Flow Cytometry

Flow Cytometry is a laser based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second.

Flow Cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in basic research, clinical practice and clinical trials. A common variation is to physically sort particles based on their properties, so as to purify populations of interest.

• The basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted and sorted. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths.

• Once the sample is injected into a stream of sheath fluid within the flow chamber, they are forced into the center of the stream forming a single file by the PRINCIPLE OF HYDRODYNAMIC FOCUSING.

• 'Only one cell or particle can pass through the laser beam at a given moment.'

Flow Cytometry technique is applied in two major field......

• 1. Clinical

• 2. Research

• Absolute CD4 counts HIV/AIDS

• HLA B27 assay Joint Pain

• Diagnosis and Classification of leukemia

• Detection of MRDHematological Malignancies

• DNA Ploidy• S Phase fraction Solid Tumours

• TBNK• Phagocytic function defect

Primary Immunodeficiency

disorders

Cont.. • Reticulocyte count• PNH• Osmotic fragility assay

Hemolytic anaemia

• Feto- maternal Hemorrhage• treatment response in Sickle Cell

AnemiaFetal Hb detection

• Platelet receptor assays (Platelet count, GT, BSS)

• Platelet function assay (CD62P, PAC-1)Bleeding Disorders

• CD34 STEM CELL COUNTS• Residual WBC count in leukodepleted

blood packs• Flow cytometry Crossmatch

Transfusion and Transplant

• Surface markers in PMN, Monocytes• Cytokine responseHost Immune

response in Sepsis

•FluidicsProvide a constant stream of sheathTransport the sample to the interrogation pointArrange and focus the cells to the laser intercept•Optics Focus the excitation light Collect the emitted light• ElectronicsConvert the optical signals into electronic signalsSend the signals to the analysis computer•Computer Display data graphically Control instrument settings

BD FACSCanto-II flow cytometer currently has 2 lasers.....

• 1. Red laser• 2. Blue laser

• Violet laser also to be added when required.

MULTIPARAMETRIC RAPID ANALYSIS OF LARGE NUMBER OF CELLS INFORMATION AT A SINGLE CELL LEVELDETECTION OF ATYPICAL CELL POPULATIONS ALLOWS PHYSICAL ISOLATION OF CELLS OF

INTEREST

Specimens accepted for Flow Cytometry are :

1. Peripheral Blood (if for leukemia panel, atypical cell population should be >20%)

2. Bone Marrow

3. Ascitic fluid & Pleural fluid (If adequate cells are present)

Both EDTA & Heparin Tube should be collected.

Following parameter of the cells has to be identified by flow cytometry…..

• Size• granularity or internal complexity• fluorescence intensity

1. HLA-B27 for arthritis and spondylitis.2. CD3/4/8 for monitoring T-cells in auto-immune diseases like HIV.3. Natural Killer cells enumeration for infertility cases.4. PNH clone detection for deficiency of GPI-linked protein.5. Immunophenotyping for leukemia and lymphoma panel on peripheral

blood and bone marrow specimen6. HPV (Human Papiloma Virus) for cervical cancer7. HLA T & B crossmatch for Kidney transplant (serotyping to be done by

PCR)8. Stem cell enumeration for transplant cases

Clinical Purpose and Scope:• HLA-B27 is a major histocompatibility complex (MHC) class-I

molecule. MHC class I molecules are cell surface glycoproteins that are expressed on most nucleated human cells and platelets. There is a strong association between the presence of the HLA-B27 antigen and an increased incidence of ankylosing spondylitis as well as other disorders such as Reiter’s syndrome, psoriatic arthritis and arthropathies associated with inflammatory bowel diseases. These disorders are collectively known as seronegative spondylo-arthropathies. An HLA-B27 positive patient is more likely to exhibit spondylo-arthropathies than an HLA-B27 negative patient.

Clinical purpose and scope:Helper T-lymphocytes (CD3+, CD4+) and suppressor lymphocytes (CD8+) percentages are used to characterize and monitor some forms of immunodeficiency and autoimmune diseases. Determining percentages of helper T lymphocytes may be useful in monitoring human immunodeficiency virus (HIV) infected individuals. These individuals typically exhibit a steady decline in the number of helper T lymphocytes as the infection progresses. The loss of CD4+ helper cells reduces the body’s ability to fight infections especially viruses bacteria, fungi and certain parasites. The CD8 increases in cases of AIDS and the CD4/CD8 ratio gets altered. These counts are useful for predicting prognosis and initiating antibiotic therapy against opportunistic infections in T cell Immunodeficient individuals. Measurement of CD4+ T cell levels has been used to establish decision points for initiating prophylaxis for Pneumocystis caninii pneumonia and other opportunistic infections.

• Clinical purpose and scope

• The Primary use of this antibody is to enumerate the number or percentage of lymphocytes in blood. The Natural Killer cells identified as being CD3-ve/CD16+56+ve have been shown to mediate non MHC restricted cytotoxicity against certain tumors and virus infected cells. They play a crucial role in immunological defence and regulation. Changes in NK cell activity have been useful for monitoring disease outcome or progression. NK cell measurement is a useful prognostic tool for women with recurrent spontaneous abortions.

• Clinical Purpose and Scope:PNH is a rare acquired clonal haematological disorder. Protein manifestations such as haemolytic anaemia, thrombopenia, neutropenia, aplastic anaemia and deep thrombosis evoke PNH. A proportion of the patient’s neutrophils have been demonstrated to be part of the PNH clone in all patients with PNH. The presence of a population of cells with GPI- linked protein is diagnostic of PNH. It is very important to analyze more than one protein because there are rare cases in which an inherited deficiency of one protein has been described. The primary use of these antibodies is to evaluate the percentage of granulocytes in blood which express these antigens (CD55 & CD59) and those which do not express them. The CD55 and CD59 antigens are present on all human leucocytes including lymphocytes, monocytes, granulocytes, eosinophils, basophils, red blood cells and reticulocytes. CD55 and CD59 antigens are absent on the hemopoietic cells of patients suffering from paroxysmal nocturnal hemoglobinuria.The presence of negative population above 5% is normally adopted as a criteria for recognition of the PNH clone.

An overview of PNH

PNH – Clinical FeaturesPNH – Clinical Features

HaemoglobinuriaHaemoglobinuria

Intravascular haemolysisIntravascular haemolysis disabling symptomsdisabling symptoms

- abdominal painabdominal pain- dysphagiadysphagia- erectile failureerectile failure- severe lethargysevere lethargy

Budd-ChiariBudd-Chiarisyndromesyndrome

ThrombosisThrombosis- liver, cerebral- liver, cerebral- 50% of patients- 50% of patients- 33% of patients - 33% of patients is fatalis fatal

Aplastic anaemiaAplastic anaemia

Bone Marrow FailureBone Marrow Failure- often precedes PNHoften precedes PNH- selects for PNH cloneselects for PNH clone

Proteins Deficient from PNH Blood Cells

CD59, CD90, CD109

CD55CD58CD59 CD48CD52PrPcCD16

CD24 CD55CD58 CD59 CD48 PrPC

CD73 CD108

CD55CD58CD59

CD109PrPC

GP500Gova/b

CD55CD58CD59PrPCAChE

JMH AgDombroch

HG Ag

CD55 CD58*CD59 CD14CD16 CD24CD48 CD66bCD66c CD87CD109 CD157LAPNB1 PrPC

p50-80 GPI-80ADP-RT NA1/NA2

CD14 CD55 CD58*CD59 CD48 CD52

CD87 CD109 CD157Group 8 PrPC GPI-80

CD16

CD55 CD58*CD59 CD48CD52 CD87 CD108 PrPcADP-RT CD73CD90 CD109

CD16*

Haematopoietic Stem Cell

Platelets

RBC

PMN

B cells

Monocytes

T cells

NK cells

QuickTime™ and aGIF decompressor

are needed to see this picture.(Courtesy of Lucio Luzzatto)(Courtesy of Lucio Luzzatto)

Why does PNH occur?

PNH clones– Lack complement regulatory molecules and therefore

probably “weakened”– Have no malignant potential– Occur at low levels in normal individuals

BUT:– PNH “always” occurs with aplastic anaemia– Both rare disorders (1 in 100,000+) so unlikely to be

chance

Relative Growth Advantage in PNHNormal stem cells GPI-deficient (PNH) stem cells

GPI-linked GPI-linked antigenantigen

Relative Growth Advantage in PNH

Relative Growth Advantage in PNH

Intense growth factor driven Intense growth factor driven expansionexpansion

Relative Growth Advantage in PNH

Normal red blood cells are Normal red blood cells are protected from complement protected from complement attack by a shield of attack by a shield of terminal complement terminal complement inhibitors inhibitors (2,3)(2,3)

Without this protective Without this protective complement inhibitor complement inhibitor shield, PNH red blood shield, PNH red blood cells are destroyed cells are destroyed (2,3)(2,3)

Intact RBCIntact RBCFree Haemoglobin in Free Haemoglobin in

the Blood from Destroyed the Blood from Destroyed PNH RBCsPNH RBCs

ComplementComplementActivationActivation

Significant Significant Impact on Impact on Morbidity Morbidity (3)(3)

Significant Significant Impact on Impact on Survival Survival (3)(3)

Anaemia Anaemia

HaemoglobinuriaHaemoglobinuria

ThrombosisThrombosis

FatigueFatigue

Renal FailureRenal Failure

Pulmonary Pulmonary HypertensionHypertension

Erectile DysfunctionErectile Dysfunction

DyspnoeaDyspnoea

DysphagiaDysphagia

Abdominal PainAbdominal Pain

PNH is a Progressive Disease of Chronic Haemolysis (1-4)

Renal Damage in PNH

• Chronic haemolysis and cell-free plasma haemoglobin lead to chronic kidney disease in PNH (1,2)

• Renal damage in PNH may be due to repetitive exposure of tissue to cell-free haemoglobin (3,4)

• 64% of patients with PNH have stage 1-5 chronic kidney disease (5)

• Renal failure has been identified as the cause of death in approximately 8 – 18% of PNH patients (6,7)

Laboratory Investigation of PNH

• Flow cytometry immunophenotyping is the method of choice for PNH testing

• Diagnosis or identification of PNH cells by demonstrating deficiency of GPI-linked proteins from granulocytes/monocytes/red cells

Red Cell Analysis: Routine testing

ADVANTAGES Relatively

straightforward Best way to identify

Type II cells RBC clone size

associated with symptoms

DISADVANTAGES Often underestimates

clone size because of transfusion or haemolysis

False negatives common

To detect clone sizes of at least 1%

Routine Red Cell Analysis: Reagents

For historical reasons, CD55 and CD59 are most commonly used

CD59 is strongly expressed, while CD55 is weak− CD55 may not be necessary− Rare congenital CD59 deficiency cases− Some variation in CD59 clones

Other GPI-anchored reagents (CD58) exist, but limited experience

Anti-glycophorin (CD235a) may be used to identify red cells, but this may not be necessary for routine analysis− Can guard against failure of antibody to contact cells

Red cell testing

CD58PECD58PE

CD55 PECD55 PE

CD55 PECD55 PE

CD59 FitcCD59 Fitc

CD59 PECD59 PE

CD59 FitcCD59 Fitc

Leucocyte Analysis: Routine testing

Granulocyte PNH clone probably gives most accurate estimate of PNH clone size

Monocyte clones can usually be determined in same tube and confirms granulocyte result, though because monocytes are less numerous, precision is lower

Type II granulocytes can occasionally be recognized but red cells are typically better for this purpose

Lymphocytes are not a suitable target for testing

Guideline Summary

Granulocyte analysis provides better estimate of size of PNH clone than RBC analysis

Thus, routine red cell analysis not recommended without white cell analysis, though a granulocyte screening assay may be viable, especially in labs with low prevalence of PNH

Lymphocyte analysis not recommended because of lifespan of lymphocytes

CD34+ STEM CELL ENUMERATION

Clinical purpose and scope

The cell surface protein CD34 is frequently used as a marker for positive selection of engrafting human hematopoietic stem and progenitor cells, both in research and in clinical transplantations. Since the small population of cells that bear the CD34 antigen are thought to be responsible for multilineage engraftment, graft assessment by flowcytometric quantitation of CD34 positive cells provide a rapid, reliable and reproducible assay.

Apheresis is a technique by which particular component of blood is removed from the blood and the main volume being returned to the body

SPECIMEN FOR STEM CELL ENUMERATION

Leukemia and Lymphoma

Leukemia

Historic Perspective

•1945•The initial description of leukemia as a clinical entity was made by Bennett in Scotland and in Germany.

Leukemia

• A group of malignant disorders affecting the blood and blood-forming tissues of – Bone marrow– Lymph system– Spleen

• Occurs in all age groups

Leukemia

• Results in an accumulation of dysfunctional cells because of a loss of regulation in cell division

• Fatal if untreated– Progressive

Leukemia

• Often thought of as a childhood disease• The number of adults affected with

leukemia is 10 times that of children

Environmental factors for leukemia

Ionizing radiation• Leukemia is associated with exposure to

ionizing radiation such as nuclear weapons in Hiroshima and Nagasaki.

• Both acute and chronic forms of leukemia including AML, ALL and CML were associated.

Chemical drugs

• A variety of chemicals and drugs have been associated with the development of leukemic transformation

• Examples: Benzene, Chloramphenecol, Phenylbutazone and Cytotoxic alkylating chemotherapeutic agents.

Viruses

• The human T-cell leukemia-lymphoma virus-I (HTLV-I) has been implicated as a causative agent of adult T-Cell leukemia-lymphoma.

• Another related virus HTLV-II has been isolated from patients with atypical hairy cell leukemia (CLL)

• The Epstein’s Barr virus has been linked to Burkitt’s lymphoma.

Acute ChronicAge All ages Adults

Clinical onset Sudden Insidious

Leukemic cells Immature Mature

Anemia Mild to severe Mild

Thrombocytopenia Mild to severe Mild

WBC Variable Increased

Organomegaly Mild prominent

Comparison of acute and chronic leukemia

• Panel of monoclonal antibodies is designed as a screening panel for testing Blood and Bone marrow samples in patients suspected to be suffering from acute leukemias as well as chronic leukemias.

FAB vs WHO Classifications of Hematologic Neoplasm

• FAB criteria– Morphology– Cytochemistry

• WHO criteria– Morphology– Immunophenotypi

ng– Cytogenetic

• Karyotyping• Molecular testing

– Clinical features

Classification of leukemiaMain classification

Chronic leukemia Acute leukemia

Lymphoid LymphoidMyeloid Myeloid

FAB

AMLM0M1M2M3M4M5M6M7

ALL is further divided into 2 categories:

• 1. B – ALL

• 2. T - ALL

WHO Lymphoid Neoplasms

• B cell neoplasms• T/NK cell neoplasms• Hodgkin lymphoma (disease)

Mature B Cell Neoplasms

• B cell CLL/SLL• B prolymphocytic

leukemia• Burkitt’s lymphoma /

leukemia• Splenic marginal

zone B lymphoma• Extranodal marginal

B lymphoma

• Hairy cell leukemia• Lymphoplasmocytic

leukemia• Mantle cell lymphoma• Plasma cell myeloma /

plasmacytoma• Follicular lymphoma• Diffuse large B

lymphoma

T/NK Cell Neoplasms

• T prolymphocytic leukemia

• T granular lymphocytic leukemia

• Aggressive NK cell leukemia

• Adult T lymphoma / leukemia

• Mycosis fungoides (Sezary syndrome)

• Anaplastic large cell lymphoma

• Hepatosplenic T lymphoma

• Peripheral T lymphoma• Immunoblastic T

lymphoma

Leukemia Etiology and Pathophysiology

• No single causative agent • Most from a combination of factors

– Genetic and environmental influences

Leukemia Etiology and Pathophysiology • Associated with the development of

leukemia – Chemical agents– Chemotherapeutic agents– Viruses– Radiation – Immunologic deficiencies

Leukemia Classification • Acute versus chronic

– Cell maturity• Acute: clonal proliferation of immature

hematopoietic cells (the formation of blood or blood cells )

• Chronic: mature forms of WBC; onset is more gradual

– Nature of disease onset

Acute Lymphocytic Leukemia (ALL)

• Most common type of leukemia in children• 15% of acute leukemia in adults• Immature lymphocytes proliferate in the

bone marrow

Acute Lymphocytic Leukemia

• Signs and symptoms may appear abruptly– Fever, bleeding

• Insidious with progressive– Weakness, fatigue

• Central nervous system manifestations

Chronic Myelogenous Leukemia (CML)• Excessive development of mature

neoplastic granulocytes in the bone marrow– Move into the peripheral blood in massive

numbers– Ultimately infiltrate the liver and spleen

Chronic Myelogenous Leukemia

• Philadelphia chromosome– The chromosome abnormality that causes

chronic myeloid leukemia (CML) (9 &22)– Genetic marker

• Chronic, stable phase followed by acute, aggressive (blastic) phase

Chronic Lymphocytic Leukemia (CLL)

• Production and accumulation of functionally inactive but long-lived, mature-appearing lymphocytes

• B cell involvement• Lymph node enlargement is noticeable

throughout the body– ↑ incidence of infection

Chronic Lymphocytic Leukemia

• Complications from early-stage CLL is rare– May develop as the disease advances– Pain, paralysis from enlarged lymph nodes

causing pressure

Hairy Cell Leukemia

• 2% of all adult leukemias• Usually in males > 40 years old• Chronic disease of lymphoproliferation

– B lymphocytes that infiltrate the bone marrow and liver

Hairy Cell Leukemia

• Cells have a “hairy” appearance• Symptoms from

– Splenomegaly, pancytopenia, infection, vasculitis

• Treatment– alpha-interferon, pentostatin, cladribine

Unclassified Leukemias

• Subtype cannot be identified• Malignant leukemic cells may have

– Lymphoid, myeloid, or mixed characteristics• Frequently these patients do not respond

well to treatment– Poor prognosis

Leukemia Clinical Manifestations• Relate to problems caused by

– Bone marrow failure• Overcrowding by abnormal cells• Inadequate production of normal marrow elements• Anemia, thrombocytopenia, ↓ number and function

of WBCs

Leukemia Clinical Manifestations• Relate to problems caused by

– Leukemic cells infiltrate patient’s organs• Splenomegaly• Hepatomegaly• Lymphadenopathy• Bone pain, meningeal irritation, oral lesions

(chloromas)

Leukemia Diagnostic Studies • To diagnose and classify

– Peripheral blood evaluation (CBC and blood smear)

– Bone marrow evaluation• To identify cell subtype and stage

– Morphologic, histochemical, immunologic, and cytogenic methods

Leukemia Collaborative Care

• Goal is to attain remission (when there is no longer evidence of cancer cells in the body)

• Chemotherapeutic treatment– Induction therapy

• Attempt to induce or bring remission• Seeks to destroy leukemic cells in the tissues,

peripheral blood, bone marrow• Patient may become critically ill

– Provide psychological support as well

What is remission?

The main aim of treatment for acute lymphoblastic leukaemia is to give a remission. This means that the abnormal, immature white cells or blasts can no longer be detected in your blood or bone marrow, and normal bone marrow has developed again.

• However, once you are in remission there may still be a very small number of abnormal lymphoblasts left. To destroy these, your doctor may prescribe maintenance or continuation chemotherapy which may last for several years. These drugs are mainly taken as tablets and you will need to have regular check-ups to monitor their effect. Very specialised blood tests to find particular proteins present on the surface of the leukaemia cells can show if any leukaemia cells are still present in the body.

• For many people with acute lymphoblastic leukaemia the remission lasts indefinitely and the person is said to be cured.

Leukemia Collaborative Care • Chemotherapeutic treatment (cont.)

– Intensification therapy• High-dose therapy• May be given after induction therapy• Same drugs at higher doses and/or other drugs

Leukemia Collaborative Care • Chemotherapeutic treatment (cont.)

– Consolidation therapy• Started after remission is achieved• Purpose is to eliminate remaining leukemic cells

that may not be evident– Maintenance therapy

• Lower doses of the same drug

Leukemia Chemotherapy

• Combination chemotherapy– Mainstay treatment – 3 purposes

• ↓ drug resistance• ↓ drug toxicity to the patient by using multiple

drugs with varying toxicities • Interrupt cell growth at multiple points in the cell

cycle

Leukemia - Bone Marrow and Stem Cell Transplantation

• Goal– Totally eliminate leukemic cells from the body

using combinations of chemotherapy with or without total body irradiation

Leukemia - Bone Marrow and Stem Cell Transplantation

• Eradicates patient’s hematopoietic stem cells• Replaced with those of an HLA-matched

(Human Leukocyte Antigen)• Sibling (is a brother or a sister; that is, any person

who shares at least one of the same parents )• Volunteer• Identical twin• Patient’s own stem cells removed before

PATIENT DETAILS REQUIREMENT

Currect patient name Age and sex Reffering doctor’s name & contact details Clinical history Recent CBC & PBS report Type of specimen Collection time of specimen

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• HPV BY FLOW CYTOMETRY

HPV infection and dysplastic transformation

Cancer

Normal

Low-grade squamous

intraepithelial lesion (LSIL)

High-grade squamous

intraepithelial lesion (HSIL)

Modified from Palefsky JM 2011

• Clinical Purpose and Scope:The primary use of this test is to detect circulating donor specific antibodies in the serum of potential allograft recipients. FCM testing detects these preformed anti donor antibodies if present in the recipient’s serum. It is a sensitive test capable of detecting low levels of clinically relevant HLA antibodies. FCXM is most useful when the potential recipient is highly sensitized to HLA antigens or has had previous renal transplantation with rejection/graft failure. It can independently evaluate T and B cell activity in mixed cell populations. The detection of circulating anti HLA antibodies in the serum of potential renal allograft recipients is generally considered to be contradiction to transplantation.