laboratory procedures
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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Laboratory ProceduresLaboratory ProceduresSpecimen collection, Specimen collection,
transportation and handlingtransportation and handlingSpecimen preparationSpecimen preparationPanelsPanelsReporting of resultsReporting of resultsData storage and retrievalData storage and retrieval
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Appropriate SpecimensAppropriate Specimens for Analysis for Analysis
Peripheral blood, bone marrow aspirates, Peripheral blood, bone marrow aspirates, body fluids, cerebrospinal fluidbody fluids, cerebrospinal fluid
Lymphoid tissue biopsies, skin and Lymphoid tissue biopsies, skin and mucosal biopsies, fine needle aspirates mucosal biopsies, fine needle aspirates of tumorsof tumors
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Unacceptable SpecimensUnacceptable Specimens Specimens > 48 hours old Specimens > 48 hours old
(dead cells)(dead cells)
Severe hemolysisSevere hemolysis(loss of cells)(loss of cells)
Clotted specimens Clotted specimens (loss of cells)(loss of cells)
If the specimen is not rejected, a comment If the specimen is not rejected, a comment is included in the interpretation. is included in the interpretation. (irretrievable specimens)(irretrievable specimens)
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Specimen CollectionSpecimen Collection Blood:Blood: 10 cc drawn into an EDTA or 10 cc drawn into an EDTA or
heparin. Minimum requirement is 5 cc.heparin. Minimum requirement is 5 cc. Bone Marrow:Bone Marrow: 2-3 cc in EDTA or 2-3 cc in EDTA or
heparin.heparin. Fluids:Fluids:10-50 cc if cell count is unknown. 10-50 cc if cell count is unknown.
Anticoagulation is not necessary.Anticoagulation is not necessary. CSF:CSF: Any volume is acceptable. Any volume is acceptable.
Anticoagulation is not necessaryAnticoagulation is not necessary Tissue:Tissue: Sterile container, covered with Sterile container, covered with
sterile saline or tissue culture media. sterile saline or tissue culture media.
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Specimen TransportationSpecimen Transportation Immediate transportation to the lab Immediate transportation to the lab
is bestis best
Short-term storage (24-48 hrs) at Short-term storage (24-48 hrs) at room temperatureroom temperature
Refrigerate fluids/tissues if Refrigerate fluids/tissues if analysis takes place >24 hours analysis takes place >24 hours after collectionafter collection
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Specimen PreparationSpecimen PreparationErythrocyte lysis.Erythrocyte lysis.
The lysing agent should remove only The lysing agent should remove only mature red cells with minimal effect mature red cells with minimal effect on the remaining cells.on the remaining cells.
Density gradient separationDensity gradient separation
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Whole Blood BenchWhole Blood Bench
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Erythrocyte LysisErythrocyte Lysis
•Lyses red cells quickly Lyses red cells quickly and with minimal hands and with minimal hands on.on.
•Doesn’t enrich for Doesn’t enrich for mononuclear cells so a mononuclear cells so a small population of small population of abnormal cells could be abnormal cells could be missed.missed.
Beckman Coulter TQ PrepBeckman Coulter TQ Prep
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Density GradientDensity Gradient
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Density Gradient PreparationDensity Gradient Preparation Ficol-hypaque density Ficol-hypaque density
gradient separationgradient separation Enriches mononuclear Enriches mononuclear
cell populationscell populations Removes red cells, Removes red cells,
platelets, dead cells, platelets, dead cells, stromal elements, etc.stromal elements, etc.
Is labor-intensive and Is labor-intensive and time-consumingtime-consuming
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Cell Count and ViabilityCell Count and Viability A high proportion of dead cells can A high proportion of dead cells can
alter phenotyping resultsalter phenotyping results Target is 150,000-200,000 Target is 150,000-200,000 viableviable
cells/tube cells/tube Manual count in trypan blue vital Manual count in trypan blue vital
stain yields count of viable cells prior stain yields count of viable cells prior to staining in order to adjust to staining in order to adjust concentrationconcentration
Viability can be done on a flow Viability can be done on a flow cytometer using 7-AAD, a fluorescent cytometer using 7-AAD, a fluorescent dye which stains the dead cells.dye which stains the dead cells.
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Bone Marrow Staining BenchBone Marrow Staining Bench
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Sample AnalysisSample Analysis Side by side Side by side
placement of placement of cytometers allows a cytometers allows a single technologist to single technologist to operate two operate two instruments instruments simultaneously if simultaneously if needed.needed.
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AnalysisAnalysisReview of histogramsReview of histograms
Morphologic assessmentMorphologic assessment
Correlation between the Correlation between the morphologic and phenotypic morphologic and phenotypic findingsfindings
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Morphologic AssessmentMorphologic Assessment Smear prepared prior to lysis or density Smear prepared prior to lysis or density
gradient separation.gradient separation.
Cytospin prepared following density gradient Cytospin prepared following density gradient separation.separation.
For tissue specimens, a smear or touch prep For tissue specimens, a smear or touch prep can be prepared prior to disaggregation. A can be prepared prior to disaggregation. A cytospin is always prepared following cytospin is always prepared following disaggregation.disaggregation.
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Reporting of ResultsReporting of ResultsPatient Information: Demographics, Patient Information: Demographics,
referring physician and institution, history referring physician and institution, history and clinical findings, prior therapyand clinical findings, prior therapy
Indications for testing and previous flow Indications for testing and previous flow results if availableresults if available
Sample Information: Identification Sample Information: Identification number, source, date and time of number, source, date and time of collectioncollection
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Reporting of ResultsReporting of ResultsListing of antibodies usedListing of antibodies used
Descriptive summary of the Descriptive summary of the phenotype of the abnormal cells, phenotype of the abnormal cells, including fluorescence intensity including fluorescence intensity when appropriatewhen appropriate
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Reporting of ResultsReporting of ResultsFraction of abnormal cells in the Fraction of abnormal cells in the
samplesample
Morphologic findings when Morphologic findings when appropriateappropriate
Interpretation of the phenotype, and Interpretation of the phenotype, and differential diagnosisdifferential diagnosis
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Lymphocyte SubsetsLymphocyte Subsets
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Quantitation of T-cell subsetsQuantitation of T-cell subsets Assesses the immune system of HIV infected Assesses the immune system of HIV infected
persons.persons.
Recommended that the helper/inducer T-cells be Recommended that the helper/inducer T-cells be monitored every 3 to 6 months in all HIV positive monitored every 3 to 6 months in all HIV positive persons.persons.
Helper/inducer T-cell (CD4+ cell) level is a Helper/inducer T-cell (CD4+ cell) level is a criterion for surveillance case definition for AIDS.criterion for surveillance case definition for AIDS.
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Viral LoadViral LoadUsed to see if drug therapy is workingUsed to see if drug therapy is working
Does not assess immune systemDoes not assess immune system
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ImmunophenotypingImmunophenotyping
CD3CD3CD4CD4
CD # = cluster designation number
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CD4/CD8CD4/CD8Gating on LymphocytesGating on Lymphocytes
LYMPHOCYTESLymphocytesLymphocytes
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CD4/CD8 CD4/CD8
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CD4/CD8 Ratio CalculationCD4/CD8 Ratio Calculation CD4/CD8 Ratio = CD4/CD8 Ratio = # of CD4 cells# of CD4 cells # of CD8 cells# of CD8 cells Example:Example:
WBC 6100, 31% lymphocytesWBC 6100, 31% lymphocytesAbs lymph. = 1891 cells/mmAbs lymph. = 1891 cells/mm33; CD4% = 42.6 ; CD4% = 42.6 CD8% CD8%
=35.6%=35.6%CD4/CD8 RatioCD4/CD8 Ratio = = 806806
673673== 1.21.2
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CD4/CD8 CD4/CD8 Gating on lymphocytesGating on lymphocytes
LymphocytesLymphocytes
LymphocytesLymphocytes
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CD4/CD8 CD4/CD8
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CD4/CD8 Ratio CalculationCD4/CD8 Ratio Calculation CD4/CD8 Ratio = CD4/CD8 Ratio = # of CD4 cells# of CD4 cells # of CD8 cells# of CD8 cells Example:Example:
WBC 4300, 24% lymphocytesWBC 4300, 24% lymphocytesAbs lymph. = 1032 cells/mmAbs lymph. = 1032 cells/mm33; CD4% = 3.2; CD4% = 3.2 CD8% CD8%
=81.3%=81.3%CD4/CD8 RatioCD4/CD8 Ratio = = 33 33
839839== 0.00.0
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Lymphocyte SubsetsLymphocyte Subsets
T cells + B cells + NK T cells + B cells + NK cells = Lymphocytescells = Lymphocytes
13.7 + 74.1 + 9.5 = 97.513.7 + 74.1 + 9.5 = 97.5
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Immunophenotyping of Immunophenotyping of Lymphomas and LeukemiasLymphomas and Leukemias
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CD NomenclatureCD Nomenclature
CD45CD45 Leukocyte Common Leukocyte Common AntigenAntigen
CD2, CD3, CD5, CD7CD2, CD3, CD5, CD7 T cellsT cells CD4, CD8CD4, CD8CD19, CD20, CD24 CD19, CD20, CD24 B cellsB cellsCD10CD10 CALLA (B cells)CALLA (B cells)CD56CD56 NK cellsNK cellsCD34 CD34 Stem cell markerStem cell markerCD13, CD33CD13, CD33 Myeloid cellsMyeloid cellsCD14CD14 MonocytesMonocytes
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CASE STUDY #1CASE STUDY #1
The patient is a 66-year-old male who presented with WBC of The patient is a 66-year-old male who presented with WBC of 66,000. The differential showed 79% lymphocytes. A peripheral 66,000. The differential showed 79% lymphocytes. A peripheral
blood sample was sent for flow cytometric analysis.blood sample was sent for flow cytometric analysis.Antigen Positive or Negative (?)
CD2 CD5
CD10 CD19 CD20
HLA-DR CD23 CD24 CD25 Kappa
Lambda
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CASE STUDY #1CASE STUDY #1
The patient is a 66-year-old male who presented with WBC of The patient is a 66-year-old male who presented with WBC of 66,000. The differential showed 79% lymphocytes. A 66,000. The differential showed 79% lymphocytes. A
peripheral blood sample was sent for flow cytometric analysis.peripheral blood sample was sent for flow cytometric analysis.
Antigen Positive or Negative (?) CD2 N CD5 P
CD10 N CD19 P CD20 P, Dim
HLA-DR P CD23 P CD24 P CD25 N kappa N
lambda P
Diagnosis: Chronic Lymphocytic Leukemia
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Case Study #2Case Study #2
An 81-year-old man was found to have a WBC of 141,000 with 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 96% lymphocytes. A peripheral blood sample was sent for flow
cytometry studies.cytometry studies.Antigen Positive or Negative (?)
CD2 CD5
CD10 CD11c CD19 CD20
HLA-DR CD23 CD24 CD25 kappa lambda
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Case Study #2Case Study #2
An 81-year-old man was found to have a WBC of 141,000 with 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 96% lymphocytes. A peripheral blood sample was sent for flow
cytometry studies.cytometry studies.Antigen Positive or Negative (?)
CD2 N CD5 P
CD10 N CD19 P CD20 P
HLA-DR P CD23 N CD24 P CD25 N kappa N
lambda P
Diagnosis: B-Cell Lymphoma (Mantle Cell Lymphoma)
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Case Study #3Case Study #3
A 65-year-old woman presented with a WBC of 30,000. The A 65-year-old woman presented with a WBC of 30,000. The differential showed 90% blasts. A bone marrow was submitted for differential showed 90% blasts. A bone marrow was submitted for
flow cytometry.flow cytometry.
Antigen Positive or Negative (?) CD3 CD7 CD13 CD15 CD33 CD34
HLA-DR
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Case Study #3Case Study #3
A 65-year-old woman presented with a WBC of 30,000. The A 65-year-old woman presented with a WBC of 30,000. The differential showed 90% blasts. A bone marrow was submitted differential showed 90% blasts. A bone marrow was submitted
for flow cytometry.for flow cytometry.
Antigen Positive or Negative (?) CD3 N CD7 P CD13 P CD15 N CD33 N CD34 P
HLA-DR P Diagnosis: Acute myelogenous leukemia
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M0 M1 M2 M3 M4 M5 M6 M7M0 M1 M2 M3 M4 M5 M6 M7 CD13CD13 CD33CD33 CD15CD15 CD14CD14 CD11cCD11c CD4CD4 GlycoGlyco CD61CD61 CD34CD34 HLA-DRHLA-DR
Antigen Distribution in AML
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Case Study #4Case Study #4
A 5-year-old female presented with bone pain and a WBC of A 5-year-old female presented with bone pain and a WBC of 50,000. A bone marrow sample was submitted for flow cytometry.50,000. A bone marrow sample was submitted for flow cytometry.
Antigen Positive or Negative (?) CD3
CD10 CD15 CD19 CD20 CD24 CD34
HLA-DR
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Case Study #4Case Study #4
A 5-year-old female presented with bone pain and a WBC of A 5-year-old female presented with bone pain and a WBC of 50,000. A bone marrow sample was submitted for flow cytometry.50,000. A bone marrow sample was submitted for flow cytometry.
Antigen Positive or Negative (?) CD3 N
CD10 P CD15 N CD19 P CD20 N CD24 P CD34 N
HLA-DR P
Diagnosis: Acute lymphoblastic leukemia
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Case Study #5Case Study #5A 17 year old girl presents with “pancytopenia”: WBC=2.3, Plt Ct=31.A 17 year old girl presents with “pancytopenia”: WBC=2.3, Plt Ct=31.
AntigenAntigen Positive or Negative(?)Positive or Negative(?)CD3CD3
CD4CD4
CD8CD8
CD10CD10
CD13CD13
CD19CD19
CD20CD20
CD24CD24
CD33CD33
CD34CD34
CD117CD117
HLA-DRHLA-DR
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Case Study #5Case Study #5
AntigenAntigen Positive or Negative(?)Positive or Negative(?)CD3CD3 NN
CD4CD4 NN
CD8CD8 NN
CD10CD10 PP
CD13CD13 NN
CD19CD19 PP
CD20CD20 NN
CD24CD24 PP
CD33CD33 NN
CD34CD34 PP
CD117CD117 NN
HLA-DRHLA-DR PP
Diagnosis: Acute lymphoblastic leukemia
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Antigen Distribution in B Lineage ALLAntigen Distribution in B Lineage ALL pro-B pre-pre-B pre-B pro-B pre-pre-B pre-B Burkitt’s Burkitt’s
HLA-DRHLA-DR CD34CD34 CD19CD19 CD24CD24 CD10CD10 CD20CD20 TdTTdT CyIgMCyIgM SIgMSIgM Ig Genes RIg Genes R
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DNA Cell Cycle Analysis by DNA Cell Cycle Analysis by Flow CytometryFlow Cytometry
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Clinical applications of DNA cell cycle Clinical applications of DNA cell cycle analysisanalysis
The DNA content and/or the synthetic phase The DNA content and/or the synthetic phase fraction provide prognostic information in:fraction provide prognostic information in:Breast carcinomaBreast carcinomaColon carcinomaColon carcinomaPediatric acute lymphoblastic leukemiaPediatric acute lymphoblastic leukemia
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GG22MM GG00
GG11
ss
0 200 400 600 800 1000
GG00 GG11
ss GG22 MM
DNA AnalysisDNA Analysis
DNA content
Count
2N2N 4N4N
Normal Cell Cycle Normal Cell Cycle
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Definitions & TermsDefinitions & Terms
PloidyPloidyRelated to the number of chromosomes in a cellRelated to the number of chromosomes in a cell
HaploidHaploid: Number of chromosomes in a : Number of chromosomes in a gamete (germ cell) is called the HAPLOID gamete (germ cell) is called the HAPLOID number for that particular species (N)number for that particular species (N)
DiploidDiploid: The number of cells in a somatic : The number of cells in a somatic cell for a particular species (2N)cell for a particular species (2N)
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Definitions & TermsDefinitions & TermsHyperdiploidHyperdiploid: greater than the normal (2N) : greater than the normal (2N)
number of chromosomesnumber of chromosomes
HypodiploidHypodiploid: Less than the normal (2N) : Less than the normal (2N) number of chromosomesnumber of chromosomes
DNA TetraploidyDNA Tetraploidy: Containing double the : Containing double the number of chromosomes (4N)number of chromosomes (4N)
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Definitions & TermsDefinitions & TermsDNA IndexDNA Index: The ratio between the relative DNA : The ratio between the relative DNA
content of the test cells (in G0/G1phase) to the content of the test cells (in G0/G1phase) to the relative DNA content in normal G0/G1 diploid relative DNA content in normal G0/G1 diploid cellscells
S-phase fraction: The percentage of the test S-phase fraction: The percentage of the test cells that are in the synthetic phase of the cell cells that are in the synthetic phase of the cell cyclecycle
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Normal DNANormal DNA
Diploid Peak G0G1
G2M
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DNA ANALYSISDNA ANALYSIS
Channels0 50 100 150 200 250
Num
ber
080
160
240
320
400
Diploid Peak
DNA Index=1.00
Aneuploid Peak
DNA Index=1.85