This document discusses flow cytometry and its application in cancer diagnosis and molecular testing. It provides details on several tests performed using flow cytometry, including immunophenotyping for different types of leukemia and lymphoma. It also discusses the classification of acute leukemias according to the FAB and WHO systems and provides information on different FAB subtypes of acute myeloid leukemia such as M0, M1, M2, M3, M4, and M5. The document aims to inform clinicians on the diagnostic and clinical utility of various flow cytometry tests.

1. Dr Tanjina Afrin
Consultant Hematologist
DMFR
World-class
Diagnostics
Healthcare
CANCER DIAGNOSIS-
MOLECULAR TESTING

2. Flow Cytometry
&
its Application

36. Flow cytometry
Name of Test Method Specimen Type
Turn Around
Time
Immunophenotyping for Acute
Leukemia
Flow
Cytometry
Blood/Bone
Marrow
5 Days
Immunophenotyping for
Lymphoma
Flow
Cytometry
Blood/Bone
Marrow
5 Days
Immunophenotyping for
Primary Immunodeficiency
(PID)
Flow
Cytometry
Blood/Bone
Marrow
5 Days
Immunophenotyping for CLL
Flow
Cytometry
Blood/Bone
Marrow
5 Days
CD4 Count Flowcytometry
Blood/Bone
Marrow
7 Days
CD8 Count Flowcytometry
Blood/Bone
Marrow
7 Days
CD4/CD8 Ratio Flowcytometry
Blood/Bone
Marrow
7 Days
Stem Cell Enumeration
(CD34)
Flowcytometry
Blood/Bone
Marrow
5 Days

37.  Adult Hematology
 Pedi Hematology
 Medicine/Virology
 Blood Transfusion
 Transplantation
The Marketing
Opportunity

39.  Flow Cytometry measures multiple characteristics of individual
particles flowing in single file in a stream of fluid.
 Light scattering at different angles can distinguish differences in size
and internal complexity ,here as light emitted from fluorescently
labelled antibodies can identify a wide array of cell surface and
cytoplasmic antigens,
 This approach makes flow cytometry a powerful tool for detail
analysis of complex populations in a short period of time.
APPLICATIONS

40.  Immunophenotyping by flow cytometry is an important tool in the diagnosis and staging of
patientswith a haematological neoplasm. It is used in conjunction with classical morphology.
 In the bone marrow, normal blood cells develop from stem cells in a progressive series
ofdifferentiations, branching off to give different lineages .of cells
 Malignancies can arise at different stages in the development of a cell. A leukacmia or lymphoma
willexprass a specific set of markers depending on the stage and pathway of differentiation and
they areclaśsified accordingly.
 The initial evaluation is made with a panel of antibodies usually using them in three or more
colourcombinations. The basic markers include:
B cell: CDS, CD1O. CD19, CD20, CD45, Kappa, Lambda;T cell: CD2, CD3,CD4, CDS, CD7, CD8,
CD45, CD56:Myelomonocytic: CD7, CDIlb, CDI3, Cd14, CdIs, CDi6, Cd33. Cd34, cd45, cd56,
Cd117. HLA-dr;Plasma cell: CDI9. CD38, CD45, CDS6. CDI38
ANALYSIS OF LEUKAEMIAS AND LYMPHOMAS

41.  Minimal residual disease (MRD) was defined as disease beyond the limit
ofmorphological detection using conventional microscopy.
 Patients with acute leukaemia were considered to be in remission when
bonemarrow samples contained <5% neoplastic cells,
 Flow cytometric methods can detect far lower levels of disease, which can
beimportant in the clinical management of leukaemia.
 The residual tumour cells are detected using immune&luorescence of
surfacemarkers.
 A panel of at least three antibodies is used, the antibodies being selected on
thebasis of the immunophenotype of the original leukaemia.
ETECTION OF MINIMAL RESIDUAL DISEASE

42.  Hematopoietic stem cells in the bone marrow can be identified by their expression of
CD34
 Normally, the number of such cells in bone marrow is low and is negligible inperipheral
blood.
 Autologous transplantation of circulating hematopoietic progenitors cells revealed
considerable clinical advantages in comparison with bone marrow derived
hematopoietic progenitors.
 These cells can be used to repopulate a depleted bone tfrrow after, for cxample,high
dose chemotherapy distinguished by flow
 Using immunofluorescence, progenitors cells can becytometry in less than 1 h based
on the expression of surface membrane molecules.
STEM CELL ENUMERATION

43. T Cell cross-
match
 Flow cytometry can be used to crossmatch a
recipient's serum with donor lymphocytes to
detect antibodies
that could interfere with engraftment.
Priọr to organ transplantation, the organ
lymphocytes are incubated with donor'sSerum
from the potential recipient of the graft.
 After washing. bound immunoglobulins are
detected using an FITC-conjugated anti-cells
are antibody. The human IkgG identified using
a PE-CD3 conjugatc.
SOLID ORGAN TRANSPLANTATIONT
Post-operative
monitoring
 After the organ transplant, analysis of the
peripheral blood lymphocytes may help to
indicate early rejection and bone marrow
toxicity during immunosuppressive therapies,
and to help in the differentiation of infections
from transplant rejection.
 A variety of cell surface marker s and
activation antigens can be used depending
on organ and the clinical condition & the
Organ transplanted.

44.  Autoantibodies to leucocytes, platelets and erythrocytes may be found in a variety of
autoimmune conditions and can cause anaemia, leukopenia, or thrombocytopenia.
 They are detected by immunofluorescence in either a direct or an in direct assay
 In the former, anti-human Ig are used to detect Ig on the surface of the patient's cells.
 In the indirect assay, the reaction of antibodies in the patient's serum with cells from a
normal person is observed.
 The procedures are similar to those used for a T cell cross match
DETECTION OF AUTOANTIBODIEs

45.  Foeto-materal bleeding can scnsitive a Rhesus blood group D-ve mother to D+ve blood cells from the
foctus.
 In a subsequent pregnancy, hacmolytic discase of the new born child can be caused by the destruction
of Rhesus D +ve blood cells of the foetus by matermal anti-D antibodies.
 Prophylactic anti-D given to the Rhesus D -ve mother shortly after delivery of a Rhesus D+ve child
significantly reduces the incidence of anti-D sensitization in the mother and has led to the virtual
elimination of the disease from mothers so treated.
 Since the dose of anti-D given is related to the size of the focto-matcral hacmorrhage quantitation of
foctal-maternal hacmorrhage is therefore important
 Quantitation is achieved by labelling the erythrocytes in a sample of maternal blood with FITC-
conjugated, non-agglutinating anti-D antibodies.
 A population of as few as 0.1% foctal cells is sufficient to sensitize the parent
FOETO-MATERNAL HAEMORRHAGEF

46.  Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired disease characterized by the
development of an abnormal clone of precursor cells in the bone marrow.
 The white cells and red cells produced are dysfunctional and are susceptible to lysis
 Conventional laboratory tests for the diagnosis of PNH include the sugar water testand the Ham's acid
haemolysis test. Problems associated with these tests include stringent specimen requirements and
limited specificity.
 Analysis of this clonal abnormality by flow cytometry, in general, can be accomplished by analysis of
CD55 and CD59 on red cells specific for decay-accelerating factor and membrane-inhibitor of reactive
lysis, respectively.
PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

47. ACUTE LEUKAEMIAS

48. LEUKAEMIA
The leukemia's are a group of disorders
characterized by the accumulation of
malignant white cells in the bone marrow and blood.

49. CLASSIFICATION
A ) According to the clinical course of disease-
● Acute leukaemia.
● Chronic leukaemia.
B) According to the cell line predominance:
● Myeloid leukaemia.
●Lymphoid leukaemia.

50. ACUTE LEUKAEMIA
Definition :
Presence of ≥ 20 % blast cells in the blood or bone
marrow at clinical presentation (Except M3 variety)
However, it can be diagnosed with less than 20%
blasts if specific leukaemia-associated cytogenetic or
molecular genetic abnormalities are present.

51. Acute leukemia
Aggressive disease
Symptoms are due to :
1. Bone marrow failure
2. Infiltration of organs
Proliferation of cells which do not mature.
Acute myeloid leukemia - more common in adults.
Lymphoblastic variety - more common in
children.

52. Classification
French- American- British (FAB) Classification:
Based on cell morphology on May-Grunwald-Giemsa
(MGG) staining of peripheral blood and bone
marrow smears with the addition of simple
cytochemical techniques
WHO (World Health Organization)classification:
Based on clinical data, morphology, cytochemistry,
immunophenotype, cytogenetic and molecular
biology.

53. French-American-British
(FAB) Classification
 Acute myeloid leukaemia :
M0 - Minimally differentiated AML.
M1 - AML without maturation
M2 - AML with maturation
M3 - Acute promyelocytic leukaemia.
M4 - Acute myelomonocytic leukaemia.
M5 - Acute monocytic leukaemia.
M6 - Acute erythroleukaemia.
M7 - Acute megakaryocytic leukaemia.
 Acute lymphoblastic leukaemia :
L1- Homogeneous small lymphoblasts;
L2 -Heterogeneous lymphoblasts;
L3 -Large homogeneous lymphoblasts

54. French-American-British
Classification of AML
Class Incidence Marrow morphology and comments.
M0 2 – 3% Blasts
• lack definite cytologic & cytochemical
markers (e.g. myeloperoxidase -ve)
• express myeloid lineage antigens
. resemble myeloblast ultrastructurally.
M1 20% •Very immature
• ≥3% are peroxidase +ve.
•Few granules or Auer rods.
•little maturation beyond the myeloblast stage.

55. Mo

56. Figure : M1 blast cells show few granules but may show
Auer rods, as in this case

57. Class Incidence Marrow morphology and comments
M2 30 – 40 % • Full range of myeloid maturation.
• Auer rods present in most cases.
• Myeloid cells myeloperoxidase positive.
• Presence of t (8;21) defines a prognostically
favourable subgroup.
M3 5 – 10% • Hypergranular promyelocytes.
• Many Auer rods per cell,often in parallel bundles
( faggots )
• Most cell myeloperoxidase positive.
• Younger patients ( 35 – 40 yr.)
• DIC .
• t (15;17) translocation - characteristic.

58. Figure : M2 cells show multiple cytoplasmic granules;

59. Figure : AML, M3 subtype: microgranular variant. The usually
bilobed cells contain numerous small azurophilic granules.

61. Class Incidence Marrow morphology and comments
M4 15 – 20% • Myelocytic and monocytic differentiation
• Immatue and mature cells of both myeloid and
monocytic lineage.
• Myeloid elements resemble M2 AML.
• Myeloid elements +ve for myeloperoxidase.
• Monoblasts: + ve for nonspecific esterases.
M5 10% • M5a (poorly differentiated ) :
monoblasts & promonocytes predominate
in marrow and blood.
( monoblasts : peroxidase - ve,
nonspecific esterase +ve)
• M5b(diferentiated) :
mature monocytes predominate in the
peripheral blood.

62. Figure : M4 blasts have some monocytoid differentiation

63. Figure : M5a-monoblastic
leukaemia in which >80% of
blasts are monoblasts;
Figure : M5b- monocytic but
<80% of blasts are
monoblasts.

64. Class Incidence Marrow morphology and comments
M6 5% • Erythroblasts > 50% of marrow nucleated cells
• Dysplastic erythroid precursors.
Some are megaloblastoid
Others with giant or multiple nuclei.
• Myeloblasts and promyelocytes increased.
• Periodic acid schiff stain positive
• Nonerythroid precursors –
>30% are myeloblasts;
M7 1% • Megakaryoblast,some with cytoplasmic budding
• Positive platelet peroxidase reaction
• Myelofibrosis or increased marrow reticulin.

65. Fig : M6 showing preponderance of erythroblasts

66. Fig : M7-megakaryoblastic leukaemia showing
cytoplasmic blebs on blasts.

67. FAB Classification of acute
lymphoblastic leukaemia:
● L1 Homogeneous small lymphoblasts;
scanty cytoplasm,
regular round nuclei,
inconspicuous nucleoli.
● L2 Heterogeneous lymphoblasts;
variable amounts of cytoplasm,
irregular or cleft nuclei,
large nucleoli.
● L3 Large homogeneous lymphoblasts,
Burkitt cell-type;
basophilic cytoplasm,
round nuclei,
prominent nucleoli,
cytoplasmic vacuolation.

68. Fig. ALL, L1 subtype: rather small, uniform blast cells with scanty cytoplasm,
and rounded or cleft nuclei with usually a single nucleolus.

69. Fig. ALL, L2 subtype: blast cells which vary considerably in size and amount
of cytoplasm; the nuclear/ cytoplasmic ratio is rarely as high as In L1. The
nuclei are variable in shape and often contain many nucleoli.

70. Figure: ALL, L3 subtype: blast cells with deeply staining, cytoplasm
containing numerous small perinuclear vacuoles.

71. Lymphoma
It is a defined as lymphoma is a cancer
of . lymph node
lymphoma is a type of cancer that
begins in immune system cells called
lymphocytes .production of one or
more abnormal cells in one or more of
the lymph nodes.

72. Abnormal lymphocytes collect in one or more
lymph node or in lymph tissues such as spleen
,tonsils and eventually they from mass of cells called
tumor.
Cancer that forms in the germ-fighting
lymphatic system is called lymphoma.
Lymphoma: A tumor of the lymphoid tissue. The major
types of lymphoma are Hodgkin's disease and non-
Hodgkin's lymphoma

73. Lymphoma
Main types:
1)Hodgkin lymphoma
2) Non Hodgkin lymphoma
# B-cell lymphoma
# T-cell lymphoma

74. B lymphocytes (B cells): B cells make •
proteins called antibodies to help protect
the body from germs (bacteria and
viruses).
T lymphocytes (T cells): There are several
•
types of T cells. Some T cells destroy
germs or abnormal cells in the body. Other
T cells help boost or slow the activity of
other immune system cells.

75. TYPES OF H L
Nodular Sclerosis HL is the most common • subtype
of HL, accounting for 60 to 80 percent of all HL cases.
In nodularsclerosis CHL, the involved lymph nodes
contain RS cells (Reed/Sternberg ) mixed with normal
white blood cells. The lymph nodes often contain a lot
of scar tissue, which is where the name nodular
sclerosis (scarring) originates.

76. Lymphocyte-Depletion
. Involvement of lymph node by HIV- •
associated classic Hodgkin lymphoma of the
lymphocyte depletion subtype. Large Hodgkin Reed-
Sternberg (HRS) cells with multiple nuclei and
prominent nucleoli are present. HRS cells express
the typical phenotype with intense staining

77. The Revised European American
Lymphoma Classification (REAL)
I. Precursor B-cell neoplasm:
# Precursor B-lymphoblastic leukemia/lymphoma
II. Mature (peripheral) B-cell neoplasms
# B-cell chronic lymphocytic leukemia / small lymphocytic
lymphoma
# Lymphoma of mucosa-associated lymphoid tissue type
(MALT) # Follicular lymphoma
# Mantle cell lymphoma
# Diffuse large cell B-cell
lymphoma # Burkett's lymphoma

78. The Revised European American
Lymphoma Classification (REAL)
I. Precursor T cell neoplasm:
# Precursor T-lymphoblastic lymphoma/leukemia
II. Mature (peripheral) T cell and NK-cell neoplasms
# Adult T cell lymphoma/leukemia (HTLV1+) # Mycosis fungoides/Sézary's
syndrome
# Peripheral T cell lymphoma, not otherwise characterized
# Anaplastic large cell lymphoma, T/null cell, primary systemic type
T-Cell and Natural Killer Cell Neoplasms

79. Hodgkin lymphoma
1) Nodular lymphocyte
predominance Hodgkin's
lymphoma
2) Classical Hodgkin's lymphoma
Nodular sclerosis Hodgkin's lymphoma
Lymphocyte-rich classical Hodgkin's
lymphoma
Mixed cellularity Hodgkin's lymphoma
Lymphocyte depletion Hodgkin's lymphoma

80. Hodgkin lymphoma
 Definition:
A neoplastic transformation of lymphocytes particularly in
lymph nodes.
Characterized by:
1) the presence of Reed-Sternberg cells on histology
2) spreading in an orderly fashion to contagious lymph nodes
( For example, Hodgkin lymphoma that starts in the cervical lymph nodes
may
spread first to the supraclavicular nodes then to the axillary nodes )

81. Epidemiology

82. Clinical presentation
 Enlarged, painless, rubbery, non- erythematous, nontender lymph
nodes are the hallmark of the disease.
 May become painful after drinking alcohol
 Patients may develop ‘’B’’symptoms which are:
# Drenching night sweats.
# 10% weight loss
# Fever
 25% have ''B'' symptoms
 Although pruritus is common in the disease it is not one of the ‘’B’’
symptoms.
 Cervical, supraclavicular and axillary lymphadenopathy are the
most common initial signs of the disease.

83. Clinical
presentation
Extralymphatic sites may be involved such
as: # Spleen
# Liver
# Bone
marrow # Lung
# CNS
Extralymphatic involvement is more common with
non- hodgkin lymphoma.

84. Clinical presentation
Emergency presentation:
Infections
SVC obstruction ( facial edema, increased JVP
and Dyspnea)

85. Staging
The doctor considers the following to determine
the stage of Hodgkin lymphoma:
The number of lymph nodes affected.
Whether these lymph nodes are on one or both sides of
the diaphragm.
Whether the disease has spread to the bone
marrow, spleen, liver, or lung.
Each stage is divided into A or B symptoms according to
the presence of systemic symptoms.

86. Investigations used for staging
 Chest x-ray : X-ray pictures may show swollen lymph nodes or other signs of disease in the
chest .
 CT: Chest, abdomen and pelvis ( CT is sensitive enough to detect any
abnormal nodes)
 MRI
 PET scan
 LP for CSF cytology if any CNS signs
 Lymphangiography and laparotomy are no longer used for staging.
 A bone marrow biopsy is used when :
1) B symptoms
2) Stage3 or 4

87. Abnormal lab tests ( don’t alter
the stage of the disease)
CBC: anemia and high WBC ( Eosinophilia is
common)
LDH: high ( poor prognostic factor)
ESR: high ( poor prognostic factor)
LFTs: help determine the need for liver biopsy

88. After lymphoma is diagnosed, a variety of tests may be carried
out to look for specific features characteristic of different types
of lymphoma. These include:
1) Immunophenotyping
2) Flow cytometry
3) FISH testing.
The classification of lymphoma can affect treatment and prognosis.
Classification systems generally classify lymphoma according
to:
1) Whether or not it is a Hodgkin lymphoma.
2) Whether the cell that is replicating is a T cell or B cell.
3) The site that the cell arises from.

89. Histoogy
Hodgkin has several histological subtypes.
Lymphocyte-predominant has the
Best prognosis.
Lymphocyte-depleted has the Worst
prognosis.