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PubMed2003

Differential leukocyte count method for bovine low somatic cell count milk.

Whereas many differential leukocyte count methods for high somatic cell count (SCC) milk from mastitic cows are available, only a few have been developed for low SCC milk. We have developed a flow cytometric differential leukocyte count method for low SCC milk. The procedure consists of 1) 1.5 ml of diluted milk sample (30%, vol/vol dilution with PBS), 2) centrifugation, 3) leukocyte labeling with SYTO 13 and 4) flow cytometric analysis. Four major leukocyte populations can be clearly identified in the green fluorescence-side scatter dot plot: lymphocytes and monocytes (LM), polymorphonuclear neutrophils (PMN), mature macrophages (Mphi), and cells with apoptotic features based on chromatin condensation and nuclear fragmentation. The optimal processing temperature was 20 degrees C. Significant differences among samples with similar differential leukocyte counts were found. Storage of milk samples during 2 d at 7 degrees C had no effect on differential leukocyte count. Using the new method, differential leukocyte count was performed in low SCC milk samples from cows in early, mid, and late lactation. In accordance with previous studies, PMN and Mphi percentages were lower and LM percentages were higher in early lactation than in the other stages of lactation. The percentage of cells with apoptotic features was higher in early lactation than in mid and late lactation. In conclusion, a rapid, simple, accurate, and reproducible standard procedure was developed to determine the differential leukocyte count (Mphi, PMN, LM, and cells with apoptotic features) of bovine low SCC milk.

Animals↗

PubMed1991

[Changes in the blood cell counts with aging].

We analyzed the blood cell counts and serum levels of total protein (TP), total cholesterol (TC) and triglyceride (TG) of 2,231 healthy subjects (1,295 men and 936 women) between age 20 and 99 years in order to clarify the following two subjects. (1) In the approximately 10 years since the report of Shirakura et al in 1978, eating habits have improved and the average life expectancy has extended in Japan. Is there any effect of such betterment on blood cell counts of the aged? (2) It has been pointed out that quality of everyday life, such as staying at home but not in an old-age home, working, traveling, and so forth, had an influence on the blood cell counts of aged. Is there any difference between the blood cell counts of people under 60 years and those of people older than 60 years who have a good quality of life as mentioned above? The hemoglobin concentration, red blood cell count, and hematocrit value began to decrease in men in their sixth decade and in women in their seventh decade and the change was more prominent with advancing age, especially in men. The white blood cell count and platelet count tended to decrease with advancing age. The serum levels of TP, TC, and TG also declined with age in those over 60 years of age. These results confirmed that the hemoglobin concentration, red blood cell count, and hematocrit value decrease in the elderly subjects as they grow older and it may be considered that reduced ingestion of protein is one of the causes of the phenomenon.

Adult↗

PubMed1996

Reduced CD4 cell counts in blood do not reflect CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1 infection.

OBJECTIVE: To investigate whether the loss of CD4 cells seen in peripheral circulation of HIV-1-positive individuals reflects a similar depletion of CD4 cells from lymphoid tissue. DESIGN: CD4 and CD8 cells in tonsillar mononuclear cell suspensions were quantified relative to tonsillar B cells, as these were thought to remain numerically unchanged in the course of HIV infection. Results were related to the CD4 cell counts in blood and to the clinical status of the patients. METHODS: Blood samples and tonsillar tissue were obtained from 13 HIV-1-seropositive individuals and six seronegative controls. B cells and T-cell subsets in mononuclear cells were quantified using a three-colour flow cytometry protocol. Histological sections were morphologically classified and B-cell areas were quantified by morphometry. RESULTS: The B-cell fraction was confirmed to be relatively unchanged in asymptomatic HIV-1-seropositive individuals compared with controls. The tonsillar CD4 : B-cell ratios in asymptomatic individuals was similar to those seen in controls, whereas the CD4 : B-cell ratios in symptomatic HIV-1-infected individuals were greatly reduced. The tonsillar CD4 : CD8 cell ratios in HIV-1-infected individuals were much lower than those seen in controls, in the asymptomatic group due to a considerable expansion of the tonsillar CD8 cell subset, and in the symptomatic group also due to a loss of CD4 cells. CONCLUSIONS: We found no evidence of CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1-infected individuals despite low CD4 cell counts in blood. Loss of CD4 cells from this lymphoid tissue seems to occur as a late-stage phenomenon correlated with the onset of clinical symptoms.

Adult↗

PubMed2001

Inverse relationship between patient peripheral blood CD34+ cell counts and collection efficiency for CD34+ cells in two automated leukapheresis systems.

BACKGROUND: The purpose of this study was to analyze the CD34 cell collection efficiency (CE) of automated leukapheresis protocols of two blood cell separators (Spectra, COBE [AutoPBSC protocol] and AS104, Fresenius [PBSC-Lym, protocol]) for peripheral blood progenitor cell (PBPC) harvest in patients with malignant diseases. STUDY DESIGN AND METHODS: PBPCs were collected by the Spectra AutoPBSC protocol in 95 patients (123 collections) and the AS104 PBSC-Lym protocol in 87 patients (115 harvests). Patients underwent a median of one (range, 1-4) conventional-volume apheresis procedure of 10.8 L (9.0-13.9) to obtain a target cell dose of > or =2.5 x 10(6) CD34+ cells per kg. RESULTS: The median overall CD34 CE was significantly better on the AS104 than on the Spectra: 55.8 percent versus 42.4 percent (p = 0.000). This was also true below (59.2% vs. 50.1%; p = 0.022) and above (51.2% vs. 41.3%; p = 0.001) the preleukapheresis threshold of 40 CD34+ cells per microL needed to collect a single-apheresis autograft. However, at > or =40 circulating CD34+ cells per microL, both cell separators achieved the target of > or =2.5 x 10(6) CD34+ cells per kg. The CD34 CE dropped significantly, from 59.2 percent at <40 cells per microL to 51.2 percent at > or =40 cells per microL on the AS104 (p = 0.017) and from 50.1 percent to 41.3 percent on the Spectra (p = 0.033). CONCLUSION: Whereas the CD34 CE was significantly different with the AS104 and the Spectra, the CD34 CE of both machines correlated inversely with peripheral blood CD34+ cell counts, showing a significant decline with increasing numbers of circulating CD34+ cells. Nevertheless, at > or 40 preapheresis CD34+ cells per microL, sufficient hematopoietic autografts of > or =2.5 x 10(6) CD34+ cells per kg were harvested by a single conventional-volume (11 L) leukapheresis on both cell separators.

Adolescent↗

PubMed1997

The effects of disposable daily wear contact lenses on goblet cell count.

PURPOSE: The number of goblet cells on the inferior bulbar conjunctiva increases in response to daily wear contact lens use. We conducted a study to examine whether daily wear soft contact lenses worn on a 2-week disposable schedule would also induce the same goblet cell response. METHODS: Twenty-eight subjects who had never worn contact lenses were fit with 38% water polymacon lenses. The lenses were worn on a daily wear basis and replaced every two weeks over a 6-month period. Goblet cell count was determined by impression cytology of the inferior bulbar conjunctiva. RESULTS: No significant increase in goblet cell count was observed in the subjects during the 6 months period; the 3.23% baseline goblet cell count was never exceeded. In our previous study, after 6 months of conventional daily wear lens use, the goblet cell count increased from a baseline of 4.19% to 8.0%. CONCLUSIONS: It appears that wearing daily wear soft contact lenses on a disposable basis may be less irritating to the ocular surface than wearing soft contact lenses on a conventional daily wear basis.

Adolescent↗

PubMed1989

Guinea pig lung cells. Method of isolation and partial purification, identification, ultrastructure, and cell count.

Guinea pig lung cells (over 700 x 10(6) cells/lung) were obtained following gentle digestion of lung tissues with a solution of protease type VII (50 micrograms/ml). The viability of these cells was over 86% as estimated by the trypan blue exclusion technique. The cell suspensions were elutriated into eight fractions, which were characterized by selected staining techniques (Alcian blue, esterase, and Papanicolaou) and by electron microscopy. Differential cell counts were done to establish the percentages of each type of cells in the overall population. Electron microscopy analyses of the cell populations have allowed the identification of most of the various isolated cell types and showed that the cellular organelles and ultrastructures were well preserved. These cell populations will be used for characterizing lung immunologic, metabolic, and endocrine functions, as well as for studying cell interactions.

Animals↗

PubMed1997

Centenary of Gaule and Lewin, pioneers in cell counting methodology.

We commemorate the one hundredth anniversary of the publication of a pioneering paper on cell counting, by Gaule and Lewin. Their paper describes a new method for counting cells in tissue sections. First they found the mean number of cell profiles per cell by examining 50 selected cells in serial sections. Then they counted the total number of cell profiles in the ganglion, and finally divided this total profile number by the mean number of profiles per cell. They thought this method more accurate than counting cells by counting only profiles that showed the nucleolus because they noted that a cell's nucleolus sometimes appeared in more than one section and that a single cell could have more than one nucleolus.

Animals↗

PubMed2005

CD4 cell-monitored treatment interruption in patients with a CD4 cell count > 500 x 106 cells/l.

BACKGROUND: Little is known about CD4 cell count changes in patients with high CD4 cell counts who interrupt antiretroviral therapy, especially in those with a nadir of 250-350 x 10 cells/l. METHODS: Data derived from 139 patients from seven prospective cohorts who had > 12 months highly active antiretroviral therapy (HAART), CD4 cell count nadir of > 250 x 10 cells/l and at pre-interruption of > 500 x 10 cells/l. Endpoint was time to CD4 cell count < 350 x 10 cells/l or reinitiation of treatment. RESULTS: At interruption, the median CD4 cell count was 800 x 10 cells/l, median viral load was 1.7 log10 copies/ml. At the time of analysis, 63 (45.3%) had resumed therapy or experienced < 350 x 10 cells/l CD4 cells over a median interruption of 75 weeks. Of these, 33 (52.4%) experienced a decline to < 350 x 10 cells/l and 30 (47.6%) restarted therapy before their CD4 cell count had fallen below this level. In 43 patients with CD4 cell nadir of 251-350 x 10 cells/l, median time to therapy resumption or CD4 cell count < 350 x 10 cells/l was 61 weeks. Higher CD4 cell count nadir, longer duration of viral load suppression on therapy, and higher viral load level at interruption were independently associated with longer time to restart therapy. The risk of clinical events was 5 per 1000 person-years of follow-up. CONCLUSIONS: Patients who started therapy with CD4 cell count of 250-350 x 10 cells/l and who later interrupted therapy appear able to remain off therapy with a CD4 cell count > 350 x 10 cells/l for a substantial period of time.

Adult↗

PubMed2006

Low CD4+ T-cell counts in HIV patients receiving effective antiretroviral therapy are associated with CD4+ T-cell activation and senescence but not with lower effector memory T-cell function.

The adverse effects of immune activation on CD4(+) T-cell recovery and the relationship between CD4(+) T-cell counts and effector T-cell function were examined in HIV-1 patients receiving long-term effective ART. Patients with nadir CD4(+) T-cell counts <100/microl, > 12 months on ART and >6 months with <50 HIV RNA copies/ml were stratified by current CD4(+) T-cell counts and patients from the lowest (n = 15) and highest (n = 12) tertiles were studied. We assessed proliferation (Ki67), activation (HLA-DR, CD38) and replicative senescence (CD57) by flow cytometry and CD4(+) T-cell responses to CMV by IFN-gamma ELISpot. Proportions of CD4(+) T-cells expressing HLA-DR or CD57 were strong univariate predictors of total (P = 0.0002 and P = 0.002) and naive (P < 0.0001 and P < 0.0001, respectively) CD4(+) T-cell counts, suggesting that CD4(+) T-cell activation drives the depletion of naive CD4(+) T-cells. This was clearest in patients with a small/undetectable thymus. IFN-gamma responses to CMV were similar in patients with low or high CD4(+) T-cell counts.

Adult↗

PubMed1984

Processing of lung lavage fluid causes variability in bronchoalveolar cell count.

To study the effects on cell counts of different ways of processing bronchoalveolar lavage (BAL) fluid, we performed 77 BAL in 19 healthy control subjects and in 58 patients with sarcoidosis. We investigated the role of readers, hemocytometers, mode of identification of macrophages, cell washing, and speed of cytocentrifugation. No significant effect of reader or hemocytometer was observed. The percentage of macrophages, determined as large cells stained with neutral red in a Malassez hemocytometer (63.9 +/- 24.6%, mean +/- SD) was lower than the percentage of macrophages determined by May Grunwald Giemsa staining (76.3 +/- 19.2%, p less than 10(-9)). Cell counts decreased 34% after 2 washings (p less than 0.001), and more lymphocytes were counted after cytocentrifugation at 90 g (33.2 +/- 25.3%) than at 23 g (27.7 +/- 22.1%). We conclude that bronchoalveolar cell counts vary with changes in processing lung lavage fluid and that this variability should be considered when using BAL cell counts.

Bronchi↗

PubMed2000

Association of ABO incompatibility with elevation of nucleated red blood cell counts in term neonates.

OBJECTIVE: Nucleated red blood cells in the circulation in term neonates have been associated with a wide range of pathologic conditions. We sought to examine the relationship between nucleated red blood cells in the circulation of term neonates and maternal-neonatal blood type compatibility. STUDY DESIGN: We prospectively collected umbilical blood from all live-born neonates delivered at our institution. Venous blood was analyzed for nucleated red blood cells and is reported as the number of nucleated red blood cells per 100 white blood cells. We reviewed maternal and neonatal records for neonates born at > or =37 weeks' gestation for correlative clinical data. Statistical analysis was performed with the SAS statistical software package (version 6.12; SAS Institute, Inc, Cary, NC). Kruskal-Wallis analysis was used as a nonparametric test. RESULTS: We evaluated 1661 neonates delivered during the study period and found a mean (+/-SD) of 9.29 +/- 18.56 nucleated red blood cells per 100 white blood cells (range, 0-327 nucleated red blood cells per 100 white blood cells). Nucleated red blood cell counts were lower in ABO-compatible maternal-fetal dyads (mean +/- SD, 8.29 +/- 12.84 nucleated red blood cells per 100 white blood cells; range, 0-216 nucleated red blood cells per 100 white blood cells) than in ABO-incompatible dyads (mean +/- SD, 13.16 +/- 13.16 nucleated red blood cells per 100 white blood cells; range, 0-327 nucleated red blood cells/100 white blood cells; P =.006). Neonates of mothers with blood groups A and B had significantly lower nucleated red blood cell counts (P <.05). Dyads with maternal type O and neonate type B had significantly higher nucleated red blood cell counts (P <.002). Nonparametric testing determined that type O mother and type B neonate combinations had significantly higher umbilical cord nucleated red blood cell counts (P <.001). CONCLUSION: Maternal-fetal ABO incompatibility is associated with elevation of nucleated red blood cell count in term neonates. Nucleated red blood cell elevation does not always connote a serious pathologic process, however, because ABO incompatibility usually does not adversely affect neonatal outcome. The clinical significance of an elevated nucleated red blood cell count thus is limited.

ABO Blood-Group System↗

PubMed1990

Counting cells with stereology: random versus serial sectioning.

Counts of cells and nuclei from sections provide information central to studying structural changes in cells, tissues, and organs. This study considers some of the practical problems associated with counting cells with the newer random and serial sectioning methods of stereology and tests the hypothesis that similar cell counts can be obtained with both random and serial sectioning methods. Using irregularly shaped nuclei from alveolar cells of the goat lung, we compared cell counts derived from random (electron microscopic) and serial sectioning (light microscopic) methods. The results showed that both sectioning methods gave similar cell counts (10(7)/cm3 of parenchyma) for type 1 epithelial cells (5.0 vs. 5.0; P=1.0), type 2 epithelial cells (8.6 vs. 9.8; P= 0.42) and interstitial cells (34.6 vs. 33.4; P=0.64), provided that corrections were introduced for section-related biases and that the nuclei of the random sectioning method were corrected for shape. We found counting biases of 5%-7% for nuclear shape and 16% for section compression. These observations support the hypothesis that similar cell counts can be obtained with random and serial sectioning, even when nuclei have irregular shapes.

Animals↗

PubMed2006

Comparison of capillary based microflurometric assay for CD4+ T cell count estimation with dual platform Flow cytometry.

The CD4+ T cell count estimation is an important monitoring tool for HIV disease progression and efficacy of anti-retroviral treatment (ART). Due to availability of ART at low cost in developing countries, quest for reliable cost effective alternative methods for CD4+ T cell count estimation has gained importance. A simple capillary-based microflurometric assay (EasyCD4 System, Guava Technology) was compared with the conventional flow cytometric assay for estimation of CD4+ T cell counts in 79 HIV infected individuals. CD4+ T cell count estimation by both the assays showed strong correlation (r = 0.938, p < 0.001, 95% CI 0.90 to 0.96). The Bland Altman plot analysis showed that the limits of variation were within agreeable limits of +/- 2SD (-161 to 129 cells/mm3). The Easy CD4 assay showed 100% sensitivity for estimating the CD4+ T cell counts < 200 cells/mm3 and < 350 cells/mm3 and 97% sensitivity to estimate CD4+ T cell count < 500 cells/mm3. The specificity ranged from 82 to 100%. The Kappa factor ranged from 0.735 for the CD4+ T cell counts < 350 cells/mm3 to 0.771 for < 500 cells/mm3 CD4+ T cell counts. The system works with a simple protocol, is easy to maintain and has low running cost. The system is compact and generates minimum amount of waste. Hence the EasyCD4 System could be applied for estimation of CD4+ T cell counts in resource poor settings.

Journal Article↗

PubMed1990

Significance of blasts in low-cell-count cerebrospinal fluid specimens from children with acute lymphoblastic leukemia.

The purpose of this study was to determine whether the presence of more than 5% blasts in a differential count of cytocentrifuged cerebrospinal fluid (CSF) with less than 6 leukocytes/microliter was predictive of central nervous system (CNS) relapse in children with acute lymphoblastic leukemia (ALL). A double concentrate method of cytocentrifuge preparation was used to analyze 4543 consecutive CSF specimens from 349 children with ALL between January 1, 1982, and September 30, 1988. One hundred nine CSF specimens from 58 evaluable children had less than 6 leukocytes/microliter and more than 5% blasts on cytocentrifuge differential count (low-cell-count specimen with blasts [LCB]). During the study period, 25 of 332 evaluable children (7.5%) had CNS leukemic recurrence. In 22 of 25 (88%), the CNS relapse was preceded by at least one abnormal low-cell-count CSF specimen. One of 34 patients with a single LCB at diagnosis (3%) had subsequent CNS relapse compared with five of eight patients (62.5%) with a single LCB during remission (P = 0.0002). Of 16 children with two or more LCB during remission, nine (56%) had CNS relapse defined by standard criteria, whereas six additional patients in this group were declared to be in CNS relapse on the basis of their repetitive LCB. Whether diagnosing CNS recurrence earlier in its course based on a modification of the definition of CNS leukemia will change the frequency of subsequent adverse events or make possible decreased intensity of CNS retreatment remains to be determined.

Adolescent↗

PubMed1983

Breakdown of caseins by proteinases in bovine milks with high somatic cell counts arising from mastitis or infusion with bacterial endotoxin.

Milk obtained from cows which were either infected by clinical mastitis or had been subjected to intramammary infusion of Escherichia coli endotoxin possessed high counts of somatic cells and very high levels of proteinase activity which hydrolysed the caseins almost completely in a few hours at 37 degrees C. The rate of hydrolysis of beta-casein was slightly greater than that of alpha S1-casein, but in both cases hydrolysis was enhanced by 6 cycles of freezing and thawing to disrupt somatic cell membranes. A study of the relationship between proteinase activity and cell count suggested that only some of the proteinase activity originated in the somatic cells and also that the identity of the cells making up the total cellular population was important. Maximum proteolysis occurred at 50-60 degrees C, but the temperature-activity curve was a broad peak. Likewise the pH versus activity plot was very broad and was almost flat over the pH range 6-9. Experiments with a number of inhibitors of proteinases failed to give a clear cut pattern of inhibition. All evidence obtained was consistent with the view that several different enzymes with different pH and temperature optima and different specificities contributed to the overall hydrolysis of caseins in these milks. From electrophoretic band patterns one of these enzymes was clearly plasmin, but in high cell count milks other proteinases also became significant.

Animals↗

PubMed2000

Variability in CD34+ cell counts in umbilical cord blood: implications for cord blood transplants.

OBJECTIVE: To determine if total nucleated cell counts alone are sufficient for predicting the efficacy of cord blood units for transplant from neonatal umbilical cord blood samples. METHODS: Umbilical cord blood samples were collected from 200 mothers at delivery and the cord blood units processed. The total nucleated cells and CD34+ cells were enumerated and compared for each sample. RESULTS: Despite an apparent linear correlation between total nucleated cell counts and CD34+ cell counts, each group of total nucleated cell counts demonstrated a high degree of variation in CD34+ cell counts and could be as low as 0.1% of total nucleated cell counts. CONCLUSIONS: Large variations in CD34+ cell counts per total nucleated cell count are present for cord blood units from neonatal umbilical cord samples. Hence a CD34+ cell count for each cord blood unit would improve selection of samples for transplant.

Antigens, CD34↗

PubMed2001

Application of differential inflammatory cell count as a tool to monitor udder health.

A flow cytometric technique called differential inflammatory cell count was standardized by staining bovine peripheral blood leukocytes with a combination of DNA binding dyes SYBR green 1 and propidium iodide in water. Leukocytes were also stained with propidium iodide in detergent to determine total cell count. Differential inflammatory cell count assay was evaluated with individual quarter milk samples from 13 cows. Cows were sampled at weekly intervals for 3 wk and assayed for total cell count, mononuclear leukocyte count, and polymorphonuclear leukocyte count. Simultaneously, milk samples were evaluated by the conventional electronic somatic cell count (SCC) technique. Somatic cell count positively correlated with total cell count (r = 0.9), mononuclear leukocyte count (r = 0.8), and polymorphonuclear leukocyte count (r = 0.89). Quarters with SCC > log10 5.4 had a higher total cell count, mononuclear leukocyte count, and polymorphonuclear leukocyte count and were more often culture positive compared with quarters with SCC < log10 5.4. Quarters that were culture positive on all three test occasions had a higher proportion of polymorphonuclear leukocytes (33 to 49%) compared with quarters that were culture negative on all three test occasions (17 to 25%). The findings of this study suggest that differential inflammatory cell count assay has the potential to evolve as a new technique for evaluation of udder health status.

Animals↗

PubMed1989

Relationship between test-day measures of somatic cell count and milk production in California dairy cows.

The relationship between test-day measures of milk somatic cell count and milk yield was evaluated using the November 1985 test data from 8352 Holstein cattle (2923 primiparous and 5429 multiparous cows) located in ten Tulare County, California dairies. Following correction for herd and stage of lactation effects, design variable regression was used to create separate models for primiparous and multiparous cows predicting the changes in milk production associated with milk somatic cell count class. Cell counts were stratified by 1/2 loge cell count (x1000 cells/mL) units, permitting comparisons with previous studies. Cell counts less than 148,000/mL were not found to be associated with significant reductions in milk yield when compared to the reference class (cell counts less than 20,000/mL). Consistent incremental decreases in milk production were not noted with increasing cell count strata, even following the natural log transformation. The most dramatic production losses were noted in the range of 148,000 to 665,000 cells/mL. Primiparous cattle in the 403,000 to 665,000 cell count strata experienced a 5.22 kg (19.72%) decrease in test-day milk yield. Multiparous cattle in the same class experienced 3.01 kg (7.82%) reductions in milk production. Primiparous and multiparous cows had similar production losses. The study population differed from previous studies on the basis of herd size, milk production and the level of udder health, measured by milk somatic cell count. These differences and the choice of experimental design may in part explain differences in study results and conclusions.

Animals↗