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Laboratory Procedures Specimen collection, transportation and handling Specimen preparation Panels Reporting of results Data storage and retrieval 1 Appropriate Specimens for Analysis Peripheral blood, bone marrow aspirates, body fluids, cerebrospinal fluid Lymphoid tissue biopsies, skin and mucosal biopsies, fine needle aspirates of tumors 2 Unacceptable Specimens Specimens > 48 hours old (dead cells) Severe hemolysis (loss of cells) Clotted specimens (loss of cells) If the specimen is not rejected, a comment is included in the interpretation. (irretrievable specimens) 3 Specimen Collection Blood: 10 cc drawn into an EDTA or heparin. Minimum requirement is 5 cc. Bone Marrow: 2-3 cc in EDTA or heparin. Fluids:10-50 cc if cell count is unknown. Anticoagulation is not necessary. CSF: Any volume is acceptable. Anticoagulation is not necessary Tissue: Sterile container, covered with sterile saline or tissue culture media. 4 Specimen Transportation Immediate transportation to the lab is best Short-term storage (24-48 hrs) at room temperature Refrigerate fluids/tissues if analysis takes place >24 hours after collection 5 Specimen Preparation Erythrocyte lysis. The lysing agent should remove only mature red cells with minimal effect on the remaining cells. Density gradient separation 6 Whole Blood Bench 7 Erythrocyte Lysis •Lyses red cells quickly and with minimal hands on. •Doesn’t enrich for mononuclear cells so a small population of abnormal cells could be missed. Beckman Coulter TQ Prep 8 Density Gradient 9 Density Gradient Preparation Ficol-hypaque density gradient separation Enriches mononuclear cell populations Removes red cells, platelets, dead cells, stromal elements, etc. Is labor-intensive and time-consuming 10 Cell Count and Viability A high proportion of dead cells can alter phenotyping results Target is 150,000-200,000 viable cells/tube Manual count in trypan blue vital stain yields count of viable cells prior to staining in order to adjust concentration Viability can be done on a flow cytometer using 7-AAD, a fluorescent dye which stains the dead cells. 11 Bone Marrow Staining Bench 12 Sample Analysis Side by side placement of cytometers allows a single technologist to operate two instruments simultaneously if needed. 13 Analysis Review of histograms Morphologic assessment Correlation between the morphologic and phenotypic findings 14 Morphologic Assessment Smear prepared prior to lysis or density gradient separation. Cytospin prepared following density gradient separation. For tissue specimens, a smear or touch prep can be prepared prior to disaggregation. A cytospin is always prepared following disaggregation. 15 Reporting of Results Patient Information: Demographics, referring physician and institution, history and clinical findings, prior therapy Indications for testing and previous flow results if available Sample Information: Identification number, source, date and time of collection 16 Reporting of Results Listing of antibodies used Descriptive summary of the phenotype of the abnormal cells, including fluorescence intensity when appropriate 17 Reporting of Results Fraction of abnormal cells in the sample Morphologic findings when appropriate Interpretation of the phenotype, and differential diagnosis 18 Lymphocyte Subsets 19 Quantitation of T-cell subsets Assesses the immune system of HIV infected persons. Recommended that the helper/inducer T-cells be monitored every 3 to 6 months in all HIV positive persons. Helper/inducer T-cell (CD4+ cell) level is a criterion for surveillance case definition for AIDS. 20 Viral Load Used to see if drug therapy is working Does not assess immune system 21 Immunophenotyping CD4 CD3 CD # = cluster designation number 22 CD4/CD8 Gating on Lymphocytes LYMPHOCYTES Lymphocytes 23 CD4/CD8 24 CD4/CD8 Ratio Calculation CD4/CD8 Ratio = # of CD4 cells # of CD8 cells Example: WBC 6100, 31% lymphocytes Abs lymph. = 1891 cells/mm3; CD4% = 42.6 CD8% =35.6% CD4/CD8 Ratio = 806 673 = 1.2 25 CD4/CD8 Gating on lymphocytes Lymphocytes Lymphocytes Lymphocytes 26 CD4/CD8 27 CD4/CD8 Ratio Calculation CD4/CD8 Ratio = # of CD4 cells # of CD8 cells Example: WBC 4300, 24% lymphocytes Abs lymph. = 1032 cells/mm3; CD4% = 3.2 CD8% =81.3% CD4/CD8 Ratio = 33 839 = 0.0 28 Lymphocyte Subsets T cells + B cells + NK cells = Lymphocytes 13.7 + 74.1 + 9.5 = 97.5 29 Immunophenotyping of Lymphomas and Leukemias 30 CD Nomenclature CD45 CD2, CD3, CD5, CD7 CD4, CD8 CD19, CD20, CD24 CD10 CD56 CD34 CD13, CD33 CD14 Leukocyte Common Antigen T cells B cells CALLA (B cells) NK cells Stem cell marker Myeloid cells Monocytes 31 CASE STUDY #1 The patient is a 66-year-old male who presented with WBC of 66,000. The differential showed 79% lymphocytes. A peripheral blood sample was sent for flow cytometric analysis. Antigen CD2 CD5 CD10 CD19 CD20 HLA-DR CD23 CD24 CD25 Kappa Lambda Positive or Negative (?) 32 33 34 35 CASE STUDY #1 The patient is a 66-year-old male who presented with WBC of 66,000. The differential showed 79% lymphocytes. A peripheral blood sample was sent for flow cytometric analysis. Antigen CD2 CD5 CD10 CD19 CD20 HLA-DR CD23 CD24 CD25 kappa lambda Positive or Negative (?) N P N P P, Dim P P P N N P Diagnosis: Chronic Lymphocytic Leukemia 36 Case Study #2 An 81-year-old man was found to have a WBC of 141,000 with 96% lymphocytes. A peripheral blood sample was sent for flow cytometry studies. Antigen CD2 CD5 CD10 CD11c CD19 CD20 HLA-DR CD23 CD24 CD25 kappa lambda Positive or Negative (?) 37 38 39 40 Case Study #2 An 81-year-old man was found to have a WBC of 141,000 with 96% lymphocytes. A peripheral blood sample was sent for flow cytometry studies. Antigen CD2 CD5 CD10 CD19 CD20 HLA-DR CD23 CD24 CD25 kappa lambda Positive or Negative (?) N P N P P P N P N N P Diagnosis: B-Cell Lymphoma (Mantle Cell Lymphoma) 41 Case Study #3 A 65-year-old woman presented with a WBC of 30,000. The differential showed 90% blasts. A bone marrow was submitted for flow cytometry. Antigen CD3 CD7 CD13 CD15 CD33 CD34 HLA-DR Positive or Negative (?) 42 43 44 Case Study #3 A 65-year-old woman presented with a WBC of 30,000. The differential showed 90% blasts. A bone marrow was submitted for flow cytometry. Antigen CD3 CD7 CD13 CD15 CD33 CD34 HLA-DR Positive or Negative (?) N P P N N P P Diagnosis: Acute myelogenous leukemia 45 Antigen Distribution in AML M0 CD13 CD33 CD15 CD14 CD11c CD4 Glyco CD61 CD34 HLA-DR M1 M2 M3 M4 M5 M6 M7 46 Case Study #4 A 5-year-old female presented with bone pain and a WBC of 50,000. A bone marrow sample was submitted for flow cytometry. Antigen CD3 CD10 CD15 CD19 CD20 CD24 CD34 HLA-DR Positive or Negative (?) 47 48 49 Case Study #4 A 5-year-old female presented with bone pain and a WBC of 50,000. A bone marrow sample was submitted for flow cytometry. Antigen CD3 CD10 CD15 CD19 CD20 CD24 CD34 HLA-DR Positive or Negative (?) N P N P N P N P Diagnosis: Acute lymphoblastic leukemia 50 Case Study #5 A 17 year old girl presents with “pancytopenia”: WBC=2.3, Plt Ct=31. Antigen Positive or Negative(?) CD3 CD4 CD8 CD10 CD13 CD19 CD20 CD24 CD33 CD34 CD117 51 HLA-DR 52 53 54 55 Case Study #5 Antigen Positive or Negative(?) CD3 N CD4 N CD8 N CD10 P CD13 N CD19 P CD20 N CD24 P CD33 N CD34 P CD117 N HLA-DR P Diagnosis: Acute lymphoblastic leukemia 56 Antigen Distribution in B Lineage ALL pro-B HLA-DR CD34 CD19 CD24 CD10 CD20 TdT CyIgM SIgM Ig Genes R pre-pre-B pre-B Burkitt’s 57 DNA Cell Cycle Analysis by Flow Cytometry 58 Clinical applications of DNA cell cycle analysis The DNA content and/or the synthetic phase fraction provide prognostic information in: Breast carcinoma Colon carcinoma Pediatric acute lymphoblastic leukemia 59 Normal Cell Cycle G2 M G0 DNA Analysis G1 G0 G1 s C o u n t G2 M s 0 200 400 600 800 1000 4N 2N DNA content 60 Definitions & Terms Ploidy Related to the number of chromosomes in a cell Haploid: Number of chromosomes in a gamete (germ cell) is called the HAPLOID number for that particular species (N) Diploid: The number of cells in a somatic cell for a particular species (2N) 61 Definitions & Terms Hyperdiploid: greater than the normal (2N) number of chromosomes Hypodiploid: Less than the normal (2N) number of chromosomes DNA Tetraploidy: Containing double the number of chromosomes (4N) 62 Definitions & Terms DNA Index: The ratio between the relative DNA content of the test cells (in G0/G1phase) to the relative DNA content in normal G0/G1 diploid cells S-phase fraction: The percentage of the test cells that are in the synthetic phase of the cell cycle 63 Normal DNA Diploid Peak G0G1 G2M 64 400 DNA ANALYSIS Diploid Peak 320 DNA Index=1.00 240 Aneuploid Peak 0 80 160 Number DNA Index=1.85 0 50 100 150 Channels 200 250 65