Use of flow cytometry in non neoplastic hematologic conditions
- 1. USE OF FLOW CYTOMETRYIN NON- MALIGNANT HEMATOLOGIC DISORDERS - Muneerah Saeed
- 2. - INTRODUCTION • Multiparameter flow cytometry (FCM) has become an integral part of the diagnosis and classification of hematological malignancies. • However, several nonmalignant or premalignant disorders also benefit from this technology in hematology lab. • Study of normal immunophenotypic characteristics of peripheral blood cell subsets and their modifications in several clinical conditions 2
- 3. - BRIEF OVERVIEW -BASIC PRINCIPLES • A flow cytometer is an instrument that evaluates the physical and/or chemical characteristics of single cells / particles as they pass individually in a fluid stream through a measuring device and sensing point illuminated by an incident light. • The ability of various components of the cells, such as surface receptors, intracellular molecules, and DNA, to bind with fluorescent probes allows their detection and evaluation. • The flow cytometer consists of four systems: • Fluidics • Optics • Electronics • Computer interface 3
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- 5. - CLINICALAPPLICATIONS IN HEMATOLOGY NON- ONCOLOGIC RBC evaluations Cell surface analysis 1. Identification of PNH cells 2. Identification of RBC phenotypes such as Rh D type Intracytoplasmic applications 1. Enumeration of reticulocytes 2. Detection of hemoglobin F in RBCs, such as for fetomaternal hemorrhage 5
- 6. - Platelet evaluations Cell surface analysis 1. Detection of platelet activation 2. Diagnosis of Bernard-Soulier syndrome and Glanzmann thrombasthenia 3. Diagnosis of GpIa and GpVI deficiencies 4. Diagnosis of α granule deficiency 5.Detection of antiplatelet antibodies Intracytoplasmic applications α or δ granule deficiency (eg, Hermansky-Pudlak syndrome) 6
- 7. - WBC evaluations I. Cell surface analysis I. Leukocyte subset identification and enumeration such as CD4+ T-cells for HIV+ patients II. Detection of specific antigen expression on leukocytes, such as HLA-B27 III. CD34+ and other lineage stem cell detection and enumeration IV. Identification of paroxysmal nocturnal hemoglobinuria (PNH) cells in suspected cases II. Intracytoplasmic applications I. Detection of cytoplasmic enzymes, such as myeloperoxidase (MPO) in MPO deficiency II. Detection of cytoplasmic CD3, CD22, CD79, immunoglobulin heavy and light chains, and bcl-2 expression III. Detection of intracytoplasmic cytokines IV. Detection of bound antibodies to intracytoplasmic antigens III. Intranuclear applications Detection of terminal deoxynucleotidyl transferase, cyclins, Ki-67, and other antigens 7
- 8. - • Reticulocyte count • PNH • Hereditary Spherocytosis • Estimation of F cells (HbF) • Malaria detection RBC disorders & Erythropoiesis
- 9. - RETICULOCYTE COUNT • Can be differentiated from mature RBC on RNA content • The laboratory protocol is easily and rapidly performed • It is adequate for blood samples with increased or normal number of reticulocytes; • It is comparable with the automated methods available Commonly used dyes • Thiazole orange • Thioflavin T • Auramine O • Coriphosphine O Gating- FSC vs Fluorescence
- 10. - PAROXYSMAL NOCTURNAL HEMOGLOBINURIA • A rare acquired clonal hematopoietic stem cell defect; an acquired somatic mutation in PIG-Agene. • Affected PNH cells have absent or diminished expression of GPI-linked proteins(including CD55 & CD59) on the cell membrane • FCM analysis can both determine the percentage of abnormal cells and identify discrete populations with different degrees of deficiency( Particularly on Erythrocytes) 1. PNH III- Complete deficiency of GPI-Aps 2. PNH II - Subtotal deficiency (usually ∼10% of normal expression) 3. PNH I- Normal expression
- 11. - 11 • Quantitation of at least 2 GPI- Aps- • exclude the possibility of an inherited, isolated deficiency of a single GPI- AP • No single GPI-anchored protein is always expressed on all hematopoietic cell • FLAER binds directly to the GPI and, hence, more sensitive. • RBCs - underestimate extent of the PNH clone • ↓ PNH RBC survival, subsequent enrichment of the sample for normal RBCs especially if done after a hemolytic episode • Transfusion • Granulocyte - samples >2 days old not reliable • Monocytes - usually smaller numbers compared to granulocytes. Problem in analyzing pancytopenic patient. Antibodies Useful in PNH Testing-ICCS Type of analysis Target cell Gating strategies Informative reagents Routine Red cells Log FSC/SSC; glycophorin A CD59 (CD55) Granulocyte s CD45/SSC or CD15 (or equivalent)/SSC FLAER, CD24, CD66b, CD16; two reagents preferred. CD55/CD59 combination not recommended. Monocytes CD45/SSC or CD33/SSC or CD64/SSC or CD163/SSC FLAER, CD14, CD48, CD55, CD157 High sensitivity Red cells Glycophorin A + scatter CD59+/- CD55 in same or different colors Granulocyte s CD15/SSC FLAER, CD24, CD66b, CD16a; two reagents essential. CD55/CD59 combination not recommended
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- 14. - RecommendationsforflowcytometricanalysisindiagnosisandmanagementofPNH 14 For patients with clinical evidence of hemolysis (classic PNH and PNH/aplastic anemia) • At diagnosis, flow cytometric analysis of both erythrocytes and granulocytes/monocytes is recommended. • After establishment of the diagnosis, flow cytometric analysis is recommended every 3months until the clone size is shown to be stable for 2 years • AA patients with no detectable clone should be screened every 6 months for 2 years and yearly thereafter if the parameters are stable For patients with aplastic anemia or RCUD without clinical evidence of hemolysis • At diagnosis, analysis of erythrocytes and granulocytes using high-sensitivity flow cytometry. • There is no role for serially monitoring the size of the PNH clone in MDS
- 15. - HEREDITARY SHEROCYTOSIS 15 • Among the Most Common causes of Inherited Hemolytic Anemia • HS results from the deficiency or dysfunction of red blood cell membrane proteins, such as α spectrin, β spectrin, ankyrin, Band-3 protein, etc • Current guidelines recommend the use of flow cytometry-based methods as a screening test for the diagnosis of HS. • There are two types of tests using flow cytometry: flow cytometric osmotic fragility (FCM OF) and eosin-5′-maleimide (EMA) binding test
- 16. - FCM OSMOTIC FRAGILITY 16 • Based on the susceptibility or resistance of red cells to lysis when exposed to deionized water (DW) • A red cell suspended in isotonic NS undergoes hemolysis when exposed to DW, Then the red cell count is measured sequentially by FCM in real-time before and after DW spiking Advantages • time and labor-saving; quantitative and objective; • cost-effectiveness; no need for pre-incubation of blood samples; • high test efficiency rates; and it may be performed with K2 EDTA • ~85.7% sensitivity and 97.2% specificity • Can be utilized to indicate clinical severity in HS patients • Studies- method of choice for routine diagnostics to screen for HS
- 17. - EOSIN-5′-MALEIMIDE (EMA) BINDING TEST 17 • Based on the principle that the dye binds to band 3 protein, an important red cell membrane component • A positive result for HS is indicated by a reduction in the percentage of the mean channel fluorescence(MCF) Positive: % fluorescence reduction is > 21% Negative: when < 16% Equivocal: between 16 and 21% • High sensitivity (92.7%–96.6%) and specificity (99.1%) for HS • Decreased fluorescence : Hereditary pyropoikilocytosis, cryohydrocytosis, congenital dyserythopoietic anemia type II and South-East Asian ovalocytosis It is recommended that EMA staining should be performed on the day of blood sample collection in patients with significant hemolysis, because this may affect test results
- 18. - 18 Advantages: Time-efficient, laborsaving, quantitative, objective and cost-effective. Results not influenced by storage for up to 6 days Discriminates between different red cell populations, so not affected by recent transfusions Disadvantages: EMA dye is relatively expensive and its working solution is unstable to light Lack of universal reference ranges for normal controls and HS individuals. The results of the EMA test, obtained from different laboratories, can vary significantly and are not comparable with one another due to the different fluorescence scales from different flow cytometer models It is also important to standardize the software on which the results will be evaluated.
- 19. - HBF CELLS ESTIMATION • For conditions such as: Fetomaternal hemorrhage (FMH) β- thalassemia Sickle cell disease treated with hydroxyurea (to boost hemoglobin F production), etc • Detection by only anti–Hb F antibody for FMH underestimates massive FMH and the use of anti-D Ab more accurately quantifies FMH • The advantage is that no Rh D cells exist in Rh D– mothers, unlike the presence of low quantities of HbF in healthy adults • The availability of phycoerythrin-labeled anti-D and fluorescein isothiocyanate–labeled anti–HbF permits the use of both for improved sensitivity and specificity. • However, determination and quantification of “F cells” and intensity of hemoglobin F per RBC can be done using only anti–HbF. Other markers such as anti–glycophorin A and anti-CD71 (transferrin receptor) can be used to isolate RBCs
- 20. - 20 Fetomaternal hemorrhage detection using anti‐HbF antibody
- 21. - RBC IMMUNOPHENOTYPING 21 • Provide information about specific antigens on the cell surface. • Stem cell transplant patients or for serial quantification of RBC engraftment • Confirmation of a true Rh-null RBC phenotype also has been achieved using FCM. • Early RBC progenitors (esp. when numerous) may simulate other progenitors.. especially in cytopenias. Cells initially mistaken for myeloblasts but later identified as erythroid precursors using anti–glycophorin A in severe vitamin B12 deficiency
- 22. - MALARIA DETECTION • Disadvantages • Not as rapid & cheap as QBC & immunochromatography • Not as accurate as PCR • False positive in high reticulocytes “UNDESIRED LUXURY OF LABORATORY” • Future directions • Calculation of IC50, LD50 • Detecting antibodies that block RBC invasion- Vaccine development
- 23. - 23 • Primary platelet disorders • Disorders of thrombopoiesis • Detection of anti-platelet antibodies • Monitoring drug-induced platelet dysfunction (Heparin-induced thrombocytopenia) • Platelets production and turnover Platelet disorders
- 24. - PRIMARY PLATELET DISORDERS • Glanzmann Thrombasthenia (Deficiency of integrin αIIb β3) - αIIb (GPIIb, CD41) and β3 (GPIIIa, CD61) • Bernard Soulier syndrome (GpIb-IX-Vcomplex Defect)-Measurement of platelet GPIb(CD42b), GPIX (CD42a), and GPV (CD42d) expression • Dense granule storage pool deficiency: Inherited Myeloproliferative disorders and renal failure • Collagen receptor deficiency (GpIa/IIa & GPVI) • Pseudo vWD/platelet type vWD (GpIba)
- 25. - DISORDERS OF THROMBOPOIESIS 25 • Identification of young platelets (i.e., those containing mRNA) according to staining properties with thiazole orange - Reticulated platelets. • Thrombocytopenic patients- • Bone marrow contains normal/increased megakaryocytes- elevated proportions of circulating reticulated platelets • Impaired platelet production (reduced bone marrow megakaryocytes)- lowered reticulated platelets • An aid in assessing bone marrow recovery after bone marrow transplantation • May be useful in evaluating both treatment response and thrombotic risk in patients with thrombocytosis
- 26. - DETECTION OFANTI-PLATELET ANTIBODIES 26 Immunity against platelets: • Autoimmune thrombocytopenia Caused by platelet-associated antibodies (PAIgG) • Fetal / neonatal alloimmune thrombocytopenia: Antibodies against HPA-1a or HPA-5b alleles are most common • Post transfusion purpura • Platelet transfusion refractoriness: Antibodies against HLA-A or -B alleles are the most common cause for immune-mediated platelet transfusion failure
- 27. - PLATELET ACTIVATION • Under physiologic and pathologic condition platelets become activated • Major surgery • HIT • PNH • Atheroembolic diseases- MI, CVA • Markers • Classical- • CD62p, CD63, CD40L, GPIIb/IIIa receptor (PAC-1 binding) • Alternative- • PMP, heterotypic aggregates, FXIII, Phosphatidyl serine
- 28. - HEPARIN INDUCED THROMBOCYTOPENIA FCM detect the following: (1) platelet microparticles formed as a result of platelet activation; (2) platelet monocyte aggregates and platelet- neutrophil aggregates; (3) expression of antigens expressed on activated platelets only, such as P- selectin (CD62p) (4) heparin-PF4 complexes; and (5) serotonin content of platelets
- 29. - • Enumeration of CD34+ Stem Cells • Immunodeficiency • HLA cross-match & organ transplantation • Detection of Specific Antigen Expression • Intra-cellular cytokine production WBC Evaluation
- 30. - ENUMERATION OF CD34+ HEMATOPOIETIC STEM CELLS • SCT- • Minimum number of CD34+ stem cells must be infused to repopulate the bone marrow after chemotherapy ablation • FCM- absolute and relative CD34+ cell count • Gating- CD34+ dim45
- 31. - IMMUNODEFICIENCY • Primary immunodeficiency (PID) • Secondary immunodeficiency • Calculation of CD4/CD8 in HIV patients
- 32. - PRIMARY IMMUNODEFICIENCY 32 • Primary immunodeficiency disorders (PIDs) are a heterogeneous group of inherited disorders that affect different components of the immune system. • Predisposes affected individuals - increased frequency and severity of infection, autoimmunity, and aberrant inflammation and malignancy.
- 33. - CLASSIFICATION TCell Disorders • Severe Combined Immunodeficiency • Wiskott Aldrich Syndrome • Ataxia Telangectasia • Digeorge Anomaly B Cell Disorders • XL Agammaglobinemia • Common variable immunodeficiency • Selective IgA deficiency • AR Agammaglobinemia • Hyper IgM Syndrome-XL 33 Phagocyte Disorders Compliment Disorders • Chronic Granulomatous Disease • Leukocyte Adhesion Defect • Chediac Hegashi Syndrome • Myeloperoxidase Deficiency • Cyclic Neutropenia(Elastase defect) • Deficiency in early complement pathway components (C1q, C1r, C2, C4) • Deficiency in late complement pathway components (C5, C6, C7, C8, C9) • C3 and regulatory components
- 34. - 34 CLINICAL PRESENTATION ROUTINE LABORATORY INVESTIGATIONS- CBC, CRP, CULTURE, X-RAY BIOCHEMICAL AND FLOW CYTOMETRY CONFIRMATORY TEST- GENETIC
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- 37. - SEVERE COMBINED IMMUNODEFICIENCY • SCID disorders, the most severe forms of PID, are generally characterized by complete absence of T-cell mediated immunity and impaired B-cell function T-B-NK- SCID :ADA deficiency T-B-NK+ SCID suggest mutations in RAG1&2,Artemis, DNA ligase IV, and Cernunnos T-B+NK- phenotype is characteristic for X-SCID and JAK3 deficiency • X-SCID have impaired tyrosine phosphorylation of STAT5 and STAT3 in response to stimulation with IL-2 and IL-21, respectively 37
- 38. - X-LINKEDAGAMMAGLOBULINEMIA • XLA is characterized by the absence of circulating B cells and severe reduction of all serum immunoglobulins due to mutations in the BTK gene • Because patients with XLA have no B cells, intracellular BTK expression has to be evaluated in monocytes or platelets 38
- 39. - 39 Hyper IgM syndromes HIGM syndromes are a group of genetic disorders affecting molecules involved in B cell class switch recombination and somatic hypermutation. CD40L (CD154) expression by activated CD4+ T cells is absent/ reduced.
- 40. - 40 Wiskott-Aldrich syndrome and X-linked thrombocytopenia A rare X-linked disorder characterized by microplatelets, eczema, immunodeficiency,& increased risk of autoimmune disease and hematologic malignancy. Caused by mutations in the WASgene encoding the WASp
- 41. - Chronic Granulomatous Disease • A genetically heterogeneous PID affecting bactericidal function of phagocytes • CGD is caused by defects in the NADPH oxidase complex, which is responsible for the phagocyte respiratory burst leading to the generation of superoxide and other reactive oxygen species 41
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- 44. - ENUMERATION OF CD4 COUNTS • CD4 COUNTS used for assessing patient’s immunity, when to start treatment and response to treatment risk of non- AIDS related events and mortality • CD4/8 ratio associated with clinical progression, independent of CD4 counts 44
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- 46. - DETECTION OF SPECIFIC ANTIGEN EXPRESSION • HLA B27 Overexpressed in • Ankylosing Spondylitis, • Psoriatic Arthropathy, • Acute anterior uveitis • PCR remains the gold standard FCM have • comparative sensitivity and specificity as PCR • TAT is short. • FCM+PCR- 99.6% & 100%
- 47. - CONCLUSION • The availability of an ever-expanding array of fluorescent probes and monoclonal antibodies and improvements in instrumentation and protocols is leading to expansion in the menu of clinical tests by FCM • The research applications of today to study the expression of various molecules and physiologic processes will give birth to clinical tests of tomorrow for the diagnosis, follow-up, and prognostication of malignant and nonneoplastic Disorders 47
- 48. THANK YOU
- 49. - REFERANCES • Flow Cytometry In Hematological Nonmalignant Disorders; N. ROBILLARD Et Al, Int. Jnl. Lab. Hem. 2016 • Clinical Applications Of Flow Cytometry For Nonneoplastic Disorders, Zahid Kaleem, MD; Am J Clin Pathol 2006 • Flow Cytometry-based Diagnosis Of Primary Immunodeficiency Diseases, H. Kanegane, Allergology Int’l (2017) • Advances In Laboratory Diagnosis Of Hereditary Spherocytosis; M. G. Farias; Clin Chem Lab Med 2016; • Detection Of Fetomaternal Hemorrhage Y. A. Kim & R. S. Makar; American. Jrnl Of .Hemat., Nov,2011 • Flow Cytometry And Platelet Disorders/Linden Et Al, Seminars In Thrombosis And Hemostasis/Volume 30, Number 5 2004 • Multicolor Flow Cytometry For Evaluation Of Platelet Surface Antigens And Activation Markersj.F. Van Velzen Et Al. / Thrombosis Research 130 (2012)
Editor's Notes
- #4: Cellular suspensions (usually derived from blood or tissue) are labeled using fluorochrome-labeled monoclonal antibodies. Optics consists of the light-amplification source (laser). The laser emits light of a specified wavelength. Several types of lasers, each capable of exciting specific fluorochromes and causing them to emit light. The electronics involve the conversion of photons into interpretable data
- #11: As the defect is acquired and not inherited. Thus, only a fraction of the total hematopoietic cells is affected Knowing both the percentage and type of deficient red cells is helpful in managing the anemia of PNH
- #12: Intl clinical cytometry society
- #15: REFRACTORY CYTOPENIA SINGLE LINEAGE DYSPLASIA
- #16: Conventionally, HS diagnosis is established through a series of tests, which include spherocytes identification in peripheral smear, reticulocyte count, osmotic fragility, et
- #17: have demonstrated that the FCM OF test could become the
- #18: Binding with other proteins of red cell membrane, such as Rh protein, Rh glycoprotein, and CD47, is less frequent On the other hand, Bianchi consider the test as positivity when the percentage of fluorescence reduction is 11% …. diagnosis The use of different cut-off values can change the test sensitivity and specificity [30].
- #21: Positive anti‐fetal hemoglobin (anti‐HbF) flow cytometry: Depending on anti‐HbF fluorescence intensity, HbF‐negative cells, F‐cells, and fetal red blood cells (RBCs) can be distinguished. Various samples showing increased fetal RBCs of 1.7% and 6.5% (panels A and C, respectively), increased adult F cells (panel B), and no fetal red cells (panel D)
- #22: the presence or absence of ….. and may raise the suspicion of acute leukemia,
- #23: Inhibitory conc, lethal dose
- #25: Flow cytometric analysis diagnosis of both homozygous and heterozygous states of Glanzmann thrombasthenia.
- #27: The more platelet antibody bound, the higher the fluorescence intensity, and the farther the histogram shifts to the right on the log scale. (A) Platelets incubated with normal serum showing low fluorescence and little to no antibodies detected (blue histogram), (B) the same platelets incubated with patient’s serum containing weakly reactive antibodies and a modest shift in fluorescence (red histogram), (C) the same platelets incubated with patient’s serum containing strongly reacting antibodies and a high level of fluorescence intensity (black histogram).
- #28: Platelet activation results in a series of changes in the platelet structure and biochemistry. These changes can be measured as markers of platelet activation by flow cytometry. Activation of platelets can lead to vesiculation of the platelet membrane and the formation of platelet derived microparticles which are therefore a marker of platelet activation and are the site of the assembly of plasma coagulation complexes
- #29: A-negative control- PRP+heparin alone ,,,, b- positive control-PRP STIMULATED BY Ca ionophore C- PRP+patient plasma,,,, d- prp+ patient plasma+ heparin
- #38: Flow cytometry in a patient with X-linked severe combined immunodeficiency. A, Patient T cells (CD3þ), B cells (CD19þ), and NK cells (CD16þCD56þ) were separated based on the lymphocyte gate. T and NK cells are absent, and the number of B cells are increased. B, CD132 (common g chain) expression by CD19þ B cells. Patient cells lack CD132 expression. Gray shaded area, isotype control; black line, anti-CD132 staining. C, Flow cytometric evaluation of STAT5 phosphorylation following IL-2 stimulation. Patient cells fail to respond. Gray shaded area, no stimulant; black line, 20 min post-IL-2 stimulation. D, Flow cytometric evaluation of STAT3 phosphorylation following IL-21 stimulation. Patient cells fail to respond. Gray shaded area, no stimulant; black line, 20 min post-IL-21 stimulation
- #39: Flow cytometry in a patient with X-linked agammaglobulinemia (XLA). A, CD19þCD20þ B cells are markedly reduced. B, Bruton's tyrosine kinase (BTK) expression is absent in patient monocytes. A bimodal or mosaic pattern of BTK expression is demonstrated in the heterozygous carrier. Gray shaded area, isotype control; black line, anti-BTK monoclonal antibody.
- #41: Flow cytometric detection of WASp in a patient with Wiskott-Aldrich syndrome. Cytoplasmic WASp expression was markedly reduced in patient CD3þ T cells, CD19þ B cells, andCD14þ monocytes. Gray shaded area, isotype control; black line, anti-WASp