Correlation of CD4⁺ CD25⁺ Foxp3⁺ Treg with the recovery of joint function after total knee replacement in rats with osteoarthritis.

In this study, we observed changes in CD4(+) CD25(+) Foxp3(+) Treg expression in rats with osteoarthritis (OA) to explore the role that CD4(+) CD25(+) Foxp3(+) Treg plays in the decline in the condition of OA rats. Thirty rats were randomly divided into 2 groups equally and OA was induced in rats in the model group by injection of papain and l-cysteine into the right knee joint. Cartilage lesions were scored by the modified Mankin scale; pulmonary function was assessed by spirometry; interleukin (IL)-17 and IL-4 levels were evaluated by the enzyme-linked immunosorbent assay; and the levels of CD4(+) CD25(+) Foxp3(+) Treg in peripheral blood were measured by flow cytometry. The left knee joints of the model rats appeared palpable swelling and osteophytes, while the body weight, heart and lung function of these rats decreased. The serum IL-4 level was lower, whereas the serum IL-17 level was higher in the model group (P < 0.05). The peripheral blood CD4(+) CD25(+) Foxp3(+) Treg of CD4(+)T cells was significantly lower. Correlation of the changes in the levels of IL-4, IL-17, and Treg suggests that the underlying mechanism may be a reduction of the regulatory effect of Treg. The specific mechanism still requires further study.

Systemic mastocytosis in adults: 2013 update on diagnosis, risk stratification, and management.

DISEASE OVERVIEW: Systemic mastocytosis (SM) results from a clonal proliferation of abnormal mast cells (MC) in one or more extracutaneous organs. DIAGNOSIS: The major criterion is presence of multifocal clusters of morphologically abnormal MC in the bone marrow. Minor diagnostic criteria include elevated serum tryptase level, abnormal MC expression of CD25 and/or CD2, and presence of KITD816V. RISK STRATIFICATION: The 2008 World Health Organization (WHO) classification of SM has been shown to be prognostically relevant. Classification of SM patients into indolent (SM), aggressive SM (ASM), SM associated with a clonal non-MC lineage disease (SM-AHNMD) and mast cell leukemia (MCL) subgroups is a useful first step in establishing prognosis. MANAGEMENT: SM treatment is generally palliative. ISM patients have a normal life expectancy and receive symptom-directed therapy; infrequently, cytoreductive therapy may be indicated for refractory symptoms. ASM patients have disease-related organ dysfunction; interferon-α (±corticosteroids) can control dermatological, hematological, gastrointestinal, skeletal, and mediator-release symptoms, but is hampered by poor tolerability. Similarly, cladribine has broad therapeutic activity, with particular utility when rapid MC debulking is indicated; the main toxicity is myelosuppression. Imatinib has a therapeutic role in the presence of an imatinib-sensitive KIT mutation or in KITD816-unmutated patients. Treatment of SM-AHNMD is governed primarily by the non-MC neoplasm; hydroxyurea has modest utility in this setting. INVESTIGATIONAL DRUGS: Dasatinib's in vitro anti- KITD816V activity has not translated into significant therapeutic activity in most SM patients. In contrast, recently updated data confirms Midostaurin's significant anti-MC activity in patients with advanced SM.

Evaluation of the effect of IL-22 on human cord blood CD4+ T cells.

IL-22 is a member of IL-10 cytokine family which is believed to play an important role in inflammatory responses. IL-22 has similarities with IL-10 including conserved sequences with IL-10. IL-22 receptor is also comprised of two chains known as L-22R1 and L-10R2; supporting the speculation that the two cytokines may have similar effects. The aim of this study was to shed some light on the biological activity of IL-22 upon the cord blood CD4+CD25- T cells. In this research, cord blood T CD4+CD25- cells were cultured in presence of anti CD2/CD3/CD28 coated beads, IL-2 and IL-22 for two weeks at 37 degrees C and 5% CO2. Flow cytometry analysis showed that IL-22 has no effect upon CD25 and Foxp3 expression. Also, the results indicated that IL-22 is not involved in CD4+ T cell proliferation. Moreover, the results of suppression assay did not show any suppression effect on the cultured T cells. Thus, it seems that umbilical cord blood T cells probably do not express IL-22R1 on their surface.

[Clinical and laboratory features of patients with CD34(+) acute promyelocytic leukemia].

OBJECTIVE: To explore the expression of CD34 in patients with acute promyelocytic leukemia (APL) and investigate the clinical and laboratory features of CD34(+) APL patients. METHODS: 262 APL patients diagnosed by chromosome analysis and/or fusion gene examination in the last five years were retrospectively analyzed in this study. To survey the expression of CD34 in those patients, all the cases were divided into two groups (CD34(+) APL vs. CD34(-) APL). The clinical features including age, gender, abnormal values of the peripheral hemogram before treatment, the complete remission (CR) rate and the incidence of DIC and laboratory data such as the results of morphology, immunology, cytogenetics and molecular biology (MICM) between those two groups were compared. RESULTS: Of the 262 APL patients, 38 (14.5%) cases were positive for CD34 expression. There were no statistically significant differences between CD34(+) APL and CD34(-) APL groups in gender and age (P > 0.05). Before treatment, the median level of WBC in CD34(+) APL was 25.92 x 10(9)/L, which was significantly higher than that of CD34(-) APL (5.3 x 10(9)/L, P < 0.05). CD34(+) APL by morphology classification were mostly of the subtypes M3b and M3v (65.8%), while these subtypes in CD34(-) APL (40.3%) were significantly less (P < 0.01). There were no statistically significant differences between the two groups compared in respect of complete remission (CR) rate and the incidence of DIC (P > 0.05). The expression level of CD34 in APL had correlation to the expression level of CD2, CD7 and CD117; the latter three phenotypes in CD34(+) APL were significantly higher than those in CD34(-) APL (P < 0.01). No significant difference was found between those two groups by chromosome analysis, but there was more PML-RAR-alpha transcript short form in CD34(+) APL than that in CD34(-) APL (P < 0.05). CONCLUSION: CD34(+) acute promyelocytic leukemia is a unique subtype of APL with different biological characteristics.

Natural killer cell number and phenotype in bovine peripheral blood is influenced by age.

Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3(-)CD2(+) and NKp46(+) largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3(-)CD2(+)NKp46(+). The remainder of the NK-like cells comprised two minor populations, CD3(-)CD2(+)NKp46(-) and CD3(-)CD2(-)NKp46(+); the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3(-)CD2(+) and NKp46(+) NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8alphaalpha and CD8alphabeta and did not express CD21, WC1, CD14 or gammadelta TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3(-)CD2(-)NKp46(+) population expressed CD8alpha compared to CD3(-)CD2(+)NKp46(+) cells. Furthermore, a significantly greater proportion of the CD3(-)CD2(+)NKp46(-) population expressed CD8 compared to total CD3(-)CD2(+) cells. Adult cattle had a significantly higher proportion of perforin(+) cells compared to calves aged

Association of CD2 with fibrinogen in human plasma: depletion of the soluble E-receptor in blood clotting.

The soluble E-receptor (SER) of lymphocytes that is related to CD2 was detected in human plasma and serum using immunoelectrophoresis with sheep antiserum. All plasma samples (n=18) demonstrated reactivity with antiserum, whereas the reactivity of the corresponding sera remained low or undetectable. The depletion of SER in clotting is associated with fibrinogen, as shown by crossed-affinity immunoelectrophoresis with antisera to plasma proteins. The SER-associated fibrinogen was purified and analysed by the SDS-polyacrylamide gel electrophoresis and immunoblotting. A band close to 66 kDa was detected with monoclonal antibodies to CD2. The association of CD2 and other soluble receptors with fibrinogen via domains is suggested. It is recommended that the fresh plasma, not serum, should be used to study circulating receptors because coagulation may appreciably diminish their physiological level in blood samples.

Changes in circulating lymphocyte subpopulations following administration of the leucocyte function-associated antigen-3 (LFA-3)/IgG1 fusion protein alefacept.

Alefacept, a recombinant leucocyte function-associated antigen-3 (LFA-3)/IgG1 fusion protein approved for the treatment of psoriasis, is reported to reduce selectively the numbers of circulating CD4(+) CD45RO(+) and CD8(+) CD45RO(+) T cells, while sparing the naive cells. The purpose of the present study was to elucidate further the effect of alefacept on various circulating lymphocyte subsets. Sixteen patients, 12 with chronic plaque psoriasis and four with pustular psoriasis, received alefacept 7.5 mg once weekly for 12 weeks. Blood samples collected at study entry and after 12 weeks of treatment were analysed by four-colour flow cytometry. There were statistically significant reductions in the total number of conventional memory (CD45RA(-) CD27(+)) and effector (CD45RA(-) CD27(-) or CD45RA(+) CD27(-)) T cells, including CD4(+) and CD8(+) T cells expressing CD161 and CD8(+) T cells expressing cutaneous lymphocyte-associated antigen (CLA). Natural killer (NK) T cells were also reduced significantly, while no statistically significant changes were seen in NK cells and CD4(+) CD25(high) cells. The affected subpopulations were all characterized by a high expression of CD2. However, CD4(+) CD25(low), and CD4(+) CLA(+) cells, which also expressed relative high levels of CD2, were not reduced significantly. Our results suggest a heterogeneous effect of alefacept on the circulating memory T cell population, indicating that high expression of CD2 may not, by itself, be sufficient to explain the reduction in cell count for a specific subpopulation.

Bovine CD2-/NKp46+ cells are fully functional natural killer cells with a high activation status.

BACKGROUND: Natural killer (NK) cells in the cow have been elusive due to the lack of specific NK cell markers, and various criteria including a CD3-/CD2+ phenotype have been used to identify such cells. The recent characterization of the NK-specific NKp46 receptor has allowed a more precise definition of bovine NK cells. NK cells are known as a heterogeneous cell group, and we here report the first functional study of bovine NK cell subsets, based on the expression of CD2. RESULTS: Bovine CD2- NK cells, a minor subset in blood, proliferated more rapidly in the presence of IL-2, dominating the cultures after a few days. Grown separately with IL-2, CD2- and CD2+ NK cell subsets did not change CD2 expression for at least two weeks. In blood, CD2- NK cells showed a higher expression of CD44 and CD25, consistent with a high activation status. A higher proportion of CD2- NK cells had intracellular interferon-gamma in the cytoplasm in response to IL-2 and IL-12 stimulation, and the CD2- subset secreted more interferon-gamma when cultured separately. Cytotoxic capacity was similar in both subsets, and both carried transcripts for the NK cell receptors KIR, CD16, CD94 and KLRJ. Ligation by one out of two tested anti-CD2 monoclonal antibodies could trigger interferon-gamma production from NK cells, but neither of them could alter cytotoxicity. CONCLUSION: These results provide evidence that bovine CD2- as well as CD2+ cells of the NKp46+ phenotype are fully functional NK cells, the CD2- subset showing signs of being more activated in the circulation.

[Cytology and immunophenotyping of acute promyelocytary leukaemia]. / Cytologie et immunophénotypage des leucémies aiguës promyélocytaires.

Acute promyelocytic leukaemia (AML3) is characterized by particular clinical and biological features. We report the cytology and the immunophenotype of 14 AML3 from which 3 were AML3v. A double negativity of HLA-DR and CD34 is found in 12 cases and aberrant expression of CD2 in 2AML3v. Aberrant expression of CD56 and CD22 was shown in, respectively, one case, CD15, CD65 and CD117 expressions were variable. Cytological diagnosis is often evident, although in some cases, it is not typical and immunophenotype will contribute to the diagnosis.

Expression of cell surface antigens in diabetic patients and healthy controls after injury.

AIMS: Due to the systemic character of Type 2 diabetes, cellular disturbances paralleled by an altered expression of various growth factors constitute the basis for impaired wound healing. Cell-surface antigens are altered in chronic wounds and may also have an effect on the persistance of diabetic foot lesions. METHODS: We investigated blood samples of diabetic patients with diabetic foot ulcers (n=21) in comparison with those from healthy control patients subsequent to an injury (n=9). A blood sample (EDTA) was taken from each participant (in the trauma control group on the third day after injury) and examined by flow cytometry [fluorescence-activated cell sorter (FACS)]. Typical cell surface antigens involved in wound healing were studied [cluster of differentiation (CD)2, CD3, CD4, CD25 and human leukocyte antigen (HLA)-diabetic retinopathy (DR)]. RESULTS: known to adversely affect wound healing were elevated in diabetic patients (CD2 p<0.001; CD3 p=0.016, CD4 p=0.22, CD25 p<0.001). HLA-DR expression was also decreased in diabetic foot patients (p=0.023). CONCLUSIONS: Cell-surface antigens appear to be altered in diabetic patients when compared to healthy controls. Thus, due to the systemic character of Type 2 diabetes, cellular disturbances may well constitute the basis for impaired wound healing in diabetes.

Efficacy of dendritic cell generation for clinical use: recovery and purity of monocytes and mature dendritic cells after immunomagnetic sorting or adherence selection of CD14+ starting populations.

Immunotherapy with monocyte-derived dendritic cells (Mo-DCs) is applied to an increasing number of patients requiring large-scale production of clinical-grade dendritic cells with standardized Mo-DC generation protocols. In many countries, e.g., in Germany, Mo-DCs are legally considered medicinal products, which must be produced under Good Manufacturing Practice (GMP) conditions by an institution holding an official production license. Plastic adherence, immunomagnetic selection of CD14(+) monocytes and depletion of CD2(+) and CD19(+) cells are used to enrich monocytes for Mo-DC culture. The latter two have received approval of the European Union (CE). However, enrichment by plastic adherence is well-established and commonly used for clinical and research Mo-DC applications. The various plastic materials, nevertheless, have not been officially approved for monocyte selection for clinical use. In the present study therefore, we compared three methods for enrichment of CD14(+) monocytes with regard to efficiency of enrichment, yield of monocyte-derived functional mature dendritic cells, cost effectiveness, and handling. We demonstrate that CD14 selection and CD2 and CD19 depletion yield similar results regarding purity of mature DEs MoDCs (97-99% vs. 64-97%) and their immunostimulatory capacity. However, cell preparations cultured after CD14 selection possessed 91% to 97% CD14(+) cells, whereas CD2-/and DC19-depleted preparations contained only 8% to 57% CD14(+) cells. Thus, positive selection requires smaller culture volumes to generate equal numbers of Mo-DCs. Both methods gave better results than plastic adherence. In conclusion, of the techniques examined, CD14 selection of monocytes gave the best results regarding reproducibility, yield, and purity of the resulting monocytes and mature Mo-DCs.

Peripheral immature CD2-/low T cell development from type 2 to type 1 cytokine production.

Immature myeloid and NK cells exist, and undergo cytokine-induced differentiation, in the periphery. In this study, we show that also immature CD2(-/low) T cells exist in peripheral blood. These cells produce the type 2 cytokines IL-13, IL-4, and IL-5, but not IFN-gamma or IL-10, and, upon culture with IL-12- and TCR-mediated stimuli, differentiate to IL-13(+)IFN-gamma(+) cells producing high IL-2 levels, and finally IL-13(-)IFN-gamma(+) cells. The monokine combination IL-12, IL-18, and IFN-alpha substitutes for TCR-mediated stimulation to induce the same differentiation process in both immature CD2(-/low) and primary mature CD2(+) IL-13(+) T cells. IFN-alpha is needed to maintain high level IL-2 production, which is confined to type 2 cytokine-producing cells and lost in the IFN-gamma(+) ones. Upon TCR-mediated stimulation, IFN-gamma(+) cells are then induced to produce IL-10 as they undergo apoptosis. These data indicate that peripheral type 2 cytokine(+) T cells are immature cells that can differentiate to effector IFN-gamma(+) cells following a linear monokine-regulated pathway identical with that previously described for NK cells. They define the cellular bases to support that cell-mediated immune responses are regulated not only via Ag-induced activation of mature effector cells, but also via bystander monokine-induced maturation of immature T cells.

[Surface CD2, CD4, CD8 markers and IL-2 and TNF-alpha cytokines in amyotrophic lateral sclerosis]. / Markery powierzchniowe CD2, CD4, CD8 oraz cytokiny: IL-2 i TNF-alpha w stwardnieniu bocznym zanikowym.

The cause of amyotrophic lateral sclerosis is still unknown. In the paper CD2, CD4 and CD8 markers on mononuclear cells as well as levels of TNF-alpha and IL-2 in sera from 15 patients with ALS were evaluated. There was a significant increase of TNF-alpha in sera of ALS patients in comparison with control group. This is the first such observation. It supports the concept that immune mechanisms may play a role in the pathogenesis of ALS.

Expression of CD2 and activation markers on blood T-helper cell subsets in patients with transfusional iron overload.

The aim of the present study was to investigate the relationship between different measures of iron status, and the expression of CD2, and the activation markers CD25, CD71, CD45RO, HLADR CD38 within the Th-cell subset in patients with progressive transfusional iron overload. We estimated the expression of the activation surface markers on the Th cells of peripheral blood by flow cytometry from 22 multiply transfused patients. The number of CD2 binding sites (BS) on Th cells was significantly higher in the patients (82 917 +/- 30 801) than in age-matched normal controls (41 145 +/- 6989, P < 0.0001). When investigating whether this difference could be due to the iron overload we found the number of CD2 BS closely related to the iron saturation of serum transferrin (TfS) (R2 = 0.78, P < 0.001). The relationship to the serum ferritin concentration and to the number of blood units given was weaker, but also significant (R2 = 0.22, P < 0.027, respectively, R2 = 0.21, P < 0.032). Also the fraction of mature memory Th cells which express CD45RO at a high level was directly related to the TfS (R2 = 0.57, P < 0.0001), while the expression of CD38 within the Th cell fraction was inversely related to the TfS (R2 = - 0.43, P = 0.009). The expression of HLA-DR (but not of CD25 and CD71) was also directly related to the TfS (R2 = 0.29, P = 0.01). Our results show a clear, statistical relationship between the iron status and the expression of surface markers within Th cells in multiply transfused patients.

Multiple binding sites for melatonin on Kv1.3.

Melatonin is a small amino acid derivative hormone of the pineal gland. Melatonin quickly and reversibly blocked Kv1.3 channels, the predominant voltage-gated potassium channel in human T-lymphocytes, acting from the extracellular side. The block did not show state or voltage dependence and was associated with an increased inactivation rate of the current. A half-blocking concentration of 1.5 mM was obtained from the reduction of the peak current. We explored several models to describe the stoichiometry of melatonin-Kv1.3 interaction considering one or four independent binding sites per channel. The model in which the occupancy of one of four binding sites by melatonin is sufficient to block the channels gives the best fit to the dose-response relationship, although all four binding sites can be occupied by the drug. The dissociation constant for the individual binding sites is 8.11 mM. Parallel application of charybdotoxin and melatonin showed that both compounds can simultaneously bind to the channels, thereby localizing the melatonin binding site out of the pore region. However, binding of tetraethylammonium to its receptor decreases the melatonin affinity, and vice versa. Thus, the occupancy of the two separate receptor sites allosterically modulates each other.

Relation between lymphocyte subpopulations of peripheral blood and immune responses of modified live hog cholera virus vaccine in pigs treated with an ionized alkali mineral complex.

Thirty-nine healthy pigs (28-32 days old) were purchased from a commercial swine farm and housed at swine pens of the College. The animals were vaccinated intramuscularly (1 ml) with an attenuated live hog cholera virus (HCV, LOM strain) and then boostered at 5 weeks after the first vaccination. The animals were divided into 4 experimental groups: 0.05% (w/w) PowerFeel-supplemented diet (T-1, n = 10); 3% (w/w) SuperFeed-supplemented diet (T-2, n = 10); diluted PowerFeel solution (1 : 500, v/v) as drinking water (T-3, n=9); control (n=10). PowerFeel is an original form of ionized alkali mineral complex (IAMC) and SuperFeed is a commercial product of IAMC. The subpopulation of lymphocyte in blood was assayed by a flow cytometry and HCV-specific antibody was determined by an indirect immunofluorescence assay. In IMAC-treated groups, the proportions of subpopulation expressing MHC-class II, CD2+, CD4+, CD8+, and surface IgM+ B lymphocytes were significantly decreased at 5-weeks after the first vaccination. Significant decreases were also observed in the proportions of MHC-class II, CD2+ and CD8+ lymphocyte at 3-weeks after the booster injection. The humoral immune responses in T-1 and T-2 groups were greater than those in T-3 or control group. These results suggest that IAMC-supplemented diets may have an HCV-specific immunostimulatory effect in pigs.

Alterations in expression and in serum activity of dipeptidyl peptidase IV (DPP IV, CD26) in patients with hyporectic eating disorders.

The notion that patients with eating disorders maintain a functional immunosurveillance in spite of severe malnutrition has attracted researchers for years. Dipeptidyl peptidase IV (DPP IV), a serine protease with broad tissue distribution and known activity in serum, operates in the cascade of immune responses. Membrane-bound DPP IV expressed on lymphocytes, also known as the leucocyte antigen CD26, is considered to participate in T-cell activation. We hypothesized that the activity of DPP IV in serum and expression of CD26 in lymphocytes may be altered in patients with eating disorders. Serum DPP IV activity and the number of CD26 (DPP IV)-positive peripheral blood lymphocytes were measured in 34 patients [anorexia nervosa (AN): n = 11, bulimia (B): n = 23] in four consecutive weekly analyses. In addition, the expression of CD25 (interleukin-2 receptor alpha chain) was evaluated to estimate the degree of T-cell activation. The same analyses were carried out in healthy female volunteers (HC, n = 20). CD2-CD26-positive cells were reduced in patients compared with healthy controls [mean 40.2% (AN) and 41.1% (B) versus 47.4% (HC), P < 0.01], while the DPP IV activity in serum was elevated [mean 108.4 U/l (AN) versus 91.1 U/l (B) and 80.3 U/l (HC), P < 0.01]. The potential implications of our observations on, and beyond, immune function are discussed.

Cytogenetic clonality analysis in monosomy 7 associated with juvenile myelomonocytic leukemia: clonality in B and NK cells, but not in T cells.

It remains unclear which lymphoid lineages are involved in juvenile myelomonocytic leukemia (JMML). We report a JMML patient who acquired monosomy 7 after intensive chemotherapy. In this case, the expression of monosomy 7 was analyzed in T, B and natural killer (NK) cells highly purified from peripheral blood mononuclear cells of the patient. The fluorescence in situ hybridization method revealed the expression of monosomy 7 in B cells, but not T cells. Half of the NK cells expressed monosomy 7; when NK cells were divided into CD2- and CD2+ populations, this abnormality was positive in 91.1% of CD2- NK cells but in only 14.7% of CD2+ NK cells. These results suggest that, in this JMML patient who acquired monosomy 7 after intensive chemotherapy, B cells and half of NK cells, but not T cells, have monosomy 7.

Characterization of leukemic cells in CD2/CD19 double positive acute lymphoblastic leukemia.

In the diagnosis of leukemia, CD2 which is a T-cell associated marker and CD19 which is a B-cell associated marker are widely used to determine the lineage of leukemic cells. It is known that the cells of acute lymphoblastic leukemia (ALL) express both CD2 and CD19 in some cases. The origins of these cells are generally thought to be a common precursor for T- and B-lymphocytes. However, cytoplasmic staining of CD3 which is a more specific marker for T-lineage and cytoplasmic staining of mb-1 (CD79a) which is more specific for B-lineage were not performed in previous reports and the determination of the cell lineages of these cells was unclear. We had two cases of ALL whose blasts were CD2/CD19 double positive. The first case was assessed as B-lineage because the cells expressed cytoplasmic CD79a and lacked cytoplasmic CD3. The immunoglobulin (Ig) heavy chain gene was rearranged. The other cell surface markers including CD22 and HLA-DR also suggested that these cells were B-lineage. The CD2 expression may be a coincidence and should not be taken as a T-cell marker in this case. It was difficult to determine the lineage in the second case because both cytoplasmic CD79a and cytoplasmic CD3 were expressed and neither TCR beta chain nor Ig heavy chain genes were rearranged. The other surface markers were not useful to determine the lineage. We concluded that this case was really an unclassified ALL. Accordingly, cytoplasmic staining of CD3 and CD79a should be carried out in the diagnosis of leukemia when it is difficult to determine the cell lineage.