W.M. Comans-Bitter

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 103 to 105 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 (rs = 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 (rs = 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 (≤10−4). Furthermore, in the 48 double-positive pairs, MRD levels in BM and PB varied enormously with MRD levels in BM being up to 1000 times higher than in the corresponding PB samples. Consequently, BM samples cannot easily be replaced by PB sampling for MRD analysis in childhood precursor-B-ALL, in line with their BM origin. In T-ALL, which are of thymic origin, BM sampling might be replaced by PB sampling, because the dissemination of T-ALL cells to BM and PB appears to be comparable.

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 ∼4 h, and can be run in parallel to routine immunophenotyping.

Triple immunological staining with colloidal gold, fluorescein and rhodamine as labels☆

Colloidal gold particles are detectable by light microscopy with polarized light in the same epi-illumination system as for fluorescence microscopy. Colloidal gold particles can thus be used in combination with a fluorochrome for the combined immunological detection of surface membrane and cytoplasmic markers. We analyzed human bone marrow cells by a triple immunological staining for 3 different markers, using colloidal gold, fluorescein and rhodamine as labels. Our results demonstrate that such a triple immunological staining provides a powerful tool for study of the heterogeneity of small cell populations.

Analysing the developing lymphocyte system of neonates and infants.

Abstract 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.

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.

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.

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.

The Small Subpopulation of T Cell Marker+/Tdt+ Cells in the Human Bone Marrow may Represent Prothymocytes

Most normal haematopoietic differentiation stages have malignant counterparts in the form of a leukaemia or non-Hodgkin lymphoma (NHL) (Foon et al., 1982). The acute lymphoblastic leukaemias (ALL) may be regarded as the malignant counterparts of normal immature lymphoid cells (Janossy et al., 1980, 1981). These ALL can be characterized immunologically by use of several markers such as: terminal deoxynucleotidyl transferase (TdT), the HLA-DR non-polymorphic antigen, the common ALL antigen (cALL), cytoplasmic immunoglobulin μ heavy chain (cIgM), surface membrane Ig (SmIg) and the T cell markers Tp41, T11, T1, T6, T4, T8 and T3 (Foon et al., 1982; Vodinelich et al., 1983).