Murine cytomegalovirus DNA in peripheral blood of latently infected mice is detectable only in monocytes and polymorphonuclear leukocytes

Cytomegalovirus (CMV), as do other herpesviruses, establishes a lifelong latent infection in its natural host. While in immunologically intact hosts most CMV infections are subclinical, clinical disease follows severe immunosuppression and immunodeficiency. In these situations CMV may produce serious life-threatening disease, and virus reactivated from the latent state is often responsible. Essential to understanding this virus and its pathogenesis is the need to define particular tissue and cell types harboring viral DNA. We searched for viral DNA and RNA in subpopulations of blood cells from mice latently infected with murine CMV by using differential centrifugation and fluorescent antibody cell sorting followed by polymerase chain reaction analysis. Following intravenous inoculation, the viral DNA was found to be present in the buffy coat at and after 21 days postinfection, and both granulocytes and peripheral blood mononuclear leukocytes (PBML) were reservoirs. Further analysis of the PBML fraction by separation into Mac-1+ and Mac-1- cells revealed that monocytes harbored the DNA while lymphocytes were not sites of persistence. We conclude that in buffy coat of latently infected mice the viral DNA is present only in cells of the myeloid lineage. The relationship of this DNA to the latent infection is discussed.     

Anticoagulant selection influences flow cytometric determination of CD11b upregulation in vivo and ex vivo

We recently devised three-colour flow cytometric assay for evaluating expression of CD11b on neutrophils and monocytes in circulation. Artefactual upregulation of CD11b ex vivo was minimized by cooling blood samples on ice. In this communication we further characterize the method in terms of different anticoagulants. EDTA was less optimal than ACD or heparin because (i) saturating concentrations of CD11b antibody (clone D12) were not achieved with resting cells; (ii) CD11b fluorescence intensity of synovial fluid cells, i.e., in vivo activated cells expressing CD11b at high levels, was significantly lower in EDTA plasma, and (iii) EDTA mediated more cell damage at 37 degrees C, as determined by PI staining. The fluorescence data suggested that D12 antibody binding was dependent on divalent cations. Saturating concentrations in the presence of EDTA in medium were easily obtained with synovial fluid cells and peripheral blood phagocytes activated with chemotactic peptide FMLP, suggesting that cell activation decreased cation concentrations required for D12 antibody binding. Using another CD11b antibody (2MPL19c), whose binding proved to be cation independent, it was shown that CD11b upregulation was not affected by EDTA. ACD was superior to heparin and phenylalanylprolylarginyl chloromethyl ketone (PPACK), a thrombin inhibitor, because cell counts were significantly lower in heparinized samples in cold, and in PPACK-anticoagulated samples treated with LPS at 37 degrees C. Kinetics of L-selectin shedding was similar in heparin and ACD, suggesting that cell loss did not derive from differences in cell activation. In comparison of buffy coat cell assay and whole blood assay, neutrophil CD11b expression was similar but background fluorescence was significantly higher in whole blood preparations. This implies that nonspecific antibody binding may occur more in whole blood assay, whereas in the buffy coat cell assay, sample manipulation procedures may slightly increase CD11b antibody binding, but not control antibody binding. Finally, it was confirmed that warming from 0 degrees C, but not from room temperature, to 37 degrees C increased CD11b expression significantly on neutrophils, and it was further shown that monocytes undergo similar changes. Cooling did not upregulate CD11b, and completely prevented LPS-induced upregulation. In conclusion, the results support use of ACD in evaluating CD11b expression; if EDTA is used, it is important to make sure that binding of CD11b antibody selected does not require presence of divalent cations in medium.     

Studies of the use of L-leucyl-L-leucine methyl ester in canine allogeneic marrow transplantation

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is a lysosomotropic agent that selectively kills cytotoxic T cells and their precursors, natural killer cells, and monocytes but not helper T cells or other cells of hematopoietic origin. In this study, the effects of treatment of bone marrow and peripheral blood buffy coat with Leu-Leu-OMe on the outcome of allogeneic marrow transplantation were studied in several canine models. Whereas incubation of autologous marrow with Leu-Leu-OMe had no adverse effects on subsequent engraftment, incubation of marrow from dog leukocyte antigen (DLA)-identical littermates resulted in a high rate of graft failure. Previous studies have demonstrated that the addition of peripheral blood buffy coat allows engraftment of unrelated DLA-nonidentical marrow, and in this study we found that incubation of buffy coat with Leu-Leu-OMe did not alter this graft promoting effect. In a final experiment it was demonstrated that incubation of both marrow and peripheral blood buffy coat did not prevent the development of graft-versus-host disease in recipients of marrow from DLA-haploidentical littermates. In considering the eventual application of Leu-Leu-OMe in the clinic, these results are less encouraging than those previously reported using murine models.     

Attitudes of oncologists, family doctors, medical students and lawyers to euthanasia

OBJECTIVE: To evaluate clearance of the vaccine strain, immunologic responses, and potential shedding of Brucella abortus strain RB51 organisms after vaccination of bison calves. ANIMALS: Fourteen 7-month-old female bison calves. PROCEDURE: 10 bison calves were vaccinated SC with 1.22 x 10(10) colony-forming units of B abortus strain RB51. Four bison calves were vaccinated SC with 0.15M NaCl solution. Rectal, vaginal, nasal, and ocular swab specimens were obtained to evaluate potential shedding by vaccinated bison. The superficial cervical lymph node was biopsied to evaluate clearance of the vaccine strain. Lymphocyte proliferative responses to strain RB51 bacteria were evaluated in lymph node cells obtained from biopsy specimens and also in peripheral blood mononuclear cells. RESULTS: Strain RB51 was recovered from superficial cervical lymph nodes of vaccinates examined 6, 12, and 18 weeks after vaccination (4/4, 3/4, and 1/4, respectively) but not in vaccinates examined at 24 weeks (0/3) after vaccination or nonvaccinates examined at all sample collection times (n = 1 bison/sample period). Serologic, immunologic, and bacterial culture techniques failed to reveal shedding of strain RB51 by vaccinates or infection of nonvaccinated bison. Lymphocyte proliferative responses were evident in lymph node cells and blood mononuclear cells from strain RB51-vaccinated bison beginning 12 weeks after vaccination. CONCLUSION: Strain RB51 was cleared from bison by 18 to 24 weeks after vaccination. Bison vaccinated with strain RB51 did not shed the vaccine strain to nonvaccinated bison housed in close proximity. Strain RB51 did not induce antibody responses in bison that would interfere with brucellosis surveillance tests, but did stimulate cell-mediated immunity.     

Rapid method for the identification of esterase-positive cells in suspensions of mononuclear cells separated by a Ficoll-Paque gradient

Cytochemical staining for acid alpha-naphthyl acetate esterase (ANAE) activity is widely used for identifying monocytes in mixed cell populations; esterase staining is usually performed on slide prepartions of whole blood, buffy coat or Ficoll-Paque separated mononuclear cells (MC). In this paper we propose a method for staining MC while they are in suspension, which utilizes, with slight modifications, the same reagents used in the procedure for esterase staining on smears. Suspension method is rapid and costless; furthermore, it overcomes the loss of esterase stainability observed in slide preparations of Ficoll-Paque separated MC.U     

Leukocytes in the cervix: a quantitative evaluation of cervicitis

OBJECTIVE: To quantify the numbers of leukocytes in the normal cervix and relate these numbers to the diagnosis of cervicitis. METHODS: Isolated cell suspensions were prepared from cervical tissue recovered at hysterectomy from 37 women who had no obvious cervical disease. The percentages of CD45+ cells (leukocytes) in these preparations were determined using immunofluorescence-based flow cytometric analysis. These percentages were compared with the pathologist's assessment of cervicitis. RESULTS: Leukocytes were present in all cervical samples tested. For endocervical samples, the mean (+/- standard error of the mean [SEM]) percentage of CD45+ cells was 12.4 +/- 1.9% of cells in patients with a diagnosis of cervicitis (n = 16) and 9.1 +/- 1.1% in patients without cervicitis (n = 17). For ectocervical samples, the mean (+/- SEM) percentage was 14.8 +/- 3.0% in those with cervicitis (n = 16) and 9.5 +/- 1.6% in those without cervicitis (n = 19). The differences between samples from patients with cervicitis and those without cervicitis were not statistically significant at the .05 level. Intra- and interassay variabilities were 5.7 +/- 1.2% and 7.3 +/- 1.6%, respectively. CONCLUSION: Our study demonstrates there is a resident population of leukocytes in the cervix. Leukocyte number did not relate clearly and consistently to the diagnosis of cervicitis made by the pathologist. We suggest that the resident population of leukocytes, in the absence of other indicators of infection, may confuse determinations of cervicitis.     

Effects of high calcium intake on fat digestion and bile acid excretion in feces of veal calves

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.     

Disclosing the cancer diagnosis: the patients'' experiences

To elucidate whether leukocytes in the surface secretion on the adenoid have a potential immunological function, imprint samples of surface secretion were obtained from the adenoid of 11 children and the corresponding mucosal area of 10 adults. May-Grünewald-Giemsa staining was used for evaluation of cell morphology and spatial relations, and immunohistochemical staining for identification of granulocytes, B cells, T cells and macrophages. The children's imprints showed large numbers of leukocytes, with mononuclear cells in the majority. The adults' imprints were characterized by moderate numbers of epithelial cells and few leukocytes. In six out of seven adenoid secretions successfully analysed with all four CD antigens, there was the simultaneous presence of granulocytes, B cells, 1 cells and macrophages. This was the case in one of nine analysable adult secretions. The CD-positive cells were often seen in juxtaposition, in clusters of two to 10 cells, as well as in contact with leukocytes of unknown CD antigenicity and epithelial cells. The simultaneous presence in the secretion of morphologically intact and CD antigen-presenting leukocytes in juxtaposition could indicate a potential immunological function of these cells.     

Epidural analgesia for labour and delivery: informed consent issues

We analyzed the clade distribution of B and E in HIV-1 isolates in Japan by a nested PCR method using 5'-CCCACAAGATTTAAATATG-3' of the gag gene as clade B primer and 5'-CCCACAAGATTTAAACTCC-3' of the gag gene as clade E primer. Seventy-two anti-HIV-1 confirmatory positive serum samples were collected during a period of 1991-1996 in two hospitals in Yokohama City. Peripheral blood mononuclear cells were obtained from the buffy coat of these samples and extracted DNA were used for nested PCR. The 72 cases comprised of 11 Japanese hemophiliacs, 14 Japanese male homosexuals, 19 Japanese male heterosexuals, 5 Japanese female heterosexuals and 23 Thai female heterosexuals. Of these 36 were clade B and 35 were clade E and one case showed positive PCR results for both B and E primers. Almost all male Japanese hemophiliacs and homosexuals in our sample have clade B, while the female Thai heterosexuals have clade E, irrespective of the year of isolation. As for Japanese male heterosexuals, through 1993, clade B was predominant but since 1994, the predominate clade switched to clade E. Although the number of Japanese female heterosexuals in our sample is small, clade B was isolated in 2 cases even after 1994.     

Modulation of the T cell compartment by blood transfusion. Effect on cytotoxic and helper T lymphocyte precursor frequencies and T cell receptor Vbeta usage

Recent data suggest that the favorable effect of pretransplant blood transfusion (BT) on transplant outcome depends on the HLA match. HLA-DR or haplotype shared transfusions lead to transplantation tolerance, and HLA-mismatched BT leads to immunization. The immunological mechanism involved is still unknown. To investigate the effect of HLA compatibility between blood donor and recipient on the T cell compartment, we determined the frequency of cytotoxic and helper T cell precursors specific for blood donor cells (n=20) and the T cell receptor Vbeta (TCRBV) repertoire of the CD4- and CD8-positive peripheral blood mononuclear cells before, at 2 weeks after, and at more than 10 weeks after BT (n=10). Patients had received one transfusion of a nonstored (<24 hr after withdrawal) erythrocyte concentrate without buffy coat containing on average 6x10(8) leukocytes. Eight patients shared an HLA-B and -DR antigen, nine patients shared one HLA-DR antigen, and three patients shared no HLA class II antigens with the blood donor. All patients showed a significant increase in both cytotoxic and helper T cell precursor frequencies against the blood donor 2 weeks after BT. In most patients, the frequencies reached pretransfusion levels again long after BT. In 5 of 10 patients, an expansion of one or more TCRBV families was observed in either the CD4 or CD8 compartment. This study demonstrates that BT, irrespective of the degree of HLA matching, induces activation of the T cell compartment. The degree of sharing of HLA antigens was not correlated with quantitative changes in cytotoxic T lymphocyte precursor or helper T lymphocyte precursor frequencies, or changes induced in the TCRBV repertoire. Cytotoxic and helper T lymphocyte precursor frequencies and TCRBV repertoire determined after BT do not give an indication for a state of tolerance prior to transplantation.     

Interleukin-10 inhibits the in vitro proliferation of human activated leukemic CD5+ B-cells

Umbilical cord blood mononuclear cells isolated by density centrifugation are contaminated by erythrocytes and nucleated erythroid precursors which may exceed 50% of the total cell population, and thus interfere with phenotypic, functional and mRNA analyses. Lysis with hypotonic ammonium chloride can overcome this problem, but interferes with lysosomal function and should be avoided when cell preparations are intended for functional studies. The aim of this study was to develop a technique for removing erythroid cells from cord blood mononuclear cell preparations that would be as effective as ammonium chloride lysis but would not affect cellular function. This paper describes a method using 10F7, a mouse monoclonal antibody against human glycophorin A, and magnetic beads coated with anti-mouse immunoglobulin. The population of cord blood mononuclear cells recovered using this technique was of high purity, good yield and viability, and the cells responded appropriately to stimulation in vitro. To maximise cost-effectiveness, purification with magnetic beads could be performed after two density separations to reduce the quantity of beads required.     

Haemophilus ducreyi elicits a cutaneous infiltrate of CD4 cells during experimental human infection

Human subjects were experimentally infected with Haemophilus ducreyi for up to 2 weeks. Bacterial suspensions were delivered into the epidermis and dermis through puncture wounds made by an allergy-testing device. Subjects developed papular lesions that evolved into pustules resembling natural disease. Some papular lesions resolved spontaneously, indicating that host responses may clear infection. Bacteria were shed intermittently from lesions, suggesting that H. ducreyi may be transmissible before ulceration. Host responses to infection consisted primarily of cutaneous infiltrate of polymorphonuclear leukocytes, Langerhans cells, macrophages, and CD4 T cells of alpha beta lineage. Expression of HLA-DR by keratinocytes was associated with the presence of interferon-gamma mRNA in the skin. There was little evidence for humoral or peripheral blood mononuclear cell responses to bacterial antigens. The cutaneous infiltrate of CD4 cells and macrophages provides a mechanism that facilitates transmission of human immunodeficiency virus by H. ducreyi.     

Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes

Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4(+) T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4(+)CD8(-) single-positive T cells and CD4(+)CD8(+) double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4(+) T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-gamma production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8(+) T cells were the major producers of IFN-gamma. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4(+) LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.     

Activation and adherence of lyophilized human platelets on canine vessel strips in the Baumgartner perfusion chamber

The Baumgartner perfusion technique was used to test the ability of rehydrated lyophilized human platelets to adhere to the thrombogenic surface of a denuded arterial vessel segment and to undergo platelet activation under conditions of high shear. Twenty preparations of washed platelets were stabilized by 1-hour or 2-hour exposure to paraformaldehyde before freeze-drying in a Virtis 600 lyophilizer. The response of these fixed-dried preparations was compared with that of non-fixed platelets in fresh citrated whole blood. The outcome of each perfusion experiment was quantified in photomicrographs by morphometric estimation of the percent area of the vessel segment covered by adherent platelets after immunofluorescent staining with monoclonal antibodies to glycoprotein Ib (CD42) or glycoprotein IIbIIIa (CD41a). Evidence of activation in nonadherent platelets was examined by flow cytometry for CD62 and GP53 expression. In addition, thromboxane B2 was measured by radioimmunoassay as an index of platelet responsiveness to activation conditions during perfusion. The percent vessel coverage observed with lyophilized platelets in recombined whole blood was somewhat less than that of platelets in fresh whole blood (39% vs 73%, respectively). In other perfusion experiments performed with non-denuded vessel segments, the percent coverage was reduced by half or more for both types of platelet preparation. Scanning electron microscopy confirmed that the lyophilized platelets did not adhere to areas of intact endothelium. Furthermore, the lyophilized platelets showed a small-but-significant rise in CD62 or CD63 activation antigen expression and generated thromboxane B2 at about one third the rate of fresh platelets in these perfusion experiments. The thromboxane generation during perfusion was inhibited in fresh or lyophilized platelet preparations by pretreatment with indomethacin or PGE-1. We interpret these data as evidence of the ability of our lyophiilized platelet preparations to respond at least partially to physiologic stimuli and to adhere to appropriate thrombogenic sites to support hemostasis.     

Attenuation of canine nephrotoxic glomerulonephritis with an extracorporeal immunoadsorbent

It was the purpose of this study to establish and evaluate a freezing-and-thawing method for preservation of hemopoietic stem cells from the peripheral blood. Blood leukocytes collected by means of an IBM Blood-Cell-Separator were frozen in plastic bags using 10% DMSO and controlled cooling rates. Thawing was performed rapidly, and DMSO was diluted and removed prior to the in-vitro and in-vivo assays. The mean recovery of mononuclear cells collected from 82 leukaphereses was 86%. To assess the recovery of cryopreserved hemopoietic stem cells, the soft agar culture method adapted for the dog was used. There was no significant difference in the CFUc recovery per 1 X 10(6) mononuclear cells or in per leukapheresis after different cryopreservation times (1--6 and 7--27 months). To evaluate the hemopoietic repopulation capability of cryopreserved blood stem cells, leukapheresis-derived leukocytes were transfused into 1200 R whole body x-irradiated dogs. The hemopoietic repopulation pattern at day 10 after transfusion of comparable numbers of fresh or frozen leukocytes was not significantly different, as measured in bone marrow smears and sections and by granulocyte concentration in the peripheral blood.     

Electron microscopy of surface structures of Blastocystis sp. from different hosts

Samples of Blastocystis sp. obtained from humans, monkeys, pigs and chickens were examined by scanning electron microscopy and transmission electron microscopy to compare surface structures. The surface coat of Blastocystis sp. cells from each host species showed some morphological variations, but these were not sufficiently different to allow judgement to be made on speciation. The surface structure morphology appeared similar for samples of Blastocystis sp. from the same host species. The surface coat of the cultured human isolate of B. hominis was much thinner than that of cells from fresh human faecal material, and the cell surface appeared to be smoother and without the small projections seen in the fresh forms. Bacteria were frequently found in association with the surface coat of Blastocystis sp. from all fresh faecal material. Possible functions of the surface coat, especially in relation to protection against osmotic shock, are discussed.     

Treatment of chronic myeloid leukemia in relapse after umbilical cord blood transplantation

Umbilical cord blood (UCB) is increasingly used as a source of hematopoietic progenitor cells for allotransplantation. Donor-derived buffy coat cells are considered optimal treatment for leukemia relapses after transplantation of allogeneic bone marrow. Experience with relapses after UCB transplants are sparse. Here we report a girl who received an UCB transplant for chronic myeloid leukemia, relapsed after three years, failed to respond to donor buffy coat cells, but achieved a complete hematologic, cytogenetic, and molecular remission on interferon-alpha.     

Dermal microvascular injury in the human peripheral blood lymphocyte reconstituted-severe combined immunodeficient (HuPBL-SCID) mouse/skin allograft model is T cell mediated and inhibited by a combination of cyclosporine and rapamycin

We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model, split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin, when subsequently introduced by intraperitoneal injection, caused destruction of the human dermal microvasculature by day 16, evident as endothelial cell sloughing and thrombosis. In the same specimens, mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4+ and CD8+ T cells, some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human, but not mouse, macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3+ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8+ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4+ T cells. These data suggested a cytolytic T cell-dependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage. Neither cyclosporine nor rapamycin alone effectively reduced either mononuclear cell infiltration or vascular injury. However, a combination of the two agents reduced both parameters. We conclude that the huPBL-SCID/skin allograft model may be used both to study cytolytic T cell-mediated rejection and to test the effect of immunosuppressive drug strategies in vivo in a small-animal model of human immune responses.     

Ultrastructural localization of DNA in leukemic cells using osmium ammine B

In order to determine whether there were any differences in distribution of nuclear DNA between acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML), the localization of DNA in blasts from the bone marrow or buffy coat of 30 patients with ALL and 30 patients with AML was examined ultrastructurally by staining with osmium ammine B. By the ultrastructural cytochemistry, DNA in ALL cells was clumped in the nuclei, while in AML cells, it was dispersed. DNA had accumulated around the nucleoli of some blasts, and flecks of DNA were observed in nucleoli of a majority of blasts. The perinucleolar and intranucleolar DNA distribution could be classified into four types. The types with abundant perinucleolar DNA were frequently observed in ALL blasts, while the majority of AML blasts showed scant perinucleolar DNA. The types with intranucleolar flecks of DNA were more prominent in leukemic cells than in normal immature leukocytes. In conclusion, the pattern of distribution of DNA in the nuclei of leukemic cells differs between ALL and AML.