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Flow-cytometry is a laboratory technology derived from the concept of cell measurement in fluid flow. This technique is based on laser, which is used to identify and check the physical and chemical properties of cells and particles.
During the process, a sample of cells or particles is suspended in a liquid and injected into the flow cytometer. The most important advantage of this method is that approximately ten thousand cells can be analyzed, classified and processed by the computer in less than one minute in terms of the presence of different markers (at least 4 types). Flow cytometry is usually used to evaluate bone marrow, peripheral blood and other fluids of the body.
Laboratory technicians or pathologists perform flow cytometry. This test can be performed on a sample of blood, bone marrow, tissue, or other body fluids.
The blood sample can be taken in the same laboratory that performs the flow cytometry technique, but some other samples including body fluids (pleura, ascites, etc.) and bone marrow aspiration must be taken by a specialist doctor. Briefly, the sample of blood, bone marrow or tissue cells is placed in a suspension, stained using a specific method with fluorescent dyes (monoclonal antibodies conjugated with fluorescent dye) and injected into the flow cytometer.
The cells are arranged linearly according to the hydrodynamic rules of the device and then pass in front of the laser beam which results in the production of scattered fluorescent light. Finally, the cells are counted and sorted by the machine. The data is stored at the computer, analyzed and reported by the laboratory through a histogram or dot chart. In this way, the laboratory interprets your flow cytometry results and puts its findings into a comprehensive laboratory report.
The laboratory takes into account the results of the flow cytometry analysis as well as the medical history, symptoms and last physical examination of the patient, and for this reason it is common for the laboratory to obtain the history, patient records and the results of other tests.
Flow cytometry is requested and used by medical professionals for various purposes, including: evaluation of cell count, type, function, characteristics, along with the identifying microorganisms such as bacteria, fungi, or yeast, finding biomarkers (characteristics showing normal function), diagnosis and follow-up of potential treatment of blood and bone marrow cancers. Flow cytometry may be used to identify and count the types of white blood cells in the evaluation of infectious diseases, autoimmune disorders and immunodeficiencies. It is also used to diagnose and classify leukemia or lymphoma.
Flow cytometry is usually used as a follow-up test after a complete blood count (CBC) or differential white blood cell count (WBC). This follow up is essential if the initial test shows an increased lymphocyte count, abnormal cell count, or the presence of immature blood cells and whether the cancer responds to a particular treatment. It can also be used to detect the recurrence of the disease after treatment.
The specialist doctor looks at the markers (antigens) on the cells by receiving the flow cytometry results report. A healthy cell displays a pattern of antigens that matches the type and maturity of the cell. An abnormal cell shows different patterns that may indicate the presence of leukemia, lymphoma, or other diseases. Abnormal results are usually seen in the presence of:
1. Acute Leukemia is a type of blood cancer in which the bone marrow makes a lot of abnormal blood cells. Leukemia may affect red blood cells, white blood cells, and platelets. Based on cell surface markers, acute leukemia is divided into B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL) and acute myeloid leukemia (AML).
Flow cytometric evaluation of CD13, CD33, CD117, CD65, and anti-MPO markers are required for diagnosis and determination of AML subgroups (M0-M5), disease course, and treatment. Flow cytometric evaluation of markers CD10, CD19, CD20, CD22, CD23, CD25, CD79a, and TdT is usually performed to detect B-ALL. Additionally, sIgG, sIgM, and kappa light chains and Lambda are also required for confirmation of B-ALL.
Flow cytometry evaluation of CD2, CD3, CD4, CD5, CD6, CD7 and TdT markers are necessary for T-ALL.2. Myelodysplastic syndrome includes a heterogeneous group of hematopoietic stem cell diseases with variable clinical courses, characterized by dysplasia of one or more types of blood cells, as well as cytopenia and functional abnormalities of the bone marrow lineages. MDS is a progressive clonal disorder that affects mostly adult men aged 60-70.
The disease begins as a treatment-resistant anemia that may progress to AML. Flow cytometric assessment of CD45, CD34, CD117, HLA-DR, and CD123 for myeloid progenitor cells (myeloblasts) to differentiate them from other cell populations, such as hematopoietic stem cells, B-cell progenitors, monoblasts, basophils, erythroblasts and plasmacytoid dendritic cells.
3. Lymphoproliferative disorders (LPD) of B cells are relatively common disorders that can be chronic or more aggressive. The diagnosis of this category is usually made following the observation of large lymph nodes (LN) or spleen, asthenia and cytopenia or even incidentally as a lymphocytosis reported in a routine peripheral blood count (PB) and complete blood count (CBC).
Morphological examination is the first laboratory finding which indicates an increase in percentage and/or absolute number and/or abnormal lymphocytes. This prompts further immunophenotypic investigations using flow cytometry, which in most cases show the presence of monotypic B cells. Therefore, multiparametric flow cytometry plays an important role in the diagnosis and identification of B-cell LPDs with PB and/or bone marrow (BM) involvement. Flow cytometric evaluation of CD19, CD20, CD10 lymphocytes and classification based on CD5 along with surface immunoglobulins are essential for the physician’s diagnosis.
4. Chronic leukemia is a form of leukemia that is less severe and progresses more slowly. The most common type of leukemia is B-CLL, which is characterized by lymphadenopathy, peripheral blood and bone marrow lymphocytosis. In patients with B-CLL, flow cytometric examination of CD19, CD20, CD22, CD23, CD25, CD5, CD43 and some cell surface immunoglobulins is performed. Chronic myeloid leukemia (CML) begins with hepatosplenomegaly, extra-bone marrow hematopoiesis, and increased proliferation of myeloid cells and detection of immature cells in the peripheral blood.
After the initiation of the blastic stage, CML cannot be distinguished from AML in terms of cytology, phenotype and clinical features. Flow cytometry of patients; cells in terms of CD33, CD10, CD7 and CD34 is requested to diagnose CML. Flow cytometric markers CD13, CD15 and CD16 (neutrophils), CD38 (basophils), etc. can also be used to differentiate CML types.
5. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder that occurs when immune system damages red blood cells and platelets. If left untreated, PNH can cause hemolytic anemia, chronic kidney disease, or thrombosis (blood clots). An altered and abnormal population of bone marrow stem cells gives rise to circulating mature hematopoietic cells that lack the expression of certain cell surface proteins that have in common their association with cell membranes from through a glycosyl-phosphatidyl-inositol (GPI).
GPI deficiency is easily assessed in erythroid, granulocyte, and monocyte cells, and flow cytometry is the method of choice for their detection. Flow cytometry assesses the loss of expression of the following GPI- linked antigens: CD59 on erythrocytes, CD14 and FLAER on monocytes, and CD24 and FLAER on granulocytes.
6. Immunodeficiency disorders impair the ability of the immune system to defend the body against foreign factors that attack the body (such as bacteria, viruses, fungi, and cancer cells). As a result, unusual bacterial, viral, fungal, or lymphoma infections or other cancers may develop in an immunocompromised person.
Immune system defects are a wide range of disorders that are classified into two categories based on their origin: primary and secondary/acquired. The primary defects of the immune system are caused by congenital genetic defects and are mostly evident during childhood and after birth (Table 1). Acquired immunodeficiencies are secondary to other causes including human immunodeficiency virus (HIV) (the most common), immunosuppressive drugs, chronic conditions of some diseases (such as cancers and diabetes) and rarely radiation therapy.
Primary immunodeficiency disorders:
Type of immunodeficiency based on the affected part of the immune system
Humoral immunodeficiency: disorders of B lymphocytes and their products (antibodies).
Diseases
• Common variable immunodeficiency (CVID)
• Deficiency of a specific antibody (selective immunoglobulin deficiency), such as IgA deficiency
•Transient hypogammaglobulinemia of infancy
• X-linked agammaglobulinemia
Cellular immunodeficiency: problems with T lymphocytes
• Chronic mucocutaneous candidiasis
• D George syndrome
• X-linked lymphoproliferative syndrome
Combined humoral and cellular immunodeficiency: problems with B and T cells
• Ataxia – telangiectasia
• Hyper-immunoglobulinemia E syndrome
• Severe combined immunodeficiency
• Wiskott-Aldrich syndrome Defects of phagocytic cells: difficulty in moving or killing the activity of these cells
• Chediak Higashi syndrome (rare)
• Chronic granulomatous disease (CGD)
• Periodic neutropenia
• Leukocyte adhesion defect
Deficiency or lack of complement proteins • Component 1 (C1) inhibitor deficiency (hereditary angioedema)
• Deficiency of C2, C3, C4, C5, C6, C7, C8 and/or C9
Some immunodeficiency disorders shorten life span. Others persist throughout life but do not affect life expectancy, and a few resolve with or without treatment. Early diagnosis is necessary to prevent infection and death in these patients, which is done with blood tests to count white blood cells and their differential count, flow cytometry and genetic tests. The number of flow cytometry markers for diagnosing these diseases is very wide, but among the most common ones, we can mention CD3, CD4, CD8, CD11, CD15, CD18, CD62 and some cell surface
immunoglobulins.
7. Infertility: Primary infertility (PI) is defined as the inability of a woman or a man to conceive after one year of regular intercourse without using contraceptive methods. Infertility is a serious problem that can ruin a couple's life. Flow cytometric examination of immunological factors including CD3, CD4, CD8, CD16 and CD56 in infertile women is common to diagnose NK cell disorders. Also, in men, examining sperm DNA and apoptotic changes caused by programmed cell death are necessary to diagnose infertility. In this case, the flow cytometry examination of DNA fragmentation index (DFI) as well as involved proteins such as p53 and Bcl-2 helps to determine the cause and treatment of infertility.
8. Platelet Dysfunction Syndrome: Platelets are one of the blood components that help stop bleeding. In patients with platelet dysfunction, it is more difficult than normal to form a blood clot to stop bleeding. Platelet dysfunctions can be classified based on hereditary (transmitted from parents) and non-inherited disorders.
Von Willebrand disease is the most common inherited disorder related to platelets. There are a number of other rare inherited disorders that affect platelets, including Glanzmann disease, Wiskott-Aldrich syndrome, Chédiak-Higashi syndrome, and Bernard-Soulier syndrome.
Some of these syndromes include skin disorders, abnormal functioning of the immune system and decreased kidney function. Acquired platelet disorders are usually caused by certain drugs and conditions.
The most common drugs that affect platele function are aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), along with antiplatelet drugs such as clopidogrel and similar drugs used to prevent strokes and heart attacks. Diseases that can affect platelet function include cirrhosis, multiple myeloma, kidney disease, and systemic lupus erythematosus (SLE).
Some people may develop platelet dysfunction after cardiopulmonary bypass during open heart surgery. Flow cytometry allows in vivo quantification of platelet surface receptors and activation markers in citrated whole blood.
The main advantage of flow cytometry to assess platelet function is that it requires only a minimal amount of blood and platelets, especially in children. Second, it can be used for severe thrombocytopenic patients due to flow cytometry-based single-cell analysis independent of platelet count. Third, uncentrifuged whole blood is used for flow cytometry, which can prevent platelet activation. Flow cytometry is used to assess the expression of platelet surface glycoproteins (GP), including GPIIb/IIIa, GPIb/IX/V, GPIa/IIa, P-selectin (CD62p), CD63, lysosomal-associated membrane protein 1, and mepacrin.
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