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SPAM583
- Thread starter AlphaLung
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BlazerBoy
New member
Study design
Three techniques were assessed for their ability to identify AM. First, the standard FC techniques for identifying AM, i.e. intrinsic forward scatter and autofluorescence. Second, the ability of the LSC to capture and contour around AM nuclei, stained with the nucleic acid dye propidium iodide (PI). Thirdly, capture of whole AM when the cytoplasm was stained with a fluorescently conjugated monoclonal antibody to CD68, which reacts selectively with a specific 110-kDa cytoplasmic glycoprotein present in mononuclear phagocytes. Slides were processed as outlined below and where data was obtainable for any of the above techniques the "sensitivity" and "positive predictive value" of the technique was tested as well as the repeatability of the analysis.
Methods
[size=-1]Bronchoalveolar lavage[/size]
All children received a general anaesthetic. At induction, either an inhalational agent or propofol was used according to the preference of the anaesthetist. Immediately after intubation and within 5 min of induction of anaesthesia, children underwent nonbronchoscopic BAL as previously described 16. Briefly, the child's head was turned to the left and a suction catheter (six French Gauge (FG) <1 yrs, eight FG 1–5 yrs and 10 FG >5 yrs) was wedged in the lower airway. A total 1 mL·kg body weight–1 (maximum 20 mL) of sterile saline at room temperature was instilled and immediately aspirated into a suction trap using gentle negative pressure (100–200 mmHg). The procedure was repeated twice. Pooled bronchoalveolar lavage fluid (BALF) was stored on ice prior to processing.
[size=-1]Processing of samples[/size]
BALF was immediately centrifuged at 250[font=arial,helvetica]x[/font]g for 5 min at 4°C. After removal of the supernatant the cell pellet was resuspended in phosphate-buffered saline (PBS) (Sigma-Aldrich Company, Dorset, UK). This procedure was repeated twice and the cells resuspended in PBS at a final concentration of 1[font=arial,helvetica]x[/font]105 cells·mL–1. A total 1[font=arial,helvetica]x[/font]104 unfixed cells were cytocentrifuged at 800 rpm for 5 min onto several glass microscope slides (Shandon Scientific, Runcorn, Cheshire, UK). Slides were then air-dried for 1 h before fixing in chloroform:acetone mix at a ratio of 1:1 for 10 min. After fixation, slides were air dried for a further hour prior to storing for up to 18 months at –20°C until immunoassayed. The BALF cell differential count was determined prior to fixation by staining a slide with Diff-Quik (Dade Behring, Deerfield, IL, USA) and counting >300 leukocytes. Slides were brought up to room temperature and washed in PBS for 10 min. Slides were processed for each child in one of three ways.
[size=-1]Capture using autofluorescence and forward scatter[/size]
Slides were left unstained in order to assess contouring around forward scatter and autofluorescent properties of the cell.
[size=-1]Capture using nuclear staining[/size]
To one slide, PI (Sigma-Aldrich) was added at a concentration of 20 µg·mL–1 with Ribonuclease H (Sigma-Aldrich) at a concentration of 15 µg·mL–1 for 30 min at room temperature in the dark. Preliminary work using concentrations of PI ranging from 20–100 µg·mL–1 achieved the same intensity of staining.
[size=-1]Capture using a pan-macrophage monoclonal antibody[/size]
Two slides from each child were processed using standard immunostaining techniques after blocking with goat serum for 25 min. One slide was incubated with a mouse monoclonal antibody to human CD68 (BD Pharmingen, San Diego, CA, USA) in goat serum at a final concentration of 10 µg·mL–1, and one with an approriate isotypic control (mouse immunoglobulin (Ig)G2b) (Serotec Ltd, Oxford, UK) in goat serum for 1 h. Both slides were then washed twice in PBS before incubation with the secondary antibody Alexa Fluor 633 goat anti-mouse IgG2b (Molecular Probes, Leiden, the Netherlands) in goat serum at a final concentration of 13 µg·mL–1 at room temperature for 30 min in the dark.
[size=-1]Laser scanning cytometry[/size]
All slides were mounted in Immunomount (Shandon Scientific) and processed on the LSC within 48 h. Slides were scanned on the LSC (CompuCyte, Cambridge, MA, USA) and cells detected by contouring around one of the following: intrinsic cellular autofluorescence, forward scatter, nucleic acid PI and cytoplasmic CD68. For each slide at the start of scanning the threshold photomultiplier tube (PMT) sensitivity was adjusted such that the maximum detection of stained areas was achieved with a minimum detection of background staining. Once adjusted, the slide was scanned and data collected. A minimum of 1,000 cells per slide were analysed by the LSC using the [font=arial,helvetica]x[/font]20 objective.
During scanning, images of the area being scanned were randomly selected and displayed on the data display window and inspected visually. This window shows pixelated areas and the presence of software-generated contours. This enables an assessment of whether contours have appropriately identified single AM, or have inappropriately characterised debris or clumps of cells as individual AM with a single contour, or even failed to detect and hence contour around AM. For each child a minimum of three data display windows were examined. AM were identified by the operator according to either the nuclear or cell size and contour shape and the following parameters were calculated: 1) the proportion of pixelated clusters that were morphologically single AM and were contoured around (sensitivity), and 2) the proportion of contours associated with pixelated clusters that were morphologically single AM (positive predictive value).
For AM stained with CD68, a qualitative assessment of specificity and negative predictive value for the sample was made by comparing the amount of pixelated clusters contoured around on the isotypic control slide compared to the CD68 stained slide. The variation in repeated measurements on the same subject (repeatability) was determined in AM from three randomly selected children. Two sets of slides obtained from a single sampling procedure and stored in an identical manner were immunostained with CD68 on two separate occasions and then scanned on the same LSC by the same observer each within 48 h of immunostaining.
After imaging, a presumed population of single AM were selected according to homogeneity and contour area from the software produced graphs of contour area versus pixel intensity. These "cells" were then displayed using the "relocation" feature on the software. For each sample, once scanning was recommenced, the first 36 contoured pixelated clusters that were within the selected area were visualised and the positive predictive value of the test for selecting single AM was calculated. This determined whether selecting a homogeneous population of cells with similar contour size would exclude debris or clumps of cells that had been inappropriately contoured around and increase the positive predictive value of the test for selecting single cells.
To determine whether it was possible to obtain fluorescent data within the contours, the integrated fluorescence was determined for each of the fluorochromes used. The integrated fluorescence for the active sensor is the background-corrected sum of the pixel values within the data contour (equivalent to FC-integrated fluorescence). The integrated fluorescence was corrected for contour area.
Confocal microscopy
To independently assess the specificity of CD68 fluorescent staining of AM and the degree of background staining, CD68-immunostained AM were imaged using the laser scanning confocal microscope (Bio-Rad Radiance 2000, Hertfordshire, UK). Positively stained slides were compared to the isotypic control and a qualitative assessment of the specificity and negative predictive value of CD68 immunostaining was estimated. Statistics
The data are presented as medians and interquartile ranges (IQR). Sensitivities and positive predictive values were calculated according to the methods by Altman and Bland 17, 18. In order to assess the repeatability of the test, the Bland-Altman repeatability coefficient was calculated. This defines the range in which 95% of measurements for sensitivity and positive predictive value would be expected to fall if the test was repeated on the same subject under the same conditions of measurement and is the probability of technical error. It was calculated by plotting the difference between the test and retest results for the three children studied and then calculating the mean and sd of the differences. The repeatability coefficient was calculated as twice the sd of the difference of the repeated measures 19.
Three techniques were assessed for their ability to identify AM. First, the standard FC techniques for identifying AM, i.e. intrinsic forward scatter and autofluorescence. Second, the ability of the LSC to capture and contour around AM nuclei, stained with the nucleic acid dye propidium iodide (PI). Thirdly, capture of whole AM when the cytoplasm was stained with a fluorescently conjugated monoclonal antibody to CD68, which reacts selectively with a specific 110-kDa cytoplasmic glycoprotein present in mononuclear phagocytes. Slides were processed as outlined below and where data was obtainable for any of the above techniques the "sensitivity" and "positive predictive value" of the technique was tested as well as the repeatability of the analysis.
Methods
[size=-1]Bronchoalveolar lavage[/size]
All children received a general anaesthetic. At induction, either an inhalational agent or propofol was used according to the preference of the anaesthetist. Immediately after intubation and within 5 min of induction of anaesthesia, children underwent nonbronchoscopic BAL as previously described 16. Briefly, the child's head was turned to the left and a suction catheter (six French Gauge (FG) <1 yrs, eight FG 1–5 yrs and 10 FG >5 yrs) was wedged in the lower airway. A total 1 mL·kg body weight–1 (maximum 20 mL) of sterile saline at room temperature was instilled and immediately aspirated into a suction trap using gentle negative pressure (100–200 mmHg). The procedure was repeated twice. Pooled bronchoalveolar lavage fluid (BALF) was stored on ice prior to processing.
[size=-1]Processing of samples[/size]
BALF was immediately centrifuged at 250[font=arial,helvetica]x[/font]g for 5 min at 4°C. After removal of the supernatant the cell pellet was resuspended in phosphate-buffered saline (PBS) (Sigma-Aldrich Company, Dorset, UK). This procedure was repeated twice and the cells resuspended in PBS at a final concentration of 1[font=arial,helvetica]x[/font]105 cells·mL–1. A total 1[font=arial,helvetica]x[/font]104 unfixed cells were cytocentrifuged at 800 rpm for 5 min onto several glass microscope slides (Shandon Scientific, Runcorn, Cheshire, UK). Slides were then air-dried for 1 h before fixing in chloroform:acetone mix at a ratio of 1:1 for 10 min. After fixation, slides were air dried for a further hour prior to storing for up to 18 months at –20°C until immunoassayed. The BALF cell differential count was determined prior to fixation by staining a slide with Diff-Quik (Dade Behring, Deerfield, IL, USA) and counting >300 leukocytes. Slides were brought up to room temperature and washed in PBS for 10 min. Slides were processed for each child in one of three ways.
[size=-1]Capture using autofluorescence and forward scatter[/size]
Slides were left unstained in order to assess contouring around forward scatter and autofluorescent properties of the cell.
[size=-1]Capture using nuclear staining[/size]
To one slide, PI (Sigma-Aldrich) was added at a concentration of 20 µg·mL–1 with Ribonuclease H (Sigma-Aldrich) at a concentration of 15 µg·mL–1 for 30 min at room temperature in the dark. Preliminary work using concentrations of PI ranging from 20–100 µg·mL–1 achieved the same intensity of staining.
[size=-1]Capture using a pan-macrophage monoclonal antibody[/size]
Two slides from each child were processed using standard immunostaining techniques after blocking with goat serum for 25 min. One slide was incubated with a mouse monoclonal antibody to human CD68 (BD Pharmingen, San Diego, CA, USA) in goat serum at a final concentration of 10 µg·mL–1, and one with an approriate isotypic control (mouse immunoglobulin (Ig)G2b) (Serotec Ltd, Oxford, UK) in goat serum for 1 h. Both slides were then washed twice in PBS before incubation with the secondary antibody Alexa Fluor 633 goat anti-mouse IgG2b (Molecular Probes, Leiden, the Netherlands) in goat serum at a final concentration of 13 µg·mL–1 at room temperature for 30 min in the dark.
[size=-1]Laser scanning cytometry[/size]
All slides were mounted in Immunomount (Shandon Scientific) and processed on the LSC within 48 h. Slides were scanned on the LSC (CompuCyte, Cambridge, MA, USA) and cells detected by contouring around one of the following: intrinsic cellular autofluorescence, forward scatter, nucleic acid PI and cytoplasmic CD68. For each slide at the start of scanning the threshold photomultiplier tube (PMT) sensitivity was adjusted such that the maximum detection of stained areas was achieved with a minimum detection of background staining. Once adjusted, the slide was scanned and data collected. A minimum of 1,000 cells per slide were analysed by the LSC using the [font=arial,helvetica]x[/font]20 objective.
During scanning, images of the area being scanned were randomly selected and displayed on the data display window and inspected visually. This window shows pixelated areas and the presence of software-generated contours. This enables an assessment of whether contours have appropriately identified single AM, or have inappropriately characterised debris or clumps of cells as individual AM with a single contour, or even failed to detect and hence contour around AM. For each child a minimum of three data display windows were examined. AM were identified by the operator according to either the nuclear or cell size and contour shape and the following parameters were calculated: 1) the proportion of pixelated clusters that were morphologically single AM and were contoured around (sensitivity), and 2) the proportion of contours associated with pixelated clusters that were morphologically single AM (positive predictive value).
For AM stained with CD68, a qualitative assessment of specificity and negative predictive value for the sample was made by comparing the amount of pixelated clusters contoured around on the isotypic control slide compared to the CD68 stained slide. The variation in repeated measurements on the same subject (repeatability) was determined in AM from three randomly selected children. Two sets of slides obtained from a single sampling procedure and stored in an identical manner were immunostained with CD68 on two separate occasions and then scanned on the same LSC by the same observer each within 48 h of immunostaining.
After imaging, a presumed population of single AM were selected according to homogeneity and contour area from the software produced graphs of contour area versus pixel intensity. These "cells" were then displayed using the "relocation" feature on the software. For each sample, once scanning was recommenced, the first 36 contoured pixelated clusters that were within the selected area were visualised and the positive predictive value of the test for selecting single AM was calculated. This determined whether selecting a homogeneous population of cells with similar contour size would exclude debris or clumps of cells that had been inappropriately contoured around and increase the positive predictive value of the test for selecting single cells.
To determine whether it was possible to obtain fluorescent data within the contours, the integrated fluorescence was determined for each of the fluorochromes used. The integrated fluorescence for the active sensor is the background-corrected sum of the pixel values within the data contour (equivalent to FC-integrated fluorescence). The integrated fluorescence was corrected for contour area.
Confocal microscopy
To independently assess the specificity of CD68 fluorescent staining of AM and the degree of background staining, CD68-immunostained AM were imaged using the laser scanning confocal microscope (Bio-Rad Radiance 2000, Hertfordshire, UK). Positively stained slides were compared to the isotypic control and a qualitative assessment of the specificity and negative predictive value of CD68 immunostaining was estimated. Statistics
The data are presented as medians and interquartile ranges (IQR). Sensitivities and positive predictive values were calculated according to the methods by Altman and Bland 17, 18. In order to assess the repeatability of the test, the Bland-Altman repeatability coefficient was calculated. This defines the range in which 95% of measurements for sensitivity and positive predictive value would be expected to fall if the test was repeated on the same subject under the same conditions of measurement and is the probability of technical error. It was calculated by plotting the difference between the test and retest results for the three children studied and then calculating the mean and sd of the differences. The repeatability coefficient was calculated as twice the sd of the difference of the repeated measures 19.
BlazerBoy
New member
This study reports, for the first time, a way of studying stored paediatric AM using the LSC. Contouring around the macrophage-specific cellular antigen CD68 is a sensitive and specific technique for detecting AM, with excellent test retest repeatability for sensitivity. The advantages of this technique are three-fold. First, the AM is completely contoured around allowing specific cellular or surface antigens to be further studied without the need for a software generated peripheral contour, as the authors have demonstrated with CD68. Second, AM can be easily discriminated from other cell populations within the BALF sample. Third, most cells are contoured around singly; those that are contoured around as clumps can be easily excluded according to contour size.
The two previously reported methods for detecting cells using the LSC involve either staining the nucleus with a nucleic acid dye 11–13 or contouring according to scatter properties 14. Nuclear contouring has been successfully employed for cells with single nuclear profiles and can discriminate different cell populations in peripheral blood according to the nuclear size and density of chromatin condensation 12. The nucleus of the AM however is a less attractive cell localising and contouring parameter since AM are large cells (10–20 µm diameter) 20 with large eccentrically placed nuclei that are often horseshoe shaped and frequently appear bi- or even multi-nucleated when flattened on cytospins 21. When the nucleus is used as the triggering parameter, the software often identifies binucleated cells as two separate AM. Furthermore, AM have a high propensity to homotypically aggregate 22 and contours from neighbouring nuclei frequently overlap (fig. 2
). An additional problem with nuclear staining is that the chromatin within the nucleus is not densely packed 23 and its density is dependent on cell age and nuclear size; hence, staining with PI is weaker at standard concentrations and contouring less consistent. As a result of the above problems, the authors' attempts to use the AM nucleus as the contouring parameter were not successful.
Adult AM (especially those obtained from smokers) are intensely autofluorescent and this, combined with their intrinsic forward scatter properties, have allowed them to be easily identified by FC 7. AM sampled from young rats are much less autofluorescent than those sampled from older animals 24 and this may explain the weak autofluorescent properties of the paediatric samples. In addition, the process of storing cells first by applying centrifugal force, then fixing and freezing cells undoubtedly causes leaching of natural fluorochromes from cells 25, as well as altering AM morphology 26 and thus their forward scatter properties. For these reasons AM cannot be easily identified by these "intrinsic" properties. Light scatter has successfully been used as the LSC contouring parameter on live lymphocyte preparations 14 adherent to the slide, and the possibility for exploiting the forward scatter and autofluorescent properties of live AM requires further study before these can be excluded as useful contouring parameters.
What are the limitations of the present study? First, the accepted gold standard for determining the presence and intensity of cellular and surface antigens is currently FC. Ideally any new technique should be compared against this gold standard. Unfortunately, a direct comparison with FC was not made as many of the samples, especially from infants, contained insufficient cells to process them using both techniques. Second, although LSC and confocal microscopy confirm the absence of background staining and hence no contouring around cells which were not AM, it is possible that some AM were CD68 negative and not detected. Reports suggest that this accounts for
3% of AM 6.
To obtain data on other immune receptors, dual staining with CD68 and the receptor of interest will be necessary. One potential disadvantage of LSC, and indeed FC when dual staining, is the problem of "spill-over" of one fluorochrome into a neighbouring PMT that is being used to detect a different fluorochrome (i.e. "spectral overlap"). For a fluorochrome to be a useful contouring parameter there has to be minimal spill-over into neighbouring PMT so that the presence and intensity of the antigens under test can be studied. In order to avoid spectral overlap the authors suggest using a fluorochrome such as Alexa Fluor 633 as the contouring parameter since it is excited by the HeNe laser at 633 nm and emits at a wavelength of 670 nm far beyond the detection parameters of the other PMTs. These dyes also have the benefits of low fluorescence quenching upon binding to proteins and hence can be stored for up to 48 h with minimal bleaching. However, one disadvantage of Alexa 633 is that it is not visible to the naked eye and hence an assessment of staining using standard immunofluorescence is not possible. Therefore, the authors also immunostained some slides with R-phycoerythrin, which is excited by the argon laser and detected by the orange PMT and can be viewed with the naked eye. This detects fluorescent spectra close to that of the green and far-red PMT. The degree of compensation required for spill-over into these neighbouring PMT (compensation factor) was tested. For orange spill-over into green the compensation factor was 0.03% and for orange into far red it was 0.02% (data not shown) thus making this an acceptable contouring fluorochrome. To conclude, laser scanning cytometry is suited to the study of paediatric alveolar macrophages since it uses very small numbers of cells (
5,000 per slide) and can be applied to stored slides. By using a pan-macrophage marker as the triggering parameter the authors were able to overcome the limitations of laser scanning cytometry and could both accurately identify alveolar macrophages and obtain fluorescence data from these cells. It is speculated that laser scanning cytometry will be an important methodology in the study of developmental changes in the immune profile of the human alveolar macrophages.
The two previously reported methods for detecting cells using the LSC involve either staining the nucleus with a nucleic acid dye 11–13 or contouring according to scatter properties 14. Nuclear contouring has been successfully employed for cells with single nuclear profiles and can discriminate different cell populations in peripheral blood according to the nuclear size and density of chromatin condensation 12. The nucleus of the AM however is a less attractive cell localising and contouring parameter since AM are large cells (10–20 µm diameter) 20 with large eccentrically placed nuclei that are often horseshoe shaped and frequently appear bi- or even multi-nucleated when flattened on cytospins 21. When the nucleus is used as the triggering parameter, the software often identifies binucleated cells as two separate AM. Furthermore, AM have a high propensity to homotypically aggregate 22 and contours from neighbouring nuclei frequently overlap (fig. 2
). An additional problem with nuclear staining is that the chromatin within the nucleus is not densely packed 23 and its density is dependent on cell age and nuclear size; hence, staining with PI is weaker at standard concentrations and contouring less consistent. As a result of the above problems, the authors' attempts to use the AM nucleus as the contouring parameter were not successful. Adult AM (especially those obtained from smokers) are intensely autofluorescent and this, combined with their intrinsic forward scatter properties, have allowed them to be easily identified by FC 7. AM sampled from young rats are much less autofluorescent than those sampled from older animals 24 and this may explain the weak autofluorescent properties of the paediatric samples. In addition, the process of storing cells first by applying centrifugal force, then fixing and freezing cells undoubtedly causes leaching of natural fluorochromes from cells 25, as well as altering AM morphology 26 and thus their forward scatter properties. For these reasons AM cannot be easily identified by these "intrinsic" properties. Light scatter has successfully been used as the LSC contouring parameter on live lymphocyte preparations 14 adherent to the slide, and the possibility for exploiting the forward scatter and autofluorescent properties of live AM requires further study before these can be excluded as useful contouring parameters.
What are the limitations of the present study? First, the accepted gold standard for determining the presence and intensity of cellular and surface antigens is currently FC. Ideally any new technique should be compared against this gold standard. Unfortunately, a direct comparison with FC was not made as many of the samples, especially from infants, contained insufficient cells to process them using both techniques. Second, although LSC and confocal microscopy confirm the absence of background staining and hence no contouring around cells which were not AM, it is possible that some AM were CD68 negative and not detected. Reports suggest that this accounts for
To obtain data on other immune receptors, dual staining with CD68 and the receptor of interest will be necessary. One potential disadvantage of LSC, and indeed FC when dual staining, is the problem of "spill-over" of one fluorochrome into a neighbouring PMT that is being used to detect a different fluorochrome (i.e. "spectral overlap"). For a fluorochrome to be a useful contouring parameter there has to be minimal spill-over into neighbouring PMT so that the presence and intensity of the antigens under test can be studied. In order to avoid spectral overlap the authors suggest using a fluorochrome such as Alexa Fluor 633 as the contouring parameter since it is excited by the HeNe laser at 633 nm and emits at a wavelength of 670 nm far beyond the detection parameters of the other PMTs. These dyes also have the benefits of low fluorescence quenching upon binding to proteins and hence can be stored for up to 48 h with minimal bleaching. However, one disadvantage of Alexa 633 is that it is not visible to the naked eye and hence an assessment of staining using standard immunofluorescence is not possible. Therefore, the authors also immunostained some slides with R-phycoerythrin, which is excited by the argon laser and detected by the orange PMT and can be viewed with the naked eye. This detects fluorescent spectra close to that of the green and far-red PMT. The degree of compensation required for spill-over into these neighbouring PMT (compensation factor) was tested. For orange spill-over into green the compensation factor was 0.03% and for orange into far red it was 0.02% (data not shown) thus making this an acceptable contouring fluorochrome. To conclude, laser scanning cytometry is suited to the study of paediatric alveolar macrophages since it uses very small numbers of cells (
BlazerBoy
New member
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, ultimately leading to destruction of joint cartilage and bone.1 Joint destruction in RA is perpetuated by an aggressive, invasive pannus tissue, a vascular and fibrous granulation tissue consisting of macrophages (M
), synovial fibroblasts (SFB), T lymphocytes, and B lymphocytes/plasma cells. Immunotyping with specific antibodies has an important role in identifying and localising each cell type in tissue specimens by immunohistochemistry (IHC) and in characterising the purity of cell populations isolated from the synovial membrane (SM). T cells are easily and unequivocally detected by their expression of CD3, a molecule co-expressed with the T cell receptor in an obligatory fashion. For FB, several markers have been described—for example, prolyl 4-hydroxylase,2 CD90/Thy-1,3 or CD55.4 For immunotyping of M
, monoclonal antibodies (mAbs) directed against different epitopes of CD68 are widely used markers.5
M
play a critical role in the course of RA owing to their abundance in the inflamed SM and at the cartilage-pannus junction and to their activation status (as shown by overexpression of major histocompatibility complex class II (MHC II) molecules, proinflammatory cytokines, and matrix degrading enzymes).6 Also, articular destruction correlates with the density of synovial M
(as assessed by CD68 staining)7; this density, in turn, is reduced after successful antirheumatic treatment.8–10
CD68 (the human homologue of mouse macrosialin) is a heavily glycosylated, 110 kDa membrane protein; its transcription is regulated by a promoter containing multiple GGAA sequences instead of a TATA box or an INR sequence.11 CD68 is closely related to the family of lysosomal associated, mucin-like membrane proteins (lamps). Although CD68 is predominantly located in lysosomal membranes, a small fraction is also found on the cell surface.12,13 Although the biological function of CD68 has not been fully defined, CD68 serves as a scavenger receptor for oxidised low densitylipoprotein14 and may also be involved in cell-cell interactions.12
Although prolyl 4-hydroxylase is widely used as a FB marker and CD14/CD68 are employed as M
markers, their specificity for the respective cell type remains to be established. Prolyl 4-hydroxylase, a tetramer consisting of two
and ß subunits, shares the ß subunit with disulphide isomerase, a multifunctional polypeptide expressed in many different cell types.15 Therefore, the specificity of the selected anti-prolyl 4-hydroxylase mAbs has to be carefully checked. On the other hand, the monocyte/M
marker CD14 is also found on gingival FB isolated from inflamed gingiva,16 and CD68 is expressed in retinal epithelial cells,17 osteoblasts,18 and FB-like cells from the bone marrow.19 This study therefore aimed at further defining the usefulness of CD68 as a reliable monocyte/M
marker for IHC and flow cytometry (FACS). CD68 expression was compared with the expression of other M
and FB markers by IHC in sections of the SM (single and double labelling), as well as in isolated SFB, skin FB, gingival FB, and monocytic cell lines by FACS (surface and intracellular staining).
M
CD68 (the human homologue of mouse macrosialin) is a heavily glycosylated, 110 kDa membrane protein; its transcription is regulated by a promoter containing multiple GGAA sequences instead of a TATA box or an INR sequence.11 CD68 is closely related to the family of lysosomal associated, mucin-like membrane proteins (lamps). Although CD68 is predominantly located in lysosomal membranes, a small fraction is also found on the cell surface.12,13 Although the biological function of CD68 has not been fully defined, CD68 serves as a scavenger receptor for oxidised low densitylipoprotein14 and may also be involved in cell-cell interactions.12
Although prolyl 4-hydroxylase is widely used as a FB marker and CD14/CD68 are employed as M
BlazerBoy
New member
For double labelling after completion of single staining, sections were first incubated with 20% human serum/PBS for 20 minutes. Then the specific antibodies, diluted in PBS/10% horse serum, were added for 45 minutes. For detection, sections were incubated for 45 minutes with an alkaline phosphatase coupled goat antimouse antibody (Dako, Hamburg, Germany). The alkaline phosphatase was disclosed using a solution containing FAST Blue BB (1.0 mg/ml), and naphthol AS-MX phosphate (0.3 mg/ml; Sigma) in 0.2 M Tris-HCl, pH 8.4. Endogenous alkaline phosphatase was blocked with 0.24 mg/ml levamisole (Sigma). For isotype controls, no positive staining was seen in single staining or double labelling experiments.
[font=arial,verdana,helvetica]Evaluation of tissue sections after immunohistochemistry[/font]
The percentage of positively stained cells was scored semiquantitatively by two observers (EK, RWK) in a "blinded" manner. Single-positive cells were identified by unequivocal brown (peroxidase) or blue (alkaline phosphatase) staining, whereas double-positive cells showed a mixture of both colours (dirty brown-blue colour).
[font=arial,verdana,helvetica]Tissue digestion and cell culture[/font]
Synovial cells were obtained as previously published.21 Briefly, SFB were isolated by trypsin/collagenase digestion (Roche, Mannheim, Germany), short term in vitro adherence (7 days) to remove non-adherent cells, and negative isolation using magnetobead coupled anti-CD14 mAbs (Dynal, Hamburg, Germany). This procedure resulted in high enrichment of SFB with a contamination of <2% leucocytes or endothelial cells.
[font=arial,verdana,helvetica]Isolation of skin fibroblasts[/font]
Primary-culture skin FB were prepared as published previously.21
[font=arial,verdana,helvetica]Isolation of gingival fibroblasts[/font]
Samples of gingival tissue were obtained during removal of granulation tissue from chronically inflamed dental roots. Gingival tissue samples were finely minced with scissors and digested for 30 minutes at 37°C in 10 ml PBS containing 0.25% trypsin (Gibco). After trypsin treatment, tissue samples were digested in 10 ml 0.1% collagenase P (Boehringer Mannheim) in Dulbecco’s modified Eagle’s medium (DMEM; Gibco)/10% fetal calf serum (FCS; Gibco) for 2 hours at 37°C. The tissue was dispersed by repeated pipetting and the cells were collected by centrifugation and washed with serum-free DMEM. Thereafter, the cells were cultured in DMEM/10% FCS, 12.5 mM HEPES, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (2.5 mg/ml; all Gibco). The medium was changed every 2–3 days.
[font=arial,verdana,helvetica]THP-1 and U937 cell lines[/font]
The human monocytic cell lines THP-1 and U937 (both from the German collection of micro-organisms and cell cultures (DMSZ), Braunschweig, Germany) were grown in suspension culture in RPMI 1640 medium containing 10% FCS (both Gibco) without antibiotics.
[font=arial,verdana,helvetica]Flow cytometry[/font]
FACS analysis of negatively purified RA SFB, OA SFB, and skin FB was performed to characterise their purity and their CD68 expression. Table 2
indicates the concentrations of mAbs used. For immunofluorescence labelling, 2[font=arial,helvetica]x[/font]105 cells were suspended in 100 µl PBS/1% FCS/0.02% NaN3. The cells were incubated with unconjugated primary mAbs, followed by incubation with FITC labelled secondary mAb (each for 30 minutes at 4°C). After every step, cells were washed three times with PBS/1% FCS/0.02% NaN3. Specificity of staining was confirmed using equal concentrations of isotype matched control mAbs.
For intracellular staining, cells were washed twice with PBS/1% FCS/0.02% NaN3 and fixed for 10 minutes at 4°C in 4% PFA (Fluka, Deisenhofen, Germany). After washing twice with PBS/1% FCS/0.02% NaN3, the pellet was resuspended in permeabilisation buffer (PBS/1% FCS/0.02% NaN3, and 0.5% saponin; Serva, Heidelberg, Germany) and incubated for 10 minutes at RT. Unlabelled primary mAbs were added at saturating concentrations and detected with a secondary FITC labelled goat antimouse antibody (Dako), both for 45 minutes at 4°C in permeabilisation buffer.
Analyses were performed on a FACS-Calibur using the software Cell Quest (Becton Dickinson, San Jose, CA, USA). Forward and side scatter gates were set to include all viable cells. A gate was set to exclude 99% of the cells stained with control immunoglobulins. To determine the percentage of THP-1 and U937 cells positive for CD68 (surface and intracellular staining), a gate was placed at the intercept of the curves obtained with specific mAbs and control immunoglobulins; the percentage of cells stained with control immunoglobulin was then subtracted from the percentage of cells stained with the specific mAb.
[font=arial,verdana,helvetica]Statistical analysis[/font]
The non-parametric Mann-Whitney U test was applied for data analyses using the software SPSS 10.0 (SPSS Inc; Chicago, IL, USA). Significant differences were accepted for p
[font=arial,verdana,helvetica]Evaluation of tissue sections after immunohistochemistry[/font]
The percentage of positively stained cells was scored semiquantitatively by two observers (EK, RWK) in a "blinded" manner. Single-positive cells were identified by unequivocal brown (peroxidase) or blue (alkaline phosphatase) staining, whereas double-positive cells showed a mixture of both colours (dirty brown-blue colour).
[font=arial,verdana,helvetica]Tissue digestion and cell culture[/font]
Synovial cells were obtained as previously published.21 Briefly, SFB were isolated by trypsin/collagenase digestion (Roche, Mannheim, Germany), short term in vitro adherence (7 days) to remove non-adherent cells, and negative isolation using magnetobead coupled anti-CD14 mAbs (Dynal, Hamburg, Germany). This procedure resulted in high enrichment of SFB with a contamination of <2% leucocytes or endothelial cells.
[font=arial,verdana,helvetica]Isolation of skin fibroblasts[/font]
Primary-culture skin FB were prepared as published previously.21
[font=arial,verdana,helvetica]Isolation of gingival fibroblasts[/font]
Samples of gingival tissue were obtained during removal of granulation tissue from chronically inflamed dental roots. Gingival tissue samples were finely minced with scissors and digested for 30 minutes at 37°C in 10 ml PBS containing 0.25% trypsin (Gibco). After trypsin treatment, tissue samples were digested in 10 ml 0.1% collagenase P (Boehringer Mannheim) in Dulbecco’s modified Eagle’s medium (DMEM; Gibco)/10% fetal calf serum (FCS; Gibco) for 2 hours at 37°C. The tissue was dispersed by repeated pipetting and the cells were collected by centrifugation and washed with serum-free DMEM. Thereafter, the cells were cultured in DMEM/10% FCS, 12.5 mM HEPES, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (2.5 mg/ml; all Gibco). The medium was changed every 2–3 days.
[font=arial,verdana,helvetica]THP-1 and U937 cell lines[/font]
The human monocytic cell lines THP-1 and U937 (both from the German collection of micro-organisms and cell cultures (DMSZ), Braunschweig, Germany) were grown in suspension culture in RPMI 1640 medium containing 10% FCS (both Gibco) without antibiotics.
[font=arial,verdana,helvetica]Flow cytometry[/font]
FACS analysis of negatively purified RA SFB, OA SFB, and skin FB was performed to characterise their purity and their CD68 expression. Table 2
indicates the concentrations of mAbs used. For immunofluorescence labelling, 2[font=arial,helvetica]x[/font]105 cells were suspended in 100 µl PBS/1% FCS/0.02% NaN3. The cells were incubated with unconjugated primary mAbs, followed by incubation with FITC labelled secondary mAb (each for 30 minutes at 4°C). After every step, cells were washed three times with PBS/1% FCS/0.02% NaN3. Specificity of staining was confirmed using equal concentrations of isotype matched control mAbs. For intracellular staining, cells were washed twice with PBS/1% FCS/0.02% NaN3 and fixed for 10 minutes at 4°C in 4% PFA (Fluka, Deisenhofen, Germany). After washing twice with PBS/1% FCS/0.02% NaN3, the pellet was resuspended in permeabilisation buffer (PBS/1% FCS/0.02% NaN3, and 0.5% saponin; Serva, Heidelberg, Germany) and incubated for 10 minutes at RT. Unlabelled primary mAbs were added at saturating concentrations and detected with a secondary FITC labelled goat antimouse antibody (Dako), both for 45 minutes at 4°C in permeabilisation buffer.
Analyses were performed on a FACS-Calibur using the software Cell Quest (Becton Dickinson, San Jose, CA, USA). Forward and side scatter gates were set to include all viable cells. A gate was set to exclude 99% of the cells stained with control immunoglobulins. To determine the percentage of THP-1 and U937 cells positive for CD68 (surface and intracellular staining), a gate was placed at the intercept of the curves obtained with specific mAbs and control immunoglobulins; the percentage of cells stained with control immunoglobulin was then subtracted from the percentage of cells stained with the specific mAb.
[font=arial,verdana,helvetica]Statistical analysis[/font]
The non-parametric Mann-Whitney U test was applied for data analyses using the software SPSS 10.0 (SPSS Inc; Chicago, IL, USA). Significant differences were accepted for p
BlazerBoy
New member
CHOPPING 'EM OFF AGAIN!
Yep.., here we go again! I was called into my project manager's office last Friday at around 9:40 a.m. and was layed off at the sound of a lame excuse! I was frustrated, angry, sad, and yet relieved all at the same time. As I mentioned in the past, I was diagnosed with a spinal injury requiring future surgery as a result of the type of work I do. Whenever all this went down, I asked my pr-mangr. if it was going to be a problem with my staying employed. His reply was absolutely not because I was a model employee whose services would always be on high demand with the company and that they would work with me by whatever means necessary. BULLSHIT!!!
I suppose his response as well as the best benefits package I'd ever had with any company, were the sole reasons I remained an employee despite the constant dread and anguish of reporting for work there everyday. Many times I worked on things which had absolutely nothing to do with my trade but was still expected to be top notch at it. With the new divisional supervisor in place, almost everything he would place a bid on had nothing to do with electricity. His trade was boilers and steam propulsion plants and was straight out of the NAVY and had never been in charge at a civilian type of company. I've done both to which I can verify they are not to be dealt with in the same manner...., a fact which he has yet to realize!
Anyway, pr-mgr. (Mr. "C") started out by saying, "Well you know a couple of weeks back we sent you for the medical evaluation and with those results as they stand I really can't use you on any jobs, so as much as I hate to do it I-....,"
(It was here where I abruptly cut him off!)
Yep.., here we go again! I was called into my project manager's office last Friday at around 9:40 a.m. and was layed off at the sound of a lame excuse! I was frustrated, angry, sad, and yet relieved all at the same time. As I mentioned in the past, I was diagnosed with a spinal injury requiring future surgery as a result of the type of work I do. Whenever all this went down, I asked my pr-mangr. if it was going to be a problem with my staying employed. His reply was absolutely not because I was a model employee whose services would always be on high demand with the company and that they would work with me by whatever means necessary. BULLSHIT!!!
I suppose his response as well as the best benefits package I'd ever had with any company, were the sole reasons I remained an employee despite the constant dread and anguish of reporting for work there everyday. Many times I worked on things which had absolutely nothing to do with my trade but was still expected to be top notch at it. With the new divisional supervisor in place, almost everything he would place a bid on had nothing to do with electricity. His trade was boilers and steam propulsion plants and was straight out of the NAVY and had never been in charge at a civilian type of company. I've done both to which I can verify they are not to be dealt with in the same manner...., a fact which he has yet to realize!
Anyway, pr-mgr. (Mr. "C") started out by saying, "Well you know a couple of weeks back we sent you for the medical evaluation and with those results as they stand I really can't use you on any jobs, so as much as I hate to do it I-....,"
(It was here where I abruptly cut him off!)
BlazerBoy
New member
Me- "Let me just stop you right there...., so you're letting me go..., after I busted my ass for you..., and put up with all the promises I was told yet never recieved.., worked out of my field, put my life at risk over and over, made you look good by making this division alot of damn money, designed and built things that people in positions higher than me couldn't...., and you don't feel I meet your F****** criteria?!! Screw you!..., you no good son of a bitch.., and by the way thanks for one helluva Christmas present!"
C- "But what choice did I have.., I have to do what's best for the company and don't get me wrong you're a good guy and damn good at your job and I hate to loose you and-.."
Me- "If you hated to loose me so damn much, then WHY did you request the medical evaluation in the first place?!! If you really felt bad about it you would've given more than just two hours notice. A descent boss who had a trustworthy employee would've been man enough to have the balls to give enough notice to allow time to look for another job. Seeing as how your plan was to shitcan me I really don't see how it could've been a problem!"
"Besides, the results said FIT FOR WORK WITH MINOR LIMITATIONS.., and was a TEMPORARY situation at best! The limitations only said to give a required break after 15 minutes of physically demanding activities and no lifting over 50 lbs unassisted until my back had healed. This has nothing to do with my future surgery! Everybody except myself and one other person in the division smokes..., and as a federal law are given mandatory smoke breaks. So, in reality I really wouldn't be doing anything different than I am now because whenver they took breaks the rest of us got a break as well. The 50 pound rule is already company policy to begin with."
"Not only that, NOONE at this company ever works more than 15 to 30 minutes without taking a break anyhow and you and I both know it! The real reason is so YOU can have a reason on a piece of paper to "justify" yourself so you don't have to try and accept any blame for any of it. I understand with no electrical jobs to charge my time to that most of my electrical work gets paid to me from company overhead which cuts into YOUR divisional budget. I also understand some Texas corporate executive who doesn't know me veiws me as a liability instead of an asset. I can also recall expressing my concerns about it to you over a year ago to make it clear to corporate as to reason I charged to overhead via facilities electrical supervisor to prevent this very action!! So don't sit here and try and convince me you had no f***ing part in today's actions because if anybody did it's YOU!!"
He started trying to justify his excuse by trying to show me the paperwork, etc. to which I just told him to save his breath because I didn't care to hear anymore. I turned and told him "I'll be in the trailer collecting my tools, paperwork and personal belongings when you're ready to check me out..., otherwise I don't recommend you saying anything else to me EVER! As a matter of fact you can kiss my sorry ass!!"
C- "But what choice did I have.., I have to do what's best for the company and don't get me wrong you're a good guy and damn good at your job and I hate to loose you and-.."
Me- "If you hated to loose me so damn much, then WHY did you request the medical evaluation in the first place?!! If you really felt bad about it you would've given more than just two hours notice. A descent boss who had a trustworthy employee would've been man enough to have the balls to give enough notice to allow time to look for another job. Seeing as how your plan was to shitcan me I really don't see how it could've been a problem!"
"Besides, the results said FIT FOR WORK WITH MINOR LIMITATIONS.., and was a TEMPORARY situation at best! The limitations only said to give a required break after 15 minutes of physically demanding activities and no lifting over 50 lbs unassisted until my back had healed. This has nothing to do with my future surgery! Everybody except myself and one other person in the division smokes..., and as a federal law are given mandatory smoke breaks. So, in reality I really wouldn't be doing anything different than I am now because whenver they took breaks the rest of us got a break as well. The 50 pound rule is already company policy to begin with."
"Not only that, NOONE at this company ever works more than 15 to 30 minutes without taking a break anyhow and you and I both know it! The real reason is so YOU can have a reason on a piece of paper to "justify" yourself so you don't have to try and accept any blame for any of it. I understand with no electrical jobs to charge my time to that most of my electrical work gets paid to me from company overhead which cuts into YOUR divisional budget. I also understand some Texas corporate executive who doesn't know me veiws me as a liability instead of an asset. I can also recall expressing my concerns about it to you over a year ago to make it clear to corporate as to reason I charged to overhead via facilities electrical supervisor to prevent this very action!! So don't sit here and try and convince me you had no f***ing part in today's actions because if anybody did it's YOU!!"
He started trying to justify his excuse by trying to show me the paperwork, etc. to which I just told him to save his breath because I didn't care to hear anymore. I turned and told him "I'll be in the trailer collecting my tools, paperwork and personal belongings when you're ready to check me out..., otherwise I don't recommend you saying anything else to me EVER! As a matter of fact you can kiss my sorry ass!!"
BlazerBoy
New member
As a company rule, the supervisor (mr. "E") had to accompany me until I was done at the front office and out the gate. I had an entire workshop set up there so, needless to say my truck was fully loaded whenever I left. Before I left however Mr.C made at least 5 more attempts to explain his position to which I was totally unreceptive. His first attempt he said, "Now just a damn minute! I did everything possible to-...,"
"I told you to back off and leave me alone and that's what I meant!", I said. As I hate to do, I lost my temper and smashed my cup of coffee against the side of the truck as a result splattering Mr.C with the contents. OH WELL..., he was warned! I wallked into the trailer and began gathering my stuff. Shortly thereafter, "C" makes a second attempt! I didn't even respond. Instead I almost knocked him down with a box full of tools as I walked past him to my truck bed. He then had the ghaul to ask me to reassemble a special type of power panel I had almost finished refurbishing before I was wisked in for the kill. Me- "You mean that panel sitting there on the floor?" C- "Yes, that's the one. I'd really appreciate it since noboby else has any experience on panels that old. Besides, we ARE paying you a full 8 hours!" Me- "Sure Mr. C..., I'll fix it right up for you!!...,"
WHAM!!!!!! As my right foot bounces the metal panel off 3 walls of the trailer and pieces go flying everywhere!
Me- "That ought to fix it right up for you..., oops..., I almost forgot the rest of the parts!" (they too, bounced in all directions!) It was at this point he showed his TRUE colors---"I could fire you if that's what you want!" "No you can't "C", I've already signed the lay-off notice and sent it up to the office! In order to fire me you'd have to first re-hire me..., and let me tell you.., after the way you just screwed me over I wouldn't work for you if my life depended on it so f*** off!"
C- "Well what amI supposed to do about the panel?"
Me- "I have some suggestions, but I'm sure you won't like any of them..., I suppose you'll have to hire an electrician so you can screw him over like you did me.., I really couldn't tell you and dont care one way or the other!"
He finally walked out of the trailer coming back a third time with the next in charge to which I took the "written excuse" and tore it up and handed it to Mr. "C's" boss. I said, "As I told "C"..., save it because I could care less what the lame excuse is because I know the real truth and it's not on that paper. Now if you'll excuse me I have to finish getting my things."
Then "C" says, "We didn't have to give you 8 hours!"
"I told you to back off and leave me alone and that's what I meant!", I said. As I hate to do, I lost my temper and smashed my cup of coffee against the side of the truck as a result splattering Mr.C with the contents. OH WELL..., he was warned! I wallked into the trailer and began gathering my stuff. Shortly thereafter, "C" makes a second attempt! I didn't even respond. Instead I almost knocked him down with a box full of tools as I walked past him to my truck bed. He then had the ghaul to ask me to reassemble a special type of power panel I had almost finished refurbishing before I was wisked in for the kill. Me- "You mean that panel sitting there on the floor?" C- "Yes, that's the one. I'd really appreciate it since noboby else has any experience on panels that old. Besides, we ARE paying you a full 8 hours!" Me- "Sure Mr. C..., I'll fix it right up for you!!...,"
WHAM!!!!!! As my right foot bounces the metal panel off 3 walls of the trailer and pieces go flying everywhere!
Me- "That ought to fix it right up for you..., oops..., I almost forgot the rest of the parts!" (they too, bounced in all directions!) It was at this point he showed his TRUE colors---"I could fire you if that's what you want!" "No you can't "C", I've already signed the lay-off notice and sent it up to the office! In order to fire me you'd have to first re-hire me..., and let me tell you.., after the way you just screwed me over I wouldn't work for you if my life depended on it so f*** off!"
C- "Well what amI supposed to do about the panel?"
Me- "I have some suggestions, but I'm sure you won't like any of them..., I suppose you'll have to hire an electrician so you can screw him over like you did me.., I really couldn't tell you and dont care one way or the other!"
He finally walked out of the trailer coming back a third time with the next in charge to which I took the "written excuse" and tore it up and handed it to Mr. "C's" boss. I said, "As I told "C"..., save it because I could care less what the lame excuse is because I know the real truth and it's not on that paper. Now if you'll excuse me I have to finish getting my things."
Then "C" says, "We didn't have to give you 8 hours!"
BlazerBoy
New member
Me- "According to the company handbook..., you most certainly do in a lay-off situation!"
At that point they both walked away but not before Mr."B" (mr. C's boss) came over and shook my hand and told me it REALLY WASN"T his doing and wished me luck in my future saying I'd be missed by many people there. (true/false-who knows-who cares! But as mr. C's boss, I find it VERY hard to believe he didn't have a hand in it as well). I went around and said my goodbyes as I got along well with just about everyone there. The supply manager, a very nice middle aged lady, started crying and got up from her desk and came and hugged me goodbye.., cursing my boss in a low muttered voice. (all the girls in the company made it a point to hug me goodbye so that was OK (some huggable, some not! ha! ha!). (DISCLAIMER: no guys were hugged in the making of this post!)
The machine shop supervisor actually started crying as he shook my hand so long. He turned in the direction of "C" and said, "If that bastard can't see how much you've done for this company and how good of an asset you are he can go f*** himself before he'll ever get any favors from me again! You're certainly going to be missed by alot of people. You ever need anything you just let me know. To hell with "C!" "C" tried to have his say another time in passing to which I showed him the international sign and did an about face in the opposite direction!
Mr. E, on the other hand, surprised me. He actually called several places and put in "words" for a possible management popsition for me. He apologized for our differences stating he now understood my anamocity toward Mr. C and as a result feared for his job as well. He said, "I always figured I would go before you..., just shows you can't trust anybody these days." Sincere or not, I think he meant well and even though he had a small part in the medical evaluation taking place, I don't feel like he considered the fallout of his actions beforehand.
Basically, I pulled a muscle in my back as a result of having no help and a heavy work load. My back hurt on a Wednesday night, I called in on Thursday morning to see if I could just come in and catch up on paperwork w/out any strenuous work, but was denied. Mr. "E" TOLD ME to take the day and make sure I had a doctor's note (there's the navy talking!). So, I went and the doctor made me take Friday as well....., the day I was to lead a job for Mr. E putting him in a bad situation with the ship's captain and chief engineer. But, as I said..., it was his decision for me to go to the doctor, not mine.
When I came in that Monday Mr. C said, "I'm concerned about your ability to do this kind of work anymore. I'm going to request that the company send you for a medical evaluation to determine where we stand on the issue." More was said, but that's the short of it basically. So as far as I see it was Mr. C who actually put the ball into play. Mr. E was witnessed by my co-workers as complaining to C that he "couldn't depend on me" when he needed to. Two weeks after that Monday conversation was when the axe fell.
First of all, I'm an electrician, not a boiler technician, machinist, welder, sheet metal mechanic, painter, errand runner, or a supply person..., but an ELECTRICIAN! So technically, I shouldn't have been the one to be his "flunky" to start with. After his "outburst" was when C decided to do the med-eval.
Mr. E also apologized to me for that, however I never even brought it up to him that I knew he sad it! He said, "I had no idea Mr. C was going to give you the axe because I was blowing off some steam.., and for that I feel responsible and I really want to personally apologize to you as it wasn't my intension for you to loose your job.., man I'm really sorry."
Me- "Don't worry about it E..., I hated this place anyway so maybe it's probably a good thing. Trust me..., I've got enough high level experience I can be selective about where I want to work. Maybe all I needed was a little push..., so try not to beat yourself up over it. Now that you see C's true colors though I'd be more carefull in the future. No hard feelings, OK?"
E- "Sure thing, you've got it."
Finally, I had all my stuff done and was outside the front office getting ready for the humiliating journey out the gate never to be allowed re-admittance (I have the combination though-HA! HA! doh!) I turned over all of my many keys as 20 to 30 people came out of the offices to see me off.., shaking my hand, some hugged or gave the high five signal. Low and behold "C" gives it one more try and walks over to my truck and starts thumbing through the contents of the bed as if he thinks he's going to find something I'm not "supposed to have."
At that point they both walked away but not before Mr."B" (mr. C's boss) came over and shook my hand and told me it REALLY WASN"T his doing and wished me luck in my future saying I'd be missed by many people there. (true/false-who knows-who cares! But as mr. C's boss, I find it VERY hard to believe he didn't have a hand in it as well). I went around and said my goodbyes as I got along well with just about everyone there. The supply manager, a very nice middle aged lady, started crying and got up from her desk and came and hugged me goodbye.., cursing my boss in a low muttered voice. (all the girls in the company made it a point to hug me goodbye so that was OK (some huggable, some not! ha! ha!). (DISCLAIMER: no guys were hugged in the making of this post!)
The machine shop supervisor actually started crying as he shook my hand so long. He turned in the direction of "C" and said, "If that bastard can't see how much you've done for this company and how good of an asset you are he can go f*** himself before he'll ever get any favors from me again! You're certainly going to be missed by alot of people. You ever need anything you just let me know. To hell with "C!" "C" tried to have his say another time in passing to which I showed him the international sign and did an about face in the opposite direction!
Mr. E, on the other hand, surprised me. He actually called several places and put in "words" for a possible management popsition for me. He apologized for our differences stating he now understood my anamocity toward Mr. C and as a result feared for his job as well. He said, "I always figured I would go before you..., just shows you can't trust anybody these days." Sincere or not, I think he meant well and even though he had a small part in the medical evaluation taking place, I don't feel like he considered the fallout of his actions beforehand.
Basically, I pulled a muscle in my back as a result of having no help and a heavy work load. My back hurt on a Wednesday night, I called in on Thursday morning to see if I could just come in and catch up on paperwork w/out any strenuous work, but was denied. Mr. "E" TOLD ME to take the day and make sure I had a doctor's note (there's the navy talking!). So, I went and the doctor made me take Friday as well....., the day I was to lead a job for Mr. E putting him in a bad situation with the ship's captain and chief engineer. But, as I said..., it was his decision for me to go to the doctor, not mine.
When I came in that Monday Mr. C said, "I'm concerned about your ability to do this kind of work anymore. I'm going to request that the company send you for a medical evaluation to determine where we stand on the issue." More was said, but that's the short of it basically. So as far as I see it was Mr. C who actually put the ball into play. Mr. E was witnessed by my co-workers as complaining to C that he "couldn't depend on me" when he needed to. Two weeks after that Monday conversation was when the axe fell.
First of all, I'm an electrician, not a boiler technician, machinist, welder, sheet metal mechanic, painter, errand runner, or a supply person..., but an ELECTRICIAN! So technically, I shouldn't have been the one to be his "flunky" to start with. After his "outburst" was when C decided to do the med-eval.
Mr. E also apologized to me for that, however I never even brought it up to him that I knew he sad it! He said, "I had no idea Mr. C was going to give you the axe because I was blowing off some steam.., and for that I feel responsible and I really want to personally apologize to you as it wasn't my intension for you to loose your job.., man I'm really sorry."
Me- "Don't worry about it E..., I hated this place anyway so maybe it's probably a good thing. Trust me..., I've got enough high level experience I can be selective about where I want to work. Maybe all I needed was a little push..., so try not to beat yourself up over it. Now that you see C's true colors though I'd be more carefull in the future. No hard feelings, OK?"
E- "Sure thing, you've got it."
Finally, I had all my stuff done and was outside the front office getting ready for the humiliating journey out the gate never to be allowed re-admittance (I have the combination though-HA! HA! doh!) I turned over all of my many keys as 20 to 30 people came out of the offices to see me off.., shaking my hand, some hugged or gave the high five signal. Low and behold "C" gives it one more try and walks over to my truck and starts thumbing through the contents of the bed as if he thinks he's going to find something I'm not "supposed to have."
BlazerBoy
New member
Now I try most of the time to watch my language as my parents didn't teach or allow me to talk that way in thier presense or otherwise. But I've journeyed long and far and have crossed alot of real turds and associated a-holes in my travels. Sometimes, a reality check or two suddenly asserts the need for a little bit of color in the good old English language so as to "get the point across!"Me- "What the hell are you looking for..., because it's not in there so get the f*** away from my truck!"
C- "I suggest you don't rock the boat..., you might need a reference.., I'd hate to have to paint the wrong picture!"
Me- "You see all these people here "C"..., I don't need you or your damn reference so shove it! I have all the references I'll ever need. Now I'm not going to ask you again to get the f*** away from my truck....., NOW DAMMIT!"
I started walking toward him and he finally backed away. I waved to all the descent folks, and gave "the international gesture" to "C". I peeled out as I was both frustrated and humiliated throwing dust and gravel in his direction! THIS was my payment for all my hard work and efforts...., THIS as it has been other times in the past..., THIS now a scar reaching deep within me once again making me doubt trusting anyone..., THIS now having taken it's toll upon me in a way I can't quite put my finger on..., but most of all THIS having physically and mentally ruined my spirit of wanting to do anything for anyone with the velocity of effort thrusted forward in the past. The people who seem to launch forward in life always seem to be the ones noone can tolerate. So..., maybe the next job I get I'll just have to be more of cold hearted asshole like all the jerks I've had the pleasure of working for. Maybe only then will I be able to trust someone..., the only one I can in THIS is me.
C- "I suggest you don't rock the boat..., you might need a reference.., I'd hate to have to paint the wrong picture!"
Me- "You see all these people here "C"..., I don't need you or your damn reference so shove it! I have all the references I'll ever need. Now I'm not going to ask you again to get the f*** away from my truck....., NOW DAMMIT!"
I started walking toward him and he finally backed away. I waved to all the descent folks, and gave "the international gesture" to "C". I peeled out as I was both frustrated and humiliated throwing dust and gravel in his direction! THIS was my payment for all my hard work and efforts...., THIS as it has been other times in the past..., THIS now a scar reaching deep within me once again making me doubt trusting anyone..., THIS now having taken it's toll upon me in a way I can't quite put my finger on..., but most of all THIS having physically and mentally ruined my spirit of wanting to do anything for anyone with the velocity of effort thrusted forward in the past. The people who seem to launch forward in life always seem to be the ones noone can tolerate. So..., maybe the next job I get I'll just have to be more of cold hearted asshole like all the jerks I've had the pleasure of working for. Maybe only then will I be able to trust someone..., the only one I can in THIS is me.
