An empirical evaluation of the performance of antibody identification tasks

Four empirical studies were conducted for better understanding of the nature of problem-solving activities by medical technologists and medical technology students when performing antibody identification tasks. The results indicated the importance of strategies that ensure the collection of converging evidence, as these strategies protect against the fallibility of commonly used heuristics and against errors due to simple slips. The results also indicate that not only do students make significant numbers of errors, but so do practicing technologists. In one of the studies covering a 1-year period, for instance, a group of 16 technologists made a total of 41 errors in 1057 cases. On the basis of these findings, several alternatives are proposed to reduce errors.     

Analysis of p53 mutations in a large series of lymphoid hematologic malignancies of childhood

p53 mutations are found in a wide variety of cancers, including hematologic malignancies. These alterations apparently contribute to development of the malignant phenotype. We analyzed a large series of lymphoid (330 cases) and a smaller series of myeloid (29 cases) malignancies of childhood for p53 mutations by single-strand conformational polymorphism (SSCP) following polymerase chain reaction. Samples with abnormal SSCP were reamplified and analyzed by direct sequencing method. p53 mutations were detected within the known mutational hotspots (exons 5 to 8) in 8 of 330 lymphoid malignancies, and in none of 29 myeloid malignancies, showing that the frequency of p53 mutations in childhood lymphoid malignancies was very low (8 of 330 cases [2%]). Four of these patients had very aggressive, fatal acute lymphocytic leukemia (ALL). None of 13 infants and none of 48 patients with T-lineage leukemia had detectable p53 mutations in their ALL cells. Exceptionally, p53 mutations were comparatively frequent in a small sample of B-cell non-Hodgkin's lymphomas (2 of 8 cases). Mutations were detected in samples from two patients with ALL at relapse; these were not detected in samples at initial diagnosis from the same patients, suggesting that p53 mutations may be associated with progression to a more malignant phenotype. Seven of eight alterations of p53 were missense mutations, and seven of eight samples may be heterozygous for the mutant p53, indicating that p53 protein may act in a dominant negative fashion.     

Production of Colony-stimulating Factor by Malignant Leukocytes

There is considerable evidence supportinga role for colony-stimulating factor (CSF)as a humoral regulator of leukopoiesis.Data on CSF levels in the serum and urineof patients with leukemia and on the invitro responsiveness of leukemic cells toCSF have suggested a basis for consideringleukemia as a primary disorder of leukopoietic regulation. We examined the question of leukemic cell production of CSF.Conditioned medium from cultured leukemic cells was tested for colony-stimulating activity against normal human bonemarrow using a two-layer agar colonyassay technique. The cells from patientswith acute myelogenous leukemia and apatient with chronic myelogenous leukemia in blast crisis did not elaborate CSFnor did acute lymphocytic leukemia cells.CSF production was documented with cellsobtained from patients with chronicmyelogenous leukemia in the chronicphase and two patients with acute myelomonocytic leukemia. In acute leukemiathe cellular production of CSF correlatedclosely with morphologic and functionalmaturation along the monocyte-macrophage line. Evidence was obtained thatthe adherent cells within the leukemicpopulation were primarily responsible forCSF production. We interpret these data toindicate that neoplastic hematopoieticcells may produce CSF in relation to theircapacity for mononuclear leukocytedifferentiation.

Submitted on July 11, 1973 Revised on November 9, 1973 Accepted on November 13, 1973     

Infections in hairy-cell leukemia.

In order to determine the nature of infectious complications in hairy-cell leukemia we studied 20 consecutive patients seen at UCLA and analyzed the available literature. The incidence of serious infection in our series was 40%, and pneumonia and septicemia due to Pseudomonas and E. coli organisms were the leading types of infections. Fungal infections with Cryptococci and Histoplasma organisms were documented, and a single case of Pneumocystis carinii pneumonia was observed. Noninfectious fever occurred in 30% of our patients. There was a clear relationship between fungal disease and corticosteroid therapy, and the overall incidence of infection was correlated with the degree of neutropenia and corticosteroid treatment. No relationship was found between age, duration of disease, or the use of cytotoxic chemotherapy and infectious complications. Of the 13 infectious episodes, 11 occurred in patients prior to splenectomy. Only two episodes were seen in splenectomized patients, both occurring in the immediate postoperative period. We conclude that splenectomy has a beneficial effect in reducing the incidence of infections in hairy-cell leukemia and that corticosteroids should be used cautiously, since they predispose to opportunistic infection in this disease.     

Trimethoprim and Sulphamethoxazole Inhibition of Haematopoiesis in Vitro

The effect of trimethoprim and sulphamethoxazole on haematopoiesis was studied in vitro using cloning techniques for human and murine erythroid and granulocytic precursor cells. Trimethoprim was found to inhibit granulopoiesis and erythropoiesis in vitro in a dose-dependent fashion with approximately 50% inhibition of human erythroid and granulocytic colonies at a therapeutically achievable concentration of 7 micrograms/ml. Sulphamethoxazole was also shown to impair haematopoiesis in vitro. The inhibition caused by both these constituents of co-trimoxazole was completely reversed by folinic acid. The data suggest that co-trimoxazole can impair human haematopoiesis by inhibition of tetrahydrofolate synthesis. These observations suggest that the clinical haematopoietic toxicity of trimethoprim-sulphamethoxazole can be abrogated by simultaneous administration of folinic acid.     

Proliferation of human malignant hematopoietic cells in immunodeficient mice: suppression by antibody to pluripotent K-562 leukemia cells involves direct cytolysis and effector cells

Six human hematopoetic cell lines were successfully heterotransplanted into athymic (nude) and asplenic-athymic (lasat) neonatal mice. The tumors arising from leukemia and lymphoma cells could then be serially transplanted into adult nude mice. Seven days after the fourth serial mouse passage, each mouse was treated with goat immune gamma globulin against K-562 cells. One control group was treated similarly, but with nonimmune (normal) gamma globulin, while another control group was not treated. The goat gamma globulin was not toxic for nude and lasat mice, and the immune, but not the normal, gamma globulin suppressed local subcutaneous growth of myelosarcomas, lymphosarcomas, and Burkitt lymphoma cells. On the other hand, the growth of lung, breast, and prostatic carcinomas and a melanoma of human origin were not altered by the immune gamma globulin. Since suppression of cell growth occurred equally well in decomplemented mice, a complement-mediated cytotoxicity apparently cannot be considered as responsible for the abrogation. The Fab fragment of the immunoglobulin did not suppress the growth of the myelosarcomas. We conclude that antibody suppression of the in vivo proliferation was specific for malignant hematopoietic cells and that the Fc portion of IgG is necessary for in vivo cytolysis of leukemia cells. The most probable mechanisms are direct antibody cytolysis and antibody-dependent macrophage-mediated cytotoxicity.     

Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain sequences

Human T-cell receptor (TCR) delta-chain diversity mainly originates from high junctional variability, since only a limited number of germline elements is available. This extraordinary diversity at the V.J junction, due to the use of two D delta elements and extensive incorporation of N nucleotides, constitutes a specific clonal marker for cell populations exhibiting rearranged TCR delta genes. To this end we amplified in vitro by polymerase chain reaction (PCR) the TCR delta junctional region of five acute lymphoblastic leukemias (ALL), isolated respective DNA fragments, and used them directly as clonospecific probes. The combination of PCR technology and hybridization to clonospecific probes permitted the detection of leukemia DNA at dilution of 1:100,000 in all five cases. Moreover, we were able to investigate one of the ALL patients 11 months after achieving continuous complete remission. Conventional Southern blot analysis failed to detect rearranged TCR genes at this stage. However, residual leukemic cells could readily be detected by PCR technique. We conclude that the strategy proposed here is a very sensitive tool to detect minimal residual disease in a significant proportion of human lymphoid neoplasias.     

A point mutation in the integrin beta 3 cytoplasmic domain (S752-->P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa)

Agonist-induced inside-out signaling results in an increased affinity of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) for soluble ligands (fibrinogen [Fg] and PAC1). Ligand binding to integrins initiates outside-in signaling that leads to cellular responses such as cell spreading and focal adhesion formation. A point mutation in the beta 3 cytoplasmic domain (S752-->P) is associated with blocked inside- out alpha IIb beta 3 signaling in a variant Glanzmann's thrombasthenia. This mutation was introduced into beta 3 and cotransfected into Chinese hamster ovary cells with a chimeric alpha subunit consisting of the alpha IIb extracellular and transmembrane domains and the alpha 6B cytoplasmic domain. The substitution of the alpha IIb cytoplasmic domain with that of alpha 6 led to activation of alpha IIb beta 3 to bind PAC1, mimicking inside-out signaling. This effect was reversed by the S752-->P mutation, indicating a disruption of inside-out signaling by the mutation. In addition, transfectants expressing this beta 3 variant showed reduced alpha IIb beta 3-mediated cell spreading on immobilized Fg, focal adhesion, and fibrin clot retraction, suggesting an impairment in outside-in alpha IIb beta 3 signaling. Therefore, a single point mutation in the beta 3 cytoplasmic domain impaired bidirectional signaling through alpha IIb beta 3.