H. J. Adriaansen

Immunological marker analysis of cells in the various hematopoietic differentiation stages and their malignant counterparts.

The characterization of cells in the various hematopoietic differentiation stages can be performed morphologically. Additional characterization is obtained by immunological marker analysis (1–4). The expression of a particular set of immunological markers designates a cell to a particular differentiation stage. The various markers are detectable by means of antibodies or by rosette techniques (5–7).

Detection of Minimal Residual Acute Lymphoblastic Leukemia by Immunological Marker Analysis: Possibilities and Limitations

The detection of small numbers of malignant cells is a major problem at diagnosis and during follow-up of malignancies (1). The possibilities and limitations of methods for the detection of minimal residual disease in general depend on their specificity (malignant cells have to be identified among many normal cells), sensitivity (detection limit of the method) and reproducibility. Although the method as such may have an excellent reproducibility, the cell sample may not always be representative for the whole cell population.

Quality assessment of immunological marker analysis and the immunological diagnosis in leukaemia and lymphoma: a multi-centre study

Summary In order to standardize and assess the quality of immunophenotyping of leukaemias and lymphomas for diagnostic purposes, a cooperative study group in the Netherlands, SIHON, has formulated guidelines for the composition of antibody panels to be applied and guidelines for the interpretation of the marker analysis. To assess the value of these guidelines frozen cell samples of three patients with different haematological malignancies were sent to the 26 participating laboratories twice a year. Here we present the results with respect to the marker analysis and to the immunological diagnosis on 387 samples from 18 patients. A large inter‐laboratory variation was seen in the percentage of positive cells for each marker, which influenced the valuation of a marker to be discordant positive in up to 23% and discordant negative in up to 40%. No single major factor could be traced to explain the large variation in the results. However, probably due to the balanced composition of the antibody panel and to the application of the guidelines for interpretation, this variation did not much influence the agreement in immunological diagnosis. In only 13/387 samples (3.3%) differences in the percentage of positive cells caused disagreement in the final diagnosis. In 23 samples (5.9%) the disagreement was due to an incorrect application of the guidelines. Quantitative data of single observations obtained from different laboratories, in which the materials and methods are not standardized, cannot be compared; but standardization of guidelines for marker sets and for interpretation contributes to a high grade of agreement in immunological diagnosis.

TdT positive B-cell acute lymphoblastic leukaemia (B-ALL) without Burkitt characteristics.

Summary The leukaemic cells in a 23‐year‐old man were small to medium‐sized lymphoblasts with no cytoplasmic vacuoles and negative with PAS as well with peroxidase and acid phosphatase staining. Cytogenetic analysis showed ‐6,+12,‐22,+mar(6p::22q), resulting in a trisomy 12 and monosomy of the long arm of chromosome 6. Immunological marker analysis revealed that the majority of the blasts was positive for terminal deoxynucleotidyl transferase (TdT) as well as surface membrane immunoglobulin (SmIg, ü, λ), although B‐ALL are supposed to be negative for TdT. The blasts were also positive for HLA‐DR, CD9 (BA‐2), CD10 (VIL‐A1) and CD24 (BA‐1), but negative for the B‐cell markers CD20 (B1) and Y29/55. Double immunofluorescence staining confirmed that almost all TdT+ cells were also positive for Smμ, Smλ, HLA‐DR and CD10. We thus made a diagnosis of TdT+ B‐ALL without Burkitt characteristics. Since we could not detect SmIg+/TdT+ cells in bone marrow samples from adult healthy volunteers and from 10 children with ALL in complete remission, we conclude that TdT+ B‐ALL cells may not have a normal counterpart in bone marrow or represent a malignant counterpart of a very rare cell in an intermediate differentiation stage between the pre‐B‐cell and the early B lymphocyte.

Ultrasound examination of pathological cervical lymph nodes in patients with non-Hodgkin's lymphoma and Hodgkin's disease

Summary. The choice of treatment of malignant lymphoma may vary considerably and is mainly determined by the pathology and clinical stage. Ultrasonography is currently one of the most sensitive techniques for evaluation of the cervical area. We set out to assess the value of ultrasound imaging when added to conventional staging (physical examination, laryngoscopy, computer tomography of thorax and abdomen, bone marrow cytology and histology) of maliganant lymphoma in 47 patients with untreated lymphoma. Hodgkins disease was present in 14 patients and non‐Hodgkins lymphoma in 33 individuals. The ultrasound results were independently compared with physical examination of the neck. Ultrasonography revealed additional pathological lymph nodes in 6/47 cases (13%). Furthermore, the diagnosis non‐Hodgkins lymphoma could be established in one patient merely as a result of ultrasonography. Ultrasonography of the neck may reveal more pathological lymph nodes in a significant number of patients and may be of value in the initial staging of patients with malignant lymphoma.

Pitfalls in the immunophenotyping of leukaemia and leukaemic lymphomas: survey of 9 years of quality control in The Netherlands

During the last decade, biannual quality controls were performed in The Netherlands focusing on the immunophenotyping of leukaemic haematological malignancies. All results on 48 specimens obtained by 18–34 laboratories were analysed. The interlaboratory variability and percentages of discordant results from 30 markers were measured by assessing false positive or negative (cut‐off 10%) results in comparison with median results of the group. The quality of the immunophenotypic diagnoses obtained from the interpretation of these markers in relation to clinical data was evaluated by scoring them as ‘correct’, ‘minor fault’, ‘major fault’, ‘not based upon the markers used’, and ‘no diagnosis’. CD3, CD8, CD19, CD61 and Smλ had the lowest percentage discordancy (sum of total negative and positive discordant values 5–7.5% of assays); CD13, CD15, cyCD22, CD33 and TdT scored worst with 14–20% cumulative discordancy. The analysis of each diagnosis yielded 78% acceptable immunophenotypic conclusions (correct 54% and minor fault 24%). It appeared that the major faults in immunophenotyping were caused by suboptimal antibody selection and erroneous interpretation of the results obtained, rather than by technical errors. Large differences per diagnostic category were observed, with the best scores for mature B‐cell leukaemias, AMLs and common‐ALL, and the poorest scores for T‐cell malignancies which were correctly diagnosed in only 24–60% of specimens. Mature T‐NHL and T‐PLL were mistakenly diagnosed as T‐ALL by 40% of the centres. Misinterpretation of TdT immunofluorescence or omitting this marker contributed significantly to these wrong diagnoses. A median of 4% of immunophenotypic diagnoses were not based on a correct panel of antibodies, but upon the morphology of the accompanying blood smear, and was often flawed by overinterpretation.

Double Marker Analysis for Terminal Deoxynucleotidyl Transferase and Myeloid Antigens in Acute Nonlymphocytic Leukemia Patients and Healthy Subjects

The enzyme terminal deoxynucleotidyl transferase (TdT) is expressed on the nuclear membrane of normal precursor B and T cells as well as their malignant counterparts, i.e., acute lymphoblastic leukemias (ALLs) and some lymphoblastic lymphomas [1, 2]. TdT expression has also been found in 5%–46% of acute nonlymphocytic leukemias (ANLLs) [3–9]. In ANLL there is a large variability in the percentage of TdT+ cells, and also the intensity of TdT expression varies per cell. In most studies a limit of at least 10% of TdT+ cells was adopted for the diagnosis of a TdT+ ANLL. However, it is likely that in some ANLLs smaller TdT+ leukemic subpopulations are present.

Gold-Induced Pneumonitis

Gold-induced pneumonitis is a rare complication of gold salt therapy. We describe a patient with rheumatoid arthritis treated with gold salts, who developed bilateral interstitial pulmonary abnormalities and showed a dramatic response on corticosteroid therapy. Although after 4 weeks of treatment with corticosteroids the chest X-ray and lung function were still abnormal, bronchoalveolar lavage showed a normal cell distribution. Corticosteroid therapy was continued for 8 months since there was still improvement of pulmonary function studies. This case supports the view that in gold-induced pneumonitis a prolonged treatment with corticosteroids may be necessary, as lung function continued to improve.

Persistent polyclonal B-cell lymphocytosis: extensively proliferated CD27+IgM+IgD+ memory B cells with a distinctive immunophenotype.

Persistent polyclonal B-cell lymphocytosis: extensively proliferated CD27+IgM+IgD+ memory B cells with a distinctive immunophenotype

Immunophenotypic and Immunogenotypic Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia

Despite major improvements in the treatment of patients with acute lymphoblastic leukemia’s (ALL) during the last two decades, still 20%–30% of children with ALL and 60%-75% of adult ALL patients will develop a relapse (Hoelzer et al. 1984; Linker et al. 1987; Riehm et al. 1990; Veerman et al. 1990; Ellison et al. 1991). Apparently, the current treatment protocols are not capable of killing all leukemic cells in these patients, although they seemed to be in complete remission according to cytomorphological criteria. Since the detection limit of cytomorphological techniques is 1%–5% leukemic cells, it is obvious that such techniques can only provide superficial information about the effectiveness of leukemia treatment. More sensitive techniques for the detection of low numbers of leukemic cells are needed to obtain better insight into the reduction of tumor mass during induction treatment and further eradication of the leukemic cells during maintenance treatment.