Assessment and selection of the recipient cows' corpus luteum at the time of embryo transfer, and its influence on conception rate.

OBJECTIVES: To describe the influence of physical traits of the corpus luteum (CL), as described by transrectal ultrasonography on day 6 post-oestrus, on the conception rate following embryo transfer (ET) in recipient beef cows. To investigate if higher recipient utilisation rates were achievable, without compromising conception rates to ET. DESIGN/RESULTS: Data were analysed from Holstein Friesian embryos (n = 1075) frozen in ethylene glycol thawed for direct transfer into one herd of Angus recipient cows. For pregnancies achieved in the program (n = 693), no statistically significant effect was found for the physical traits of the recipients' CL on conception rate (CL volume (P = 0.20), CL side (P = 0.14). Conception rates were similar for recipients with a central lacuna (62%, n = 245) and recipients with no central lacuna (66%, n = 448) (P = 0.10). Of the pregnant recipients with a central lacuna (n = 245), 98.3% had no remaining luteal cavity by the 30-day pregnancy ultrasound. No effect on conception rate was found with either the small (<50% of CL diameter) or large (>50% of CL diameter) central lacunae (P = 0.18). For recipients with CLs that did not meet previous industry selection guidelines (n = 172, 16% of study population), the conception rate (63%) was not significantly different to the routinely selected recipient CLs (n = 903, conception rate 65%) (P = 0.83). CONCLUSIONS: The suitability of a potential ET recipient is determined by observing an appropriately timed oestrus and a detectable CL, regardless of size or quality.

Predictive power of the relative lymphocyte concentration in patients with advanced heart failure.

BACKGROUND: The physiological stress suffered by patients with heart failure results in an increased production of cortisol and a shift in the leukocyte differential toward a decreased percentage of lymphocytes (%L). The purpose of this study was to determine the prognostic significance of a low %L in advanced heart failure. METHODS AND RESULTS: Patients evaluated in our cardiac transplantation clinic between April 1988 and July 1995 were retrospectively reviewed (n=263). Fifty-two patients were excluded because they had recent trauma, infection, surgery, myocardial infarction, corticosteroid use, or history of malignancy. In the remaining 211 patients, we used Cox proportional hazards analysis to examine the association between survival and transplant-free survival with baseline variables. Univariate analysis showed a significant association between time to death and %L (P=.004), New York Heart Association (NYHA) class (P=.002), and maximal oxygen uptake (P=.05). Univariate analysis of the end point of survival free from transplantation yielded similar results. One- and 4-year survival rates for patients with a low %L (<20.3%) were 78% and 34% compared with 90% and 73% for those with a normal %L. Multivariate analysis showed NYHA class (P<.008) and %L (P<.01) were independent predictors of survival and survival free from cardiac transplantation. CONCLUSIONS: The relative lymphocyte concentration is an inexpensive, readily available, simple prognostic marker in patients with symptomatic heart failure who do not have recent trauma, infection, surgery, myocardial infarction, corticosteroid use, or history of malignancy. It could be incorporated into clinical models to predict patient outcome and to aid in the selection of patients for cardiac transplantation.

Usefulness of the lymphocyte concentration as a prognostic marker in coronary artery disease.

The prognostic utility of the relative lymphocyte concentration was tested in a population-based study of chronic coronary artery disease. This inexpensive, readily available test was found to be significantly related to survival (p = 0.03) in 211 patients followed for a mean of 45 months.

Incremental value of the leukocyte differential and the rapid creatine kinase-MB isoenzyme for the early diagnosis of myocardial infarction.

OBJECTIVE: To test whether automated measurements of cortisol-induced changes in the leukocyte differential can provide an early marker of myocardial infarction, especially when combined with the rapid creatine kinase-MB isoenzyme. DESIGN: A prospective, blinded study of these measurements at the time of initial assessment in the emergency department. SETTING: Large multispecialty clinic hospital. PATIENTS: 511 consecutive patients presenting to the emergency department with chest pain. One hundred twenty-seven patients with infection, trauma, or metastatic cancer or receiving myelosuppressive or glucocorticoid therapy were excluded. MEASUREMENTS: Automated leukocyte differentials, rapid creatine kinase-MB levels, cortisol levels, and routine clinical measurements. RESULTS: Of 69 patients with myocardial infarction, only 39% had diagnostic electrocardiographic ST-segment elevation. ST-segment elevation had a specificity of 99% and a positive predictive value of 93%. A relative lymphocytopenia (lymphocyte decrease < 20.3%) or elevated rapid creatine kinase-MB level (> 4.7 ng/mL) was more sensitive than ST-segment elevation (sensitivities of 58% and 56%, respectively) but less specific (specificities of 91% and 93%, respectively). The presence of both a relative lymphocytopenia and an elevated rapid creatine kinase-MB level had a sensitivity of 44%, a specificity of 99.7%, and a positive predictive value of 97% (95% Cl, 80% to 99%). Both a relative lymphocytopenia and an elevated rapid creatine kinase-MB level were independent (P < 0.001) predictors of infarction in patients without ST-segment elevation. If myocardial infarction was suspected by the presence of both abnormal markers or ST-segment elevation, the sensitivity for early diagnosis increased from 39% (ST elevation alone) to 65% (Cl, 52% to 76%); the specificity was 99%; and the positive predictive value was 94% (Cl, 82% to 98%). CONCLUSIONS: The presence of both a relative lymphocytopenia and an elevated rapid creatine kinase-MB level was an accurate early marker of myocardial infarction that appeared to improve the sensitivity of early diagnosis compared with that of ST-segment elevation alone.

Replating efficiency of metastatic melanoma cells from lymph node and subcutaneous sites does not predict patient survival.

The efficiency of replating of cells from primary colonies grown in semisolid medium has been used to detect and quantitate self-renewal in vitro. A positive correlation has been found by others between the replating efficiency of cells from myelogenous leukemia and patient survival. In the current study we measured primary and secondary replating efficiency of metastatic melanoma cells from subcutaneous tissues or lymph nodes of twelve patients and related these results to patient survival from time of biopsy. No relationship was found between primary and secondary plating efficiency nor for primary or secondary replating efficiency and survival. These results suggest that colony-forming melanoma cells grown under anchorage-independent conditions do not identify a stem cell population important for survival distinct from highly proliferative cells. These studies do not, however, rule out the possibility that a non-clonogenic transitional cell population exists in the tumor.

Leukocyte concentrations in discrimination of benign from malignant lung lesions.

Malignant lung lesions are associated with significant changes in leukocyte concentrations. However, overlap of values between normal subjects and patients with cancer has limited the clinical utility of this determination. To decrease the overlap, 2,000 cells per differential determination were counted, and replicate automated cell counts were performed in blood samples obtained on different mornings between 7 and 9 A.M. Seventy-five patients who presented with undiagnosed lung lesions were analyzed. Benign and malignant lung lesions could be accurately distinguished by either relative granulocyte or lymphocyte counts. With 65.5 percent granulocytes as the cutoff, 86 percent of patients (38 of 44) proved to have lung cancer had higher values, and 97 percent of patients (30 of 31) with benign lesions had lower values. Likewise, with 28 percent lymphocytes as the cutoff, 87 percent of patients with lung cancer (39 of 44) had lower values, and 94 percent of patients (29 of 31) with benign disease had higher values. Relative monocyte counts were not different. Absolute leukocyte concentrations, although different for each group, had considerable overlap and thus were poor discriminators. These data suggest that precise analytical techniques and the use of relative leukocyte concentrations can improve the clinical utility of the leukocyte count as a discriminator of benign and malignant lung lesions.

Serum C-reactive protein concentration in the management of infection in patients treated by continuous ambulatory peritoneal dialysis.

In a prospective study over 21 months, serum C-reactive protein (CRP) concentration was measured serially in 39 consecutive patients undergoing continuous ambulatory peritoneal dialysis. All patients with peritonitis mounted a CRP response, and the height of the response correlated well with the severity and extent of the peritoneal damage. Patients who recovered uneventfully after antimicrobial treatment showed a prompt fall in CRP from its peak value towards normal. In contrast, each patient in whom the serum CRP value remained raised after antimicrobial treatment had a complicated course. During routine outpatient follow up the serum CRP value remained within the normal range in the absence of intercurrent complications. These results, together with the commercial availability of rapid and precise assays for CRP, indicate that serial CRP measurements may be useful in monitoring the efficacy of antimicrobial treatment during episodes of peritonitis and in the recognition of intercurrent complications in patients undergoing continuous ambulatory peritoneal dialysis.

Similar self-renewal properties for different sizes of human primary melanoma colonies replated in agar.

Clonogenic assays currently define colonies as multicellular growth units above an arbitrarily designated cutoff size rather than by the biological function of different-sized growth units. To define the cutoff size between clusters and colonies in terms of the biological function of the cells within the growth units, we directly measured the self-renewal and proliferative capacity of cells from different-sized melanoma colonies. Primary colonies formed from cells of two patients were removed, pooled according to size, and replated, and the frequency and size distribution of the secondary colonies were analyzed. Cells from primary melanoma colonies that resulted from four to eight population doublings had similar extensive proliferative and self-renewal characteristics. The results demonstrated that self-renewal was not limited to cells in large colonies and suggested that the cutoff may be below 16 cells/growth unit. These data support the use of relatively small multicellular growth units to define colonies and measure highly proliferative human melanoma tumor cells. In addition, these methods may allow the determination of the cutoff size for other tumor types in terms of the biological function of cells rather than arbitrarily designating a cutoff size.

Kinetics of clonogenic melanoma cell proliferation and the limits on growth within a bilayer agar system.

Accurate descriptions of the kinetics of cell growth in semi-solid agar clonogenic systems have been difficult because the number of cells in colonies of different sizes is largely unknown. We stained and removed tumor cell colonies from agar, directly counted their cells, and established equations to quantitate the number of cells within colonies of different sizes. We used these equations to quantitate, in terms of cell number and volume, the total amount and kinetics of clonogenic cell proliferation from biopsies of human melanoma and cell lines of several different tumor types. Daily observations of cells in agar and serial photography indicated a 0- to 4-day delay in the onset of proliferation in agar followed by rapid growth and then abrupt cessation of proliferation. We quantified the extent of proliferation of cells from melanoma biopsies of seven patients and 11 cell lines after they were allowed to proliferate in agar until they stopped. Approximately 10% of cells divided one to five times while only 0.01% divided six to nine times. The total number of cells within the colonies at the end of growth was different while the total volume of cells within the colonies per plate was similar; approximately 10(9) microns 3 cellular volume per plate represents an upper limit for proliferation within the closed, nonrefed bilayer agar system. Previous replating studies using the same biopsy cells have shown that clonogenic melanoma cells can self-renew and have more proliferative capacity than that expressed during primary colony formation. Thus, the clonogenic assay only measured initial proliferative capacities. Furthermore, variable delays in the onset of proliferation may contribute to the heterogeneity of colony size within clonogenic assays.

Immunohistochemical localization of actinomycin D in human melanoma tumor cells.

Human melanoma tumor colonies and derived cell lines (81-46a) were exposed to Actinomycin D (Act D) in vitro in order to study subcellular sites of drug-cell interaction. Light microscopy of 13 day-old colonies previously treated for 24 hours with 0.01 microgram/ml, 0.1 microgram/ml, and 1 microgram/ml Act D revealed a decrease in colony size and organization, an increase in pyknotic nuclei, and an accumulation of cell debris concomitant with an increase in Act D concentration. The 81-46a cells were treated with 0.1 microgram/ml Act D for 15, 30, and 60 minute intervals, followed by Act D antibodies. Immunofluorescence microscopy revealed both cytoplasmic, vesicular, filamentous, and nuclear variations in drug distribution with increased exposure. Cell viability studies indicated a decrease in viable cells as exposure time to Act D increased.

Quantitation of the number of cells within tumor colonies in semisolid medium and their growth as oblate spheroids.

The number of cells within tumor colonies has not been determined accurately in prior reports because, in all but small clusters, cells grow too closely and stacked to allow direct counting of cells by inverted microscopy. Therefore, we stained colonies formed in agar from 38 tumor cell samples of diverse histological origin, removed them with a micropipet, and directly counted the number of cells. The number of cells within colonies increased geometrically with colony diameter and inversely with the size of the cells within the colonies. The relationship can be described using linear regression: [In(no. of cells/colony) = 0.87 - 2.80 In (colony cell diameter) + 2.38 In (colony diameter)] which gave an R2 of 0.92. Measurements of colonies in agar showed that they grew as oblate spheroids rather than spheroids. In an average sample, 60-micron-diameter colonies contained eight to 10 cells; the range for the 38 samples was 1.2 to 48.5. These results precisely define colonies in terms of cell number. They allow calculation of the total number of cells formed from clonogenic cells, a more complete estimate of proliferation than conventional cloning efficiencies which only measure initial proliferation. Furthermore, because of the dependence on the size of the colony cells, if colonies are defined only by a specific diameter then they do not contain similar numbers of cells. Calculations which assume spherical colonies, rather than the oblate spheroid shape we found, greatly overestimate the number of cells within colonies. Our data can increase the accuracy of quantitation in the clonogenic system and thus improve the interpretation of the proliferation of clonogenic tumor cells.

Improvement of human melanoma colony formation in soft agar using boiled instead of autoclaved agar.

The effect of agar sterilized by either boiling or autoclaving on human melanoma colony formation in soft agar was compared using cells from 17 biopsies of metastatic malignant melanoma. The frequency of colony formation was significantly increased for cells grown in boiled agar in 8 samples (47%), unchanged in 8 samples (47%), and decreased in only one sample (6%). There were increases in both cluster and colony formation for the melanomas which had augmented colony formation when grown in boiled agar. There was also qualitative morphological improvement, including rounder, smoother cells and less extracellular debris surrounding the colonies. These data suggest that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.

Method for measurement of self-renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma.

A procedure was developed to directly measure the self-renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. A culture of colony-forming cells was performed with bilayer agar in microtiter wells. The number of live tumor cells from biopsies of melanoma tissue was determined and was used to calculate plating efficiencies. Sequential photography showed that cells did not migrate in agar, thereby documenting that all the cells within colonies were direct descendants of clonogenic cells. A calibrated pneumatically controlled micropipet attached to a micromanipulator was used to quantitatively remove melanoma colonies without removing adjacent cells or agar. Plucked primary colonies were mechanically disaggregated into single cells; viability was greater than 95% as determined by trypan blue dye exclusion. Dose-related formation of secondary colonies was observed after replating of cells from pooled primary colonies. Cells from individual colonies were replated, and secondary colonies formed. These techniques allowed a simple and direct assessment of the self-renewal capacity of colony-forming melanoma cells.

Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar.

Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson's Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best's carmine, Page-Green method for inclusion bodies, and Snook's reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis.

Persistent lymphocytopenia as a diagnostic feature of bronchogenic carcinoma.

Patients with abnormalities due to bronchogenic carcinoma, noted on chest films, have decreased peripheral blood lymphocytes and increased total white cells compared to patients with benign lesions. Precision studies of 40 patients revealed that a low percentage of lymphocytes averaged over a three-week period distinguished bronchogenic carcinoma patients from patients with benign lesions with 95 percent overall accuracy. Lesions as small as 1.0 cm were correctly predicted to be malignant. Mean 8 AM plasma cortisol levels were elevated in patients with bronchogenic carcinoma and there was a negative correlation of 8 AM plasma cortisol levels with precentage of lymphocytes. Increased levels of endogenous cortisol may account for lymphocytopenia in bronchogenic carcinoma patients.

Stability of peripheral blood lymphocyte count and E-rosette forming lymphocytes in healthy adults.

Highly reproducible peripheral blood lymphocyte (PBL) count and percent E-rosette forming lymphocyte (%E-RFL) assays were developed by modifying existing procedures. PBL count assay variation was reduced by using replicate electronic white blood cell (WBC) counting and 2,000 WBC differentials. The %E-RFL assay modifications reduced variation and include the use of: (a) a capillary buffy coat isolation procedure that recovered more than 85% of the PBL without the use of chemical separation media, (b) centrifugation temperatures of 20--22 degrees C, and (c) toluidine blue staining that allowed enumeration of %E-RFL and not E-rosette forming cells, by exclusion of nonlymphoid cells. Day-to-day variation of PBL count and %E-RFL was defined by making serial determinations over a period of months. The data indicate that healthy adults maintain PBL counts and %E-RFL within narrow ranges that are specific for each individual.