Lawrence Kass

Monocytes With Altered Phenotypes in Posttrauma Patients

CONTEXT Posttrauma patients show impaired immune responsiveness and increased susceptibility to infections. Although monocytes in these patients have been known to express decreased HLA-DR, induction of HLA-DR using interferon gamma failed to reduce susceptibility to infection, suggesting additional factors also may be involved in the impaired immune responsiveness. CD4 plays an integral role in most of the functions of HLA-DR. In newborn infants, who have impaired immune responsiveness, we found a concomitant reduction of CD4 on monocytes with decreased HLA-DR expression. OBJECTIVE Because monocytes in posttrauma patients have not been previously studied for morphology, coexpression of CD4 and HLA-DR, and activity of alpha-naphthyl butyrate esterase, the purpose of this study was to analyze these factors in this population. DESIGN Monocyte morphology; expression of CD4, CD11b, CD13, CD16, and HLA-DR by 3-color flow cytometry; and analysis of alpha-naphthyl butyrate esterase activity by cytochemical staining were studied in 27 posttrauma patients and 20 control subjects. RESULTS Monocytes in posttrauma patients showed significant differences in the following characteristics compared with controls: (1) increase of subsets displaying the phenotypes CD4-/CD14+/HLA-DR- and CD4-/CD14+/CD16-, (2) decrease in mean fluorescence intensity of CD4 and HLA-DR expression in monocytes that were positive for these markers, (3) decrease in alpha-naphthyl butyrate esterase activity, and (4) decreased amount of cytoplasm and cytoplasmic vacuoles.Conclusion.-Our study suggests that in posttrauma patients, as in newborns, there is a marked increase of monocytes with decreased expression of CD4 and HLA-DR, as well as decreased alpha-naphthyl butyrate esterase activity. Concomitant reduction in CD4 and HLA-DR expression on monocytes may contribute to impaired immune responsiveness in these patients.

Identification of Normal and Leukemic Granulocytic Cells with Merocyanine 540

After fixation in a modified Bouins solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.

Perivascular Plasmacytosis: A Light-Microscopic and Immunohistochemical Study of 93 Bone Marrow Biopsies

In bone marrow biopsies from 13 hematologically normal persons and from 80 patients with a variety of disorders, we found perivascular plasmacytosis. In all instances except the one case of multiple myeloma, plasma cells were polyclonal and normal in morphology. This was especially pronounced in patients with HIV infection, and in individuals following chemotherapy. In the same patients, sinusoids were also prominent, and appeared dilated. In biopsy sections, small endothelial-lined vessels appeared to arise from attenuation of the sinusoidal lumen. After a short segment of only endothelial-lined vessels, perivascular plasmacytosis appeared. When smooth muscle cells began to line the endothelium, plasma cells virtually disappeared. The biological significance of this finding is unknown. Possibly, the close proximity of plasma cells to endothelial cells early in the development of blood vessels could facilitate entry of immunoglobulins into the blood.

A Selective Stain for Eosinophils Using Two Oxazine Dyes Applied Sequentially

A methanolic solution of the oxazine textile dye, C.I. basic blue 122, followed by an aqueous alkaline solution of the oxazine dye, C.I. basic blue 141, and a brief rinse in an acetate buffer at pH 3.45 produces intense black staining of eosinophil granules. This staining was selective for eosinophils while other types of peripheral blood leukocytes showed little if any staining under the same conditions. This staining procedure may be useful for detecting eosinophils in samples of blood, bone marrow, or urine when eosinophiluria results from interstitial nephritis.

Lycramine Brilliant Blue Jl: A New Stain for Human Megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte cytoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.

Identification of Normal and Abnormal Human Megakaryocytes Based on Acid Fast Metachromasia after Staining with Basic Black MSP

Megakaryocytes from normal persons and from patients with immune thrombocytopenic purpura, myelodysplastic disorders, hypersplenism, and essential thrombocythemia displayed vivid magenta metachromatic staining of the cytoplasm when stained with basic black MSP followed by brief exposure to dilute hydrochloric acid. Under the same conditions, other hematopoietic cells were completely decolorized. Acid fast metachromasia of megakaryocytes facilitates their identification, particularly in cases of small and atypical megakaryocytes found in disease states.

Rapid Identification of T Helper and T Cytotoxic/Suppressor Lymphocytes with an Oxazine Dye

Peripheral blood lymphocytes displayed a plurality of sizes and colors when exposed first to a methanolic solution of C.I. basic blue 141, then to an aqueous alkaline solution of the same dye and rinsed in a neutral HEPES buffer containing trace amounts of various salts. As confirmed with purified lymphocyte subpopulations obtained with a cell sorter, T helper cells (CD4) were small and their nuclei and cytoplasm stained deep blue. T cytotoxic/suppressor cells (CD 8) were larger than T helper cells, their nuclei stained pale green or blue green and their cytoplasm contained a cluster of magenta colored granules. From start to finish, the stain takes 15 min to perform. Used in the manner described, basic blue 141 holds promise as a rapid means of identifying and differentiating CD4 and CD8 cells under the ordinary light microscope without using monoclonal antibodies or fluorescence.

A Two Step Stain for Normal and Leukemic Monocytes Using Two Different Dyes Applied in Sequence

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.

Kallichrome: a new stain for erythroblasts.

Using the obscure dye kallichrome, erythroblasts at all stages of maturation demonstrated intense yellow to yellow-brown staining of both nucleus and cytoplasm. Staining of this type was not observed in other types of normal or pathological marrow cells. As such, kallichrome may be a valuable stain for identification of erythroblasts and their distinction from other types of blood cells.

Preferential Staining of Granulocytic Cells by Sulphonaphthyl Red

Using sulphonaphythyl red (Michrome #947), granules in mature and immature granulocytic cells stained bright red. Granules were not visualized in other types of normal or leukemic blood cells. As such, sulphonaphthyl red may be useful in distinguishing normal and abnormal granulocytic cells from other types of blood and bone marrow cells.