Leukemias with t(8;21) or inv(16)/t(16;16) belong to the core binding factor (CBF) AMLs, which are a diagnostically and prognostically distinct subgroup [9]. Both of these chromosomal rearrangements result in the formation of fusion proteins, AML1/ETO and CBFβ/MYH11, respectively, that involve the disruption of one of the CBF transcription factor genes. The two genes involved in (8;21) translocation are the AML1 transcription factor, now called RUNX1, on chromosome 21q22.3, and the Eight-Twenty One oncoprotein (ETO) (also called MTG8) on chromosome 8q22 [29]. AML1/Runx1, one of three α-subunits of the CBF family, is a sequence-specific DNA-binding protein, while the single CBFβ subunit is non-DNA binding but enhances the DNA affinity of the α-subunits [62]. The characteristic immunophenotype of t(8;21) AML allows for a correct prediction of this genetic aberration [63]. The initially recognized distinctive features were expression of CD19, a B-lineage associated antigen, and of CD56, the neural-cell adhesion molecule, by CD34^POS myeloblasts. Presence of CD56 may explain the increased incidence of granulocytic sarcomas observed in this disease [64, 65]. While CD19 is consistently present in the t(8;21) subtype and rarely found in other AMLs, expression of this antigen by myeloblasts is often very weak so that its detection can depend on the accurate choice of fluorochromes and an open mind on the part of the interpreting flow cytometrist. In fact, CD19 conjugated to fluorescein isothiocyanate (FITC) should be avoided at all costs. An example of CD19/CD56 double expression in a patient with AML1/ETO^POS AML is shown in Fig. 17.1. Although CD56 expression is promiscuous among myeloid leukemias and absent in a marked fraction of t(8;21) cases, the finding of both CD19 and CD56 against the background of a myeloid phenotype is highly suggestive of t(8;21) AML. As in the case of APL, an increasingly accurate surrogate marker profile for t(8;21) AML has evolved over time. One particularly helpful diagnostic tool is the diminished or absent expression of CD11a/CD18 [31, 66], a member of the β2 integrin subfamily [42]. With the exception of t(8;21) or t(15;17) (and its variants), >90 % of AML demonstrate expression of CD11a/CD18, albeit often with low intensity. The absence of CD11a in t(8;21) AML is explained by the inhibition of Runx1-dependent CD11a transcription by the AML1/ETO fusion product [66]. Variable expression of CD11a is seen in AML with dual expression of CD19 and CD7, a T-lineage affiliated antigen [48]. Previously, the incompatibility of t(8;21) with expression of CD7 had been reported [67]. More recently, CD19/CD7 double positive AML has been associated with a predominantly normal karyotype and FLT3-ITD and NPM1 mutations [50, 68].

Aside from CD19, t(8;21) myeloblasts can also express two other B-lineage antigens, CD79a and PAX5 [30]. The PAX5 gene encodes a paired box domain transcription factor, which is considered a crucial mediator of B-cell identity [69]. Tiacci et al. [30] hypothesized that the PAX5-dependent expression of CD19 and CD79a in t(8;21) AML results from the interaction and functional cooperation between the PAX5 and AML1/ETO proteins. Expression of CD56 is also not limited to the myeloid lineage [70] and in B-lineage ALL appears to be associated with BCR/ABL transcripts [71]. Taken together, these findings can lead to a misdiagnosis of B-lineage ALL, especially in view of the immaturity of the myeloid phenotype frequently encountered (as discussed before), as long as karyotypic and molecular data are unavailable. While in t(8;21) AML, the same blast population will co-express myeloperoxidase or surface myeloid antigens, e.g., CD13, weak CD33, and CD19, CD79a, PAX5, these blasts will not stain for cCD22. It is important to remember that dual absence of myeloperoxidase and cCD22 is consistent with AML. Given that the evaluation of intracytoplasmic antigens continues to be a challenge for many flow cytometric laboratories, this is a typical situation, in which the rapid flow cytometric assessment of AML1/ETO protein, anticipated for the near future [72], will be of invaluable help in the correct diagnosis of t(8;21) AML.

The expression of the T-cell affiliated antigen CD2 by leukemic myeloblasts is rarely observed, provided that antigen expression is viewed on gated leukemic cells exclusively. Although not (yet) part of a full surrogate marker profile, single antigens can serve as “diagnostic pointers” for genotypes. CD2 expression by leukemic cells with a myeloid phenotype should raise the immediate suspicion of APL (see previously in this chapter). The other genotypically defined AML subtype that frequently demonstrates CD2 expression is CBFβ/MYH11^POS AML derived from inv(16)(p13q22) or t(16;16)(p13;q22) [73]. APL and CBFβ/MYH11^pos AML can be easily distinguished given the characteristic APL immunophenotype (HLA-DR^NEGCD11a^NEGCD18^NEG) that is distinctly different from that of CBFβ/MYH11^POS disease.

Analogous to CD19 in t(8;21) leukemia, CD2 expression by CBFβ/MYH11^POS can be very weak and may be overlooked, definitely if FITC-conjugated CD2 is tested. Occasionally, however, CD2 expression can be quite strong, as exemplified in Fig. 17.2. This is an important, though not well-appreciated association, given that inv(16) is a subtle cytogenetic abnormality that is easily missed. The expectation of the typical morphologic picture associated with CBFβ/MYH11^POS AML, FAB M4 and abnormal eosinophils (FAB 4eo), leads pathologists to shy away from investing efforts into a refined surrogate marker for this AML subtype. However, central morphologic review of 98 CBFβ/MYH11^POS ECOG AML patients demonstrated that FAB M4eo was confirmed in only 59 % of cases, followed by M4 without eosinophilia (13 %), M2, with or without eosinophilia (19 %), M1 (5 %), and M5 (3 %) (Bennett JM, personal communication). When compared with institutional pathology reviews, the concordance with FAB M4eo was even lower, suggesting that immunophenotypic assistance in the diagnosis of this AML subtype could be useful.

The Philadelphia chromosome, t(9;22), which results in the BCR/ABL fusion gene, is the dominant negative prognostic factor in ALL [74]. Its incidence increases with age, accounting for 2–5 % in children and up to 28 % in adult patient cohorts [75]. The disappointing response of BCR/ABL^POS patients to standard therapy and the availability of specific inhibitors for the constitutively activated tyrosine kinase in BCR/ABL fusion proteins [76] have prompted separate trials for BCR/ABL^POS and BCR/ABL^NEG ALL, making it imperative that these patients are recognized accurately.

Cytogenetic analyses in lymphoblasts are hampered by suboptimal chromosome structure [77], and, furthermore, approximately 10 % of BCR/ABL^POS ALL lack evidence of the t(9;22) by chromosome banding [75]. Both karyotyping and molecular analyses by PCR require time, 1–2 weeks and 48 h, respectively. The flow cytometric bead assay for BCR/ABL fusion proteins [72], that is discussed next, is the ideal solution, though an expensive one. Long before the development of this bead assay, there was intense interest and urgency in the establishment of a surrogate marker profile for BCR/ABL^POS ALL. The first alleged antigenic signature relied solely on differences in staining intensity of antigens commonly found in B-lineage ALL, such as strong expression of CD10 and CD34 and weak expression of CD38 [78–80]. However, in 1997, a preliminary analysis of 144 patients enrolled in ECOG’s phase III adult ALL trial, E2993, demonstrated an association between BCR/ABL positivity and expression of CD25, the α-chain of the interleukin-2 receptor [81]. This observation confirmed an earlier report of the incidental finding of CD25 in four patients with BCR/ABL^POS ALL [82]. The final analysis of E2993 [83] solidified the surrogate marker profile for BCR/ABL^POS lymphoblasts as CD25^posCD34^high with dual expression of myeloid antigens, CD33 and CD13. Despite the frequent expression of two myeloid antigens by BCR/ABL^POS lymphoblasts, this phenotype must not be considered biphenotypic, since BCR/ABL^POS lymphoblasts unequivocally belong to the B-cell lineage (cCD22^POS, myeloperoxidase^NEG). Greater than 75 % of BCR/ABL_POS lymphoblasts express additional cytogenetic aberrations [83]. Primo et al. [84] reported that patients with del(9)(p21), in addition to the t(9;22), lacked both CD33 and CD13. Among 11/156 BCR/ABL^pos patients with del(9)(p11), del(9)(del(9)(p13), or del(9)(p21) on E2993, 8 lacked both CD33 and CD13, whereas the other 3 only showed decrease or loss of CD13 expression, thus supporting and expanding Primo’s observation (Paietta E, personal observation).

The dual expression of myeloid antigens, CD33 and CD13 by BCR/ABL^POS lymphoblasts, is not surprising, given that these blasts most commonly express immunophenotypic features of early Pre-B ALL (CD10^POS). Ludwig et al. [85] have proposed that the expression of myeloid antigens in adult B-lineage ALL differs according to the level of B-lymphoblast maturation, analogous to what has been reported from pediatric ALL [86, 87]. Preliminary analysis of the largest adult ALL trial to date (ECOG 2993) [88] confirmed but also expanded these associations. While Pro/Pre-Pre-B ALL, the immature CD10^NEG maturation stage, typically expressed CD65_(S) and CD15_(S), the CD10^POS early Pre-B stage preferentially expressed the pan-myeloid antigens CD33 and CD13, which in normal myelopoiesis appear before CD65_(S)/CD15_(S) on maturing myeloid cells. However, these relationships did not hold up in BCR/ABL^POS cases; as given in Table 17.2, the paired expression of CD33/CD13 persisted irrespective of the maturation stage of BCR/ABL^POS lymphoblasts. CD33/CD13 positivity was seen whether BCR/ABL^POS blasts lacked CD10 (Pro/Pre-Pre-B stage) or expressed intracytoplasmic μ chains (Pre-B stage). Importantly, BCR/ABL^POS lymphoblasts never expressed CD65_(S)/CD15_(S) even in those instances in which CD33 and/or CD13 were not expressed.

The (9;22)(q34;q11) translocation results in the actual Philadelphia chromosome, the derivative chromosome 22, in which the BCR/ABL fusion gene is located, and the derivative chromosome 9, where the ABL/BCR fusion gene resides, which does not appear to contribute to the pathogenesis of this disease. Various isoforms of the BCR/ABL fusion gene are created dependent on the variable breakpoints in the BCR gene. All translated BCR/ABL proteins share a similar carboxy terminal ABL tyrosine kinase domain (TKD), but differ in the portion of the BCR protein included in the fusion product, due to multiple breakpoint cluster regions in the BCR gene. In the majority of chronic myeloid leukemia (CML) and in one third of BCR/ABL^POS ALL, the break within the BCR gene occurs in the major breakpoint cluster region (M-bcr), resulting, when joined with a portion of c-ABL from chromosome 9, in a b2a2 or b3a2 fusion transcript encoding a protein of 210,000 Da molecular weight (p210^BCR-ABL). A break in the minor breakpoint cluster region (m-bcr) forms the e1a2 transcript encoding a 190,000 Da protein (p190^BCR-ABL), found mostly in BCR/ABL^POS ALL [89, 90]. The antigen expression profile for BCR/ABL^POS ALL varies with the BCR/ABL isoform, in that dual myeloid antigen expression of CD33+CD13 and CD25 expression are much more frequent in p210^BCR-ABL lymphoblasts [83].

The most striking observation with respect to CD25 and BCR/ABL^POS ALL is that expression levels of CD25 are of prognostic significance predicting for a lower likelihood to achieve complete remission [81, 83], and shorter overall (OS) [81, 83] or event-free survival [91] among BCR/ABL^POS patients. In other words, CD25 is unique in its dual function as a dependable marker of BCR/ABL^POS ALL and an independent prognostic factor for outcome in this disease.

Though isolated case reports of BCR/ABL^POS ALL with T-lineage phenotype are still occasionally posted, all of the larger studies have reported that BCR/ABL rearrangements in pediatric and adult ALL are restricted to the B-cell lineage [76, 92–94]. A substantial cohort of pediatric patients studied from various European groups found 3 T-ALLs among 61 BCR/ABL^POS patients [95]; unfortunately, no details were provided on the immunophenotypic diagnosis of those T-lineage cases, though it is apparent that cCD3 and cCD22 were not tested. The lack of demonstration of these lineage-specific antigens is the most likely reason for misdiagnosed BCR/ABL^POS T-cell phenotypes. A potential alternative cause is an apparent Philadelphia chromosome translocation by conventional karyotyping that does not yield the ABL-BCR juxtaposition on chromosome 9 [96]. While in the report by Fossa et al. [96] the leukemic phenotype was unequivocally T-lymphoid (cCD3^POS), an analogous case was seen in ECOG’s E2993 trial, with a (9;22)(q34;q11.2) translocation that by fluorescence in situ hybridization (FISH) did not result in the BCR/ABL fusion but, as in Fossa’s case, disrupted the BCR gene (ECOG Cytogenetics Committee, personal communication). The E2993 patient, however, expressed immunophenotypic features of Transitional Pre-B ALL. In summary, caution is definitely warranted when t(9;22)^POS or BCR/ABL^POS T-ALL cases are published.

MLL/AF4 is the typical subtype of infant ALL and the most prevalent of the diverse mixed lineage leukemia (MLL) fusion genes in adult ALL [97–99]. Despite improved complete remission rates with modern treatments, overall survival in adults remains poor due to short remission duration [98, 99]. MLL/AF4^POS ALL is associated with the immature Pro/Pre-Pre-B ALL maturation stage, defined by negativity for CD10. As a unique immunophenotypic feature among all ALL phenotypes, MLL/AF4^POS lymphoblasts show a tendency to express the more mature myeloid carbohydrate antigens, CD65_(S) and CD15_(S), while lacking CD33 and CD13, the pan-myeloid antigens expressed earlier in normal myelopoiesis [71, 99, 100]. On the other hand, CD33 and/or CD13 are found preferentially in CD10^pos early Pre-B ALL. Burmeister et al. [100] reported that lymphoblasts from MLL-rearranged adult ALL patients (55 % with MLL/AF4) demonstrated significantly lower expression of CD10, CD33, and CD13 and more frequent expression of CD65_(S) and CD15_(S) irrespective of B-lineage differentiation stage. Together with the myeloid antigen expression pattern in BCR/ABL^POSALL [88], these observations suggest that myeloid antigen expression in B-lineage ALL is determined by the underlying genetic defect rather than B-lymphoid developmental stage.

The cryptic (12;21)(p13;q22) translocation that results in the TEL/AML1 fusion gene is the most common genetic aberration in pediatric ALL (>20 %) and carries a favorable prognosis, especially with high-intensity therapy [101, 102]. In adults, this hybrid gene only accounts for <1–3 % of ALL [80, 103, 104], thus precluding prognostic predictions. In children, the TEL/AML1 fusion occurs exclusively in Early Pre-B ALL with certain characteristic markers: high-intensity of HLA-DR, CD40, and CD10 expression (Early Pre-B ­phenotype), commonly CD13^POSand/or CD33^POS, but CD9^NEG, CD20^NEG, and low expression of CD45, CD135, and CD86 [105–107]. A striking characteristic feature has recently been added, namely absence of CD11b expression [108]. Given the low incidence of TEL/AML1^POS ALL in adults, immunophenotypic information in this age group is scarce. The nine TEL/AML1^POS cases found among ECOG E2993 patients (1.4 %) demonstrated the characteristic CD10^POS Early Pre-B phenotype, lacking CD20 and predominantly CD34, and frequently expressing CD33+CD13. However, in comparison to Early Pre-B ALL with normal cytogenetics and molecularly negative for TEL/AML1, BCR/ABL, MLL/AF4, and E2A/PBX1, there was no difference in CD45, and the CD40 intensity was higher than the median value in normal karyotype controls in only half of the patients [104]. Furthermore, CD11b was expressed by all lymphoblasts in two of the seven TEL/AML1^POS E2993 cases, in which this antigen was tested (Paietta E, unpublished observation). Although based on small numbers of patients, the most predictive immune profile applicable for both pediatric and adult TEL/AML1^POS B-lineage ALL, to date, is CD10^POS,CD20^NEG, CD34^NEG, cIgM^NEG, frequently CD33+CD13^POS, and possibly CD11b^NEG.

As a result of two cryptic cytogenetic aberrations, inv(7)(p15q34) or t(7;7)(p15;q34), the TCRB locus (TRB@) at 7q34 is juxtaposed to the HOXA@ at 7p15 (approximately <5 % of pediatric and adult T-ALL), leading to transcriptional activation of several HOXA genes [109, 110]. The typical antigen expression patterns associated with the TRB@/HOXA rearrangement include negativity for CD2 and single expression of CD4, without CD8.

Up until recently, the detection of disease-specific fusion proteins or gene mutation products in hematologic malignancies was left to molecular testing mostly due to impracticality for antibody-based techniques in routine laboratories or the unavailability of proper antibodies. Nowadays, immunohistochemistry has replaced molecular studies for some proteins, e.g., the ALK protein derived from the (2;5) translocation in various lymphomas [6]. For some proteins, transformation into a hybrid state (e.g., PML to PML/RARα) or mutation (e.g., NPM1 to mutated NPM1) results in the altered distribution within the cell, which can be exploited by recognizing specific immunohistochemical staining patterns [6]. Recently, monoclonal antibodies for flow cytometry have been introduced that either solely recognize mutated NPM1 [144] or the aberrant ectopic cytoplasmic accumulation of the mutant protein [145].

The most exciting development in this area came last year when the Euroflow Consortium in cooperation with the Beckton-Dickinson company developed a commercial kit for the flow cytometric detection of all variants of BCR/ABL proteins encountered in ALL and CML [7]. Although simple in theory, in practice, the detection of fusion proteins turned out to be much more complicated than anticipated [72]. A new system had to be developed, which did not target the fusion protein itself but labeled each part of the protein (e.g., BCR protein and ABL protein) separately. Brilliantly, the investigators coupled a catching antibody to the BCR protein and a phycoerythrin (PE)-conjugated detecting antibody to ABL. To ensure the recognition of all BCR/ABL fusion ­protein variants, the antibodies were designed against unique epitopes away from all known breakpoints in each gene. The simplicity and reliable performance of this test system and the intentions of this group to expand their methodologic approach to the clinically most significant fusion proteins (e.g., PML/RARα, AML1/ETO, CBFβ/MYH11, TEL/AML1, etc.) in the format of multiplex assays (e.g., Core-Binding-Facor kit) will change routine patient testing. The successful implementation of the BCR/ABL assay in diagnostic workup has been published [146]. The ECOG Leukemia Translational Studies Laboratory has already implemented the BCR/ABL flow cytometry assay in the eligibility screening of leukemia patients for trials that accrue either only BCR/ABL^POS or BCR/ABL^NEG patients; in all 70 ALL patients tested, the flow cytometric results were confirmed by subsequent PCR analysis (Paietta E, personal observation). In the case of BCR/ABL, a molecular analysis may want to be run subsequent to the flow cytometric assay, if the BCR/ABL transcript form is of interest. Figure 17.3 demonstrates the clear separation of PE-fluorescence intensities, reflecting the detection of BCR/ABL fusion protein, in a BCR/ABL^NEG B-lineage ALL patient, a patient with 10 % BCR/ABL^POS blasts and a patient with 90 % BCR/ABL^POS lymphoblasts. In addition to recording the PE-mean fluorescence intensity and using the negative PE-mean fluorescence intensity range for determining positivity, the ECOG Leukemia Translational Studies Laboratory implemented the intensity ratio for easier comparison between assay runs. This ratio derives from the PE-mean fluorescence intensity of the patient (or positive control) divided by the PE-mean fluorescence intensity of the negative control run in every individual BCR/ABL bead assay. The reason for using this ratio is that the PE-mean fluorescence intensity of negative controls varies ever so slightly among assays (even when using the same cell line) and this variability should be incorporated in the calculation of the extent of positivity of a patient. This approach is analogous to that taken for the calculation of intensities of antigen expression by leukemic cells. While not sufficiently sensitive for MRD, the threshold level of at least 1 % (1 abnormal cell in 100 normal cells) [7] has been found useful in patients who presented with <1 % of circulating lymphoblasts by morphology and flow cytometric immunophenotyping (Paietta E, personal observation).

There are several aspects to antigens and antibodies and their usefulness in therapy. First, antibodies to carefully selected antigens expressed by the leukemic cells are administered in in vivo treatment. While purging of stem cell collections with antibodies used to be an important part of this aspect [19], it is no longer of great clinical relevance. Second, therapy may be determined by the specific phenotype of the leukemic cell population. In that case, we speak about phenotype-specific therapy. Both aspects contribute to targeted or personalized therapy in leukemia. Targeted approaches include the use of established surrogate marker profiles for genetic lesions, a subject already discussed earlier in this chapter. The multidrug resistance (MDR) phenotype represents an example of how specific antigens or antigen combinations can reflect functional characteristics of leukemic cells, such as the expulsion of chemotherapeutic drugs. Though MDR modulators have failed to fulfill their promises in the clinic, the strong prognostic association of expression of MDR proteins with outcome, at least in adult AML [147], should keep the interest of scientists alive in elucidating the relationship between MDR and other biologic parameters, such as cytogenetic aberrations. On the other hand, P-glycoprotein (Pgp) expression at initial diagnosis does not appear to be an independent prognostic factor in pediatric AML or ALL [148]. Despite diminishing therapeutic significance at this time, a short description of various MDR proteins and their flow cytometric detection is presented.

Therapy in acute leukemia nowadays is predominantly risk-adapted and rarely subtype-oriented. Subtype-oriented therapy applies, for instance, to APL (e.g., ATRA, arsenic trioxide), BCR/ABL^pos ALL (tyrosine kinase inhibitors with chemotherapy and allogeneic stem cell transplantation), and core-binding factor leukemias with their exquisite sensitivity to high-dose cytosine arabinoside. Other phenotypes of likely, though yet unproven therapeutic potential are ­leukemias with FLT3 gene mutations. Several inhibitors of FLT3-tyrosine kinase activity are currently being tested for clinical efficacy in FLT3-mutated AML, with the expectation that patients who share the same molecular defect might respond with equal sensitivity to the same inhibitor. The genetic variability of FLT3 gene mutations, however, impedes any prediction of response [235]. Nonetheless, the success of targeting the FLT3 receptor may be predicted by single cell network profiling (SCNP), which measures the effects of stimulatory or inhibitory therapeutic interventions on multiple signaling pathways [18]. SCNP combines immunophenotyping and potentiated phosphoflow cytometry of cellular responses to discern subpopulations of malignant cells based on their signaling profiles [236]. Assessing the functionality of cellular biochemical pathways in selected cell populations by flow cytometry provides a new tool in the monitoring of molecularly targeted therapy.

Risk-adapted therapy orients itself on established prognostic factors, such as age, white blood cell count, and cytogenetic/molecular aberrations at presentation or level of MRD following induction chemotherapy in morphologically defined complete remission. The powerful negative impact of MRD levels in otherwise standard-risk patients in ALL [15] and AML [16] has prompted increasing efforts to improve and facilitate methodologies for MRD determination. Although a higher sensitivity for detection of remaining leukemic cells is afforded by molecular techniques (e.g., PCR amplification for leukemia transcripts in AML or antigen receptor genes in ALL), flow cytometric methods are currently preferred, because they are applicable in the vast majority of patients, are cheaper, and are easily performed in routine laboratory settings. From scouring the literature, it appears safe to make the statement that although sensitivity levels of molecular and immunologic MRD assessments are not comparable, their prognostic significance appears to be equal. This suggests that below a certain level of residual disease, approximately at 1 abnormal cell in 10,000 normal cells (0.01 %), the remaining disease burden lacks clinical relevance.

In at least 95 % of acute leukemias, a single dominant lineage affiliation can be established through the detection of one of three lineage-specific antigens, myeloperoxidase (protein, not function), cCD22 or cCD3, for the myeloid, B- or T-lineage, respectively. The proposed specificity of cytoplasmic CD79a for B-cell-related ALL and potential pitfalls with its usefulness have been previously discussed in detail [19]. It is essential that myeloperoxidase be tested simultaneously with cCD3 or cCD22 in leukemic blasts that are gated flow cytometrically either through side-scatter versus CD45 or through a gating antibody, such as CD34 or CD117. Leukemia populations with truly “biphenotypic” features are rare and manifest themselves through dual expression of two lineage-specific antigens in the same cell, e.g., myeloperoxidase and cCD3 as seen in some cases with CD117^POS ALL [11]. An example of a biphenotypic subpopulation with dual myeloperoxidase/cCD22 expression in a case of otherwise B-lineage ALL is shown in Fig. 17.8. It is important to remember that CD22 and CD3 can both be found on the surface of mature B- and T-lymphocytes, respectively; control staining of the cell surface and gating exclusively on abnormal cells are an absolute prerequisite for meaningful results. The intracellular localization of these crucial lineage-specific antigens is unfortunate because their detection by flow cytometry requires technical skill and experience. In fact, routine clinical laboratories may shy away from testing intracellular antigens by flow cytometry. Aside from choosing optimal fixation and permeabilization media, it is essential that data on intracellular antigens reflect gated blast cells. Following fixation and permeabilization, scattergram characteristics (size and granularity) of cell populations change, often leading to the collapse of populations that could be easily distinguished in unfixed specimens. This situation creates a problem, especially if blast cells lack immature antigens, which are ­routinely used to separate leukemic from normal cells (e.g., CD34, CD123). As an example, CD5 and CD3 antibodies are useful in the detection of surface CD5^posCD3^neg T-lymphoblasts and separation from normal surface CD5^posCD3^pos T-lymphocytes. Thus, both of these antibodies must be combined with the myeloperoxidase antibody to restrict detection of antigen in T-lymphoblasts. If similar measures are not employed, it can lead to misleading reports, such as the finding of myeloperoxidase protein expression in a high percentage of ALLs.