Cytochemical Classification Research Articles

A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase.

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy-

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a technicon H‐1

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.

Reproducibility of the French‐American‐British classification of acute Leukemia: The southwest oncology group experience

After initial French-American-British (FAB) diagnosis by a multiinstitutional Southwest Oncology Group panel, slides of acute leukemia cases were recirculated to panel members for second review. The reproducibility of the FAB classification is analyzed. The classification is reproducible in the 70% range in panel reviewer hands and allows remarkable reproducibility in the morphologic and cytochemical distinction of acute lymphoid leukemia (ALL) from acute myeloid leukemia (AML). The limitations of a morphologic and cytochemical classification of acute leukemia are discussed. A simplification of the FAB system, merging M1, M2, and M4 as M7, is proposed; this simplification improves the system's reproducibility.

Individual nuclear uptake patterns for adriamycin and daunomycin in human leukemia and lymphoma cells.

Since both cytotoxicity and resistance towards the anthracycline derivatives adriamycin and daunomycin have been related to cellular uptake and intranuclear retention, the nuclear concentrations of these antibiotics were separately determined in various human leukemia cells after standardised in-vitro exposure. The study included analyses of cells from 31 patients (AML, 12; AUL, 7; T-cell leukemias, 4; leukemic B-cell lymphomas, 2; CML blast crisis, 5; CLL, 1). In all samples studied the uptake was concentrative with intranuclear concentrations exceeding the concentrations in the medium by factors of 4-10 for adriamycin and 6-13 for daunomycin. After 90 min of incubation, when daunomycin incorporation had reached steady state and 90% of the steady state concentrations was reached in the case of adriamycin, the comparative nuclear anthracycline concentrations showed a marked patient-to-patient variation without any relation to the cytological or cytochemical classification. Striking differences were noted in the ratios of DNM/ADM intranuclear concentrations ranging from 1.0 to 5.5. The DNM/ADM ratio was low in three cases with prior DNM treatment and, vice versa, increased in a case of CML blast crisis from 1.8 to 2.7 following ADM treatment.

Rapid in situ cytochemical classification of CFU-C colonies in soft agar culture: permanent whole culture preparations.

Soft agar culture CFU-C (colony-forming units in culture) are rapidly classified in situ as eosinophil, macrophage, and neutrophil-monocyte types by whole culture staining with luxol fast blue for eosinophil specific granules and acetoorcein for nuclei. Stained colonies may be picked and examined individually as wet mount cover slip preparations, or the agar culture may be air dried and mounted permanently. The whole culture stain has been variously modified for the enzyme markers alpha-naphthyl acetate esterase and peroxidase and for nuclear staining alone with acetoorcein.

A test with dexamethasone in acute leukemia

A simple test with dexamethasone (DMS) in acute leukemia (AL) is described. In peripheral blood, blast cell count is determined before 8 mg DMS are given intravenously, and 2, 4 and 6 hours afterwards. The result is expressed as the lowest blast cell count after DMS in percentage of the initial value. This test was performed on 51 adult patients with AL. The results were correlated with the morphological and cytochemical classification. Only patients with clearly classified AL were evaluated. A statistically significant difference in blast cell response between acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) was observed. In 12 out of 16 cases of ALL, but in only 1 out of 19 cases of AML, the blast cell count decreased to 50 percent or less of the initial value. The results of the test were further correlated with the results of treatment. In 11 out of 12 patients with ALL, who showed a response to DMS, glucocorticoids were included in the treatment regimen. A complete remission was obtained in 7 out of 10 patients who were treated for the first time. In the remaining 4 patients with a poor response to DMS, a complete remission after the first treatment was obtained in only one case. The number of patients examined is to small for final conclusions on the value of this test for a discrimination between glucocorticoid-responsive and non-responsive cases of AL. Nevertheless, these preliminary results indicate that further trials seem to be warranted.

Value of the combined cytological and cytochemical classification in the management of acute childhood leukemia.

50 leukemic patients were analyzed in a retrospective study in regard to their cytology and cytochemistry at the time of diagnosis. They were classified as ALL-PAS type, SCL-undifferentiated type and AML-peroxydase type. The results show that the combined cytological and cytochemical classification of childhood leukemia give more information about the expected course of a newly diagnosed leukemia in regard to duration of first remission and survival time and about its proper treatment.

Comparison of cytological and cytochemical classification in 47 cases of acute leukaemia

Comparison of cytological and cytochemical classification in 47 cases of acute leukaemia