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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
Continued……..
Continued…..
Continued…….
• 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.