ANSWERS|LATEST
It is possible to convert the plasma into serum by recalcifying the plasma and allowing
coagulation to occur. - ANSWER Calcium is added to the plasma to form a complex
with the citrate present in the anti-coagulant. This restores a normal
ionized calcium level allowing clotting to occur.
Polyspecific antiserum can be______ with lymphocytes to determine if there is a single
antibody or multiple
antibodies. - ANSWER absorbed
Another application of lymphocyte absorption is to use the patient's own cells to
remove_____. Absorption can be performed
at cold or warm temperatures depending on the type of antibody. -
ANSWER autoantibody
_________ are typically cold reacting (4oC) and________are typically warm reacting (22-
37oC). - ANSWER Autoantibodies
B cell antibodies
Patient serum may contain autoantibodies and/or IgM antibodies that may interfere the
binding of
anti-HLA IgG antibodies to the antigen. In complement dependent assays (CDC), IgM
antibodies
1
,can activate complement; potentially leading to ___________. - ANSWER false
positive reactivity.
DTT - ANSWER dithiothreitol, also known as Cleland's Reagent
DTT inactivates IgM molecules by cleaving the _________ in the
pentamer structure. - ANSWER disulphide bonds
Heat inactivation works by cleaving the disulphide bonds in the
pentamer structure, but is less specific than _____. - ANSWER DTT.
ALA - ANSWER therapeutic antilymphocyte
antibodies
Thymoglobulin - ANSWER purified, pasteurized, gamma immune globulin,
obtained by immunization of rabbits with human thymocytes
OKT3 - ANSWER a murine IgG monoclonal antibody directed against CD3
If these therapeutic antilymphocyte
antibodies are present in the patient serum, they can cause_______ reactivity in
T cell cytotoxic assays. - ANSWER false positive
ALA removal is accomplished by absorption using _____coated with anti-rabbit or anti-
mouse immunoglobulin for Thymoblobulin or OKT3, respectively. - ANSWER magnetic
beads
serum preparations - ANSWER 1. Recalcificiation of plasma
2
,2. Adsorption with lymphocytes
3. Inactivation of IgM antibodies
a. DTT treatment
b. Heat Inactivation
4. Depletion of therapeutic anti-lymphocyte antibodies from serum
Why is calcium added to plasma to convert it to serum? - ANSWER Calcium is added to
the plasma to combine with the citrate present in the anticoagulant.
This restores a normal ionized calcium level and allows clotting to occur.
At what temperature should serum absorption with lymphocytes be performed? -
ANSWER Typically, auto-antibodies are cold reacting and should be absorbed at 4oC
while alloantibodies
are warm reacting and should be absorbed at 37oC.
DTT treatment is more specific than heat treatment in
removing IgM antibodies. However, DTT can also affect the _______ in IgG antibodies. -
ANSWER disulfide bonds
What happens if we don't remove OKT3 prior to cytotoxicity assays? - ANSWER OKT3
is a monoclonal antibody directed against the CD3 molecule. Therefore, the
binding of OKT3 to any CD3+ cell will activate complement and lyse the target cell. This
results
in falsely positive reactivity.
Lymphocytes have a density of_______ - ANSWER ~1.077 g/ml.
Cells with a higher density than FH - ANSWER red cells and granulocytes
3
, cells of a lower density than lymphocytes - ANSWER platelets
isolation of cells from lymph nodes or spleen is normally fast and simple and leads to greater
_____ and ______ - ANSWER cell yield
viability.
Obtaining lymphocytes from spleen will require
an additional FH step along with purification steps due to a large number of contaminating
cells,
such as _______ ,______ and _______. - ANSWER macrophages
erythrocytes
platelets.
intrinsic cell properties - ANSWER cell size and
granularity of the cell.
One method of lymphocyte isolation (HP) utilizes an intrinsic cell property, __________, by
using a
density gradient medium. - ANSWER cell density
extrinsic cell properties - ANSWER cell surface marker, phagocytic capabilities and
adherence capabilities.
Immunomagnetic bead separations use the extrinisic property of __________ as the
isolation target. - ANSWER cell surface markers
Nylon wool isolation incorporates the extrinsic property of ___________ -
ANSWER adherence for B cells.
4
It is possible to convert the plasma into serum by recalcifying the plasma and allowing
coagulation to occur. - ANSWER Calcium is added to the plasma to form a complex
with the citrate present in the anti-coagulant. This restores a normal
ionized calcium level allowing clotting to occur.
Polyspecific antiserum can be______ with lymphocytes to determine if there is a single
antibody or multiple
antibodies. - ANSWER absorbed
Another application of lymphocyte absorption is to use the patient's own cells to
remove_____. Absorption can be performed
at cold or warm temperatures depending on the type of antibody. -
ANSWER autoantibody
_________ are typically cold reacting (4oC) and________are typically warm reacting (22-
37oC). - ANSWER Autoantibodies
B cell antibodies
Patient serum may contain autoantibodies and/or IgM antibodies that may interfere the
binding of
anti-HLA IgG antibodies to the antigen. In complement dependent assays (CDC), IgM
antibodies
1
,can activate complement; potentially leading to ___________. - ANSWER false
positive reactivity.
DTT - ANSWER dithiothreitol, also known as Cleland's Reagent
DTT inactivates IgM molecules by cleaving the _________ in the
pentamer structure. - ANSWER disulphide bonds
Heat inactivation works by cleaving the disulphide bonds in the
pentamer structure, but is less specific than _____. - ANSWER DTT.
ALA - ANSWER therapeutic antilymphocyte
antibodies
Thymoglobulin - ANSWER purified, pasteurized, gamma immune globulin,
obtained by immunization of rabbits with human thymocytes
OKT3 - ANSWER a murine IgG monoclonal antibody directed against CD3
If these therapeutic antilymphocyte
antibodies are present in the patient serum, they can cause_______ reactivity in
T cell cytotoxic assays. - ANSWER false positive
ALA removal is accomplished by absorption using _____coated with anti-rabbit or anti-
mouse immunoglobulin for Thymoblobulin or OKT3, respectively. - ANSWER magnetic
beads
serum preparations - ANSWER 1. Recalcificiation of plasma
2
,2. Adsorption with lymphocytes
3. Inactivation of IgM antibodies
a. DTT treatment
b. Heat Inactivation
4. Depletion of therapeutic anti-lymphocyte antibodies from serum
Why is calcium added to plasma to convert it to serum? - ANSWER Calcium is added to
the plasma to combine with the citrate present in the anticoagulant.
This restores a normal ionized calcium level and allows clotting to occur.
At what temperature should serum absorption with lymphocytes be performed? -
ANSWER Typically, auto-antibodies are cold reacting and should be absorbed at 4oC
while alloantibodies
are warm reacting and should be absorbed at 37oC.
DTT treatment is more specific than heat treatment in
removing IgM antibodies. However, DTT can also affect the _______ in IgG antibodies. -
ANSWER disulfide bonds
What happens if we don't remove OKT3 prior to cytotoxicity assays? - ANSWER OKT3
is a monoclonal antibody directed against the CD3 molecule. Therefore, the
binding of OKT3 to any CD3+ cell will activate complement and lyse the target cell. This
results
in falsely positive reactivity.
Lymphocytes have a density of_______ - ANSWER ~1.077 g/ml.
Cells with a higher density than FH - ANSWER red cells and granulocytes
3
, cells of a lower density than lymphocytes - ANSWER platelets
isolation of cells from lymph nodes or spleen is normally fast and simple and leads to greater
_____ and ______ - ANSWER cell yield
viability.
Obtaining lymphocytes from spleen will require
an additional FH step along with purification steps due to a large number of contaminating
cells,
such as _______ ,______ and _______. - ANSWER macrophages
erythrocytes
platelets.
intrinsic cell properties - ANSWER cell size and
granularity of the cell.
One method of lymphocyte isolation (HP) utilizes an intrinsic cell property, __________, by
using a
density gradient medium. - ANSWER cell density
extrinsic cell properties - ANSWER cell surface marker, phagocytic capabilities and
adherence capabilities.
Immunomagnetic bead separations use the extrinisic property of __________ as the
isolation target. - ANSWER cell surface markers
Nylon wool isolation incorporates the extrinsic property of ___________ -
ANSWER adherence for B cells.
4
