Laboratory Procedures

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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

2525

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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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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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

4545


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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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

5050


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

5656


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

Laboratory Procedures

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