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To investigate the heterogeneity of primary neutrophil granules, neutrophils in the bone marrow of 68 patients with hematologic and non-hematologic diseases were observed by electron microscopy. In addition to typical primary granules, 3 primary granule subtypes, i.e., parallel tubular granules (PTGs), fibrillar granules (FGs), and periodic lamellar granules (PLGs), were present in neutrophils. PTGs were found in only one case of chronic neutrophilic leukemia (CNL). On the other hand, FGs and PLGs were present in various cases at varying frequencies. Three subtype granules were seen preferentially in immature neutrophils. These subtypes were positive for myeloperoxidase, showing that they represent primary granules. In addition, hybrid PTG/FG granules, hybrid PTG/PLG granules, and hybrid FG/PLG granules were seen in the PTG-positive CNL case. This shows that a close association is present between PTGs, FGs, and PLGs.

Isotricha jalaludinii n. sp. found in the rumen of lesser mouse deer, Tragulus javanicus, in Malaysia was described and illustrated. This new species is characterized by the location and direction of the vestibulum, shape of the macronucleus, and absence of a dent at the vestibular opening. The presence of single peculiar isotrichid species in the rumen of mouse deer, which is recognized as one of the most primitive ruminants, suggests that the isotrichid ciliates similar to I. jalaludinii and Isotricha intestinalis were established at a fairly early period during the evolution of ruminants.

Fibrillar granules (FGs) represent neutrophilic primary granules containing clustered filaments. We investigated neutrophils in the bone marrow obtained from 17 patients with chronic myeloproliferative disorders (CMPD) by electron microscopy. FG-positive neutrophils were seen in 15 of the 17 CMPDs with varying frequencies. The diameter of FG-filaments was 7-8 nm, showing that they corresponded to neither intermediate filaments nor actin filaments.

Unique rounded granules consisting of clustered parallel tubules, 29-31 nm in diameter, with occasional, flocculent or amorphous electron-dense material (parallel tubular granules, PTGs) were found in immature neutrophils in the bone marrow in a case of chronic neutrophilic leukemia (CNL). PTGs were positive for electron microscopic myeloperoxidase. Since no structures similar to PTGs have been documented, we investigated the bone marrow of 65 adult patients with hematologic diseases, and 7 adult patients with miscellaneous non-hematologic diseases, by electron microscopy. The results showed that no PTGs were found in any cases other than the original CNL case. These findings suggest that PTGs are rare granules representing certain primary granules. In addition, the presence of PTGs might be helpful in diagnosing CNL.

We have constructed two-wavelength phase-shifting interferometry that is insensitive to the intensity changes in interferograms associated with the current variations in two laser-diode (LD) sources by using a newly developed phase-extraction algorithm. The tested phase at a synthetic wavelength can be measured from six interferograms with different phase shifts. The algorithm becomes a simple form for seven interferograms and reduces to a minimum of five phase-shifted data in the proper conditions. We shifted the phases equally in opposite directions to one another by separately varying the stepwise currents in dual LD's on an unbalanced interferometer. The measurement accuracy has been improved compared with that of the two-wavelength four-step method. The phase error caused by the power changes in the dual LD's has been investigated theoretically and experimentally. The experimental results are shown to measure a step object with a 4.6-µm synthetic wavelength.

Cryoultramicrotomy applying immunogold staining (immunocryoultramicrotomy) is a useful method to demonstrate the localization of antigens in biological specimens. Recently, we have developed a modified method for immunocryoultramicrotomy using gelatin as an embedding medium (gelatin embedding method). In this study, we compared the results obtained by the gelatin embedding method with those obtained by a conventional method applying polyvinyl alcohol embedding (PVA embedding method). Both methods were easy to perform, and yielded fairly satisfactory results in terms of detecting antigens and preserving ultrastructures. According to the gelatin embedding method, membranes enclosing organelles revealed a negatively stained image, and organelles were clearly delineated by an electron-lucent layer. In contrast, a positively stained image was obtained by the PVA embedding method; however, the delineation of organelles was somewhat inferior to that of the gelatin embedding method. Although these methods were useful to detect various biological antigens, they had some limitations in sensitivity. Prior glutaraldehyde fixation was requisite for cryosectioning and preserving ultrastructures, but it caused the loss of antigenicity to some extent. This should be taken into account when evaluating the results obtained by immunocryoultramicrotomy. In this paper, we present the detailed procedures of the gelatin embedding method, as well as the PVA embedding method, and demonstrate the localization of myeloperoxidase, lysozyme and CD3 molecules in human blood cells using both methods.

Localization of IgG in bone marrow after high-dose intravenous immunoglobulin therapy (IVIG) was investigated via light and electron microscopy. IgG was incorporated into the cytoplasm of various types of bone marrow cells of all the three lineages, particularly in reticulum cells, fat cells, and megakaryocytes. In addition, both reticulum cells and fat cells showed many elongated cytoplasmic protrusions, which were in contact with the various types of blood cells, especially the megakaryocytes. A filamentous structure was also seen near the point of contact between the cells. Ultrastructural changes of macrophages in bone marrow after IVIG suggest that these cells were activated by IVIG.

We present a unique case of crisis type chronic adult T-cell leukemia (ATL), in which the majority of leukemic cells had abundant periodic acid-Schiff (PAS)-positive cytoplasmic inclusions. These inclusions were found to be composed of glycogen because the PAS-positivity completely disappeared after digestion with amylase or human saliva. Electron microscopy also revealed that the inclusions consisted of aggregated beta particles of glycogen. The mechanism of glycogen accumulation in leukemic cells remains unknown; however, the presence of such inclusions in leukemic cells may be helpful diagnostically in T-lymphocyte malignancies.

The bone marrow of a 53-year-old woman with acute myeloid leukemia (AML) with disseminated intravascular coagulation was investigated by transmission electron microscopy. The patient had a preceding granulocytic sarcoma, and subclinical disseminated intravascular coagulation occurred concomitantly with the development of AML. Ultrastructural findings of the bone marrow at the onset of AML revealed the following: (1) The cytoplasm of the leukemic cells showed frequent fragmentation, resulting in the formation of abundant cytoplasmic fragments. (2) These cytoplasmic fragments were surrounded by abundant fibrin fibers, forming the fibrin-cytoplasmic fragment complex (FCF complex). (3) Slight fibrin deposition was seen around the leukemic cells and in the intercellular space of the bone marrow. Fibrin deposition in the bone marrow is thought to represent morphologic evidence of disseminated intravascular coagulation. The damage on the leukemic cell surface due to the cytoplasmic fragmentation seems to be closely related to the development of disseminated intravascular coagulation.

In neutrophils, lactoferrin is generally considered to be contained in secondary granules in which myeloperoxidase is lacking. However, it remains obscure whether lactoferrin is also contained in other organelles. We investigated subcellular localization of lactoferrin in human neutrophils by different immunoelectron microscopic methods. Lactoferrin was detected in secondary granules, and also in some large granules which could be distinguished from secondary granules based on their size and shape. These findings seem to indicate that the lactoferrin-positive large granules might represent primary ones or belong to another type of granules other than primary and secondary ones.

Immunocryoultramicrotomy is generally considered the most suitable method for observing the immunostainings by monoclonal antibodies to intracellular antigens by electron microscopy. However, this procedure with the free cells such as peripheral blood cells or cultured cells is not widely used because of the difficulty in preparing ultrathin frozen sections. We report here a new technique for making ultrathin frozen sections of peripheral blood cells by positioning the cell in a semigelatinized medium for ultrasectioning. Monoclonal antibody against myeloperoxidase (MPO) was reacted with the ultrathin sections made with this new technique to observe the ultrastructural localization of intracellular MPO. MPO was observed in the primary granules of the neutrophils.

Two-wavelength phase-measuring interferometry with dual laser-diode sources has been developed that is based on a phase-shifting method that uses two wavelengths varied stepwise by separately changing the currents in two laser diodes. A synthetic wavelength is produced by the addition of two single-wavelength interferograms without the need for auxiliary techniques. The phases are equally shifted in opposite directions to each other on an unbalanced interferometer. The fringe shifts that result from modulated phases are monitored with an electronic sensor to ensure accuracy. Our experimental result shows measurements of the profile of a step object with a 4.6-microm synthetic wavelength.

Rumen protozoa can produce lysine from free 2,2'-diaminopimelic acid (DAP). However, the quantitative importance of this transformation has been disputed; lysine contents of protozoal incubation supernatants reported by Onodera & Kandatsu and Masson & Ling show a 26-fold difference. The in vitro experimental methods of both groups were compared to determine the causes of this difference. Lysine production was proportional to DAP concentration. Results with rumen protozoa from sheep or goats were similar. The incubation medium and deproteinizing procedure of the Welsh group gave a two-fold increase in lysine production compared with Japanese protocols. Omissions of rice starch from protozoal incubations slightly increased lysine production, whereas omissions of antibacterial agents resulted in varying, yet relatively small changes. The greatest cause of the difference was the number of rumen protozoa incubated. When this factor was taken into account, the difference in the maximum rates of lysine production between the Welsh and Japanese groups was only three-fold, namely 4.5 versus 15.0 nmol lysine/10(5) protozoa/h. Adding other amino acids to the incubations suggested that DAP uptake by rumen protozoa may occur via transport system ASC. The importance of DAP metabolism by protozoa as a source of lysine for ruminant host animals is discussed.

A simple, rapid and sensitive assay method for plasma N tau-methylhistidine by isocratic high-performance liquid chromatography has been developed. The deproteinized plasma was treated with o-phthalaldehyde-2-mercaptoethanol. The derivatives were separated on a LiChrospher 100 RP-18 column within 10 min. The detection limit for N tau-methylhistidine was 0.5 pmol. The plasma N tau-methylhistidine content of beef cattle and dairy cows was 0.038 +/- 0.004 and 0.017 +/- 0.002 nmol/ml/kg, respectively.