magnetic separation helps ensure that
the isolated cells are not affected by passage through a dense column and that the
cells' native characteristics are preserved.
, ✔✔fetal hemoglobin assay - ✔✔Murine monoclonal antibodies directed against fetal
hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used
in a multiparametric flow cytometric assay developed for the quantitation of fetal red
cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton
X-100 permeabilization prior to monoclonal antibody incubation was developed, along
with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells.
The performance of the assay can be correlated with the Kleihauer-Betke acid elution
method.
✔✔Isolation of Mononuclear Cells from Peripheral or Cord Blood - ✔✔To isolate
mononuclear cells (MNCs) from peripheral blood, cord blood and bone marrow, it is
recommended to use media with a density of 1.077 g/mL. For this particular application,
diluted blood is layered on top of the density gradient medium and the samples are
centrifuged. Granulocytes and erythrocytes have a higher density than mononuclear
cells and during centrifugation they sediment through the density medium layer. The
less dense mononuclear cells remain at the plasma-density medium interface. This
process is greatly facilitated by performing the density gradient centrifugation in a
SepMate™ tube.
✔✔Multiplex Assay - ✔✔In the biological sciences, a multiplex assay is a type of
immunoassay that uses magnetic beads to simultaneously measure multiple analytes in
a single experiment. A multiplex assay is a derivative of an ELISA using beads for
binding the capture antibody.
✔✔Cell Cycle - ✔✔Cell Cycle Analysis. Cell cycle analysis is a very common flow
cytometry application. By using a DNA-specific stain, one can determine a DNA profile
e.g. find percentage of the population in G0/G1, S, and G2/M. This information can be
used to, for example, monitor the effect of an anticancer treatment.
✔✔Stem Cell - ✔✔Flow cytometry and cell sorting are absolutely indispensable
techniques for both the identification and isolation of embryonic and adult stem cells in
both bone marrow and other tissues. As with most things in biology, there is no single
characteristic that adequately identifies stem cells by itself.
✔✔Immunophenotyping of Live Human Pluripotent Stem Cells - ✔✔Human induced
pluripotent stem cells (hiPSC), which are derived from somatic cells through the ectopic
expression of a specific set of transcription factors and share the ESC properties of self-
renewal and pluripotency, have overcome many of the limitations associated with
hESC. Despite the advantages, there remain many obstacles to overcome prior to using
hiPSC in the treatment of disease.
✔✔Panel Design Uses - ✔✔selection of fluorochromes and antigenic targets
✔✔cell adhesion molecules (CAMs) - ✔✔Proteins on plasma membrane of cells that bin
similar proteins on other cells, thereby meiating cell-cell adhesion. There are 4 classes:
