Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 1119
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size

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

Click here for correspondence address and email


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]


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]


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.


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.

   References Top

1.Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A, et al . Proposals for the immunological classification of acute leukemias: European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia 1995;9:1783-6.  Back to cited text no. 1    
2.Knowles DM. Immunophenotypic markers useful in the diagnosis and classification pf hematopoietic noeplasms. In: Knowles DM, editor. Neoplastic Hematopathology 2 nd ed. Philadelphia, USA: Lippincott Williams and Wilkins; 2001. p. 93-226.  Back to cited text no. 2    
3.Knapp W, Strobl H, Majdic O. Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis. Cytometry 1994;18:187-98.  Back to cited text no. 3    
4.Praxedes MK, De Oliveira LZ, Pereira Wda V, Quintana IZ, Tabak DG, De Oliveira MS. Monoclonal antibody anti-MPO is useful in -recognizing minimally differentiated acute myeloid leukaemia. Leuk Lymph 1994;12:233-9.  Back to cited text no. 4    
5.Borowitz MJ, Shuster J, Carroll AJ, Nash M, Look AT, Camitta B, et al . Prognostic significance of fluorescence intensity of surface marker expression in childhood B-precursor acute lymphoblastic leukemia. Blood 1997;89:3960-6.  Back to cited text no. 5    
6.Orfao A, Chillon MC, Bortoluci AM, Lopez-Berges MC, Garcia-Sanz R, Gonzalez M, et al . The flow cytometric pattern of CD34, CD15 and CD13 expression in acute myeloblastic leukemia is highly characteristic of the presence of PML-RARalpha gene rearrangements. Haematologica 1999;84:405-12.  Back to cited text no. 6    
7.Kappelmayer J, Gratama JW, Karaszi E, Menendez P, Ciudad J Rivas R, et al . Flow cytometric detection of intracellular -myeloperoxidase, CD3 and CD79a: Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols. J Immunol Met 2000;242:53-65.  Back to cited text no. 7    
8.Campana D, Behm FG. Immunophenotyping of leukemia. J Immunol Met 2000;243:59-75.  Back to cited text no. 8    
9.Campana D, Coustan-Smith E, Janossy G. The immunologic detection of minimal residual disease in acute leukemia. Blood 1990;76:163-71.  Back to cited text no. 9    
10.Babusikova O, Mesarosova A, Konikova M, Kusenda J, Glasova M, Klobusicka M. Leukemia-associated marker combinations in acute leukemia suitable for detection of minimal residual disease. Neoplasma 1993;40:275-81.  Back to cited text no. 10    
11.Ciudad J, San Miguel JF, Lopez-Berges MC, Vidriales B, Velverde B, Ocqueteau M, et al . Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol 1998;16:3774-81.  Back to cited text no. 11    
12.Gujral S, Subramanian PG, Patkar N, Badrinath Y, Kumar A, Tembhare P, et al . Report of proceedings of the national meeting on "Guidelines for Immunophenotyping of Hematolymphoid Neoplasms by Flow Cytometry". Indian J Pathol Microbiol 2008;51:161-6.  Back to cited text no. 12  [PUBMED]  Medknow Journal
13.Storr J, Dolan G, Coustan-Smith E, Barnett D, Reilly JT. Value of -monoclonal anti-myeloperoxidase (MPO7) for diagnosing acute -leukaemia. J Clin Pathol 1990;43:847-9.  Back to cited text no. 13    
14.Koeffler HP, Ranyard J, Pertcheck M. Myeloperoxidase: Its structure and expression during myeloid differentiation. Blood 1985;65:484-91.  Back to cited text no. 14    
15.Lanza F, Latorraca A, Moretti S, Castagnari B, Ferrari L, Castoldi G. Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic myeloperoxidase and lysozyme in normal and leukemic cells. Cytometry 1997;30:134-44.  Back to cited text no. 15    
16.Klobusicka M, Kusenda J, Babusikova O. Myeloid enzymes profile related to the immunophenotypic characteristics of blast cells from patients with acute myeloid leukemia (AML) at diagnosis. Neoplasma 2005;52:211-8.  Back to cited text no. 16    
17.Hu ZB, Ma W, Uphoff CC, Metge K, Gignac SM, Drexler HG. -Myeloperoxidase: Expression and modulation in a large panel of -human leukemia-lymphoma cell lines. Blood 1993;82:1599-607.  Back to cited text no. 17    
18.Craig FE, Foon KA. Flow cytometric immunophenotyping for -hematologic neoplasms. Blood 2008;111:3941-67.  Back to cited text no. 18    
19.Campana D, Coustan-Smith E, Janossy G. Immunophenotyping in haematological diagnosis. Baillieres Clin Haematol 1990;3:889-919.  Back to cited text no. 19    
20.van Dongen JJ, Krissansen GW, Wolvers-Tettero IL, Comans-Bitter WM, Adriaansen HJ, Hookijkaas H, et al . Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies. Blood 1988;71:603-12.  Back to cited text no. 20    
21.Edelman J, Meyerson HJ. Diminished CD3 expression is -useful for detecting and enumerating Sezary cells. Am J Clin Pathol 2000;114:467-77.  Back to cited text no. 21    
22.Kappes DJ, Tonegawa S. Surface expression of alternative forms of the TCR/CD3 complex. Proc Natl Acad Sci USA 1991;88:10619-23.  Back to cited text no. 22    
23.Ortonne N, Buyukbabani N, Delfau-Larue MH, Bagot M, Wechsler J. Value of the CD8-CD3 ratio for the diagnosis of mycosis fungoides. Mod Pathol 2003;16:857-62.  Back to cited text no. 23    
24.Anderson P, Caligiuri M, Ritz J, Schlossman SF. CD3-negative natural killer cells express zeta TCR as part of a novel molecular complex. Nature 1989;341:159-62.  Back to cited text no. 24    
25.Kranz BR, Thierfelder S. Optimized detection of cytoplasmic -immunoglobulin and CD3 in benign and malignant lymphoid cells: enhanced sensitivity combined with differential staining of endogenous peroxidases and light microscopic morphology. J Histochem Cytochem 1993;41:1003-11.  Back to cited text no. 25    
26.Tuscano JM, Riva A, Toscano SN, Tedder TF, Kehrl JH. CD22 cross-linking generates B-cell antigen receptor-independent signals that activate the JNK/SAPK signaling cascade. Blood 1999;94:1382-92.  Back to cited text no. 26    
27.Silver K, Cornall RJ. Isotype control of B cell signaling. Sci STKE 2003;2003:pe21.  Back to cited text no. 27    
28.Campana D, Coustan-Smith E. Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 1999;38:139-52.  Back to cited text no. 28    
29.Campana D, Coustan-Smith E. Minimal residual disease studies by flow cytometry in acute leukemia. Acta Haematol 2004;112:8-15.  Back to cited text no. 29    
30.Craig FE. Flow cytometric evaluation of B-cell lymphoid neoplasms. Clin Lab Med 2007;27:487-512, vi.  Back to cited text no. 30    
31.Kozlov I, Beason K, Yu C, Hughson M. CD79a expression in acute myeloid leukemia t(8;21) and the importance of cytogenetics in the diagnosis of leukemias with immunophenotypic ambiguity. Cancer Genet Cytogenet 2005;163:62-7.  Back to cited text no. 31    
32.Lai R, Juco J, Lee SF, Nahirniak S, Etches WS. Flow cytometric detection of CD79a expression in T-cell acute lymphoblastic leukemias. Am J Clin Pathol 2000;113:823-30.  Back to cited text no. 32    
33.Bhargava P, Kallakury BV, Ross JS, Azumi N, Bagg A. CD79a is heterogeneously expressed in neoplastic and normal myeloid precursors and megakaryocytes in an antibody clone-dependent manner. Am J Clin Pathol 2007;128:306-13.  Back to cited text no. 33    
34.Herling M, Rassidakis GZ, Medeiros LJ, Vassalakopoulos TP, Kliche KO, -Nadali G, et al . Expression of Epstein-Barr virus latent membrane protein-1 in Hodgkin and Reed-Sternberg cells of classical Hodgkin′s lymphoma: Associations with presenting features, serum interleukin 10 levels, and clinical outcome. Clin Cancer Res 2003;9:2114-20.  Back to cited text no. 34    
35.Babusikova O, Stevulova L. Analysis of surface and cytoplasmic -immunoglobulin light/heavy chains by flow cytometry using a lysed-whole-blood technique: Implications for the differential diagnosis of B-cell malignancies. Neoplasma 2004;51:422-30.  Back to cited text no. 35    
36.Chang CC, Schur BC, Kampalath B, Lindholm P, Becker CG, -Vesole DH. A novel multiparametric approach for analysis of cyto-plasmic -immunoglobulin light chains by flow cytometry. Mod Pathol 2001;14:1015-21.  Back to cited text no. 36    
37.Ganick DJ, Finlay JL. Acute lymphoblastic leukemia with Burkitt cell -morphology and cytoplasmic immunoglobulin. Blood 1980;56:311-4.  Back to cited text no. 37    
38.Slaper-Cortenbach IC, Admiraal LG, Kerr JM, van Leeuwen EF, von dem Borne AE, Tetteroo PA. Flow-cytometric detection of terminal deoxynucleotidyl transferase and other intracellular antigens in combination with membrane antigens in acute lymphatic leukemias. Blood 1988;72:1639-44.  Back to cited text no. 38    
39.Huh YO, Smith TL, Collins P, Bueso-Ramos C, Albitar M, Kantarjian HM, et al . Terminal deoxynucleotidyl transferase expression in acute -myelogenous leukemia and myelodysplasia as determined by flow cytometry. Leuk Lymph 2000;37:319-31.  Back to cited text no. 39    
40.Drexler HG, Sperling C, Ludwig WD. Terminal deoxynucleotidyl transferase (TdT) expression in acute myeloid leukemia. Leukemia 1993;7:1142-50.  Back to cited text no. 40    
41.McCurley TL, Greer JP, Glick AD. Terminal deoxynucleotidyl transferase (TdT) in acute nonlymphocytic leukemia. A clinical, morphologic, -cytochemical, immunologic and ultrastructural study. Am J Clin Pathol 1988;90:421-30.  Back to cited text no. 41    
42.Sasaki R. Terminal deoxynucleotidyl transferase (TdT). Nippon Rinsho 1995;53:186-9.  Back to cited text no. 42    
43.Muehleck SD, McKenna RW, Gale PF, Brunning RD. Terminal -deoxynucleotidyl transferase (TdT)-positive cells in bone marrow in the absence of hematologic malignancy. Am J Clin Pathol 1983;79:277-84.  Back to cited text no. 43    
44.Proctor SJ, Dickinson AM, Fail B, Walker W, Serisier S. Terminal deoxynucleotidyl transferase activity in childhood and adult acute lymphoblastic leukaemia. J Clin Pathol 1981;34:892-5.  Back to cited text no. 44    
45.Boule JB, Rougeon F, Papanicolaou C. Terminal deoxynucleotidyl transferase indiscriminately incorporates ribonucleotides and -deoxyribonucleotides. J Biol Chem 2001;276:31388-93.  Back to cited text no. 45    
46.Suzumiya J, Ohshima K, Kikuchi M, Takeshita M, Akamatsu M, Tashiro K. Terminal deoxynucleotidyl transferase staining of malignant -lymphomas in paraffin sections: A useful method for the diagnosis of lymphoblastic lymphoma. J Pathol 1997;182:86-91.  Back to cited text no. 46    
47.Oshimi K, Shinkai Y, Okumura K, Oshimi Y, Mizoguchi H. Perforin gene expression in granular lymphocyte proliferative disorders. Blood 1990;75:704-8.  Back to cited text no. 47    
48.Denyer MS, Wileman TE, Stirling CM, Zuber B, Takamatsu HH. -Perforin expression can define CD8 positive lymphocyte subsets in pigs -allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells. Vet Immunol -Immunopathol 2006;110:279-92.  Back to cited text no. 48    
49.Williams NS, Engelhard VH. Perforin-dependent cytotoxic activity and lymphokine secretion by CD4+ T cells are regulated by CD8+ T cells. J Immunol 1997;159:2091-9.  Back to cited text no. 49    
50.Timonen T, Ortaldo JR, Herberman RB. Characteristics of human large granular lymphocytes and relationship to natural killer and K cells. J Exp Med 1981;153:569-82.  Back to cited text no. 50    
51.Kawasaki A, Shinkai Y, Kuwana Y, Furuya A, Ligo Y, Hanai N, et al . -Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells. Int Immunol 1990;2:677-84.  Back to cited text no. 51    
52.Kanavaros P, Boulland ML, Petit B, Arnulf B, Gaulard P. Expression of cytotoxic proteins in peripheral T-cell and natural killer-cell (NK) lymphomas: Association with extranodal site, NK or Tgammadelta phenotype, anaplastic morphology and CD30 expression. Leuk Lymph 2000;38:317-26.  Back to cited text no. 52    
53.Boulland ML, Kanavaros P, Wechsler J, Casiraghi O, Gaulard P. -Cytotoxic protein expression in natural killer cell lymphomas and in alpha beta and gamma delta peripheral T-cell lymphomas. J Pathol 1997;183:432-9.  Back to cited text no. 53    
54.Packard BZ, Telford WG, Komoriya A, Henkart PA. Granzyme B activity in target cells detects attack by cytotoxic lymphocytes. J Immunol 2007;179:3812-20.  Back to cited text no. 54    
55.Ohshima K, Haraoka S, Harada N, Kamimura T, Suzumiya J, Kanda M, et al . Hepatosplenic gammadelta T-cell lymphoma: Relation to Epstein-Barr virus and activated cytotoxic molecules. Histopathology 2000;36:127-35.  Back to cited text no. 55    
56.Felgar RE, Macon WR, Kinney MC, Roberts S, Pasha T, Salhany KE. TIA-1 expression in lymphoid neoplasms: Identification of subsets with cytotoxic T lymphocyte or natural killer cell differentiation. Am J Pathol 1997;150:1893-900.  Back to cited text no. 56    
57.Elnenaei MO, Jadayel DM, Matutes E, Morilla R, Owusu-Ankomah K, Atkinson S, et al . Cyclin D1 by flow cytometry as a useful tool in the diagnosis of B-cell malignancies. Leuk Res 2001;25:115-23.  Back to cited text no. 57    
58.Jain P, Giustolisi GM, Atkinson S, Elnenaei MO, Morilla R, Owusu-Ankomah K, et al . Detection of cyclin D1 in B cell lymphoproliferative disorders by flow cytometry. J Clin Pathol 2002;55:940-5.  Back to cited text no. 58    
59.de Boer CJ, Schuuring E, Dreef E, Peters G, Bartek J, Kluin PM, et al . Cyclin D1 protein analysis in the diagnosis of mantle cell lymphoma. Blood 1995;86:2715-23.  Back to cited text no. 59    
60.de Boer CJ, van Krieken JH, Kluin-Nelemans HC, Kluin PM, Schuuring E. Cyclin D1 messenger RNA overexpression as a marker for mantle cell lymphoma. Oncogene 1995;10:1833-40.  Back to cited text no. 60    
61.Troussard X, Avet-Loiseau H, Macro M, Mellerin MP, Malet M, Roussel M et al . Cyclin D1 expression in patients with multiple myeloma. Hematol J 2000;1:181-5.  Back to cited text no. 61    
62.Cook JR, Craig FE, Swerdlow SH. bcl-2 expression by multicolor flow cytometric analysis assists in the diagnosis of follicular lymphoma in lymph node and bone marrow. Am J Clin Pathol 2003;119:145-51.  Back to cited text no. 62    
63.Piattelli A, Rubini C, Fioroni M, Ciavarelli L, De Fazio P. bcl-2, p53, and MIB-1 in human adult dental pulp. J Endod 2000;26:225-7.  Back to cited text no. 63    
64.Lima M, dos Anjos Teixeira M, Queiros ML, Ribeiro dos Santos AH, Justica B. BCL-2 oncoprotein (p26) in splenic lymphoma with villous lymphocytes: A comparative study with other chronic B-cell disorders. Am J Hematol 1997;56:122-5.  Back to cited text no. 64    
65.Arber DA, Jenkins KA, Slovak ML. CD79 alpha expression in acute -myeloid leukemia: High frequency of expression in acute promyelocytic leukemia. Am J Pathol 1996;149:1105-10.  Back to cited text no. 65    
66.Mason DY, Cordell JL, Brown MH, Borst J, Jones M, Pulford K, et al . CD79a: a novel marker for B-cell neoplasms in routinely processed tissue samples. Blood 1995;86:1453-9.  Back to cited text no. 66    
67.Bearman RM, Winberg CD, Maslow WC, Racklin B, Carlson F, Nathwani BN, et al . Terminal deoxynucleotidyl transferase activity in neoplastic and nonneoplastic hematopoietic cells. Am J Clin Pathol 1981;75:794-802.  Back to cited text no. 67    
68.O′Brien MC, Bolton WE. Comparison of cell viability probes -compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry 1995;19:243-55.  Back to cited text no. 68    
69.Koester SK, Bolton WE. Intracellular markers. J Immunol Met 2000;243:99-106.  Back to cited text no. 69    
70.Stelzer GT, Marti G, Hurley A, McCoy P Jr, Lovett EJ, Schwartz A. US-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: Standardization and validation of laboratory procedures. Cytometry 1997;30:214-30.  Back to cited text no. 70    
71.Ormerod MG. Flow cytometry: A practical approach. 3 rd ed. USA: Oxford University Press; 2000. p. 36-8.  Back to cited text no. 71    
72.Larsen JK. Measurement of cytoplasmic and nuclear antigens. In: Ormerod MG, editor. Flow cytometry: A practical approach. 3 rd ed. New York, USA: Oxford University Press; 2000. p. 133-55.  Back to cited text no. 72    
73.Chernyshev AV, Tarasov PA, Semianov KA, Nekrasov VM, Hoekstra AG, Maltsev VP. Erythrocyte lysis in isotonic solution of ammonium -chloride: Theoretical modeling and experimental verification. J Theor Biol 2008;251:93-107.  Back to cited text no. 73    
74.Landay A, Jennings C, Forman M, Raynor R. Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry. Cytometry 1993;14:433-40.  Back to cited text no. 74    
75.Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 2005;67:4-17.  Back to cited text no. 75    
76.Bossuyt X, Marti GE, Fleisher TA. Comparative analysis of whole blood lysis methods for flow cytometry. Cytometry 1997;30:124-33.  Back to cited text no. 76    
77.Tiirikainen MI. Evaluation of red blood cell lysing solutions for the detection of intracellular antigens by flow cytometry. Cytometry 1995;20:341-8.  Back to cited text no. 77    
78.Kurki P, Ogata K, Tan EM. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by -immunofluorescence microscopy and flow cytometry. J Immunol Met 1988;109:49-59.  Back to cited text no. 78    
79.Hallden G, Andersson U, Hed J, Johansson SG. A new membrane -permeabilization method for the detection of intracellular antigens by flow cytometry. J Immunol Met 1989;124:103-9.  Back to cited text no. 79    
80.Mikulka WR, Bolton WE. Methodologies for the preservation of -proliferation associated antigens PCNA, p120, and p105 in tumor cell lines for use in flow cytometry. Cytometry 1994;17:246-57.  Back to cited text no. 80    
81.Li X, James WM, Traganos F, Darzynkiewicz Z. Application of biotin, digoxigenin or fluorescein conjugated deoxynucleotides to label DNA strand breaks for analysis of cell proliferation and apoptosis using flow cytometry. Biotech Histochem 1995;70:234-42.  Back to cited text no. 81    
82.Jacobberger JW, Fogleman D, Lehman JM. Analysis of intracellular antigens by flow cytometry. Cytometry 1986;7:356-64.  Back to cited text no. 82    
83.Van Ewijk W, Van Soest PL, Verkerk A, Jongkind JF. Loss of antibody -binding to prefixed cells: fixation parameters for -immunocytochemistry. Histochem J 1984;16:179-93.  Back to cited text no. 83    
84.Hopwood D. Fixatives and fixation: A review. Histochem J 1969;1:323-60.  Back to cited text no. 84    
85.Hoetelmans RW, van Slooten HJ, Keijzer R, van de Velde CJ, van Dierendonck JH. Routine formaldehyde fixation irreversibly reduces immunoreactivity of Bcl-2 in the nuclear compartment of breast cancer cells, but not in the cytoplasm. Appl Immunohistochem Mol Morphol 2001;9:74-80.  Back to cited text no. 85    
86.Hopwood D. Cell and tissue fixation, 1972-1982. Histochem J 1985;17:389-442.  Back to cited text no. 86    
87.Levitt D, King M. Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens. J Immunol Met 1987;96:233-7.  Back to cited text no. 87    
88.Clevenger CV, Bauer KD, Epstein AL. A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry. Cytometry 1985;6:208-14.  Back to cited text no. 88    
89.Lecoeur H, Ledru E, Gougeon ML. A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes. J Immunol Met 1998;217:11-26.  Back to cited text no. 89    
90.Stewart JC, Villasmil ML, Frampton MW. Changes in fluorescence intensity of selected leukocyte surface markers following fixation. Cytometry A 2007;71:379-85.  Back to cited text no. 90    
91.Jacob MC, Favre M, Bensa JC. Membrane cell permeabilization with saponin and multiparametric analysis by flow cytometry. Cytometry 1991;12:550-8.  Back to cited text no. 91    
92.Koester SK, Bolton WE. Strategies for cell permeabilization and -fixation in detecting surface and intracellular antigens. Methods Cell Biol 2001;63:253-68.  Back to cited text no. 92    
93.Goldenthal KL, Hedman K, Chen JW, August JT, Willingham MC. -Postfixation detergent treatment for immunofluorescence -suppresses localization of some integral membrane proteins. J Histochem -Cytochem 1985;33:813-20.  Back to cited text no. 93    
94.Scallen TJ, Dietert SE. The quantitative retention of cholesterol in mouse liver prepared for electron microscopy by fixation in a digitonin-containing aldehyde solution. J Cell Biol 1969;40:802-13.  Back to cited text no. 94    
95.Fiskum G. Intracellular levels and distribution of Ca 2+ in digitonin-permeabilized cells. Cell Calcium 1985;6:25-37.  Back to cited text no. 95    
96.Zelnickova P, Faldyna M, Stepanova H, Ondracek J, Kovaru F. Intracellular cytokine detection by flow cytometry in pigs: Fixation, -permeabilization and cell surface staining. J Immunol Met 2007;327:18-29.  Back to cited text no. 96    
97.Pizzolo G, Vincenzi C, Nadali G, Veneri D, Vinante F, Chilosi M, et al . Detection of membrane and intracellular antigens by flow cytometry following ORTHO PermeaFix fixation. Leukemia 1994;8:672-6.  Back to cited text no. 97    
98.Groeneveld K, te Marvelde JG, van den Beemd MW, Hooijkaas H, van Dongen JJ. Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes. Leukemia 1996;10:1383-9.  Back to cited text no. 98    
99.Nakase K, Sartor M, Bradstock. Detection of myeloperoxidase by flow cytometry in acute leukemia. Cytometry 1998;34:198-202.  Back to cited text no. 99    
100.Konikova E, Glasova M, Kusenda J, Babusikova O. Intracellular markers in acute myeloid leukemia diagnosis. Neoplasma 1998;45:282-91.  Back to cited text no. 100    
101.Campana D, Thompson JS, Amlot P, Brown S, Janossy G. The -cytoplasmic expression of CD3 antigens in normal and malignant cells of the T lymphoid lineage. J Immunol 1987;138:648-55.  Back to cited text no. 101    
102.Janossy G, Coustan-Smith E, Campana D. The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: A study of 500 cases. Leukemia 1989;3:170-81.  Back to cited text no. 102    
103.Hartung L, Bahler DW. Flow cytometric analysis of BCL-2 can distinguish small numbers of acute lymphoblastic leukaemia cells from B-cell precursors. Br J Haematol 2004;127:50-8.  Back to cited text no. 103    
104.Dworzak MN, Fritsch G, Froschl G, Printz D, Gadner H. Four-color flow cytometric investigation of terminal deoxynucleotidyl -transferase-positive lymphoid precursors in pediatric bone marrow: CD79a expression precedes CD19 in early B-cell ontogeny. Blood 1998;92:3203-9.  Back to cited text no. 104    
105.Roma AO, Kutok JL, Shaheen G, Dorfman DM. A novel, -rapid, -multiparametric approach for flow cytometric analysis of -intranuclear terminal deoxynucleotidyl transferase. Am J Clin Pathol 1999;112:343-8.  Back to cited text no. 105    
106.Perry A, Duenzl ML, Ansari MQ. Flow cytometric terminal -deoxynucleotidyltransferase analysis: Evaluation of Triton X-100 and methanol permeabilization methods compared with -immunofluorescence microscopy. Arch Pathol Lab Med 1994;118:1119-22.  Back to cited text no. 106    
107.Farahat N, van der Plas D, Praxedes M, Morilla R, Matutes E, Catovsky D. Demonstration of cytoplasmic and nuclear antigens in acute leukaemia using flow cytometry. J Clin Pathol 1994;47:843-9.  Back to cited text no. 107    
108.Darzynkiewicz Z, Gong J, Juan G, Ardelt B, Traganos F. Cytometry of cyclin proteins. Cytometry 1996;25:1-13.  Back to cited text no. 108    
109.Lazarus AH, Ellis J, Blanchette V, Freedman J, Sheng-Tanner X. -Permeabilization and fixation conditions for intracellular flow -cytometric detection of the T-cell receptor zeta chain and other intracellular proteins in lymphocyte subpopulations. Cytometry 1998;32:206-13.  Back to cited text no. 109    
110.He L, Hakimi J, Salha D, Miron I, Dunn P, Radvanyi L. A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells. J Immunol Met 2005;304:43-59.  Back to cited text no. 110    
111.Francis C, Connelly MC. Rapid single-step method for flow cytometric detection of surface and intracellular antigens using whole blood. Cytometry 1996;25:58-70.  Back to cited text no. 111    
112.Zamai L, Canonico B, Gritzapis A, Luchetti F, Felici C, Della Felice M, et al . Intracellular detection of Bcl-2 and p53 proteins by flow -cytometry: Comparison of monoclonal antibodies and sample preparation protocols. J Biol Regul Homeost Agents 2002;16:289-302.  Back to cited text no. 112    
113.Font P, Subira D, Mtnez-Chamorro C, Castaρón S, Arranz E, Ramiro S, et al . Evaluation of CD7 and terminal deoxynucleotidyl transferase (TdT) expression in CD34+ myeloblasts from patients with myelodysplastic syndrome. Leuk Res 2006;30:957-63.  Back to cited text no. 113    
114.Trouble shooting tips - Flow cytometry [Online]. 1998 [2 screens]. Available from: http://www.abcam.com/ps/pdf/protocols/flow_troubleshooting.pdf. [last cited on 1999].  Back to cited text no. 114    
115.Shapiro HM. Measuring Cell Surface and Intracellular Antigens. In: Shapiro HM, editor. Practical flow cytometry. 4 th ed. Hoboken, New Jersey: John Wiley and Sons, Inc; 2003. p. 345-61.  Back to cited text no. 115    

Correspondence Address:
Sumeet Gujral
Department of Pathology, Tata Memorial Hospital (TMH), Mumbai
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.48902

Rights and Permissions


  [Table 1], [Table 2], [Table 3], [Table 4]

This article has been cited by
1 Fetal RBCs Potential in Obstetric Protocols to Minimize Fetomaternal Hemorrhage
Hasnaa A. Abo-Elwafa,Salah A. Ismail,Ibrahim M. A. Hassanin,Rania M. Ahmed
Open Journal of Blood Diseases. 2017; 07(01): 51
[Pubmed] | [DOI]
2 The wealth of cytomics. Rsum of the 19th Annual Meeting of the German Society for Cytometry (Deutsche Gesellschaft Fr Zytometrie, DGfZ)
Elmar Endl, Annette Beck-Sickinger, Christian Wilhelm, Martin Schlegel, Susann Mller
Cytometry Part B Clinical Cytometry. 2010; 78b(5): 361
[VIEW] | [DOI]
3 Epidemiology of vitiligo, associated autoimmune diseases and audiological abnormalities: Ankara study of 80 patients in Turkey : Clinical, laboratory and genetic aspects of vitiligo
Y Anadolu, S Gullu, BN Akay, M Bozkir
Journal of the European Academy of Dermatology and Venereology. 2010; 24(10): 1144
[VIEW] | [DOI]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  

    Article Tables

 Article Access Statistics
    PDF Downloaded1117    
    Comments [Add]    
    Cited by others 3    

Recommend this journal