ASCP SCYM STUDY GUIDE QUESTIONS
Sample Processing - Answers - Optimized antibody concentration
Defined range of cell #
Define sample handling, time and temperature
Define acceptable time of delay for acquisition
CD2 - Answers - T cells: NK cells
CD3 - Answers - T cells
CD4 - Answers - T helper cells, monocytes (dimly expressed)
CD5 - Answers - T cells; Some B Cells
CD8 - Answers - T cytotoxic cells, macrophages
CD10 - Answers - Immature T, B Cells, PMN's
CD13 - Answers - Monocytes, PMN's
CD14 - Answers - Monocytes, PMN's (dimly expressed)
CD16 - Answers - NK cells, PMN's, monocytes
CD19 - Answers - B Cells
CD20 - Answers - B Cells
CD25 - Answers - Activated T Cells
CD33 - Answers - Monocytes
CD34 - Answers - Stem/Progenitor Cells
CD45 - Answers - Leukocytes
CD56 - Answers - NK cells, monocytes (dimly expressed)
Anti-Kappa - Answers - B Cells
Anti-Lambda - Answers - B Cells
Tdt - Answers - Immature Lymphocytes
,Monoclonal Antibody Staining Direct Staining - Answers - Rapid single step staining
Can use multiple antibodies from the same host
Monoclonal Antibody Staining Indirect Staining - Answers - Secondary amplifies single
A few labeled secondaries can detect many primaries
Clinical Applications of Flow - Answers - -Instrument Performance
-Integrity of the Specimen
-Quality in Processing of Specimens
-Appropriate Reagents and Controls
-Qualified Technical Staff
-Regulatory requirements from CAP, TJC, CLIA, and the FDA exist to ensure the
accurate reporting of flow cytometric results.
Lymphocytes Subset Enumeration (Immunophenotyping) - Answers - 2 Tubes: 1-
CD3/CD4/CD8/CD45
2- CD3/CD19/CD16&CD56/CD45
-May include analysis of CD4 and CD8 T Cells, B Cells and NK Cells.
-Results reported as proportion (%) of total lymphocyte population.
CD4 Counts in HIV Infection - Answers - -Asses degree of immunodeficiency
-Diagnose of AIDS
-Institution of Anti-Retroviral (ARV) therapy and opportunistic infection (OI) prophylaxis
-Monitoring immune reconstitution
CD4 Immunophenotyping Panels - Answers -
2 color - Answers - Isotype Ctrl
CD45/CD14
CD3/CD4
CD3/CD8
CD3/CD19
CD3/CD16 + 56
3 Color - Answers - CD3/CD4/CD45
CD3/CD8/CD45
CD3/CD19/CD16 + 56
4 Color - Answers - CD3/CD4/CD8/CD45
CD3/CD16/CD16 + 56/CD45
Determination of Absolute Counts - Answers - -Subset % X Absolute Lymphocyte Count
-Counting Beads- known concentration present in staining tube or added to tube
-Volumetric Cytometers
, Leukemia/Lymphoma Phenotyping - Answers - -Important in Diagnosis, Classification
and Monitoring of patients with hematologic malignancies.
-Utility based on the fact that hematopoiesis is characterized by the gain and loss of cell
surface and intracellular antigen during the process of maturation.
-Malignant cells may have a block at an immature stage resulting in abnormal numbers
of immune cells. With a mature phenotype may be present in abnormally high numbers.
May have an aberrant phenotype.
Leukemia/Lymphoma Phenotyping FC - Answers - -Analysis with panels of monoclonal
antibodies allows for lineage assignment and sub classification that may have further
prognostic significance.
-The identification of a unique phenotype can also facilitate post-therapy monitoring to
assess residual disease.
Relevance for the Diagnosis and Classification of Hematologic Malignancies - Answers -
-Expression of aberrant lineage associated antigen
-Absence of lineage associated antigen
-Asynchronous expression of antigens
-Abnormal level of expression of antigen
Detection of Fetal Hemoglobin - Answers - -Detection of fetal hemoglobin containing
cells in useful for the diagnosis and management of feto-maternal hemorrhage
-Traditionally assayed with the Kleihauer-Betke method
-FC offers improved sensitivity (<0.02% fetal RBCs) and reproducibility (CV <15%
-Method: Intracellular staining with a fetal hemoglobin specific monoclonal antibody
CD34 Stem Cell Enumeration - Answers - -Present on immature hematopoietic cells
-Expression is lost as cells mature
-Used as a marker to identify and enumerate stem cells in bone marrow and peripheral
blood products for transplantation
-Currently the Gold Standard for assessment of HSC products for transplant
CD3 Transplant Monitoring - Answers - -Acute graft rejection is an important
complication after solid organ transplantation that may lead to graft loss
-Severe cases of rejection may be treated with antibodies that deplete T cells from the
patient (OKT3, ATG)
-FC is used to assess the effectiveness of T cell depletion after antibody based anti-
rejection therapy
Organ Transplantation Antibody Detection - Answers - -The presence of HLA specific
antibodies in the potential recipient of a kidney transplant that are specific for the
potential donor is often a contraindication to transplantation.
-Renal transplant candidates are screened on a regular basis while on the waiting list to
monitor the presence and specificity of anti-HLA antibodies.
-When a donor is identified, recipient serum is tested for the presence of donor-specific
antibodies (crossmatch)
Sample Processing - Answers - Optimized antibody concentration
Defined range of cell #
Define sample handling, time and temperature
Define acceptable time of delay for acquisition
CD2 - Answers - T cells: NK cells
CD3 - Answers - T cells
CD4 - Answers - T helper cells, monocytes (dimly expressed)
CD5 - Answers - T cells; Some B Cells
CD8 - Answers - T cytotoxic cells, macrophages
CD10 - Answers - Immature T, B Cells, PMN's
CD13 - Answers - Monocytes, PMN's
CD14 - Answers - Monocytes, PMN's (dimly expressed)
CD16 - Answers - NK cells, PMN's, monocytes
CD19 - Answers - B Cells
CD20 - Answers - B Cells
CD25 - Answers - Activated T Cells
CD33 - Answers - Monocytes
CD34 - Answers - Stem/Progenitor Cells
CD45 - Answers - Leukocytes
CD56 - Answers - NK cells, monocytes (dimly expressed)
Anti-Kappa - Answers - B Cells
Anti-Lambda - Answers - B Cells
Tdt - Answers - Immature Lymphocytes
,Monoclonal Antibody Staining Direct Staining - Answers - Rapid single step staining
Can use multiple antibodies from the same host
Monoclonal Antibody Staining Indirect Staining - Answers - Secondary amplifies single
A few labeled secondaries can detect many primaries
Clinical Applications of Flow - Answers - -Instrument Performance
-Integrity of the Specimen
-Quality in Processing of Specimens
-Appropriate Reagents and Controls
-Qualified Technical Staff
-Regulatory requirements from CAP, TJC, CLIA, and the FDA exist to ensure the
accurate reporting of flow cytometric results.
Lymphocytes Subset Enumeration (Immunophenotyping) - Answers - 2 Tubes: 1-
CD3/CD4/CD8/CD45
2- CD3/CD19/CD16&CD56/CD45
-May include analysis of CD4 and CD8 T Cells, B Cells and NK Cells.
-Results reported as proportion (%) of total lymphocyte population.
CD4 Counts in HIV Infection - Answers - -Asses degree of immunodeficiency
-Diagnose of AIDS
-Institution of Anti-Retroviral (ARV) therapy and opportunistic infection (OI) prophylaxis
-Monitoring immune reconstitution
CD4 Immunophenotyping Panels - Answers -
2 color - Answers - Isotype Ctrl
CD45/CD14
CD3/CD4
CD3/CD8
CD3/CD19
CD3/CD16 + 56
3 Color - Answers - CD3/CD4/CD45
CD3/CD8/CD45
CD3/CD19/CD16 + 56
4 Color - Answers - CD3/CD4/CD8/CD45
CD3/CD16/CD16 + 56/CD45
Determination of Absolute Counts - Answers - -Subset % X Absolute Lymphocyte Count
-Counting Beads- known concentration present in staining tube or added to tube
-Volumetric Cytometers
, Leukemia/Lymphoma Phenotyping - Answers - -Important in Diagnosis, Classification
and Monitoring of patients with hematologic malignancies.
-Utility based on the fact that hematopoiesis is characterized by the gain and loss of cell
surface and intracellular antigen during the process of maturation.
-Malignant cells may have a block at an immature stage resulting in abnormal numbers
of immune cells. With a mature phenotype may be present in abnormally high numbers.
May have an aberrant phenotype.
Leukemia/Lymphoma Phenotyping FC - Answers - -Analysis with panels of monoclonal
antibodies allows for lineage assignment and sub classification that may have further
prognostic significance.
-The identification of a unique phenotype can also facilitate post-therapy monitoring to
assess residual disease.
Relevance for the Diagnosis and Classification of Hematologic Malignancies - Answers -
-Expression of aberrant lineage associated antigen
-Absence of lineage associated antigen
-Asynchronous expression of antigens
-Abnormal level of expression of antigen
Detection of Fetal Hemoglobin - Answers - -Detection of fetal hemoglobin containing
cells in useful for the diagnosis and management of feto-maternal hemorrhage
-Traditionally assayed with the Kleihauer-Betke method
-FC offers improved sensitivity (<0.02% fetal RBCs) and reproducibility (CV <15%
-Method: Intracellular staining with a fetal hemoglobin specific monoclonal antibody
CD34 Stem Cell Enumeration - Answers - -Present on immature hematopoietic cells
-Expression is lost as cells mature
-Used as a marker to identify and enumerate stem cells in bone marrow and peripheral
blood products for transplantation
-Currently the Gold Standard for assessment of HSC products for transplant
CD3 Transplant Monitoring - Answers - -Acute graft rejection is an important
complication after solid organ transplantation that may lead to graft loss
-Severe cases of rejection may be treated with antibodies that deplete T cells from the
patient (OKT3, ATG)
-FC is used to assess the effectiveness of T cell depletion after antibody based anti-
rejection therapy
Organ Transplantation Antibody Detection - Answers - -The presence of HLA specific
antibodies in the potential recipient of a kidney transplant that are specific for the
potential donor is often a contraindication to transplantation.
-Renal transplant candidates are screened on a regular basis while on the waiting list to
monitor the presence and specificity of anti-HLA antibodies.
-When a donor is identified, recipient serum is tested for the presence of donor-specific
antibodies (crossmatch)
