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REVIEW ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 2  |  Page : 135-144
Intracytoplasmic antigen study by flow cytometry in hematolymphoid neoplasm


Department of Pathology, Tata Memorial Hospital (TMH), Mumbai, India

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   Abstract 

Flow cytometric detection of intracellular antigens has become a standard method in establishing proper leukemic cell lineage affiliation. It has a non-debatable contribution to the diagnosis of hematolymphoid neoplasm as well as in minimal residual disease. Combination of analysis of fluorescence labeling and light scatter properties of cells allows rapid and better determination of target cell antigens. Regarding the detection of intracellular antigens, standardization of the procedure remains, however, a real challenge. Detection of intracellular antigens by flow cytometry (FCM) requires effective fixation and permeabilization of the cell membrane. In the available literature, some reports describe methodologies to achieve satisfactory results for detection of either cytoplasmic or nuclear antigens; however, no methodological consensus has yet been achieved among the laboratories. This article is an attempt to describe different approaches to detect intracellular molecules by FCM.

Keywords: Flow cytometry, hematolymphoid neoplasm, intracytoplasmic antigens

How to cite this article:
Gujral S, Tembhare P, Badrinath Y, Subramanian P G, Kumar A, Sehgal K. Intracytoplasmic antigen study by flow cytometry in hematolymphoid neoplasm. Indian J Pathol Microbiol 2009;52:135-44

How to cite this URL:
Gujral S, Tembhare P, Badrinath Y, Subramanian P G, Kumar A, Sehgal K. Intracytoplasmic antigen study by flow cytometry in hematolymphoid neoplasm. Indian J Pathol Microbiol [serial online] 2009 [cited 2020 Mar 30];52:135-44. Available from: http://www.ijpmonline.org/text.asp?2009/52/2/135/48902



   Introduction Top


Flow cytometry (FCM) allows for the simultaneous measurement of multiple correlated parameters on a single cell. When reacted with specific fluorescent-labeled antibodies, even cells with similar physical properties may be differentiated and quantified based on cell surface antigen expression. Although surface molecules provide important information about cell type, differentiation and activation, intracellular molecules can provide valuable insight into the regulation and functions of the cells. In the early stages of cell development, some antigens like CD3 and CD22 initially get expressed in the cytoplasm and, as the cell matures, appear on the cell membrane. Hence, their detection in the cytoplasm or on the membrane is used to detect the maturation status of the target cells. This is particularly relevant in the area of acute leukemia immunophenotyping because the earliest and the most specific markers for the different lymphoid and myeloid hemopoietic cell lineages are frequently absent from the cell surface but usually detectable at the intracellular level. [1],[2] Importantly, the assessment of cell lineage in acute leukemias has proven to be of significant clinical value, not only to confirm the diagnosis but also to identify the rare subset of leukemias like biphenotypic leukemias, where cells display markers that are characteristic of more than one hemopoietic cell lineage. [3],[4],[5],[6],[7] The European Group for the Immunological Characterization of Leukemias (EGILS) as well as the St. Jude's immunological classification of acute leukemia, especially biphenotypic leukemias, have given prime importance to cytoplasmic markers. [1],[8] Recognition of the major lineages viz. precursor-B, precursor-T and myeloid lineages, as well as further variants within these lineages has been proven to be of a significant clinical and therapeutic relevance. Additionally, these markers have also been proven useful for the detection of minimal residual disease (MRD). [9],[10],[11] In this respect, cytoplasmic markers like myeloperoxidase (cyMPO), cyCD3 (T cells) and cyCD79α (B cells) or cyCD22 (B cells) are most relevant. Other intracellular markers widely used in immunophenotyping of leukemia/lymphoma are terminal deoxynucleotidyl transferase (TdT), cyclin-D1, cytoplasmic Bcl-2, cytoplasmic immunoglobulins (cyIgs) heavy chain-mu () and light chains kappa (κ) and lambda (λ) for B cell and plasma cells, cytoplasmic granules TIA-1, granzyme, perforin for cytotoxic T cells and natural killer (NK) cells. Usually, the intracellular markers are needed for confirmation of lineage assignment and for MRD. [2]

In March 2008, India's first national meeting of guidelines for immunophenotyping in hematolymphoid neoplasms was held at the Tata Memorial Hospital (TMH), Mumbai. [12] It was preceded by a practice-based questionnaire survey of clinical FCM laboratories all over India regarding their adapted protocols for immunophenotyping and discussed during the guideline meeting. The overall consensus was that the demonstration of intracellular antigen is technically difficult and needs expertise. Thus, most of the laboratories prefer to use a primary panel of antibodies for surface antigens and antibodies for intracellular antigens are used as a second-line or additional panel in situations where surface marker analysis yields ambiguous results. Common intracellular markers used in the clinical cytometry laboratory with their characteristics are discussed in [Table 1]. [13],[14],[15],[16],[17],[18],[19],[20], [21],[22],[23],[24],[25],[26],[27],[28],[29],[30], [31],[32],[33],[34],[35],[36],[37],[38],[39],[40], [41],[42],[43],[44],[45],[46],[47],[48],[49],[50], [51],[52],[53],[54],[55],[56],[57],[58],[59],[60], [61],[62],[63],[64],[65],[66],[67],[68]

The procedure for intracellular antigen detection has its own challenges and limitations as compared with the surface antigens. Whereas a single method can be utilized successfully for staining surface antigens, no single method is appropriate for all intracellular antigens. [68,69] Various issues to be considered for standardization of an intracellular staining protocol include selection of fixative and permeabilization agents compatible with the marker and the antigenic site, intracellular antigen location, antibody specificity, fluorochrome selection, increased possibility of the marker sticking non-specifically to the intracellular constituents, antigen migration and solubility and troubleshooting. [69] The laboratories may perform staining of intracellular antigens separately or in combination with surface antigens. For combined analysis, a good strategy is to first stain for surface antigens, then wash, permeabilize and finally stain with cytoplasmic markers. [70],[71],[72]

In this article we share our experience and review the critical points of current knowledge of different reagents and staining protocols available for intracellular marker study.

Two common methods to separate leukocytes in a given sample are the red cell lysis method and the ficoll-hypaque (density gradient) method. The red cell lysis method is the recommended method as there is minimal manipulation and hence least risk associated with handling of the blood sample. Another advantage is that it requires a relatively low blood volume, which is beneficial in pediatric patients. [70] The ficoll-hypaque density gradient method with ficoll-sodium diatrizoate (specific gravity 1.119 and/or 1.077) may be used to obtain granulocytes and mononuclear cells separately. [71] Red blood cell lysis is routinely performed by hypotonic solutions using ammonium chloride [73] or 2% acetic acid [74] or 0.1% Triton X-100. [75] Ammonium chloride is a popular red cell lysing reagent. [76]

Common commercial kits used for whole blood lysing include [76] FACS Brand Lysing Solution (BD USA), Ortho-mune Lysing Reagent (Ortho Diagnostic USA), Serotec Erythrolyse (Serotec UK), Coulter Clone Immuno-Lyse (Coulter USA), Lyse and Fix (Immunotech USA), Optilyse B Lysing Solution (Immunotech), Lyse and Flow solution (Harlan Sera-Lab USA), Uti-Lyse reagent A (Dako Denmark) and ACK (BioWhitaker USA). These kits are based on hypotonic solution with either ammonium chloride or formic acid. [76] Tiirikainen et al. [77] have shown that the FACS Lyse method is directly applicable to the simultaneous detection of cell surface and intracellular antigens when surface antigens are labeled before the lysis. It is also shown that FACS lyse, Immunolyse and Optilyse consistently give lower FSC values than those with ACK, Ortho-mune and ImmunoPrep procedures, indicating that lysing with the former methods may result in a relative alteration of the cells as compared with the later methods. Debris generated is maximum with ACK and Ortho-mune, lesser with FACS lyse and Optilyse and least with ImmunoPrep. [76] Separation of leukocytes is followed by fixation and permeabilization for the detection of the intracellular antigens. There are many fixatives and permeabilizers available in the market. Choosing the right agent and protocol is of utmost importance for successful staining.

Selection of cell fixatives

For the identification of intracellular antigens, it is imperative that the fixation/permeabilization reagent(s) stabilize antigen expression over time and render the cell antigens, including the cytoplasmic and nuclear targets, accessible by antibody conjugates. Although there may be other desirable fixation effects, if the reagent(s) fails to meet these requirements, all other effects are rendered insignificant. Fixatives may be classified on the basis of their mechanism of action as cross-linking agents and coagulant fixatives.

Cross-linking agents

The fixatives anchor and stabilize most antigens and prevent antigen loss of antigen after addition of permeabilization agents. These cross-linking agents are formaldehyde, paraformaldehyde (PFA) and glutaraldehyde. Of these, PFA is usually the agent of choice. [68],[78],[79],[80],[81] The optimum time and temperature for the application of PFA is an important consideration for adequate protein cross-linking. Most applications show optimum results between concentrations of 0.25 and 4% at 4-25C for 2-15min.[69] Fixation causes a significant decrease in both forward and side scatter at 48h, which makes resolution of the cell population difficult for gating adjustments. This occurs as a result of cell shrinkage due to prolonged aldehyde fixation. [82] The progressive decrease in FSC due to cell shrinkage mainly takes place from 0 to 48h after fixation. Another important effect is autofluorescence, which significantly increases during 48-96h after fixation, while, during this interval, FSC remains unchanged. The cause of autofluorescence is the reaction of PFA with a variety of free amino groups that produce fluorescent products. [83] PFA has been shown to be superior to alcohol for fixation even though it is known that the reaction of formaldehyde with amino groups of antigenic sites could interfere with antibody binding and could decrease the immunoreactivity of the antigen. [84] Hoetelmans [85] showed that fixation with PFA before methanol reduced damage to the intracellular and plasma membranes.

Coagulant fixatives

Coagulant fixatives like methanol (MeOH), ethanol (EtOH) and acetone also act by cross-linking but are less efficient. The alcohols or coagulant fixatives denature proteins, resulting in permeabilization of cells, by extracting phospholipids from the cell membranes. [85],[86] EtOH or MeOH are generally administered at -20C [87] after an initial cross-linking step in PFA, which helps protect antigens from the destructive effects of alcohol and may prevent aggregation. [79],[80],[82],[85],[88] Using reflection contrast microscopy, transmission electron microscopy and scanning electron microscopy, it is demonstrated that acetone or MeOH fixation results in complete loss of integrity of intracellular structures and results in poor preservation of the plasma membrane integrity as compared with PFA or glutaraldehyde fixation. [85] Another disadvantage is that following alcohol treatment, especially EtOH, it is difficult to separate monocytes from granulocytes on a SSC/FSC plot. [89]

Following fixation, the immunofluorescence intensity generally decreases with time to varying degrees depending on the cell type and the nature of the marker. A number of markers have shown a decrease of immunofluorescence through first 24 followed by small increase at 48 or 96 h. There are a variety of possible causes for such a loss of marker expression, which include leakage from lysosomal granules in disrupted neutrophils, cleavage of the fluorochrome and/or antibody complex, internalization of the complex or quenching of the fluorochrome by fixation products. Washing out the fixative may reduce all these fixation effects to a minimum. [90]

Selection of permeabilization agents

Routinely used permeabilization agents for intracellular marker study in hematolymphoid neoplasms are lysolecithin, [69] non-ionic detergents like Tween-20, Triton X-100, N-octyl-B-D-glucopyranoside, [69],[78],[79],[80],[88] plant-derived detergents like saponin and digitonin [69],[91],[92] and alcohols like 100% MeOH and 70% ice cold EtOH. [89]

Tween 20

Tween 20 is a hydrophilic surfactant. It is a weaker dissociating agent that permeabilizes the cell membranes more slowly and gently when used at 0.2% in phosphate-buffered saline, a concentration that is well below the concentration that leads to cell disruption. Furthermore, all non-ionic detergents are chemically impure and show considerable variation among batches. Such variation is unlikely to affect the power of a weak surfactant like Tween 20 as dramatically as that of a faster-acting one like Triton X-100. This makes it easier to attain reproducible results if different batches of detergent have to be used in the course of experiments. Finally, the 15min incubation period required for penetration of the cell membrane is shown to be advantageous for processing of a large number of samples simultaneously and uniformly. [90]

Triton X-100

It is a non-ionic class A detergent that aggregates in micelles of 140 molecules. It is a fast-acting permeabilzing agent; however, it may induce dramatic modifications in the FSC/SSC parameters. After treatment with Triton X-100, it is difficult to distinguish lymphocytes from monocytes as well as dead cells from live cells. [89] Few studies have shown that permeabilization with Triton X-100 as well as lysolecithin, N-octyl-B-D-glucopyranoside and MeOH do not alter the expression of cell surface antigens and are able to maintain well-separated leukocyte subpopulations if used with optimum concentration and temperature. It may also result in the loss of a cell surface antigen of interest and significant changes of light scatter characteristics to an extent that different cell populations are not resolvable. [74]

Saponin

It is a detergent-like molecule derived from the bark of the Quillaja tree and acts mainly by solubilizing cholesterol and leaves much of the membrane structure intact. [93] Saponin permeabilization has many advantages, like it is a simple and fast method that does not require any additional step during the experiments, it does not induce any increase in autofluorescence and only a minimal increase in non-specific background fluorescence with irrelevant antibodies, it does not result in cell aggregation as shown by microscopic examination and while slightly modifying the cell morphology, it still allows detection of cell subsets differing on the basis of light scattering characteristics. It allows in distinguishing dead cells from living cells, [89] it permeabilizes the cytoplasm as well as the nuclear membranes and it does not alter the expression of most membrane antigens. It therefore enables a simultaneous analysis of the membrane and intracellular structures. [91]

Digitonin

It is a steroid glycoside derived from Digitalis purpurea, commonly known as Foxglove. [94] It has been suggested to selectively interact with cholesterol-rich domains in the plasma membranes of eukaryotic cells. [94] Digitonin-treated plasma membranes are rendered permeable to molecules of up to 200 kDa. [95] Perhaps by virtue of its preference for cholesterol domains, intracellular structures remain largely intact following digitonin treatment. These properties have made digitonin a useful reagent for the introduction of exogenous proteins such as antibodies into the cytosol of permeabilized cells. However, this reagent has been shown to reduce the resolution of the monocyte and neutrophil populations but can adequately resolve the lymphocyte populations by utilizing log fluorescence vs. SSC measurements. [74]

Most methods document the administration of detergents with or after treatment with PFA. [89],[91],[92] Furthermore, all detergents need to be carefully titrated against concentration, time and temperature to arrive at the optimum permeabilization and antigen staining intensity.

Currently available commercial kits

Various commercial kits are available and produce less non-specific binding of antibodies and low peaks of negative controls and autofluorescence. [96] Different commercial agents available in the market are enlisted in [Table 2]. However, others have shown that even commercial kits may alter the light scatter properties. [7],[15],[97],[98] The "Fix and Perm" kit is superior, with minimum changes in FSC/SSC, followed by IntraprepE and IntrastainE for leukemia/lymphoma immunophenotyping. Cytofix/CytopermE causes a significant increase in autofluorescence [7] and PermeacyteE causes a significant decrease in both FSC and SSC values and makes it impossible to differentiate between lymphocytes, monocytes and neutrophils on the basis of their light scatter characteristics. [15],[97],[98] Refer to [Table 3] for common protocols used for an individual intracellular antigen as per the literature till date. Proper selection of antibody clones along with the right fluorochrome is also important for successful yield in intracellular marker staining. [71]

Selection of antibody

To obtain a specific staining response, knowledge of the antigen location within the cell is mandatory. For example, if the target antigen is located within the nucleus, or even further compartmentalized in the nucleolus, it is advisable to use the smaller IgG (150kd) class of monoclonal antibody as opposed to IgM (950kd). The size of the pentameric IgM class antibody may restrict entry of the antibody into the proper cellular compartment or hinder binding of conformational epitopes. [71] Selection of the best antibody clone as per the literature is of utmost importance, e.g. the CD3 clone S4.1 combined with any of the above-mentioned intracellular methods gives increased autofluorescence. Hence, its use should be discouraged. [7] It is shown that clone MPO-7-phycoerythrin (PE) for MPO, UCHT-1-PE for CD3 and HM57-PE for CD79α as well as clones 124-fluorescein isothiocyanate (FITC) and Bcl-2/100-PE for Bcl-2 and clones BP53-12-FITC and G59-12-PE for p53 provided the highest specific fluorescence intensity of the respective markers independent of the cell preparation protocols. [7],[111],[112]

Fluorochrome selection

Factors important in selecting a fluorochrome include target antigen location, target antigen density as well as the fluorochrome to antibody protein ratio. [69],[92]

Target antigen location

It is a general concept about use of fluorochromes in intracellular marker staining that the very large molecular constructs like PE and allophycocynin (APC) may cause steric hindrance for access to the target antigen. [72] However, it is shown that for the target antigen located in the cytoplasm, like MPO, the larger molecular weight phycobiliprotein fluorochromes such as PE and tandem conjugates of these proteins as PE-cyanin-5.1 (PECy5), usually work better as compared with the smaller ones, i.e. FITC. [7] These fluorochromes have a large molecular weight and high quantum yield and, usually, no problems are associated with fluorochrome quenching as fluorochrome loading makes them very bright. For antigen targets located in the nucleus, the decision is generally for a smaller molecule.

Target antigen density

Antigen density can be a controlling factor in fluorochrome selection. A low-density antigen target will necessitate the use of a bright fluorochrome, like phycobiliprotein fluorochrome.

Fluorochrome to protein ratios (F:P)

The F:P ratio is a critical feature in accurate cell analysis. The intensity of the fluorescent signal is directly related to the F:P ratio, which in turn is responsible for proper integration of percent-positive events. Anti-MPO antibodies give a stronger fluorescence signal when conjugated with PE than when coupled with FITC. [92]

Selection of cell controls

Negative and positive staining cell controls set the specific fluorescence staining limits for an assay. [113] The negative staining control will allow for setting the lower integration gate for positive results and the positive staining control will determine whether the protocol successfully stained the target antigen. Positive control cells should express the target antigen and stain with the specific antibody-fluorochrome complex and the negative control should not show specific staining. The cell controls should be treated in an identical way to the sample throughout processing. [92]

Negative control antibody selection

An isotype-matched negative control should be a non-specific antibody that is closely matched in all properties to the specific antibody.

Our experience at TMH

The Hematopathology Laboratory at the TMH performs approximately 1500 cases annually for immunophenotyping by FCM (1200 acute leukemias, 300 CLPD) along with 200

CD34-stem cell counts. The intracellular staining protocol followed at the TMH cytometry laboratory starts with red cell lysis with "Cell lyse-and-wash" technique using buffered ammonium chloride (8.26 g/L). Fixation is carried out with 3% formaldehyde for 10 min at room temperature (20-24C), followed by washing with phosphate-buffered saline and then permeabilization with 0.2% saponin and incubation with the appropriate antibodies. [72] This protocol for intracellular markers works satisfactorily. However, slight changes in the FSC/SSC parameters may be observed. No comparative studies with commercial kits have been performed. The MoAb clones being used are MPO (5B8; BD), CD3 (UCHT1; BD), CD22 (HIB22; BD), TdT (E17-1519; BD), IgM (MOPC-21; BD), anti-kappa (G20-193; BD), anti-lambda (JDC-12; BD), cyclin D1 (G124-326; BD), granzyme (GB-11; BD) and perforin (δG9; BD). Regarding fluorochromes, we observed that FITC-labeled antibodies give satisfactory results for MPO and cyCD3 and for cyCD22 and TdT, PE-labeled antibodies give better results as compared with FITC.

Troubleshooting

FCM immunophenotyping is known to have a few technical problems that may lead to interpretational mistakes. These are more common with cytoplasmic marker staining as multiple chemical reactions are involved. [Table 4] describes the common problems and their troubleshoots. [72],[114],[115]


   Summary Top


Although it is impossible for any technique to work in all situations for the detection and quantitation of intracellular antigens, we recommend the following reagents and procedures:

  1. For fixation: cross-link cells before permeabilization using, as a first choice, buffered PFA at a concentration of 0.25-4.0% for 2-15min at 4-25C.
  2. To permeabilize cells: if using non-commercial, as a first choice saponin is the most recommended reagent if carefully titered for concentration - 0.05-0.5% (50-500g/mL), time (5-20min) and temperature (4-25C). The addition of low concentrations of detergent may be desirable, which assists in permeabilization and reduction of cell aggregates. If using commercial kits - the "Fix and Perm" kit (An der Grub, Vienna, Austria) is shown to be very good by many authors.
  3. Use direct staining methods (clean fluorochrome-conjugated antibodies) where free fluorochrome molecules, specifically FITC, are not present in the preparation.
  4. Use bright fluorochrome where low target antigen density is expected.
  5. Always use negative isotype-matched controls and positive and negative control cells to ensure staining specificity and adequacy.


 
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Correspondence Address:
Sumeet Gujral
Department of Pathology, Tata Memorial Hospital (TMH), Mumbai
India
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DOI: 10.4103/0377-4929.48902

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