CHT EXAM COMPLETE STUDY GUIDE
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
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. 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
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.
Nylon wool isolation procedure: - Answer- Lymphocytes are obtained and added to a
column packed with nylon wool. B cells will adhere to the nylon wool at an optimal
temperature of 37o C. The non-adherent T cells can be collected by washing the
column several times with warm media. The B cells can then be eluted by
plunging cold media through the column.
Dead cells in a flow cytometry assay can lead to
____________ of monoclonal antibodies and/or fluorochromes. - Answer-
nonspecific binding
Various cell purification methods are - Answer- Percoll and Thrombin: remove
granulocytes, platelets, and dead cells
Lympho-kwik: isolate specific populations (T &/or B lymphocytes, lymphocytes and
monocytes)
centrifugation slow/short: remove platelets in the supernatant
carbonyl iron: Granulocytes ingest altering density (heavier)
Dnase: remove dead cells
Lymphocyte interface is
indistinctive or thin - Answer- Hyperlipemic blood sample
Centrifugation force is too low and time too short
Adjust centrifugation to 2000 x g for 25 min
Presence of RBC in the
lymphocyte band - Answer- Density of RBC altered due to disease
Excessive harsh mixing of blood
Low lymphocyte yield - Answer- Low WBC of blood donor
Buffy coat left on RBC. Collect buffy coat to extend to ¼
inch of RBC layer
Cells left in interface
, Mononuclear cells (MNC) will
not pellet after washing - Answer- Insufficient volume of wash medium
Collection of ficoll layer exceeds 2.5ml
Cell viability <90% - Answer- Lack of protein in wash medium
Blood samples > 24 hrs old
>3% granulocyte contamination - Answer- Density of granulocytes altered due to
disease state
or abnormal blood sample
Specific gravity of FH is too High. Must be 1.077.
Use thrombin, ncarbonyl iron, and Lympho-Kwik™
Reagent.
Platelet contamination - Answer- Blood sample drawn in Heparin
Blood sample >24 hrs old
Cell Yield Calculations: - Answer- The main divisions separate the grid into 9 large
squares. Each square has a surface area of 1mm2, and the depth of the chamber is
0.1mm. Each square of the hemacytometer (with cover slip in place) represents a
total volume of 0.1 mm³ or 10-4 cm³. Since 1 cm³ =1 ml, the subsequent cell
concentration per ml will be determined using the following calculations:
Cells per ml = the average count per large square X the dilution factor X 104
Adjust the cell concentration using the following formula and table of recommended
cell
concentrations : - Answer-
Cells can be frozen and stored in the vapor phase of _________ indefinitely,
maintaining adequate cell viability without concerns of pH maintenance or bacterial
contamination. - Answer- liquid nitrogen
As water freezes into extracellular ice crystals, the rising solute concentration outside
the cells results in osmotic imbalance that draws water out of cells. Cells are
damaged by___________ - Answer- shrinkage and high extracellular/intracellular
solute concentrations.
a rapid rate of cooling minimizes the osmotic disturbance because ice crystals form
uniformly and less water is lost from the cells. Cells are damaged instead when the
intracellular
water forms_________ - Answer- large ice crystals which disrupt the cell membrane.
The goals of cryopreservation are: - Answer- 1) to replace some of the water with a
compound that will not form large crystals when frozen, and
2) to minimize the damaging effects of changing solute concentrations.
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
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. 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
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.
Nylon wool isolation procedure: - Answer- Lymphocytes are obtained and added to a
column packed with nylon wool. B cells will adhere to the nylon wool at an optimal
temperature of 37o C. The non-adherent T cells can be collected by washing the
column several times with warm media. The B cells can then be eluted by
plunging cold media through the column.
Dead cells in a flow cytometry assay can lead to
____________ of monoclonal antibodies and/or fluorochromes. - Answer-
nonspecific binding
Various cell purification methods are - Answer- Percoll and Thrombin: remove
granulocytes, platelets, and dead cells
Lympho-kwik: isolate specific populations (T &/or B lymphocytes, lymphocytes and
monocytes)
centrifugation slow/short: remove platelets in the supernatant
carbonyl iron: Granulocytes ingest altering density (heavier)
Dnase: remove dead cells
Lymphocyte interface is
indistinctive or thin - Answer- Hyperlipemic blood sample
Centrifugation force is too low and time too short
Adjust centrifugation to 2000 x g for 25 min
Presence of RBC in the
lymphocyte band - Answer- Density of RBC altered due to disease
Excessive harsh mixing of blood
Low lymphocyte yield - Answer- Low WBC of blood donor
Buffy coat left on RBC. Collect buffy coat to extend to ¼
inch of RBC layer
Cells left in interface
, Mononuclear cells (MNC) will
not pellet after washing - Answer- Insufficient volume of wash medium
Collection of ficoll layer exceeds 2.5ml
Cell viability <90% - Answer- Lack of protein in wash medium
Blood samples > 24 hrs old
>3% granulocyte contamination - Answer- Density of granulocytes altered due to
disease state
or abnormal blood sample
Specific gravity of FH is too High. Must be 1.077.
Use thrombin, ncarbonyl iron, and Lympho-Kwik™
Reagent.
Platelet contamination - Answer- Blood sample drawn in Heparin
Blood sample >24 hrs old
Cell Yield Calculations: - Answer- The main divisions separate the grid into 9 large
squares. Each square has a surface area of 1mm2, and the depth of the chamber is
0.1mm. Each square of the hemacytometer (with cover slip in place) represents a
total volume of 0.1 mm³ or 10-4 cm³. Since 1 cm³ =1 ml, the subsequent cell
concentration per ml will be determined using the following calculations:
Cells per ml = the average count per large square X the dilution factor X 104
Adjust the cell concentration using the following formula and table of recommended
cell
concentrations : - Answer-
Cells can be frozen and stored in the vapor phase of _________ indefinitely,
maintaining adequate cell viability without concerns of pH maintenance or bacterial
contamination. - Answer- liquid nitrogen
As water freezes into extracellular ice crystals, the rising solute concentration outside
the cells results in osmotic imbalance that draws water out of cells. Cells are
damaged by___________ - Answer- shrinkage and high extracellular/intracellular
solute concentrations.
a rapid rate of cooling minimizes the osmotic disturbance because ice crystals form
uniformly and less water is lost from the cells. Cells are damaged instead when the
intracellular
water forms_________ - Answer- large ice crystals which disrupt the cell membrane.
The goals of cryopreservation are: - Answer- 1) to replace some of the water with a
compound that will not form large crystals when frozen, and
2) to minimize the damaging effects of changing solute concentrations.
