The immunophenotyping of extramedullary myeloid cell tumors in paraffin-embedded tissue sections.

Extramedullary tissue infiltrates of acute myeloid leukemia are rare and often difficult to recognize in routine paraffin-embedded tissue sections. Since appropriate therapy for these tumors depends on their precise identification, we have studied a series of tissues infiltrated with primitive myeloid cells using monoclonal and polyclonal antibodies capable of labeling cells of the myeloid/monocytic system in paraffin-embedded tissue sections. The current retrospective study involved tissues from 15 patients (eight men and seven women) with a mean age of 51 years (range, 23-77). A diagnosis of extramedullary myeloid cell tumors had been made on the basis of routine histology, chloroacetate esterase cytochemical stain, and--in some cases--electron microscopy. Paraffin-embedded tissue sections were cut and stained employing the alkaline phosphatase antialkaline phosphatase (APAAP) immunocytochemical procedure with monoclonal antibodies against leukocyte-common antigen (PD7/26-2B11), restricted components of the leukocyte-common antigen (UCHL1, 4KB5), granulocytes (Mac-387, Leu-M1), leukocytes (MT1, MT2, LN1, LN2), HLA-DR (LN3), and elastase (NP57), as well as polyclonal antibodies against lactoferrin, lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin. Results indicate that antibodies against Mac-387, elastase, and lysozyme are most useful in the recognition of neoplastic myeloid cells. We conclude that tissues containing granulocytic tumors can be identified in paraffin-embedded tissue sections using a panel of antibodies and the APAAP procedure.

Non-Hodgkin's lymphoma with immunologic phenotype similar to non-T, non-B acute lymphocytic leukemia.

A diagnosis of diffuse poorly differentiated lymphocytic lymphoma was made from a biopsy of a scapular mass on a 24-month-old child. The bone marrow and peripheral blood were not involved in the neoplastic process. Neoplastic cells stained negatively for Sudan black B, myeloperoxidase, periodic acid-Schiff reagent, alpha-naphthyl acetate esterase, and acid phosphatase. In addition, neoplastic cells did not form nonimmune rosettes with sheep erythrocytes or contain surface membrane immunoglobulin. However, neoplastic cells were positive for terminal deoxynucleotidyl transferase and "Ia-like" antigen. We conclude that this non-Hodgkin's lymphoma has a cytochemical and immunologic phenotype similar to that of lymphoblasts from cases of non-T, non-B acute lymphocytic leukemia.

Immunophenotyping of hematologic neoplasms in paraffin-embedded tissue sections.

Several immunohistochemical methods are now available for the staining of neoplastic cells in tissue sections. The authors have found that the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method is sensitive and reliable. Murine monoclonal or nonmurine polyclonal antibodies can be used to label a variety of membranous and/or cellular constituents in tissues that have been routinely processed in a histopathology laboratory. The monoclonal antibody against leukocyte common antigen (CD45) can be used to differentiate hematologic from nonhematologic tumors. Monoclonal antibodies (L26, LN1, LN2, LN3, MB1, MB2) label B-cell lymphomas, whereas other monoclonal antibodies (UCHL1, MT1) more characteristically stain T-cell lymphomas. Polyclonal antibodies against CD3 specifically mark neoplastic cells from T-cell lymphomas and leukemias but as yet are not commercially available. Monoclonal antibodies Leu-M1 (CD15), Ber H2 (Ki-1; CD30), and LN2 label Reed-Sternberg cells from most cases of nodular sclerosis, mixed cellularity, and lymphocyte-depleted Hodgkin's disease. Monoclonal antibodies Mac 387, KP1 (CD68), and NP57 (antielastase), as well as polyclonal antibodies against lysozyme, help identify subtypes of acute myeloid leukemia and extramedullary myeloid cell tumors. Although there are now excellent reagents ready for use, there is still a significant need for more lineage-specific (particularly against CD epitopes) monoclonal antibodies capable of labeling neoplastic cells in paraffin-embedded tissue sections from patients with hematologic malignancies.

Mouse erythrocyte formation. A marker for resting B lymphocytes.

The ability of peripheral blood lymphocytes to form rosettes with mouse erythrocytes was examined using lymphocytes from normal healthy individuals of various ages and of neoplastic lymphoid cells from patients with a variety of lymphoproliferative disorders. Our results indicated that only B lymphocytes form mouse rosettes (MR). The percentage of MR varied slightly according to the strain of mouse erythrocytes used in the assay. Little variation in the mean percentage of MR was observed when lymphocytes from various age groups of healthy individuals were studied. When neoplastic lymphoid cells were incubated with mouse erythrocytes only lymphocytes from patients with chronic lymphocytic leukemia formed an increased number of MR. Peripheral blood B lymphocytes lost their ability to form MR following incubation with pokeweed mitogen. These findings supported the view that the determinant on B lymphocytes which binds to mouse erythrocytes was present only at a resting stage of B cell maturation.

Comparison of bleeding times performed on the arm and the leg.

The standardized bleeding time (SBT) is used to assess hemostatic function in patients suspected of having coagulation disorders. Because injury to the arm or the presence of intravascular cannulae often preclude the determination of SBTs on the upper extremity, the authors compared the SBT on the arm with the bleeding time on the leg to ascertain the efficacy of the lower extremity for this test. Thirty healthy volunteers were enrolled in the study. Bleeding times were performed on the forearm and on the medial aspect of the calf. The subjects then ingested 650 mg of aspirin, and the tests were repeated two hours later on the contralateral extremities. The mean preaspirin SBT (5.6 +/- 1.7 minutes did not differ significantly from the mean bleeding time on the leg (5.8 +/- 2.3 minutes) (P greater than 0.50), nor was there a significant difference between the mean postaspirin bleeding time on the arm (10.2 +/- 4.3 minutes) and that on the leg (9.9 +/- 3.7 minutes) (P greater than 0.50). On the basis of this study, the authors conclude that the arm and leg are equally reliable sites for determining bleeding times in normal persons and are equally sensitive for detection of aspirin-induced prolongation of bleeding.

Serum immunoglobulins and lymphocyte subsets in chronic lymphocytic leukemia.

The concentrations of serum immunoglobulins were correlated to the stage of disease and the proportions of peripheral blood lymphocyte subsets in 25 untreated patients with B-cell chronic lymphocytic leukemia (CLL). Diminished levels of at least one serum immunoglobulin were present in 77% of all patients with CLL and 73% of patients with Stage 0 disease. The mean concentration of IgG, IgM, and IgA decreased with advancing stage of CLL. The percentages of total T, T-helper (TH), and T-suppressor (TS) cells in the peripheral blood were less in patients with CLL than in healthy persons, but the absolute concentrations of total T, TH, and TS cells were greater in patients with CLL than controls (P less than 0.02). The absolute number of B-cells (P less than 0.01) and null cells (P less than 0.001) was also increased in patients with CLL, particularly those patients in advanced stages of CLL. These findings suggest that the hypogammaglobulinemia associated with CLL first occurs during the earliest stage of disease and may be related to the alterations in the proportion of peripheral blood lymphocytes.

Immunophenotyping of acute leukemias using paraffin-embedded tissue sections.

In a study of 55 patients with either acute lymphoid leukemia (ALL; 25 cases) or acute myeloid leukemia (AML; 30 cases), paraffin-embedded bone marrow particle sections were examined with a panel of monoclonal and polyclonal antibodies reactive toward lymphoid and myeloid-associated antigens, using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. All cases were previously classified according to the French-American-British (FAB) Co-operative Group, and cases of ALL were immunophenotyped by flow cytometry. Results indicated that myeloid-associated antibodies (Mac 387, KP 1 [CD68], antielastase, antilactoferrin, and antilysozyme) did not react with any case of ALL, M1-AML, or M6-AML, whereas at least one of these antibodies reacted with 20 of 21 (95%) cases of M2, M3, M4, and M5-AML. Anti-glycophorin C marked cases of M6-AML, whereas anti-CD3 labeled T-cell ALL. None of the antibodies tested specifically identified cases of B-cell ALL. The authors conclude that use of a selected panel of antibodies on paraffin-embedded bone marrow particle sections may be of value in the diagnosis and immunophenotypic classification of many cases of acute leukemias.

T6 monoclonal antibody reacts with blasts from cases of common antigen acute lymphocytic leukemia.

The expression of the T6 antigen on malignant lymphoid cells has been considered strong evidence in support of T-cell lineage and thymic stage of differentiation of the neoplastic cells. Thus, the authors have used the T6 monoclonal antibody for the last three years in the immunophenotyping of blasts from 60 consecutive cases of acute lymphocytic leukemia (ALL) and 8 cases of T-cell lymphoma. Blasts from 12 of 46 (26%) cases of common type ALL, 4 of 7 (57%) cases of T-cell ALL, 2 of 3 (66%) cases of lymphoblastic lymphoma, and 1 of 5 (20%) cases of peripheral (postthymic) T-cell lymphoma were positive for the T6 antigen. The authors conclude that the expression of T6 antigen on malignant lymphoid cells may not always indicate T-cell lineage.

Immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue sections. A comparison with genotypic analysis.

Several monoclonal antibodies (MoAbs) are now available for immunophenotyping non-Hodgkin's lymphomas (NHLs) in paraffin-embedded tissue sections. To determine the reliability of these reagents in predicting the genotype, 44 cases of NHL were studied with the alkaline phosphatase-anti-alkaline phosphatase technique with the use of the following MoAbs: leukocyte common antigen (CD45), Mac 387, L26, 4KB5, MB1, MB2, LN2, UCHL1, MT1, and MT2. The lineage of the neoplastic cells was determined in all cases by gene rearrangement studies for immunoglobulin heavy chain and for the T-cell receptor beta-chain. Genotypic results showed B-cell lineage in 33 cases (75%), T-cell lineage in 6 cases (14%), and mixed or undetermined lineage in 5 cases (11%). A concordance of lineage assignment by paraffin section immunophenotyping with gene rearrangement studies was observed in 37 of 39 (95%) lymphomas with an unequivocally defined genotype. MoAb L26 was the most sensitive in detecting B-cell genotype; MoAbs MT1 and UCHL1 were the most sensitive and specific, respectively, in detecting T-cell genotype. The authors conclude that lineage assignment of NHLs in paraffin sections is reflective of the corresponding genotype when an appropriate panel of MoAbs is used.

The use of monoclonal antibodies against primary myeloid granules in normal and leukemic cells.

Three monoclonal antibodies, K101, D46, and H36/71 (CD15), reactive with membrane components of primary granules of human promyelocytes, were studied to assess their binding to normal and leukemic cells. Using the alkaline phosphatase antialkaline phosphatase technique, these antibodies were applied to sections of normal organs and to peripheral blood and bone marrow films from hematologically normal individuals and patients with hematologic malignancies. In control experiments, antibodies showed reactivity with cytoplasmic constituents of granulocytes from the promyelocytic to the neutrophilic stage. In acute myeloid leukemia, antibody K101 was positive (more than 20% of blasts) in 13 of 21 (62%) cases, while antibody D46 was positive in 11 of 17 (65%) cases. Antibody H36/71 was positive in only 4 of 24 (17%) cases of acute myeloid leukemia. At least one marker was present in 6 of 8 (75%) cases of acute lymphoblastic leukemia with myeloid antigen-positive blasts and was negative in 20 cases of acute lymphoblastic leukemia with myeloid antigen-negative blasts. These results support the view that abnormal granules (with defective expression of the D46, K101, and H36/71 antigens) form in blastic and leukemic cells of patients with acute myeloid leukemia. Data also suggest that membrane components of myeloid granules are made in the cytoplasm of cells from some acute lymphoblastic leukemia patients with myeloid antigen-positive blasts.

Cell volumes of normal and malignant mononuclear cells.

The cell volumes of mononuclear cells, T lymphocytes, B lymphocytes, and monocytes from the peripheral blood of 20 normal individuals were compared to neoplastic lymphoid cells from 14 patients with chronic lymphocytic leukaemia (CLL), 20 individuals with acute lymphocytic leukaemia (ALL), and 18 cases of non-Hodgkin's lymphoma (NHL). Normal T cells were obtained by rosetting mononuclear cells with sheep erythrocytes followed by centrifugation on a gradient composed of Ficoll and diatrizoate salts. Monocyte populations were prepared by adhering mononuclear cells to plastic dishes and B cells were obtained by the depletion of T lymphocytes and monocytes from a mononuclear cell population. Cell volumes were determined on a Coulter Counter Model H4 Channelyzer. In normals, the average mean cell volume (MCV) of T lymphocytes was smaller than B lymphocytes and the average MCV of B lymphocytes was smaller than the average MCV of monocytes (p less than 0.05). The average MCV of lymphocytes from patients with CLL was smaller than the average MCV of normal B cells (p less than 0.01). The average MCV of lymphoblasts from cases of ALL was larger than the average MCV of normal peripheral blood lymphocytes (p less than 0.01). In addition, the size of lymphoblasts showed great variation within and among cases of ALL. The MCV of lymphocytes from most cases of NHL was larger than the MCV of lymphocytes from reactive lymph nodes and from the peripheral blood of normal individuals. An association was observed between the MCV of neoplastic cells and the classification according to Rappaport. We believe that the measurement of lymphoid cell volumes may be helpful in the diagnosis and prognosis of patients with a variety of lymphoproliferative disorders.

Use of the APAAP method in the classification and diagnosis of hematologic disorders.

The APAAP technique is a sensitive and relatively easy immunocytochemical method to perform. It requires only a modest amount of laboratory space and no expensive equipment with the exception of a light microscope. Staining can be performed on peripheral blood and bone marrow films as well as cryostat and paraffin-embedded tissue sections. Prior to staining peripheral blood and bone marrow films as well as cryostat sections, slides can be stored at -70 degrees C, thus allowing for batch processing of specimens. Stained slides can be kept at room temperature for prolonged periods of time without loss of label. Surface or cytoplasmic determinants can be stained with the APAAP method. Because the preparations are usually counterstained, the identification of the labeled cells can be observed and photographed. The APAAP method is a reliable procedure and a significant addition to the routine hematology and histopathology laboratory.

Lymphocytic surface markers in lymphoid leukemoid reactions.

Although mononuclear cell surface markers are primarily used to determine the lineage and stage of differentiation of leukemias and lymphomas, they can also be helpful in discriminating between some cases of neoplastic and reactive conditions. Peripheral blood lymphocytosis secondary to infection most often shows increased numbers of activated T cells of the suppressor/cytotoxic subset. A few exceptions, such as in pertussis, show predominantly T-helper cells. Although persistent B-cell lymphocytosis is most often associated with neoplastic conditions, B-cell predominant reactive conditions may also occur. Lack of light chain restrictions on B-cell membranes suggests non-neoplastic disorders. Reactive lymphadenopathy most often shows a predominance of T cells with normal to increased T-helper/T-suppressor cell ratios. In addition, normal ratios of kappa/lambda light chain surface immunoglobulin usually occur on B cells of reactive lymph nodes. Benign lymphocytic infiltrates in skin most often show a predominance of activated T-helper cells. Distinguishing reactive from neoplastic dermal infiltrates by mononuclear cell markers can be extremely difficult and may require DNA genotypic analysis. Mononuclear cell markers applied to bone marrow in patients treated for leukemia and other disorders must also be interpreted with caution. The presence of CD10 antigen (CALLA) may herald recurrence of leukemia; however, this determinant is not leukemia-specific and may be found on normal cells. Similarly, lymphoid cells bearing TdT often represent recurrent leukemia, and they must be differentiated from immature nonmalignant TdT-positive cells. Immunologic surface markers must be interpreted together with a careful review of the morphology of the tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocytic sarcoma.

Granulocytic sarcoma is a variant presentation of acute myeloblastic leukemia, occurring in extramedullary locations. It is uncommon, but it may occur at any site and at any age, which necessitates its inclusion in the differential diagnosis of all undifferentiated tumors. Histology, touch-imprint cytology, cytochemistry, immunocytochemistry, electron microscopy, and molecular studies all contribute to the diagnosis.

Terminal deoxynucleotidyl transferase activity in non-hematologic and hematologic neoplasms.

The presence of terminal deoxynucleotidyl transferase (TdT) has been determined in neoplastic cells from 50 patients with non-hematologic tumors as well as neoplastic cells from 85 patients with hematologic malignancies. The results indicate that TdT is not present in cells from non-hematologic tumors, Hodgkin's lymphoma, B cell lymphoproliferative disorders, peripheral T cell neoplasms, reactive lymphadenopathy, and acute non-lymphocytic leukemia. In contrast, TdT activity is present in non-T non-B cell acute lymphocytic leukemia, T cell acute lymphocytic leukemia, T cell lymphoblastic lymphoma and chronic granulocytic leukemia in blast crisis. It is concluded that the TdT assay is a measurement useful in the differential diagnosis of some hematologic malignancies.

Clotting, activated partial thromboplastin and coagulation times in monitoring heparin therapy.

The automated-activated coagulation time, manual-activated coagulation time and the activated partial thromboplastin time were compared to the whole blood clotting time in the measurement of hypocoagulation of heparinized blood. The normal ranges and degree of reproducibility were determined for each clotting assay. Each method was examined for its sensitivity to various concentrations of heparin. In addition, blood samples from patients treated with heparin were assayed by all four methods and their results were compared. The results indicated that the manual-activated clotting time correlated best with the whole blood clotting time, was sensitive to low concentrations of heparin, formed a discernible clot within a convenient time period in blood containing high concentrations of heparin, was reproducible and was easily performed.

Acute leukemia presenting with myeloid and lymphoid cell markers.

A case is described of acute leukemia whose neoplastic cells possessed myeloid and lymphoid characteristics. Neoplastic cells possessed cytoplasmic granules containing Sudan black B material and diaminobenzidine myeloperoxidase. In addition, these luekemic cells were positive for terminal deoxynucleotidyl transferase. Ia antigen, and the common acute lymphocytic leukemia antigen. These findings indicate that biphenotypic cell markers may exist in cases of acute leukemia. It is our belief that these results are best explained as either a mixed myeloid-lymphoid leukemia or a stem cell leukemia capable of differentiating into myeloid and lymphoid cells.

T-cell prolymphocytic leukemia with a suppressor phenotype.

A diagnosis of prolymphocytic leukemia was made from the blood and bone marrow of a 50 year old man. The neoplastic cells were studied by use of light and electron microscopy. Neoplastic cells were focally positive for acid phosphatase and alpha naphthyl acetate esterase. In addition, neoplastic cells formed rosettes with sheep erythrocytes and reacted with Leu-1 and Leu-2a but not Leu-3a antisera. No terminal deoxynucleotidyl transferase (TdT) activity was noted in these cells. It is concluded that these neoplastic cells were phenotypically mature suppressor T lymphocytes. Furthermore, T and B cell prolymphocytic leukemias were compared according to clinico-pathological, cytochemical, ultrastructural, and immunological findings derived from our review of the current literature.

Telemedicine: emerging opportunities and future trends.

Remote location, lack of specialty services, or managed care capitation contribute to limited access to health care. As reimbursement rates decline, availability to expert or high-tech care is affected. The need to "make do" with available personnel, existing technologies, or dated facilities is not uncommon and affects the quality of health care. Telemedicine accounts for these issues and serves as an alternative to providing interactive evaluation and management of patients. As computer hardware and software improve and become less costly, access to this technology will be more commonplace and establish itself as an acceptable standard of practice. The Internet may serve as a highway to resources that currently are not available in rural areas or in third-world countries. Access to Web sites that provide comprehensive and instructive material is just one part of a complex, developing area of health care.