Flow cytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia.

The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.

Detection of fusion genes at the protein level in leukemia patients via the flow cytometric immunobead assay.

Nowadays, the presence of specific genetic aberrations is progressively used for classification and treatment stratification, because acute leukemias with the same oncogenetic aberration generally form a clinically and diagnostically homogenous disease entity with comparable prognosis. Many oncogenetic aberrations in acute leukemias result in a fusion gene, which is transcribed into fusion transcripts and translated into fusion proteins, which are assumed to play a critical role in the oncogenetic process. Fusion gene aberrations are detected by karyotyping, FISH, or RT-PCR analysis. However, these molecular genetic techniques are laborious and time consuming, which is in contrast to flow cytometric techniques. Therefore we developed a flow cytometric immunobead assay for detection of fusion proteins in lysates of leukemia cell samples by use of a bead-bound catching antibody against one side of the fusion protein and fluorochrome-conjugated detection antibody. So far, we have been able to design such fusion protein immunobead assays for BCR-ABL, PML-RARA, TEL-AML1, E2A-PBX1, MLL-AF4, AML1-ETO and CBFB-MYH11. The immunobead assay for detection of fusion proteins can be performed within 3 to 4 hours in a routine diagnostic setting, without the need of special equipment other than a flow cytometer. The novel immunobead assay will enable fast and easy classification of acute leukemia patients that express fusion proteins. Such patients can be included at an early stage in the right treatment protocols, much faster than by use of current molecular techniques. The immunobead assay can be run in parallel to routine immunophenotyping and is particularly attractive for clinical settings without direct access to molecular diagnostics.

Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients.

BCR-ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR-ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR-ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR-ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR-ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR-ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR-ABL immunobead assay detects all types of BCR-ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within approximately 4 h, and can be run in parallel to routine immunophenotyping.

Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL.

Sensitive and quantitative detection of minimal residual disease (MRD) in bone marrow (BM) samples of children with acute lymphoblastic leukemia (ALL) is essential for evaluation of early treatment response. In this study, we evaluated whether the traumatic BM samplings can be replaced by peripheral blood (PB) samplings. MRD levels were analyzed in follow-up samples of 62 children with precursor-B-ALL (532 paired BM-PB samples) and 22 children with T-ALL (149 paired BM-PB samples) using real-time quantitative PCR (RQ-PCR) analysis of immunoglobulin and T cell receptor gene rearrangements with sensitivities of 10(-3) to 10(-5) (one ALL cell in 10(3) to 10(5) normal cells). In 14 of the 22 T-ALL patients, detectable MRD levels were found in 67 paired BM-PB samples: in 47 pairs MRD was detected both in BM and PB, whereas in the remaining pairs very low MRD levels were detected in BM (n = 11) or PB (n = 9) only. The MRD levels in the paired BM-PB samples were very comparable and strongly correlated (r(s) = 0.849). Comparable results were obtained earlier by immunophenotyping in 26 T-ALL patients (321 paired BM-PB samples), which also showed a strong correlation between MRD levels in paired BM and PB samples (r(s) = 0.822). In 39 of the 62 precursor-B-ALL patients, MRD was detected in 107 BM-PB pairs: in 48 pairs MRD was detected in both BM and PB, in 47 pairs MRD was solely detected in BM (at variable levels), and in 12 pairs only the PB sample was MRD-positive at very low levels (

Neonatal blood lymphocyte subpopulations: a different perspective when using absolute counts.

We compared the absolute counts of lymphocyte subpopulations in 15 neonates, and 9 adults using the whole lysed blood technique with 15 different triple immunostainings. To obtain accurate absolute lymphocyte counts in neonatal cord blood samples, the flow cytometric 'lympho-gate' was corrected for the erythroid cell contamination by normoblasts and unlysed erythrocytes. In contrast to earlier studies where relative frequencies were reported, we found that the major difference between neonatal and adult lymphocyte subpopulations concerned the much larger pool of naive 'untriggered' cells in neonates, standby for participation in primary immune responses.

Longitudinal survey of lymphocyte subpopulations in the first year of life.

Age-matched reference values for lymphocyte subpopulations are generally obtained via cross-sectional studies, whereas patients are followed longitudinally. We performed a detailed longitudinal analysis of the changes in lymphocyte subpopulations in a group of 11 healthy infants followed from birth up to 1 y of age, with special attention for early developmental markers, markers of maturation, and markers of activation. We found that T and B lymphocytes increased at 1 and 6 wk of age, respectively. In contrast, NK cells showed a sharp decline directly after birth, suggesting that they are more important during pregnancy than thereafter. CD45RA+--mainly CD4+--naive T lymphocytes were high at birth, and increased further during the first year of life; they form a large expanding pool of cells, ready for participation in primary immune responses. The absolute counts of CD45RO+ memory T lymphocytes were similar in infants and adults, albeit with a lower level of expression of CD45RO on infant T lymphocytes. Almost all infant T lymphocytes expressed CD38 throughout the first year of life. The abundant expression of CD38 on an infant's T lymphocytes might be related to a greater metabolic need of the large population of naive untriggered cells that are continually involved in primary immune responses during the first year of life. The high B lymphocyte counts in infants mainly concerned CD38+ B lymphocytes throughout the first year of life. Also, the relative frequencies of CD1c+ and CD5+ B lymphocytes were higher throughout the first year of life than in adults. Therefore, CD1c, CD5, and CD38 could be markers of untriggered B lymphocytes. In conclusion, our longitudinal survey of T and B lymphocytes, NK cells, and their subpopulations during the first year of life helps to complete the picture of lymphocyte development in infants. This information contributes to the correct interpretation of data from infants with possible immune disorders.

Autoimmune lymphoproliferative syndrome (ALPS) in a child from consanguineous parents: a dominant or recessive disease?

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by autoimmune features and lymphoproliferations and is generally caused by defective Fas-mediated apoptosis. This report describes a child with clinical features of ALPS without detectable Fas expression on freshly isolated blood leukocytes. Detection of FAS transcripts via real-time quantitative PCR made a severe transcriptional defect unlikely. Sequencing of the FAS gene revealed a 20-nucleotide duplication in the last exon affecting the cytoplasmic signaling domain. The patient was homozygous for this mutation, whereas the consanguineous parents and the siblings were heterozygous. The patient reported here is a human homologue of the Fas-null mouse, inasmuch as she carries an autosomal homozygous mutation in the FAS gene and she shows the severe and accelerated ALPS phenotype. The heterozygous family members did not have the ALPS phenotype, indicating that the disease-causing FAS mutation in this family is autosomal recessive.

Analysing the developing lymphocyte system of neonates and infants.

UNLABELLED: Technical developments in immunophenotyping and function testing have greatly facilitated studies on the developing lymphocyte system in the past decade and contributed to a better interpretation of the data obtained in these studies. This is important for the correct interpretation of data obtained in paediatric patients with possible immunological diseases. The age-related differences in lymphocyte subpopulations and function imply that the available adult reference values cannot be used in children. CONCLUSION: In this review we give an outline of the technical developments, their influence upon the interpretation of data, and the available literature about age-related changes in the developing lymphocyte system.

Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.

Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies.

Immunophenotyping of blood lymphocytes in childhood. Reference values for lymphocyte subpopulations.

OBJECTIVE: Immunophenotyping of blood lymphocytes is an important tool in the diagnosis of hematologic and immunologic disorders. Because of maturation and expansion of the immune system in the first years of life, the relative and the absolute size of lymphocyte subpopulations vary during childhood. Therefore we wished to obtain reference values for the relative and the absolute size of all relevant blood lymphocyte subpopulations in childhood. STUDY DESIGN: We used the lysed whole blood method for analysis of lymphocyte subpopulations in 429 blood samples from neonates (n = 20), healthy children (n = 358), and adults (n = 51). The following age groups were used: 1 week to 2 months (n = 13), 2 to 5 months (n = 46), 5 to 9 months (n = 105), 9 to 15 months (n = 70), 15 to 24 months (n = 33), 2 to 5 years (n = 33), 5 to 10 years (n = 35), and 10 to 16 years (n = 23). RESULTS: Our results show that the absolute number of CD19+ B lymphocytes increases twofold immediately after birth, remains stable until 2 years of age, and subsequently gradually decreases 6.5-fold from 2 years to adult age. The CD3+ T lymphocytes increase 1.5-fold immediately after birth and decrease threefold from 2 years to adult age. The absolute size of the CD3+/CD4+ T-lymphocyte subpopulation follows the same pattern as the total CD3+ population, but the CD3+/CD8+ T lymphocytes remain stable from birth up to 2 years of age, followed by a gradual threefold decrease toward adult levels. In contrast to B and T lymphocytes, the absolute number of natural killer cells decreases almost threefold in the first 2 months of life and remains stable thereafter. Our study also showed that changes in the absolute size of lymphocyte subpopulations are not always consistent with changes in their relative size. This demonstrates that the relative counts of lymphocyte subsets do not reflect their actual size and are therefore of limited value. CONCLUSION: On the basis of this study we strongly recommend that immunophenotyping of blood lymphocytes for the diagnosis of hematologic and immunologic disorders be based on the absolute rather than on the relative size of lymphocyte subpopulations. Our data can be used as age-matched reference values for blood lymphocyte immunophenotyping.

Southern blot detection of immunoglobulin lambda light chain gene rearrangements for clonality studies.

Southern blot analysis of immunoglobulin (Ig) genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig lambda (Ig lambda) gene rearrangements, we carefully determined the precise restriction map of the J-C lambda gene region, developed a suitable C lambda probe (IGLC3), and evaluated relevant restriction enzymes in combination with the IGLC3 probe. For the latter purpose, we selected 75 B cell malignancies with proven expression of Ig lambda protein chains in order to be sure that each malignancy contained at least one clonally rearranged IG lambda allele. Our extensive Southern blot analyses with the IGLC3 probe in EcoRI and/or HindIII digests revealed that combined EcoRI/HindIII digestion detected Ig lambda gene rearrangements in 95% of the 75 patients and 94% of the 98 rearranged alleles. In contrast, HindIII and EcoRI single digests allowed detection of rearrangements in only 78% and 83% of the patients, and 67% and 79% of rearranged alleles, respectively. We conclude that the use of the IGLC3 probe in combined EcoRI/HindIII digests is superior to EcoRI and HindIII single digests. This probe/enzyme combination is informative for clonality studies in approximately 95% of patients.

Transcription and protein expression of mb-1 and B29 genes in human hematopoietic malignancies and cell lines.

The transmembrane forms of all immunoglobulin (Ig) classes are associated with two glycoproteins, mb-1 and B29, that are crucial for signal transduction following antigen binding to the Ig molecule. We have investigated the transcription and protein expression of mb-1 and B29 genes during B-cell development. Sixty human continuous cell lines (35 B-lineage, 11 T-lineage, 11 myeloid-lineage and three non-hematopoietic) and 75 hematopoietic malignancies (55 B-lineage, 12 T-lineage and eight myeloid-lineage), were tested for RNA expression by Northern blotting experiments with the mb-1 pRA3 cDNA probe, and a newly isolated B29 cDNA probe. Protein expression was analyzed by immunofluorescence microscopy of cytocentrifuge preparations, which were labeled with the anti-mb-1 HM57 monoclonal antibody (mAb) and an anti-B29 polyclonal antiserum, directed against intracellular epitopes of these polypeptides. Except for two early precursor B-cell lines, mb-1 and B29 transcripts and proteins were detected in all B-cell lines and B-cell malignancies, i.e. from immature to more mature B cells, irrespective of their Ig class expression. Transcription of mb-1 genes seems to be down-regulated at the plasma cell stage, because no mb-1 transcripts and mb-1 proteins could be detected in the four plasma cell lines and two plasma cell leukemias tested. B29 transcripts were detectable in these cell samples, but low levels of B29 proteins were only detected in one plasma cell line. The HM57 mAb gave strong labeling on fresh cytocentrifuge preparations of all B-cell samples, and this mb-1 protein expression appeared to be B-cell specific. We therefore conclude that the HM57 mAb is well suited for the detection of the mb-1 molecule as a pan-B-cell marker for the diagnosis of immature and mature B-cell malignancies. The expression pattern of the mb-1 protein is comparable to that of the CD19 and CD22 antigens, but has the advantage of being B-lineage specific. Although B29 protein expression was restricted to B-lineage cells, the anti-B29 antiserum is less suitable for diagnosis of B-cell malignancies, because of the variable and generally weak signals on cytocentrifuge preparations.

The B29 and mb-1 polypeptides are differentially expressed during human B cell differentiation.

Surface immunoglobulin on mouse B cells is associated with a heterodimer comprising the products of the mb-1 and B29 genes. Here we report that antibodies raised against a peptide sequence from the intracytoplasmic C terminus of the B29 murine gene product detect the 37-kDa component of the human heterodimer, indicating that this component in man is also encoded by the B29 gene. The immunocytochemical reactivity of these anti-B29 antibodies was compared with those of antibodies to the mb-1 protein. Of 25 cases of acute lymphoblastic leukaemia (ALL), 24 were positive for mb-1 whereas B29 was expressed in only 13 cases. Most of these B29-positive ALL expressed immunoglobulin mu heavy chain in their cytoplasm (pre-B ALL). In lymphoid tissue sections, anti-B29-labeled B cell follicles in a similar fashion to anti-mb-1, with the striking exception that plasma cells were unreactive for B29, but positive for mb-1. These results suggest that the synthesis of B29 begins later in precursor B cells than that of mb-1, and ceases before the terminal plasmacyte phase.

The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells.

Recent evidence indicates that the transmembrane form of IgM on murine and human B lymphocytes is physically associated with at least two proteins, forming a disulfide-linked dimer, which may control cell surface expression of IgM and also play a role in signal transduction after Ag binding (by analogy with the TCR-associated CD3 components in T lymphocytes). We have used mAb and polyclonal antibodies against an intracytoplasmic epitope on one of these polypeptides (previously identified in murine B cells as the product of the B cell specific mb-1 gene) to study the distribution of the IgM-associated dimer in human cells. By immunocytochemical staining of normal and neoplastic B cells, we show that the human mb-1 protein appears early in B cell differentiation, probably before expression of cytoplasmic mu-chain, and persists until the plasma cell stage, where it is seen as an intracytoplasmic component. According to immunohistologic analysis of reactive lymphoid tissue and lymphoma samples, mb-1 protein is completely B cell specific. Anti-mb-1 also labels B cell areas in tissues from seven different mammalian species. Finally, the Ig-associated dimer could be isolated from human hairy-cell leukemia cells in high purity and yield by affinity chromatography using anti-mb-1 antibody. Mice immunized with this material have produced a strong polyclonal response, so that it should now be possible to prepare a panel of new mAb reactive with different epitopes on both mb-1 and on its associated polypeptide(s).

BMA031, a monoclonal antibody suited to identify the T-cell receptor alpha beta/CD3 complex on viable human T lymphocytes in normal and disease states.

Two types of T lymphocytes can be discriminated on the basis of expression of either the classical T-cell receptor (TCR) alpha beta or the more recently identified TCR gamma delta. Whereas TCR alpha beta + lymphocytes are known to respond to recognition of antigen in the context of major histocompatibility complex molecules by proliferation, lymphokine secretion, and/or cytotoxicity, the potential ligand specificities and functions of TCR gamma delta + cells have not been completely unraveled. Antibodies specific for either receptor type are important tools to elucidate the role TCR gamma delta + cells play in the immune system. They can be used to quantify TCR gamma delta + cells and TCR alpha beta + cells in normal and disease states, to isolate both T-cell subsets, and to perform in vitro functional assays. Only few antibodies reactive with common determinants on either TCR alpha beta or TCR gamma delta are available. Generally, the monoclonal antibody (mAb) WT31 is used for definition of viable human TCR alpha beta + cells. However, WT31 has recently been shown to cross-react with TCR gamma delta. We describe an mAb, BMA031, that combines the unique features of reactivity with intact viable cells and true specificity for a common determinant on the TCR alpha beta/CD3 complex. Its performance in immunofluorescence staining and immunochemistry has been compared with that of WT31 and anti-TCR gamma delta mAbs, using TCR alpha beta and TCR gamma delta expressing cells isolated from blood and bone marrow of healthy individuals and immunodeficient patients.

Antibody L26 recognizes an intracellular epitope on the B-cell-associated CD20 antigen.

Monoclonal antibody L26 is a highly selective marker of B cells and B-cell neoplasms in paraffin-embedded tissues, but it suffers from the drawback that the target molecule has not been identified. In this paper we provide evidence by two independent techniques that antibody L26 recognizes an intracellular epitope on the CD20 antigen (a pan B-cell marker). When this antigen was redistributed on the surface of unfixed viable B cells by incubation with monoclonal anti-CD20 followed by anti-mouse Ig, the diffuse cytoplasmic staining of L26 was abolished and replaced by coincident dotlike labeling for antibody L26 and the CD20 antigen. None of the other antibodies tested (covering 10 different B-cell-associated antigens) had this effect on the L26 staining pattern. Furthermore, COS-1 cells transfected with cDNA encoding the CD20 molecule gave positive staining with antibody L26 and with two other CD20 reagents, but not with antibodies to other pan B-cell markers (eg, CD19 and CD22).

Non-random expression of T cell receptor gamma and delta variable gene segments in functional T lymphocyte clones from human peripheral blood.

Human T cell receptor (TcR) gamma delta displays a variety of protein forms. Disulfide-linked (type 1) or non disulfide-linked (type 2) receptors occur, with gamma chains encoded by the C gamma 1 or the C gamma 2 gene segment, respectively. Exon 2 of C gamma 2 may either be duplicated or triplicated (type 2a or 2b receptors). TcR gamma chains differ in molecular mass and charge between type 1 and type 2 receptors. The delta chains as well as the gamma chains have different structural properties between receptor types. This cannot be due to the use of different C delta gene segments, since the genome encodes only one. To understand the genetic basis of this dichotomy in gamma/delta combinations, rearrangement and expression of V gamma, J gamma, C gamma and V delta gene segments were determined in TcR gamma/delta+ clones derived randomly from peripheral blood of normal donors. Most clones used C gamma 1, a minority C gamma 2. The different protein properties of receptor types could be explained by the non-random expression of V gamma (J gamma) and V delta gene segments. Type 1 receptors preferentially used gamma chains encoded by the V gamma 9 and J gamma 1.2 gene segments together with delta chains encoded by V delta 2. In type 2a receptors, V gamma 9 was not predominant; often other V gamma gene segments were employed, but then in high frequency in coordination with V delta 1. Reactivity of the clones with monoclonal antibodies anti-Ti gamma A, BB3 and delta-TCS-1 correlated with the expression of the V gamma 9, V delta 2 and V delta 1 gene segments, respectively. Therefore, V gamma and V delta use in TcR gamma/delta+ cells from peripheral blood of eight healthy individuals, including the two donors of the clones, could be determined tentatively by double immunofluorescence. Indeed, the V gamma 9-V delta 2 combination was predominant, while the V gamma 9-V delta 1 and particularly the V gamma 9-"V delta other" combination was rare. These data indicate that the TcR gamma delta repertoire in peripheral blood of normal individuals is largely dependent on junctional diversity and suggest that selection of receptors occurs.

Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies.

The expression of cytoplasmic CD3 (CyCD3) was analyzed in 45 leukemias, five thymus cell samples, five peripheral blood (PB) samples, and ten cell lines. All T cell acute lymphoblastic leukemias (T-ALL) that did not express surface membrane CD3 (SmCD3) appeared to express CyCD3. Furthermore, the majority of SmCD3+ T-ALL also expressed CyCD3. Analogous results were obtained with thymus cell samples in that about 95% of the thymocytes expressed CyCD3 whereas 60% to 75% of the thymocytes also expressed SmCD3. In normal peripheral blood only prominent SmCD3 expression was found. These data indicate that immature T cells express CyCD3 only, that the combined expression of CyCD3 and SmCD3 is characteristic for intermediate differentiation stages, and that mature T cells express prominent SmCD3. All (precursor) B cell leukemias, acute myeloid leukemias, and non-T cell lines tested did not express CyCD3. On the basis of these data, we conclude that CyCD3 expression is restricted to the T cell lineage and can be used as a diagnostic marker for immature SmCD3- T cell malignancies. Therefore, we evaluated which fixative is optimal for CyCD3 staining, and we determined by immunofluorescence staining and Western blotting which anti-CD3 monoclonal antibody (MoAb) can be used for the detection of CyCD3. In our opinion, acid ethanol was the best fixative for the cytocentrifuge preparations. Furthermore, we demonstrated that CyCD3 can be easily detected by use of MoAbs raised against denaturated CD3 chains such as those of the SP series (SP-6, SP-10, SP-64, and SP-78). In addition we tested 22 anti-CD3 MoAbs of the Oxford CD3 panel that were raised against native SmCD3, and it appeared that only four (UCHT1, VIT-3b, G19-41 and SK7/Leu-4) of them were able to detect CyCD3. In Western blot analysis all four MoAbs recognized the CD3-epsilon chain only.