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Laboratory Procedures

Laboratory Procedures. Specimen collection, transportation and handling Specimen preparation Panels Reporting of results Data storage and retrieval. Appropriate Specimens for Analysis. Peripheral blood, bone marrow aspirates, body fluids, cerebrospinal fluid

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Laboratory Procedures

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  1. Laboratory Procedures • Specimen collection, transportation and handling • Specimen preparation • Panels • Reporting of results • Data storage and retrieval

  2. 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

  3. 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)

  4. 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.

  5. 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

  6. Specimen Preparation • Erythrocyte lysis. • The lysing agent should remove only mature red cells with minimal effect on the remaining cells. • Density gradient separation

  7. Whole Blood Bench

  8. 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

  9. Density Gradient

  10. 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

  11. 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.

  12. Bone Marrow Staining Bench

  13. Sample Analysis • Side by side placement of cytometers allows a single technologist to operate two instruments simultaneously if needed.

  14. Analysis • Review of histograms • Morphologic assessment • Correlation between the morphologic and phenotypic findings

  15. 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.

  16. 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

  17. Reporting of Results • Listing of antibodies used • Descriptive summary of the phenotype of the abnormal cells, including fluorescence intensity when appropriate

  18. Reporting of Results • Fraction of abnormal cells in the sample • Morphologic findings when appropriate • Interpretation of the phenotype, and differential diagnosis

  19. Lymphocyte Subsets

  20. 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.

  21. Viral Load • Used to see if drug therapy is working • Does not assess immune system

  22. CD # = cluster designation number Immunophenotyping CD3 CD4

  23. CD4/CD8 Gating on Lymphocytes Lymphocytes LYMPHOCYTES

  24. CD4/CD8

  25. 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

  26. CD4/CD8 Gating on lymphocytes Lymphocytes Lymphocytes Lymphocytes

  27. CD4/CD8

  28. 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

  29. Lymphocyte Subsets T cells + B cells + NK cells = Lymphocytes 13.7 + 74.1 + 9.5 = 97.5

  30. Immunophenotyping of Lymphomas and Leukemias

  31. CD Nomenclature • CD45 Leukocyte Common Antigen • CD2, CD3, CD5, CD7 T cells CD4, CD8 • CD19, CD20, CD24 B cells • CD10 CALLA (B cells) • CD56 NK cells • CD34 Stem cell marker • CD13, CD33 Myeloid cells • CD14 Monocytes

  32. CASE STUDY #1The 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.

  33. CASE STUDY #1The 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.

  34. Case Study #2An 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.

  35. Case Study #2An 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.

  36. Case Study #3A 65-year-old woman presented with a WBC of 30,000. The differential showed 90% blasts. A bone marrow was submitted for flow cytometry.

  37. Case Study #3A 65-year-old woman presented with a WBC of 30,000. The differential showed 90% blasts. A bone marrow was submitted for flow cytometry.

  38. Antigen Distribution in AML M0 M1 M2 M3 M4 M5 M6 M7 • CD13 • CD33 • CD15 • CD14 • CD11c • CD4 • Glyco • CD61 • CD34 • HLA-DR

  39. Case Study #4A 5-year-old female presented with bone pain and a WBC of 50,000. A bone marrow sample was submitted for flow cytometry.

  40. Case Study #4A 5-year-old female presented with bone pain and a WBC of 50,000. A bone marrow sample was submitted for flow cytometry.

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