Diagnosis of haematological neoplasms
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Further classification, e.g. of AML, B‐ALL, T‐ALL
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Identification of disease spread, e.g. to the central nervous system
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Identification of a therapeutic target, e.g. CD19, CD20, CD30, CD33, CD52
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Detection of minimal residual disease (which may include identifying a leukaemia‐specific phenotype at diagnosis)
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Identification of hypodiploidy and hyperdiploidy in B‐ALL, including the detection of masked hypodiploidy when there has been duplication of a small hypodiploid clone
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Investigation of erythrocytes and their disorders
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Diagnosis of paroxysmal nocturnal haemoglobinuria (CD15, CD16, CD24, CD55, CD59, CD66b, CD157, FLAER on neutrophils; CD14, CD55, CD157 and FLAER on monocytes; CD55, CD59 and FLAER on erythrocytes) (reviewed in [1])
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Identification of a PNH clone in aplastic anaemia (predictive of better prognosis and a response to immunosuppressive therapy)
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Diagnosis of hereditary spherocytosis (eosin‐5‐maleimide binding). Binding is also reduced in hereditary pyropoikilocytosis, South‐East Asian ovalocytosis and congenital dyserythropoietic anaemia, type II
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Diagnosis of hereditary stomatocytosis due to RHAG mutation (reduced expression of CD47, which is part of the Rh protein complex)
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Detection and enumeration of fetal red cells in maternal circulation (using anti‐RhD when mother is RhD‐positive, or using permeabilised erythrocytes and an antibody to haemoglobin F) or using the two techniques in combination
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Investigation of platelets and their disorders
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Diagnosis of inherited platelet disorders: Glanzmann’s thrombasthenia, deficiency of platelet glycoprotein IIb/IIIa (CD41/CD61 absent or reduced in three quarters of patients); Bernard–Soulier syndrome, deficiency of glycoprotein I/V/IX (CD41 and CD42a/CD42b moderately reduced); Scott syndrome (annexin V not expressed on activated platelets); GFI1B mutation (CD34 expressed on platelets); Wiskott–Aldrich syndrome (deficiency of WAS protein, reduced or defective CD43 on T lymphocytes)
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Investigation of leucocytes and their disorders including investigation of immune function
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Investigation of suspected primary immunodeficiency syndromes (reviewed in [2])
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Diagnosis of autoimmune lymphoproliferative syndrome (CD3+TCRαβ+CD4–CD8– lymphocytes)
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Diagnosis of leucocyte adhesion deficiencies type I (CD18 and CD11a, 11b and 11c deficient) and type II (CD15s deficient); reduced expression of CD11b, CD18 or CD15s by phorbol esterase‐stimulated neutrophils is demonstrated
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Diagnosis of neutrophil specific granule deficiency (reduced SSC, CD15, CD16, CD66, myeloperoxidase and lactoferrin)
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Diagnosis of chronic granulomatous disease using dihydrorhodamine as a marker of H2O2 production after stimulation of neutrophils; carrier detection is also possible
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Enumeration of CD4‐positive T cells in HIV infection
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Investigation for lymphocytic variant of hypereosinophilic syndrome (aberrant phenotypes such as CD3–CD4+CD8– or CD3+CD4–CD8–)
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Diagnosis of haemophagocytic lymphohistiocytosis (HLH) (upregulation of HLA‐DR on T cells; CD57 and perforin can also be upregulated; testing for deficiency of perforin, SAP, XIAP or CD107a is used to screen for various underlying genetic defects [3, 4]
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Diagnosis of persistent polyclonal lymphocytosis
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Identification of hypersensitivity by upregulation of CD63 and CD300a on exposure of basophils to a specific allergen [5]
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Identification of sepsis by CD64 expression on neutrophils
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Other
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Enumeration and isolation of haemopoietic stem cells (CD45weak, CD34+, SSClow)
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Differential leucocyte counting; the Beckman Coulter Hematoflow, for example, can distinguish neutrophils, eosinophils, basophils, CD16– and CD16+ monocytes, B cells, CD16+ cytotoxic T cells and NK cells, CD16– T cells, myeloblasts, monoblasts, B lymphoblasts and T lymphoblasts*
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Enumeration and characterisation of reticulocytes or platelets by the binding of a fluorochrome (e.g. a proprietary mixture of polymethine and oxazine in Sysmex instruments) to RNA or the binding of a fluorescence‐labelled CD61 monoclonal antibody to platelets (CellDyn instruments)*
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AML, acute myeloid leukaemia; B‐ALL, B‐lineage acute lymphoblastic leukaemia; CD, cluster of differentiation; FLAER, fluorescent aerolysin; HIV, human immunodeficiency virus; HLA‐DR, human leucocyte antigen‐DR; PNH, paroxysmal nocturnal haemoglobinuria; RNA, ribonucleic acid; SAP, SLAM‐associated protein; T‐ALL, T‐lineage acute lymphoblastic leukaemia; XIAP, X‐linked inhibitor of apoptosis
* This is not part of conventional immunophenotyping but represents a flow cytometric immunophenotyping technique incorporated into an automated instrument for performing blood counts.
1 –, ±, +, ++;
2 negative, weak, moderate, strong;
3 negative, dim, moderate, bright;
4 hi, lo.
It should be noted that ± indicates weak expression whereas +/– indicates that expression may be positive or negative.
An immunophenotyping result will often also be subsequently incorporated into an integrated report that includes the results of other types of investigation, for example, morphological assessment and cell counts, and cytogenetic or molecular genetic analysis.
Immunohistochemistry
Immunohistochemistry predominantly employs a primary monoclonal antibody directed at the target antigen, followed by a secondary anti‐immunoglobulin antibody that is coupled to an enzyme; the enzyme can subsequently participate in an enzymatic reaction, producing a coloured product that can be visualised. The most frequently used technique is an immunoperoxidase reaction. For some purposes, for example, the detection of immunoglobulin components, polyclonal antisera may be preferred. Immunohistochemistry has an advantage over flow cytometry in that antigen expression can be related to cytological and histological features. Co‐expression of antigens can be studied by using two different enzymatic reactions (such as peroxidase and alkaline phosphatase) or by identifying the same cell population in serial sections of the tissue.
Interpretation and Limitations of Flow Cytometric Immunophenotyping
Flow cytometry must not be interpreted in isolation but in the light of the clinical history, findings on physical examination and the results of other investigations. In particular, the blood or bone marrow film should be carefully examined in the light of the clinical and laboratory findings. Specimens are frequently sent for flow studies where the referring clinician does not have a working diagnosis. For example, a patient presenting with pancytopenia could have a number of potential diagnoses including acute leukaemia, myelodysplastic syndrome, a lymphoproliferative disorder or aplastic anaemia. A morphological review is essential in order that an appropriate panel of antibodies is utilised. Not
