Transcript Med Tech Flow Cytometry Lecture

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