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Despite being essentially hepatotropic, hepatitis B virus has frequently been detected in peripheral blood leukocytes. In most cases, viral DNA has been detected by hybridisation, though, occasionally, evidence of viral RNA and virus-specific proteins has been reported. Though definitive evidence is lacking, it appears likely that the virus is able to replicate in at least a subfraction of these cells. The possible consequences with respect to the transmission and reactivation of virus infections are discussed.

It is possible accurately to distinguish lymphocytes from other leukocyte populations in peripheral blood using the combination of fluorescence associated with CD45/CD14 and forward and orthogonal light scatter. By identifying the cell population of interest based on immunofluorescence, a light scattering window can then be drawn to include all (greater than or equal to 98%) of the lymphocytes. In this manner, maximal recovery of the lymphocytes within a sample can be consistently obtained. The combination of light scattering and immunofluorescence can also be used to define the purity of the gate. The identification of nonlymphocytes within the light scattering gate can then be used to establish an accurate denominator for the percent lymphocytes stained. Once the optimal data acquisition gate has been established and characterized, it is possible to correct subsequent analyses with that particular sample since the reactivity of monoclonal antibodies on monocytes and granulocytes can be accounted for once the nonlymphocytes have been identified as being within the acquisition gate.

We report here that soluble factors from activated mononuclear leukocytes have a dramatic effect on cultured endothelial cells. While human umbilical vein endothelial cells grown under standard conditions show a polygonal, epithelial-like morphology, cells exposed to culture media conditioned by lectin-activated human mononuclear leukocytes become extremely elongated and/or send out numerous cytoplasmic processes, assuming a dendritic configuration. This effect cannot be mimicked by exogenous cyclic AMP, is reversible upon interruption of the treatment, and appears specific for endothelial cells, since it has not been observed so far with other cell types. The shape changes are accompanied by a reorganization of the endothelial cell cytoskeleton: actin microfilament bundles tend to be disposed in parallel arrays, while intermediate filaments and microtubules penetrate up to the extremity of the cytoplasmic processes. Colchicine prevents endothelial cell elongation but only slightly impairs the formation of lateral cell processes ("dendritic configuration"). Purified interleukins were tested for their ability to induce these changes of cell shape. Escherichia coli-recombinant human interleukin 2 had no effect, and gamma-interferon only a slight effect on endothelial cell morphology. Interleukin 1 induced moderate cell elongation, while combined treatment with both interleukin 1 and gamma-interferon resulted in shape changes indistinguishable from those elicited by supernatants of activated mononuclear leukocytes. The possible relevance of the observed endothelial cell changes to the reported angiogenic activity of mononuclear cell products is discussed.

A fluorescence-activated cell sorter was modified to quantify simultaneously three immunofluorescence stains on a population of cells. A dye laser containing rhodamine 6G was used to obtain 600 nm light to excite Texas Red coupled avidin. An argon ion laser operating at 488 nm excited both fluorescein and phycoerythrin directly conjugated antibodies. The emission from these fluorophores could be independently quantified as demonstrated by the histograms generated by samples labeled separately with each of the three stains. This three-color detection system was used to analyze human peripheral blood mononuclear cells for the expression of three antigens: Leu 2, Leu 7, and Leu 11. Upon reanalysis of the list mode data, several discrete subpopulations of lymphocytes could be identified based on the quantitative expression of these three antigens. Of particular interest in the normal sample studied was a population of dimly labeled Leu 2+ cells which were predominantly Leu 11+, a phenotype which is seen infrequently in normal individuals. This technique expands the combinations of antigens that can be studied at any one time and will facilitate the detection and functional analysis of cells in heterogeneous populations.

We observed postictal pleocytosis in 7 of 62 cerebrospinal fluid specimens obtained from 27 patients with epilepsy. Each patient had a known seizure disorder; none had any other cause for the pleocytosis. The maximum number of leukocytes was 12/mm3; the maximum number of erythrocytes was 190/mm3. Postictal pleocytosis was more common in samples obtained within 12 hours of the last seizure. Although previous studies have emphasized that pleocytosis is more common after repetitive generalized tonic-clonic seizures, we found increased leukocyte counts in cerebrospinal fluid after single simple, complex partial, or generalized tonic-clonic seizures.

Levels of 2' and 3' purine and pyrimidine ribonucleoside monophosphates (2'-, 3'-NMP) in leukocytes from blood and/or bone marrow were measured in three adult patients with acute non-lymphoblastic leukemias. The measurements of 2'-, 3'-NMP were made by high-performance liquid chromatography (HPLC) at various times in the course of the disease. Complete remission (CR) was obtained for all three patients but two of these have since died after relapsing at 8 and 9 months, respectively. The third patient remains in CR at 1 1/2 year. The levels of 2'-, 3'-NMP in the leukocytes of the patient remaining in remission have not changed since the beginning of his remission. However, in the patients who relapsed 2'- and 3'-NMP levels increased first in bone marrow than in blood leukocytes. These increases occurred about 3 months before the relapse was detected by morphological criteria. These data suggest that 2'-, 3'-NMP measurements may have a prognostic value if used to monitor patients with acute myeloid leukemia in CR.

We have developed a method of calculating the average local absorbance (ALA) of a nucleus from the integrated nuclear absorbance and area. One can use the ALA, along with nuclear areas measured at different point absorbance thresholds, to determine whether a nucleus is stained too lightly or too darkly for accurate absorption measurements; this allows selection of an optimal light wavelength for the performance of these measurements. The ALA can also be used for automatic and instantaneous correction of integrated absorbance values from darkly stained cells. This allows the rapid measurement of the integrated absorbances of a large number of nuclei that are heterogeneous in stain intensity. Coefficients of variation of approximately 3% are obtained for the integrated absorbances of nuclei of nontransformed G0/G1 cells. This correction method can be applied with any image densitometer that generates both integrated absorbance and area values.

Gel filtration and immunoadsorption techniques have been utilized to purify tumoricidal factors from sturgeon sera as well as human plasma. Two pure proteins with cytotoxic activity were isolated from the sturgeon serum (factors A2 and B2) and two additional cytotoxic factors were isolated from human plasma (Factors A and B). In another study, human factors A and B were quantitated in both normal donor and cancer patients' plasma. It was found that the cancer patients produced at least 50% less tumoricidal factors than did the normal donors. Furthermore, when the secretions from various lymphoid cell populations were purified utilizing immunoadsorbent techniques, it was found that the macrophages generally were producing the human tumoricidal factors. Both the sturgeon and the human factors were enzymatically cleaved into several small molecular weight fragments. Sephadex G-75 column chromatography was then utilized to isolate the enzymatic cleavage fragments. Three fragments with cytotoxic activity were isolated from both sturgeon factor A2 and B2. Similarly, human factor A was digested into two fragments with cytotoxic activity and factor B was digested into three fragments with cytotoxic activity. The molecular weights of the cytotoxic sturgeon and human fragments ranged from 14,000 to 23,000 whereas the intact sturgeon and human factors had a molecular weight slightly greater than 67,000. Ultimately, if these small molecular weight fragments can be sequenced, then it would be possible to prepare a synthetic analog which might have future potential as a mode of treatment for cancer patients.

In cytometric clinical application, it is important to obtain cell suspensions rapidly with as little cytological alteration as possible. A procedure has been achieved to prepare cell suspensions for flow cytometric analysis. The leucoconcentration technique, first described by Herbeuval for cytologic analysis, has been modified to be applied in cytometry. This technique involves Saponin lysis of red cells of peripheral blood or bone marrow samples that have been previously fixed with picric acid alcohol solution. Cells in suspension are not shifted and tinctorial affinity is not modified. Then cells have been stained with Mithramycin. Each parameter defined by Crissman has been analyzed to define the best staining conditions. The availability of Leucoconcentration with Mithramycin-DNA-staining permits determination of cell cycle with a fine resolution.

A vital, nucleic acid stain (LDS-751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS-751 were found in the fixed samples. Cells not staining or only dimly staining with LDS-751 were identified as erythrocytes and platelets, respectively. Cells staining with intermediate amounts of LDS-751 were found to be intact cells, while cells intensively stained were identified as damaged cells. Confirmation of the identity of the populations was obtained by light microscopic examination of the sorted populations and by correlating the fluorescent signals of FDA and LDS-751 in nonfixed cell preparations. Agglutinated cells could also be identified by the increased fluorescent signal in the LDS-751 channel as compared with single cells. The spectral properties of this dye permit excitation at 488 nm with emission in the far red portion of the spectrum. This allowed two-color immunofluorescence to be combined with the intact/damaged cell discrimination on fixed samples. Therefore, intact single cells could be distinguished during flow cytometric analysis, increasing the accuracy of the immunofluorescence measurements. The visualization of the multidimensional data was facilitated using color to discriminate cell populations depicted in multiple perspectives.

A purified preparation of human leukocyte interferon used at this hospital in the treatment of malignant diseases was tested for its ability to modify the spontaneous cytotoxicity of peripheral lymphocytes from healthy donors. The inhibitory effect of allogeneic lymphocytes on the (3H)thymidine incorporation of a lymphoblastoid cell line, Raji, was augmented by the presence of interferon or by pretreatment of the lymphocytes with interferon. This form of pretreatment also increased lymphocytes' capacity for reducing the number of surface-adherent tumor cells in a microassay. Moreover, lymphocytes treated with interferon exhibited an enhanced cytotoxic capacity for target cells on incubation with such cells labelled with 51CR.

Long-term storage of DNA is required for a number of genetic studies; prior to extraction, blood samples may be subject to elevated temperatures for variable intervals. We have studied the effect of temperatures ranging from -70 degrees C to +65 degrees C on human blood and on DNA extracted from it. DNA in solution stored at ambient temperatures up to 37 degrees C for 6 months was digestible by three different restriction endonucleases, whereas storage at 45 degrees C is deleterious after 6-7 weeks. DNA can be extracted from blood samples stored at -70 degrees C for at least 2 months or at 23 degrees C for a week or more, but blood stored at these temperatures may yield less high-molecular-weight DNA. Cell pellets from which plasma has been removed also can serve as a source of DNA. Isolated DNA stored dry for years (up to 30) is difficult to dissolve and may appear degraded, but a sample stored dry for 13 years and then in solution at -20 degrees C for 7 years appeared to be intact.

A human myeloid cell line, HL-60, has been shown to terminally differentiate to mature granulocytes in the presence of dimethylsulfoxide (DMSO) and other compounds. A histone polypeptide (HP) with an apparent molecular weight of 12,500 has been identified among the acid-extractable chromosomal proteins of DMSO-treated HL-60 cells. The detection of HP in total chromatin, total cell proteins or acid extracts of intact HL-60 cells treated with DMSO indicates that HP is present in intact cells and is not generated during the isolation procedures used for preparation of chromatin or histones. No radiolabelled HP was detected when chromatin was isolated from a mixture of radiolabelled mouse cells and HL-60 cells treated with DMSO. The relative increase in the amount of HP in DMSO-treated HL-60 cells correlates with a concomitant decrease of histone H2A. Fingerprint analysis of tryptic digests shows HP to be related to histone H2A. Generation of HP from endogenous and exogenous H2A. Generation of HP from endogenous and exogenous H2A is probably due to a proteolytic enzyme associated with the chromation of the differentiated HL-60 cells. HP was not observed in chromatin from a number of human or mouse hematopoietic tissues and cell lines but low levels of HP were detected in human lymphocytes and granulocytes prepared from peripheral blood of normal donors. HP was also detected in leukocytes from patients with acute leukemia. Our results suggest that: (1) HP is a proteolytic product of histone H2A and not a product of an independent gene; (2) the observed level of HP in HL-60 cells in related to the stage of differentiation; and (3) HP is a potential biological marker for human acute leukemia.

A 10-year-old boy developed progressive dystonia and dementia. His symptoms had begun at age 2 1/2 years, and he had been unable to walk by 8 years. At age 10 he was severely dystonic, unable to use his hands to feed himself, and almost anarthric . He had dysphagia and urinary incontinence, and functioned at a 4-year-old level of mental development. The mean percentages of beta-hexosaminidase A measured in serum, leukocytes, and fibroblasts by the heat denaturation method, each on three separate assays, were 5.9, 9.8, and 13.0%, respectively. These values are higher than in Tay-Sachs disease but are similar to levels seen in late-onset or adult cases of GM2 gangliosidosis. This patient appears to represent a new phenotype of juvenile GM2 gangliosidosis having dystonia as the dominant symptom.

A number of recent studies report response of patients with carpal tunnel syndrome to pyridoxine treatment. Neurological and biochemical studies were therefore performed on six patients both before and after treatment with pyridoxine for at least 9 weeks. Free pyridoxal, pyridoxal phosphate, and total pyridoxal were assayed in plasma and neutrophils. The pyridoxal status was also estimated by assaying red cell aspartate aminotransferase. No evidence was obtained to suggest that these patients were deficient in either pyridoxal or pyridoxal phosphate. Although four of the patients claimed some partial symptomatic relief, there was no consistent improvement in clinical findings or neurophysiological measurements following pyridoxine treatment.

The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes). In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the "absolute values" are 10-fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.

In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen-positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease.

We have established optimal conditions for the measurement of glucocorticoid receptors (GR) in human white cells using a whole-cell binding assay with [3H]dexamethasone as the ligand, and the subsequent determination of the GR content in normal human lymphocytes and in leukemic cells of patients with various forms of acute and chronic leukemia. A number of leukemia cell lines in continuous culture were also subjected to the GR assay, and the results were correlated with the sensitivity of these cell lines to glucocorticoid steroids in vitro. The GR content of normal human lymphocytes amounted to 4,850 +/- 1,340 (mean +/- SD) receptors/cell. The mean equilibrium dissociation constant (KD) of the interaction of [3H]dexamethasone with the GR was 1.2 x 10(-8) M. Steroidal compounds with a known glucocorticoid potency effectively competed for the binding, whereas steroids devoid of glucocorticoid activity (e.g. estradiol-17 beta and testosterone) were ineffective. The GR content of the blast cells obtained from eight patients suffering from acute leukemia and four patients with a blast crisis of chronic myelocytic leukemia was found to be highly variable (3,230-29,900 receptors/cell), while the lymphocytes of six patients with chronic lymphatic leukemia contained a rather stable GR content (2,930-5,120 receptors/cell), which was comparable with that of normal lymphocytes. GR was identified in all the 12 malignant continuous white cell lines studied. Large cells contained more GR than the smaller ones. There was no apparent correlation between the GR concentration and the sensitivity of the cells in vitro to glucocorticoids as judged by [3H]thymidine incorporation studies. Distribution of the surface markers in the leukemic cell lines did not relate to the GR concentration. We conclude that the presence of GR is probably a universal feature of the leukemic cells, and, from a clinical standpoint, probably does not alone imply steroid responsiveness.

Axial light loss (ALL) is the measurement of the total light lost from the laser beam at 0 degrees when a particle passes through the beam. Used in combination with the monoclonal antibody CD45, ALL can effectively resolve lymphocytes, monocytes, granulocytes, and dead cells in viable or fixed preparations of human peripheral blood. A bivariate display of ALL vs. CD45 clearly resolves all granulocytes from lymphocytes; although degranulated granulocytes cannot be resolved with forward-angle and right-angle light scatter, they are clearly resolved in right-angle scatter vs. CD45. A blood differential can be performed, with a single laser flow cytometer and three colors of fluorescence, when ALL is combined with fluoresceinated CD45 to resolve leukocytes, phycoerythrin-labeled NKH1 to resolve natural killer cells, and biotinylated CD3 in combination with DuoCHROME, the phycoerythrin/Texas red conjugate fluorochrome from Becton Dickinson, to resolve T-cells. B-cells are the only cells negative for both phycoerythrin and Texas red. When PE CD4 is included, the CD3+ CD4+ T-cell subset is resolved from the CD3+ CD4- subset comprising mainly the CD3+ CD8+ T-cell subset. The addition of propidium iodide is unnecessary since ALL clearly resolves dead cells in a viable preparation of human peripheral blood. Furthermore, since ALL resolves these cells even after fixation in paraformaldehyde, all samples can be fixed prior to analysis, thereby minimizing the potential biohazard risk.

A technique is described that allows direct hybridization to the DNA of cells flow sorted onto nitrocellulose filters, which obviates an intervening DNA isolation step. The feasibility of this technique for studying small numbers of cells is demonstrated with human cord blood, which has a high proportion of nucleated cells. The cells are stained with fluorescein-conjugated anti-HLe-l, a monoclonal antibody that recognizes mature leucocytes. Anti-HLe-l-positive cells are all nucleated, and a controlled, precise number of them may be sorted directly onto a nitrocellulose membrane. In cord blood, a small percentage of anti-HLe-l-negative cells are nucleated erythrocytes, which may also serve as a source of DNA. Studies were performed on male or female newborn cells flow sorted onto nitrocellulose membranes and hybridized with either a non-specific human repeat DNA probe or a Y chromosome-specific probe. Importantly, the sex of the newborn could be determined at the DNA level from as few as 50 sorted cord blood leucocytes or 5,000 HLe-l-negative cells. Since nucleated erythrocytes are common in fetal blood but rarely found in the peripheral circulation of adults, the method has potential application for the determination of fetal sex from analysis of flow-sorted nucleated erythrocytes present in the maternal circulation during pregnancy.

Glucocorticoid receptors (GCRs) were studied by the radioligand binding assay in peripheral leukocytes obtained from dogs during intestinal ischemia shock and obtained from patients with septic shock. Sixteen healthy adult mongrel dogs were divided into two groups at random. The model of intestinal ischemia shock was made by occluding both superior mesenteric artery and vein in the occlusion group. Leukocytic GCRs and plasma cortisol were measured just at the time when mean arterial pressure (MAP) was 13.3 kPa (100 mmHg), 6.6 kPa (50 mmHg), and 3.3 kPa (25 mmHg). Clinically leukocytic GCRs were measured in ten patients in septic shock during hypotensive period and in ten normal volunteers used as control subjects. The results showed that in the occlusion group of animals, the levels of GCRs decreased progressively after shock; there was a significant positive correlation between the extent of reduction of MAP and that of GCRs. Leukocytes from the occlusion group contained significantly lower levels of GCRs than those from controls. The results of clinical observation confirmed the reduction of GCR levels in patients with septic shock. This paper reports for the first time the finding of secondary disorder of GCRs during septic shock, advances a new hypothesis concerning the pathophysiology of shock at the receptor level, and offers the experimental evidence for early use of massive doses of glucocorticoid (GC) in dealing with septic shock.

Restriction fragment length polymorphism (RFLP) of Ha-ras gene was surveyed by Southern blot analysis in leukocyte DNA from 55 normal individuals and 58 urothelial cancer patients in Japan. Three common alleles and 4 rare alleles were classified. The frequency of common alleles in normal Japanese individuals differed from that in Caucasians previously reported; a 7.2-7.5-kb BamHI fragment of common allele was not observed in Japanese individuals. No significant increase in frequency of the rare Ha-ras allele was observed in the group of cancer patients. Moreover, no significant difference in frequency was observed for the 3 common alleles. Tumor DNA was compared with leukocyte DNA in 30 urothelial cancer patients: in 3 of 8 cases with heterozygous Ha-ras locus, decreased intensity of one band, indicating partial loss of one allele in tumor DNA, was observed. In 3 tumors with either deletion of one Ha-ras allele or a rare Ha-ras allele, expression of Ha-ras gene was examined by Northern blot analysis. Such genetic alterations did not always result in a marked increase in Ha-ras expression. These data suggest that these genetic alterations are not directly related to Ha-ras expression, and that RFLP of Ha-ras gene is not a useful genetic marker for urothelial cancer. On the other hand, deletion of one Ha-ras allele was observed in 1 of 5 cases of bladder cancer and in 2 of 3 cases of renal pelvic cancer, suggesting that that deletion may be important in the development of urothelial cancer.

Treatment of neoplastic guinea-pig cells for 1-14 days with 300 units of guinea-pig lymphotoxin/ml medium enhanced 2- to 5-fold their susceptibility to natural killer (NK) cell-mediated cytotoxicity as measured by 4 h 51Cr and 72 h [3H]-thymidine-release assays. The NK susceptibility of human neoplastic and guinea-pig non-neoplastic cells refractory to guinea-pig lymphotoxin growth-inhibitory activity was unchanged after lymphotoxin treatment. Lymphotoxin preparations were free of detectable interferon and the enhancing activity copurified with lymphotoxin during diafiltration and isoelectric focusing. Treatment of NK cells with lymphotoxin preparations also augmented NK-mediated lysis but that activity did not copurify with lymphotoxin. As with neoplastic cells, lymphotoxin-sensitive preneoplastic cells previously treated with ultraviolet light, a chemical carcinogen, or carcinogen and the tumor-promoting agent tetradecanoylphorbol-acetate also exhibited lymphotoxin enhancement to NK-cell destruction. Thus, the immunologic hormone lymphotoxin enhances the susceptibility of lymphotoxin-sensitive cells to NK-cell destruction, indicating that these two natural immune mediators, lymphotoxin and NK cells, have the potential in concert to regulate carcinogenesis and neoplastic growth.

Previous attempts to demonstrate abnormalities in lipid peroxidation in various forms of the neuronal ceroid-lipofuscinoses (NCL) have been unrewarding up to and including the peroxide level (peroxidase). In this experiment a survey was made in a canine model of NCL to study the relative concentration of 4-hydroxynonenal (HNE), a fragment derived from an acute oxidation product of unsaturated fatty acids. Peripheral blood cells and various tissues from an affected and a normal control dog were surveyed. HNE was assayed after reacting with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine to form the 4-hydroxynonenal (O-pentafluorobenzyl) oxime. This reaction product was then separated by capillary gas liquid chromatography (g/c) and quantitated by flame ionization. The survey showed that neutrophils isolated from affected dogs and carriers contained abnormal amounts of HNE when compared with normal control animals. Two carriers had mean values of +3,289% above normal, and neutrophils from two affected animals were +4,873% above normal. In addition, an examination of the relative HNE levels in brain, retina, retinal pigment epithelium (RPE), and kidney of an affected dog compared with a control animal also showed abnormal levels of HNE, particularly in brain (+168%) and in the RPE (+135%), the two organs exhibiting the most severe pathologic damage unique to these disorders. These findings, although preliminary, clearly document a role for HNE in this canine form of human NCLs. The well-known cytotoxic properties of HNE and other alpha,beta unsaturated aldehydes suggest a primary role in the pathogenetic events of this disorder.

Control of technique and use of biological standards in flow cytometry have become increasingly important due to the wider use of the method for ploidy determination of malignant tumors in clinical research. Trout (TRBC) and salmon erythrocytes and human buffy coat leukocytes were selected for a study of factors influencing the DNA stainability. Whether standard and test cells were mixed before or after enzymatic treatment and staining was found to be critical for the ploidy comparisons. Otherwise, artifactual differences of at least 20% may be noted, leading to an overestimation of DNA aneuploidy. The time from staining to analysis had minimal effect, with some exceptions. The proportions of different cells in the sample had no influence, and nonlinearity of measurements was negligible. Diploid cells in normal endometrium and benign ovarian tumors, as well as the diploid fraction of aneuploid tumor cells, were systematically measured to have a DNA staining 5-7% above human leukocytes.

We have radiolabelled surface glycoproteins of different types of leukemic cell. The labelled proteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. Surface glycoprotein patterns discriminatory for acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) were found. We conclude that the analysis of the surface glycoprotein profile provides a useful method for the classification of leukemic cells according to cell type and stage of differentiation.

All human hematopoietic cells seem to contain a major, heavily O-glycosylated sialoglycoprotein. Glycophorin A is specific for the erythroid lineage of cells, and leukocytes have a major sialoglycoprotein, also called leukosialin or sialophorin. Cell differentiation results in patterns of O-glycosylation in these proteins, which reflect the stage of differentiation within a cell lineage as well as lineage specificity. The altered carbohydrate compositions may influence the interactions of the cells with external ligands. Healthy individuals lacking glycophorin A in their red cells are known, whereas a deficiency of the leukocyte sialoglycoprotein may result in immunological disease. Although little is known about the physiological functions of these proteins, they form interesting models for studies on regulation of glycosylation, biosynthesis of O-glycosylated glycoproteins, and function of cell surface receptors.