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Zaha was born in the Ivory Coast but is a current England under-21 international. He plays as a forward for Crystal Palace and he's been there since 2009. Zaha is known as one of the best England starlets of our time and has huge potential. Many believe Zaha plays a bit like Red Bull star Thierry Henry but he won't live up to Henry's legacy unless he makes the move to the Premier League.
But which club should he join?
Manchester United are endowed with attacking talent and a new arrival probably isn't needed at the moment. With van Persie, Rooney, Hernandez, Welbeck, Young, Valencia and Nani United probably aren't going to punt for Zaha. That's not to mention United starlets such as Keane, Bebe and Henriquez. He wouldn't want to join a club where he's not going to get much playing time either.
Arsenal are also blessed with attacking talent. However Zaha is better than most of their options. All Arsenal fans would definitely welcome the arrival of Zaha and he would certainly fit in their squad. But if Zaha has the option to join Arsenal or United and he's given assurances of game time, he'll most definitely join the Red Devils.
Spurs have become a top six club in the past few years. They're now battling for some of the best talent in Europe and can beat better teams to certain players. This is shown in their capture of Jan Vertonghen over Arsenal. Zaha would fit in Tottenham's squad perfectly and would work well as a rotational player with Lennon, Defoe or Bale.
Liverpool have dropped in reputation over the past few years but they can still battle for some of the best players. They are also looking for vital young players to put on their wings. Zaha would fit nicely on the opposite flank to Sterling and could rotate perfectly. Although Liverpool may not finish in the best positions anymore, they still have the second best reputation in the Premier League.
Wherever Zaha goes, he'll surely flourish and become a senior England player in Roy Hodgson's set-up. That is if Hodgson still has the England job by then.


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|Gregory Campbell 'putting personal agendas aside' after healthy scratch
|04.06.15 at 12:54 pm ET
WILMINGTON — Long after his teammates had showered, fulfilled their media responsibilities, grabbed food in the team lounge and headed home, Gregory Campbell was still on the ice at Ristuccia Arena by himself.
A healthy scratch for the first time in his Bruins career Saturday, Campbell didn't feel ready to leave following Monday's approximately 40-minute practice (one for which he took the ice early). The image of him shooting pucks alone for approximately 55 minutes was fitting of his 2014-15 season: He wants to be better, but his spot in the lineup is questionable at best.
"I like being out here," Campbell said as he got off the ice, adding: "I wanted to do some things.
"It's uncharted," he said of not playing. "I've never experience it before, but at this stage of the game, it's about putting personal agendas aside and it's about honoring the team and the decisions the coaches make. It is what it is. It's about honoring the team."
Saturday's benching was perhaps overdue given the way Campbell and his fellow fourth-liners have fared this season.
After coming to Boston and centering the best fourth line in the league with Brad Marchand and Shawn Thornton, Campbell's eventual line with Daniel Paille and Thornton routinely put opponents' bottom-sixers on their heels, most notably helping change the momentum of Game 7 of the 2011 Stanley Cup finals after Vancouver took it to Boston in the early shifts.
Yet those vintage Merlot Line days, which really lasted until Campbell broke his leg in the 2013 Eastern Conference finals, are long over. Thornton is gone, Paille has been a healthy scratch in Boston's last six games, while Campbell at long last sat over the weekend.
That's where the aforementioned personal agendas may come in. A free agent at season's end who seems unlikely to return, Campbell has given a lot to this team. It can't be easy to go from a fan favorite to a scapegoat in what's been a trying season for both him and the Bruins.
|Daniel Paille uses 'positive attitude' to regain his mojo: 'When they go in, it seems you can almost do anything'
|03.08.15 at 5:20 pm ET
It's hard to tell who's more relieved at the turnaround of Daniel Paille. Both player and coach Claude Julien have to reason to be elated with the recent production of the fourth line left wing.
Since being benched for the final two games of the five-game road trip, Paille has been on fire. His two goals Sunday were the difference in a 5-3 win over the Red Wings at TD Garden. He has four goals in the six games since, including Sunday's short-handed marker.
"Sometimes when you sit out you get to reflect on what you can do better," Paille said after Sunday's offensive display. "For me, I definitely kept a positive attitude about it. Like l've said before, Claude was great with me about it. Coming back, kind of a play like you've got nothing to lose. Just keep working. If you keep working, good things will come out of it, and so far, that's what's been going right now. So it's a huge boost I guess."
"I'm sure it helped him in a good way, not necessarily as a wake-up call, more than watching the game and missing it," Julien added. "At the same time, I think there's no doubt the trade deadline's over, guys know they're here, there's a lot of players that have picked up their game I think since then. Whether it's a combination of that or combination of where we are in the standings and wanting to make sure we get ourselves into a playoff spot and doing whatever it takes, could be a lot of different things. It's nice to see a lot of those players really bring their game up a notch."
Paille was the butt of many jokes about the Bruins' lack of finish around the net. He's had the last laugh since being re-inserted into the lineup. Paille went 36 games without a goal and scored in each of his first two games back. On Sunday, he matched that total in just three shots.
"When they go in, it seems that you can almost do anything, so a big part of the game is mental and sometimes they're not going to go in and it's just staying focused on the right things that we're doing out there and for me of course it's been a frustrating time for the most part of the season, but the main point is to stay with it and having the support through the whole team here is definitely a huge boost for all of us," Paille said. Read the rest of this entry »
|Gregory Campbell not cleared for contact, eager to see what role is when he returns
|03.03.15 at 12:33 pm ET
WILMINGTON — It's not clear when or where Gregory Campbell will next play in the Bruins' lineup.
Campbell has yet to be cleared for contact, but he said he is "coming along" in his return from an upper-body injury. The veteran center, who said he got hurt in the first period of last Sunday's game against the Blackhawks, practiced with the Bruins Tuesday. He said he feels "progressively better" but is not sure whether he will be ready to play Thursday.
When Campbell is ready to return, his spot in the lineup won't be as clear as it was before. In addition to adding to the top nine with Brett Connolly, the Bruins picked up fourth-line center Max Talbot, who could potentially push Campbell out of the lineup.
"I think we added two good players to the team, so wherever they're going to play, they're going to help our team," Campbell said. "They're players that bring different things, but in Max, he's won before and he's a good role player. In Brett, he's a skilled guy, so there's a lot of potential.
"I don't really know how it's going to shake out, but I know they're going to help us."
Chris Kelly has been centering the fourth line while Campbell has been out, and the line has, perhaps not surprisingly, been better. There was talk of potentially moving to Campbell to wing this season, which never happened, but it could now given the many options that Claude Julien for his fourth line.
"Maybe, yeah. Maybe. Whatever [Julien] wants," he said. "Whatever he decides is going to benefit the team the most. It's good to have some options. It's good to have some centermen that are able to take draws and some interchangeable parts. Whatever's going to happen, I'm sure it's going to be best for the team."
|With Bruins' fourth line constantly changing, Craig Cunningham appears a safe bet to stay
|01.13.15 at 12:39 pm ET
The Bruins, injuries and a suspension aside, pretty much had one fourth line since midway through the 2010-11 season: Gregory Campbell between Daniel Paille and Shawn Thornton. Just over halfway through this season, they've had 15 different fourth lines by our count (see below).
As Scott McLaughlin recently pointed out, these fourth lines have mostly been ineffective. Yet with Simon Gagne recently informing the Bruins he will not be returning this season, the team can at least have a better idea of who will make up their bottom trio. Specifically, you can count on Craig Cunningham to stick on Gregory Campbell's right wing.
In a season that has seen the Bruins struggle to get any traction with their bottom trio(s), playing Cunningham has looked to be the right idea all along. He moves relatively well, brings an element of grit, and, when given the minutes, isn't to throw the puck on net even if it isn't the prettiest chance.
"Any time the goalie kicks out a rebound, no one really knows where it's going and it's a 50-50 puck," Cunningham said Tuesday.
Claude Julien acknowledged Tuesday that the Bruins are giving Cunningham a real shot to become a permanent member of the line, but he doesn't feel he's won anything yet.
"No. I don't think so,' Cunningham said. "For me, I'm still trying to make an impact and show that I can play at this level every night. You live day-to-day up here. For me, you never want to get comfortable. I think every day is kind of like a tryout. They're watching you and you've got to bring something every single day."
Daniel Paille moved up from the line last week as he was promoted to a new-look top-six trio with Patrice Bergeron and Milan Lucic. Chris Kelly and Jordan Caron have manned his left wing spot since, but that could continue to change.
As long as Campbell is in Boston, he figures to be on that fourth line, and it seems Cunningham stands a better chance than any of Boston's youngsters of sticking on the right side. The other wing may continue to be a revolving door, but in the meantime, Campbell and Cunningham, linemates for eight games entering Tuesday night, should seize the opportunity to prove they're the men for the job as the Bruins look to re-establish the puck-possessing, energy-providing fourth line the Bruins once had.
"I guess I was lucky — we were lucky, in a sense — to have that stability for the last four years," Campbell said. "You kind of take it for granted, because if you look around, it doesn't happen often where a line's together that long where you can create that chemistry and whatever. The thing is, our role doesn't change. We just have to take pride in that. The guys that have been on the line are more than capable of doing the job and they're good players. We'll make it work. Sometimes it takes a little bit longer."
These aren't groups that were just used for shifts at a time. From tracking the team's lineups throughout the season, these are the lines that the B's have used for a game or games at a time. Some have changed from mid-game line shakeups, but that has rarely been the case.
Paille-Cunningham-Robins: Games 1, 2, 3
Paille-Spooner-Caron: Game 4
Paille-Spooner-Gagne: Game 5
Paille-Campbell-Gagne: Games 6, 8, 9, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 25, 26, 27, 28
Paille-Campbell-Fraser: Game 7, 34, 35, 36
Paille-Campbell-Smith: Game 12
Kelly-Campbell-Paille: Game 15
Lindblad-Khokhlachev-Fraser: Game 22
Caron-Khokhlachev-Pastrnak: Game 23
Smith-Kelly-Griffith: Game 24
Paille-Campbell-Griffith: Games 29, 30, 31
Paille-Campbell-Cunningham: Game 32, 33, 38, 39, 40, 41
Lindblad-Cunningham-Griffith: Game 37
Caron-Campbell-Cunningham: Game 42
Kelly-Campbell-Cunningham: Game 43
|Why December 23 is a meaningful day for the Bruins
|12.23.14 at 1:02 pm ET
It's been a while since the Bruins approached the Christmas break as a fringe playoff team. The last time it happened, however, they won the Stanley Cup.
Dec. 23, 2010 was a critical day in that ultimately successful season. The Bruins, coming off a postseason collapse against the Flyers the previous spring, were struggling.
Offseason acquisition Nathan Horton, who was in the midst of what would be a nine-game slump with no goals and one assist, was looking like a very talented non-factor who appeared to be bringing Milan Lucic down with him.
The team was going through the motions and it was taking them nowhere. It led to the Bruins losing four of five games, punctuated by a troubling no-show in a 3-0 shutout loss to the Ducks on Garden ice. Claude Julien, who historically is a set-it-and-forget-it guy with his lines, pulled Horton off the top line and replaced him with Blake Wheeler in that game.
After that 3-0 loss, the eighth-place Bruins had two days off before they would host the Thrashers in their final game before the holiday break. Those two days were the height of "Fire Claude Mania."
In his weekly interview with CBS radio, President Cam Neely was asked if they were going to fire the coach. Neely said the Bruins weren't, but did say, "I can understand why the fans are frustrated and may be calling for a coaching change."
Dennis Seidenberg doesn't remember too many specifics about the mood of the team at that point, only saying Tuesday that "it was really dead."
Then, on Dec. 23, the Bruins came out and absolutely ran over the Thrashers. Shawn Thornton fought Eric Boulton off the faceoff and spent the next five minutes in the penalty box devising a plan to score two goals in the game. Patrice Bergeron had a shorty. Michael Ryder had a power play goal. Lucic sucker-punched Freddy Meyer and somehow didn't get suspended.
Ference fought. Horton fought. Marc freaking Savard fought. The game was an explosion of emotions and every bit the coming out party that the team had forgotten to have earlier in the season.
"I think that was definitely a defining game for us," Brad Marchand said Tuesday. "We turned it on and really didn't look back."
|Pierre McGuire: 'Hated' Bruins' schedule to open season
|10.23.14 at 2:12 pm ET
NBC Sports NHL analyst Pierre McGuire made his weekly appearance Thursday on Middays with MFB in advance of Thursday night's Bruins matchup against the Islanders. To hear the interview, go to the MFB audio on demand page.
The Bruins got off to a slow start to the season — going 1-3 in their first four games, but McGuire said some of that was because of the way their schedule was constructed — playing those four games in a six-day span.
"I hated the way their season started, not the way they were playing, but the way the schedule was set up for them," he said. "I think I talked to you guys about it, they almost had eight days where they had three games in four nights. That's crazy stuff. Then, when you compound that with a [Monday] afternoon game at home after a Saturday night loss, that's really hard. I'm not making excuses for them, but they are starting to settle into what team they want to be."
He also noted the team was coming off of trading veteran defenseman Johnny Boychuk to the Islanders, just prior to the regular season.
"I think they were all a little stunned about Johnny Boychuk being traded to the Islanders because he was an extremely popular guy on their team," said McGuire. "They started the season without Gregory Campbell, he's a very important guy on that team. I think they are feeling their way through, but they are starting heat up. I liked their game the other night against San Jose, especially the last parts of that game."
The Bruins and Boychuk will be reunited Thursday night as the Islanders will be at TD Garden. Boychuk has had a strong start to the season, posting two goals and four assists over the first six games.
|How Bruins overcame uncharacteristically bad nights from Patrice Bergeron, Zdeno Chara
|10.21.14 at 11:51 pm ET
Usually the Patrice Bergeron line and Zdeno Chara-Dougie Hamilton pairing are the Bruins' constants. They're the guys who are going to create offensive-zone possessions and not make mistakes.
That wasn't the case on Tuesday. Bergeron was on the ice for all three of the Sharks' goals, linemates Brad Marchand and Reilly Smith joined him for two of them (it is worth noting that Marchand had a nice power-play goal), and Chara was on the ice for two of them as well. Those four and Hamilton were the only Bruins who finished with Corsi-for percentages under 50 percent, meaning they were the only Bruins who were on the ice for more 5-on-5 shot attempts against than shot attempts for.
That would seemingly be a recipe for disaster for the Bruins, especially when you consider that outside of the Carl Soderberg line, the rest of the team had been one giant question mark to this point in the season. David Krejci had looked good since his return, but linemate Milan Lucic was off to a slow start and he still didn't have a set-in-stone right wing. The fourth line had featured several different combinations, and none of them had really done much. And the second and third defense pairings had been inconsistent at best, with Kevan Miller's injury raising even more questions on the back end.
At least for one night, those questions turned into answers. Lucic, Krejci and rookie right wing Seth Griffith factored into four of the Bruins' five goals, with Lucic notching three assists and Griffith scoring his first NHL goal. Two of the goals they were on the ice for — Griffith's and Torey Krug's — came as the direct result of getting bodies to the net. Krejci set a great screen on Krug's, and then Lucic created some net-front havoc that freed up Griffith on his goal.
"I think it definitely was the best game that we've played so far this season," Lucic said. "You saw we were hungry in the O-zone and hungry getting pucks to the net. We made some smart decisions in some important areas and it just seems like things are starting to head in the right direction."
The fourth line of Daniel Paille, Gregory Campbell and Simon Gagne was a positive possession line that even created some chances against the Sharks' top two lines. They scored what proved to be the game-winner midway through the third when Paille won the puck along the boards and threw a shot on net that Campbell tipped in for his first goal of the season.
Campbell and Paille were also big on the penalty kill, especially late in the game when Bergeron went to the box for a four-minute double minor. Until Krejci's empty-netter to seal the win, Campbell had the biggest play on that kill when he blocked a Joe Thornton shot that came off a Chara turnover.
"We've got to be a responsible, reliable line, and Claude [Julien] has to trust us to put us in those situations," Campbell said. "With hard work comes trust, and if we're playing our game and we're in on the forecheck and creating chances and bringing energy to the lineup, then he usually has confidence in us."
As for the bottom two defense pairings, the only glaring error was a bad miscommunication between Krug and Dennis Seidenberg that led to a goal, but as Julien pointed out after the game, Bergeron's line was just as much at fault, as Smith had failed to clear the zone and Bergeron and Marchand had gotten caught up ice.
Outside of that, the Seidenberg-Krug and Matt Bartkowski-Adam McQuaid pairings played well. Krug's goal and two assists obviously stand out, but let's not overlook the fact that Seidenberg had seven shots on goal and 12 shot attempts, and that he and Krug had Corsi-for percentages of 63 and 62 percent, respectively. McQuaid and Bartkowski weren't far behind at 61 and 57 percent, respectively, and McQuaid was also big on that final penalty kill.
Obviously this is just one game. No one should think that all of the Bruins' question marks are gone and that everyone's going to be great from here on. But on a night when the Bruins' best players were uncharacteristically unreliable, it was encouraging to see everyone else step up and show that they can lead the way, too.

subset: commoncrawl


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Irish Coffee: Banged-up Celtics 'hoping everybody is'
|04.27.12 at 1:12 pm ET
He might not be a real doctor, but he must feel like one after all the injuries he's seen in the past four months.
"We've got to be careful with them, even in the playoffs," said Celtics head coach Doc Rivers. "It just doesn't take much, it seems like, right now, for a guy to not be able to play the next night. So we have to be very careful.'
Take Paul Pierce as Exhibit A. The Celtics captain began this lockout-shortened regular season with a bone bruise in his right heel and ended it with a sprained big left toe. The original plan was to rest Pierce's ailing feet for the final two games, but his desire to stay in rhythm won out, so Rivers played him 18 minutes on Tuesday and just 2:18 on Thursday before March's Eastern Conference Player of the Month limped back to the locker room.
"He hurt it," said Rivers, referencing the toe that led the C's to list Pierce as likely unable to return. "That's why he's been sitting. And then he wanted to play. We're not sure if it was the tape, or whatever, because when he wanted to come back, he just kept saying, 'I just needed to get it loose.' So we had a long discussion, because I had no interest in putting him back in, but he really wanted to play a couple minutes just to get up and down the floor."
The Celtics dodged a bullet, as Pierce returned for the final 4:24 of the first half to score seven quick points and ease fans' fears. The same can't be said for Ray Allen, who missed his ninth straight game with bone spurs in his right ankle. On Thursday, Rivers dubbed him probable for Game 1 against the Hawks, but the Celtics announced via Twitter on Friday, "Allen will not practice today and his status for Game 1 on Sunday is still unknown."
Then again, Pierce was improbable five minutes before returning Thursday and Kevin Garnett was probable days before missing the entire 2009 playoff run. Who knows? Rest did wonders for Garnett, Rajon Rondo and Mickael Pietrus, who showed no signs of their respective hip, back and knee injuries in the win over the Bucks.
- Garnett (11 MIN, 4 PTS, 3 AST, 2 REB): "I feel really good. I'm looking forward to the playoffs."
- Pietrus (19.5 MIN, 9 PTS, 4-10 FG, 2 REB): "I was moving pretty well. Just trying to be Mickael Pietrus. I just try to get in, help my team the best way I can, try to get ready for the playoffs."
- Rondo (25 MIN, 15 AST, 4 STL, 0 PTS): "It's fine. It's as good as it's going to get, I think."
Two road games to start their first-round series against the Hawks is the downside of resting everyone down the stretch from Rondo to Greg Stiemsma, who stayed off the plantar fasciitis in his left foot and bone bruise in his right foot for a second straight game but has played through those injuries for at least a month. Should the Celtics have conceded home court advantage by playing a depleted roster in a 97-92 loss to the Hawks a week ago?
"Looking at Kevin, the way he ran [Thursday], I was happy with the choice," said Rivers of the decision. "He looked like his legs were back, and so for me that answered the question."
Count Pietrus among those whose injuries flared up as the result of this season's hectic schedule. Rivers admitted playing him three nights in a row from April 13-15 was a mistake. The good news is the playoff schedule doesn't even feature back-to-backs, let alone three games in three nights.
"I mean, listen, we're banged up," said Rivers. "I'm hoping everybody is."
The Hawks certainly are. They'll likely start the series without their top two centers in Al Horford (torn pectoral) and Zaza Pachulia (strained left foot). Even the Bulls — the C's potential second-round opponent — have battled their share of injuries to Derrick Rose, Luol Deng and Richard Hamilton. Believe it or not, the Celtics might be one of the healthier teams entering the 2012 playoffs, a reflection on the oddities of a post-lockout season.
"I always wished to play for the Celtics," said Pietrus. "Now I want to give them the best shot. I want to play hard for them to give them a chance to win the championship. And for myself, too. We have to stay focused on Celtics basketball, and that's what we've been doing. I think we can do it. It's wide open. It reminds me of '98 when we went into a lockout and San Antonio won. Everything was open."
(Have a question, concern or conception for the next Irish Coffee? Send a message to @brohrbach on Twitter.)
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The M Machine's latest offerings, 'Metropolis Pt 1' and 'Metropolis pt 2' have fast became favourites at Stoney Roads HQ, more so with the announcement of 'Metropolis Remixed' that includes a vastly abstract Proxy remix.
Don't expect any stabby "Raven" vibes here folks but a surprisingly fluid 8-bit melodic take, something you'd catch in one of your favourite arthouse flics or video games.
Proxy doesn't stand alone with a remarkably cool remix with German electronic pioneers Digitalism, dubstep dude Kill The Noise, Robotaki, Shinichi Osawa and more all having a hand in developing the The M Machine varied electronic output.
The full package will drop August 27th on Skrillexs OWSLA label
The M Machine - Metropolis Remixed Tracklisting
1. 'Moon Song' (Digitalism Remix)
2. 'Ghosts In The Machine' (Kill The Noise Remix)
3. 'A King Alone' (Robotaki Remix)
4. 'Data Palace'
5. 'Tiny Anthem' (Shinichi Osawa Remix)
6. 'Schadenfreude' (Tantrum Desire Remix)
7. 'Shadow In The Rose Garden' (Matt Lange Remix)
8. 'Faces' (Proxy Remix)
9. 'Black' (Trifonic Remix)


text: HI folks, I'm new here. Have a 91 Silveraado extended Cab 350 loaded. 240,000 km. I love this truck and am doing some A/C repairs. Replacing the compressor, accumulator, and orifice tube and converting to R134a refrigerant. I have a couple of questions as follows: 1) Where is the orifice tube located? 2) How do I purge the system, before getting it charged? Or do I drive it to a shop after installing the new components and have them do the purging and recharging? 3) How much oil goes into the new accumulator, and how much into the new compressor? I understand that I need to put the new oil in the new compressor and in the new accumulator, I'm ok with that, but I read somewhere that after doing so, I should have the system "purged" I think was the word. Then get it refilled. I do most of my own repairs, but until now have had nothing to do with A/C. Any help would be appreciated. Many thanks, Steve


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By Lisa Suennen
First Posted at Venture Valkyrie on 8/25/2013
No one should be surprised that fertility is a burgeoning area of activity for iPhone and other app providers. I mean, seriously, the love affair that so many people seem to have with their phones would suggest that human/iPhone spawn are not too far down the line. All I can say is thank God that butt dialing can't cause pregnancy. Yet.
But for now, the fertility action on cell phones is still about human-to-human interaction and there have been an increasing array of offerings in this arena. An article earlier this year in MobiHealthNews mentioned several companies offering services that track physical parameters of fertility and provide tips on how to increase chances of pregnancy, other than becoming a high school student. Kindara, Ovuline, DuoFertility and others are out there combining sensors, iPhones and the maternal instinct in the hopes of helping people create the families they will, one-day, curse under their breath at Thanksgiving dinner.
In fact, an entire consumer-focused technology ecosystem is developing around human development. Today I even saw an Indiegogo campaign from the Baby Quest Foundation, an organization that is helping to provide financial assistance to those who cannot afford the high costs of procedures like in vitro fertilization, gestational surrogacy, etc.
But my favorite entry into this race so far is a company I just read about called Glow. Brought to us by one of the PayPal founders, the company is piloting a very comprehensive approach to ensuring that 1+1-3. Here is a description from a recent article in Time:
Glow has "his" and "her" modes and collects information concerning factors such as menstrual cycles, cervical mucus and physical and emotional states. (At the moment, all the data it collects concerns the woman who hopes to get pregnant; the company plans to add factors relating to the male in the equation as well.) The app provides advice about how to maximize the chances of pregnancy, going beyond the technical issues to involve features like the ability to order flowers or make a reservation for a nice dinner.
Now if you, like me, spend any time thinking about consumer engagement in healthcare, you have to love the extra touches here. I am not sure that there is great body of scientific articles behind the idea that flowers and a nice dinner will advance the cause of getting your mate pregnant, but you gotta give these Glow guys an A for effort. While the hard science may fall short, there are centuries of qualitative experience that suggest that flowers, a nice meal and paired wine can improve your chances of having sex, which is, as I understand it, rumored to increase your chances of pregnancy. And this is important, because Glow's CEO states in the Time article that the company's goal is to double its customers chances of getting pregnant. As such, if I were in charge of product development at Glow, I would add a coaching feature to the app that helps the male participant remember to say things like, "Wow, that outfit makes your butt look so small."
The Glow app aims to use "big data" to aggregate information from its users to create an ever-smarter product. As more and more people use Glow and report their medical parameters and pregnancy outcomes, the system will continue to refine its advice to amplify the odds of success. Or so it claims anyway. Allegedly they are going to continually improve this feature by way of passive sensors that "could collect pertinent data without users having to enter it in," which sounds a little kinky if you ask me. Maybe they will do a deal with the earlier version of the FitBit, which accidentally published its customers' sex habits to a publicly readable dashboard. I'm not sure if the risk of streaming your sexual activities onto the cloud is a security nightmare in the making or a monetizable back up revenue stream, but either way it is worthy of further discussion.
And while stopping short of a money-back guarantee, Glow also features a quasi-insurance financial product to help you pay for a back-up plan (e.g., IVF) in case the whole flowers, dinner thing doesn't cut it. According to Mike Huang, Glow's Founder, "We want to be the first insurance company that everybody loves."
Wow, now that is a lofty goal. Doubling the rate of successful pregnancies is one thing, but causing consumers to love an insurance company? I'm not sure there are enough flowers and fancy dinners in the world to check that particular box. They may need to add a jewelry section.
ps-I forgot to mention another new entrant to the space which I shall not name. Taking it to a whole other level, this is a website for genetic testing to make sure you are "genetically compatible to mate" and which helps you be certain that the "man/woman you want to date is not related to you." Perhaps this risk of unintentional incest is a bigger problem than I thought. They suggest that you "…send us your and your mate's DNA and we will do the autosomal analysis and give you either the green signal or the STOP signal." I hope the results come back before you pay for the flowers.


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"Decades after most African countries received their independence, and despite tens of billions of dollars in foreign aid, they're still poor, wartorn places. Maybe there's just something different about them?" That's the basic logic behind James Watson's controversial statement, and it's been echoed by many commenters. At first glance, it doesn't seem so crazy — but it contains a grave flaw, one commonly made by people too optimistic in the power of science to explain, if not save, the world: it ignores history.
In the case of Africa, there are many social, political and economic factors to explain the continent's predicament and the failure of foreign aid. These confound — and make irrelevant — "science of intelligence" explanations. Colonialism and its legacy deserves a large chunk of blame; so does the availability of inexpensive weapons, the actions of evil or misguided rulers who have often been supported by first-world powers, the inequalities of global capitalism and — when it comes to things like violence against women and the spread of HIV/AIDS — the fundamental wrongness of many traditional beliefs.
Explaining what's wrong with Africa isn't something that can be done in a blog post — or, for that matter, hundreds of books and thousands of articles, written by the best and brightest of both Africa and the rest of the world. But it's safe to say there are many factors more important than "intelligence." For a bit of background, I suggest watching Lumumba, doing a bit of research on Robert Mugabe and reading up on the Second Congo War, also called "Africa's World War." These don't explain the whole story, but they underscore how foolish — how unscientific — explanations like Watson's are.
What's more, ignoring social and economic and historical factors in favor of science is self-serving and dangerous. Why think about the legacy of colonialism or the contemporary role of western arms dealers
— why bother to help — when you can explain away Africa's problems as unavoidable biological flaws?
Image: Mobutu Sese Seko, who ruled Zaire — now the Congo — from 1965 to 1997. He was a fabulously corrupt, brutal and anti-democratic leader, but he didn't like the Soviets, so he had our support.
Like this post? Then Digg our James Watson controversy frontpage.
See Also:
- Watson Rediscovers 1940s Attitudes Towards Race
- British Science Museum Wimps Out on Watson
- More on James "Black People Are Stupid" Watson
- The Science and Assumptions Behind Watson's Views on Blacks
- The Dog Ate Our Book Reviews: Watson's Book Given Free Pass by Press
- Watson's Mistake: Favoring Science, Ignoring History
- James Watson Suspended From Lab, but Not For Being a Sexist Hater of Fat People
- Angry IQ Tester: Watson's Critics Are Socialists!
- The Overlap Between Eugenics and a Belief in Superior White Intelligence
- Watson Resigns From Lab: Last Thoughts on a Clouded Legacy


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RCR Premium Water Systems Ltd. Moncton New Brunswick. We are committed to offering you the best water treatment equipment in the industry and service second to none, all this at an affordable price. Our best performing water softener comes with a 20 year warranty. Our philosophy is simple. We will bend over backwards to make sure you have the best experience and full satisfaction. We carry a high quality line of; Water Softeners, Iron and Sulfur Filters, Reverse Osmosis and UV lights. We provide sales and service to residential and commercial systems. So whether you have problems with your home water or commercial concerns we can provide you with options. Contact us today for more information. Call (506) 531-1006 and ask for Mike or email firstname.lastname@example.com Supply and Install OR Cash and Carry
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ED Magedson – Founder
Ripoff Report
Halton Investment CompanyLondon, Select State/Province United Kingdom
Halton Investment Company, Company cops real websites. Pretends to be a legit investment company. London United Kingdom Internet
Does your business have a bad reputation?
Fix it the right way.
Corporate Advocacy Program™
Our company was approached by Halton Investments, which asked for our bona fides and relevant documents, such as a business plan, current financials, etc. Our responding was based on their website, which indicated they had done many multi-million dollar deals with legitimate companies. We sent the docs. They took a few days to "review" and then sent a long-form application, which we were instructed to fill in. As the spelling, grammar and syntax of the accompanying email was less than professional, we did some research... and actually called one of the companies, Insalada, they listed as being under Halton Investment Company. The VP of Insalada said we were not the first to call; that his attys were looking into shutting these scam artists down. We also called the phone number given, which is a London phone, but it doesn't even ring through. We also phoned the "company attys" they listed in Dubai: an Arabic-speaking couple answered, and had no idea what we meant by calling them. NOT A LAW FIRM.
Here is the Halton email of Oct. 29, 2013:
Mr. Gregory J. Tamar
+44 703 596 5107
Managing Director,
Halton Investment Company
We responded to this email, asking them to please explain
We replied to the Halton email, asking them to explain why their website was nearly identical to that of Insalada. WE HAVE NOT RECEIVED ANY REPLY. Meanwhile, my email is constantly being bombarded with all kinds of "instant Loan" offers. Seems this was a fishing expedition, or phishing... as I replied to Halton, and they probably sold my email.
This report was posted on Ripoff Report on 10/30/2013 10:14 AM and is a permanent record located here: http://www.ripoffreport.com/reports/halton-investment-company/london-select-stateprovince/halton-investment-company-company-cops-real-websites-pretends-to-be-a-legit-investment-1095696. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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FjallRaven Keb Gaiter Trousers Regular Sand
Prize-winning Keb Trousers, now with removable gaiters. Technically-advanced trekking trousers with optimised fit and carefully-thought-through details. In warm weather the lower section of the leg can be taken off to turn the trousers into shorts, just like a regular pair of zip-off trousers. The removed section has a drawcord adjustment at the calf that together with the boot hook in the leg ending transforms it into a gaiter that protects legs in brush-filled terrain. The fabric is stretch with G-1000 Eco at the rear, the front of the legs and over the knees for extra durability. Leg endings are also reinforced with extra hardwearing G-1000 HeavyDuty so they resist wear and tear from trekking boots. The rear and knees are preshaped and have extra strong seams. The seams on the inside of the legs have been moved forward to reduce the risk of chafing. A zippered ventilation opening from knee to hip releases excess heat and even the fly has been lengthened for extra ventilation if needed. Two leg pockets with flaps, one of which has an inner mesh pocket for smaller items such as a telephone. Hand pockets are positioned high so contents do not spill out when you sit down. Strap adjustments at the leg endings and, in addition to the hidden boot hook, the leg endings are equipped with eyelets to which laces or metal hooks can be attached – yet another way to fasten the trouser leg and prevent brush or snow from finding its way into boots.
FjallRaven Keb Gaiter Trousers Regular Sand Produkteigenschaften:
- Artikelnummer: F81178R
- Geschlecht: Herren
- Konzept: Trekking
- Gewicht: 640 g in size 48
- Material: G-1000, Stretch
- Außenmaterial: G-1000 Eco: 65% polyester, 35% cotton, G-1000 HeavyDuty: 65% polyester, 35% cotton
- Beinabschlüsse: Fixe Länge
- Messertasche: Nein
- Kartentasche: Ja
- Beintyp: Zip-Off Legs
- Stretcheinsätze: Ja
- Bund: Mittelhoher Bund
- Verstärkungen: Ja
- Sicherheitstasche: Nein
- Vorgeformte Knie: Ja
- Passform: Regular Fit
|Lieferzeit - Werktage
|4 – 7
|Verarbeitete Materialien
|Materials: G-1000, Stretch
Outer materials: G-1000 Eco: 65% polyester, 35% cotton, G-1000 HeavyDuty: 65% polyester, 35% cotton
|Weltweiter Versand mit DHL Premium Air Mail


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Brandon The Vermont Symphony Orchestra's Brass Quintet and Counterpoint Holiday Concert will ring in the season at the Congregational Church in Brandon on Monday, Dec. 17, beginning at 7:00 p.m. The concert is a joint benefit for the Boys and Girls Club of Brandon and the Benjamin White Memorial Scholarship Fund at Otter Valley Union High School.
The VSO Holiday Brass and Counterpoint concert, combining shining brass and brilliant voices, celebrates its tenth anniversary this year, and is a cherished event in Brandon. For this concert Counterpoint's new director, Nathaniel G. Lew, collaborates with the VSO Brass Quintet offering a festive program from medieval to modern, including Leroy Anderson's Christmas Festival, the Canadian Brass's bravura arrangement of The Twelve Days of Christmas, Bizet's March of the Kings, and traditional carols.
Tickets cost $22 for adults, $18 for students and seniors 65 and over, and free for children under age 18 with an adult ticket purchase. Tickets are available at the Brandon and Pittsfield branches of Lake Sunapee Bank and the Boys and Girls Club of Brandon, and by telephone at the VSO office, 800-876-9293, ext. 10. Doors will open to the public for the concert at 6:30 p.m.
For more information, or a complete listing of forthcoming VSO events, please call (800) VSO-9293, ext. 10, or visit www.vso.org.
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Former child star Charlotte Church has vowed never to allow her kids to pursue a career in showbusiness, because she is "worried" about the effects of life in the spotlight.
The singer, who shot to fame at the age of 11, has two children by her ex-boyfriend Gavin Henson - daughter Ruby, four, and three-year-old son Dexter, and she reveals the tots are already showing signs of following her career path - having inherited mum's singing skills. But she's reluctant to put them on a stage.
Church tells talk show host Graham Norton, "They have lovely little voices but it's difficult, I am reticent, really worried."
When Norton then pressed her as to whether the youngsters will be child stars, she replied, "There is no way, there is not a cat in hell's chance."


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Popular property highlights
Located within a mile (2 km) of Celebration Station, Hyatt Place Greensboro is within 3 miles (5 km) of Airbound Trampoline Park Greensboro. This 3-star hotel welcomes guests with 124 rooms and conveniences like a 24-hour fitness center, free in-room WiFi, and free self parking. Also near this Greensboro hotel are Safari Nation and Greensboro Arboretum.
Free buffet breakfast is included with your stay. Entertain your taste buds with a beverage at the bar/lounge or coffee shop/café.
All 124 air-conditioned accommodations feature sitting areas. Guests can expect free WiFi and 42-inch flat-screen TVs with pay movies. Beds are dressed in premium bedding and bathrooms offer hair dryers and designer toiletries. Sofa beds, refrigerators, and coffee makers are also standard.
Property features
Guests of Hyatt Place Greensboro enjoy a 24-hour fitness center, free WiFi in public areas, and a conference center. You can put free parking to good use if you drive. The 24-hour front desk has staff standing by to help with dry cleaning/laundry, concierge services, and securing valuables. Additional amenities include a 24-hour business center, a seasonal outdoor pool, and a computer station.
- Free buffet breakfast
- Free WiFi in rooms and public areas
- Free parking
- Bar/lounge
HighlightsInformation missing or incorrect? Tell us!Opens in a new window
Hyatt Place Greensboro
Hotel Amenities
Hotel Amenities
Hyatt Place Greensboro features a 24-hour fitness center and a seasonal outdoor pool. The hotel offers a coffee shop/café. A bar/lounge is on site where guests can unwind with a drink. Guests can enjoy a complimentary breakfast. A computer station is located on site and high-speed wireless Internet access is complimentary.
A 24-hour business center is on site at this 3-star property. Event facilities measuring 357 square feet (32 square meters) include a conference center. This business-friendly hotel also offers concierge services, dry cleaning/laundry services, and express check-in. Complimentary self parking is available on site.
Hyatt Place Greensboro is a smoke-free property.
- Computer station
- Free RV, bus, truck parking
- 24-hour fitness facilities
- Total number of rooms - 124
- Number of floors - 6
- Number of buildings/towers - 1
- Outdoor seasonal pool
- Conference space size (meters) - 32
- Number of meeting rooms - 3
- 24-hour business center
- Conference space size (feet) - 357
- Conference center
- Free breakfast
- Safe-deposit box at front desk
- 24-hour front desk
- Elevator/lift
- Bar/lounge
- Coffee shop or café
- Concierge services
- Dry cleaning/laundry service
- Express check-in
- Express check-out
- Meeting rooms
- Free self parking
- Television in common areas
- Free WiFi
- Smoke-free property
Family Friendly Amenities
- Family Hotel
- Free Wi-Fi
- Rollaway/extra beds available
- Sofa bed
- Refrigerator
- Cribs/infant beds (complimentary)
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking, Free RV, bus, truck parking
Room Amenities
Hyatt Place Greensboro offers 124 air-conditioned accommodations with coffee/tea makers and designer toiletries. Accommodations offer separate sitting areas and include sofa beds. Beds feature premium bedding. 42-inch flat-screen televisions come with pay movies. Bathrooms include shower/tub combinations and hair dryers.
This Greensboro hotel provides complimentary wireless Internet access. Business-friendly amenities include desks and phones. Additionally, rooms include irons/ironing boards and blackout drapes/curtains. Housekeeping is provided daily.
- Free cribs/infant beds
- Connecting/adjoining rooms available
- Premium TV channels
- Pay movies
- Premium bedding
- Separate sitting area
- Air conditioning
- Blackout drapes/curtains
- In-room climate control (air conditioning)
- Phone
- Refrigerator
- Coffee/tea maker
- Daily housekeeping
- Designer toiletries
- Hair dryer
- Shower/tub combination
- Flat-panel TV
- Iron/ironing board
- Desk
- Sofa bed
- Rollaway/extra beds available
- Free WiFi
Where to Eat
Guests are offered a complimentary buffet breakfast.
Nearby Things to Do
Recreational amenities at the hotel include a 24-hour fitness center and a seasonal outdoor pool.
If you have requests for specific accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
- Roll-in shower
- In-room accessibility
Hotel Policies
Minimum check-in age is 21
Check-in time starts at 3 PM
Check-out time is noon
Payment types
Children and extra beds
- Children are welcome.
- Kids stay free! Children 18 years old and younger stay free when using existing bedding.
- Rollaway/extra beds are available.
- Free cribs (infant beds)!
- Pets allowed for an extra charge of USD 75 per accommodation, per stay (maximum USD 175 per stay), plus a one-time cleaning fee of USD 100
- Dogs only
- 2 per room (up to 50 lb)
- Specific rooms only
- Pet-friendly rooms can be requested by contacting the property at the number on the booking confirmation
You need to know
Extra-person charges may apply and vary depending on property policy.
Government-issued photo identification and a credit card are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
Service animals exempt from fees/restrictions
Optional extras
Hotel Name
- Greensboro Hyatt Place
- Hyatt Place Greensboro Hotel
- Hyatt Place Greensboro
- Hyatt Place Hotel Greensboro
- Hyatt Greensboro
- Hyatt Place Greensboro Hotel Greensboro
We should mention
Children 18 years old and younger stay free when occupying the parent or guardian's room, using existing bedding. The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation. The photos in this description reflect the brand standard and are provided for illustrative purposes only. This property allows pets in specific rooms only (surcharges apply and can be found in the Fees section). Guests can request one of these rooms by contacting the property directly, using the contact information on the booking confirmation.


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You missed it This great deal for Mina Studios is over. But don't despair, we've got more great deals every day.
Related Deals That You May Like
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4-Week Meal Plan with Delivery by Nutrisystem
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Integrative Wellness Academy
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Mina Studios
Complete Bathroom Demolition and Remodel
You've spent a lot of time working on your living room, your bedroom, and even on the dog house, but you seemed to have washed your hands of the situation with your bathroom. Don't let this room go, well, down the toilet -- pay $5,999 and Mina Studios will give your bathroom a complete makeover of your choosing (a $12,000 value). This deal includes demolition and trash disposal, 100 square feet of tile, preparing ceiling and walls for paint, and professional installation of a medicine cabinet, shower and bath, faucet, exhaust fan, a GFCI outlet, and toilet. You'll love your new bathroom, plus you'll save 50% on the cost -- leaving you flush with cash.
PAID VALUE EXPIRES ON May 30, 2022
PROMOTIONAL VALUE EXPIRES ON August 29, 2012


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Help. 2000 Zetec spitting out coolant from plug holes on valve cover!
View Single Post
Focus Jr. Enthusiast
Join Date: Apr 2012
Fan#: 97222
Location: Chardon, OH
What I Drive: 2000,ZX3,Blue,3DR,Auto,2.0ZETEC16
Posts: 47
FF Reputation:
Buy-Sell-Trade Rating: (
Thanks for reaching back to me.
I can see the two freeze plugs from the open holes in the valve cover where the plug wires go and one for the freeze plugs looks pretty badly corroded -- I believe this is the culprit! (
...but maybe I should R&R both, just to be certain?
) I started "treating" the plug with WD-40 to prepare it for removal.
If it is this plug (and it comes out), it should be a inexpensive repair.
(The photos with the cover removed are not from my vehicle. I need the parts first)
I went to an auto parts store (AutoZone) today and they had no clue what a core plug or freeze plug was and could not find a part. I called a Ford dealer and got the same result? WTH? Everyone within ear shot at the AutoZone told me I had a blown head gasket. (Baloney!) A mechanic who was at the store (who happened to be picking up parts to fix another 2000 Focus), offered his moonlight services and would bring over the equipment (w/ screw adapter for overflow reservoir) to pressurize the coolant system, to verify where coolant is leaking from. I will probably take him up on his offer.
I have to find some place to order the plugs! Should not be so difficult to find these!
I will follow up on this post, so others with this issue can benefit from this. Any input, thoughts, rants, raves, questions of any kind would be appreciated.
2000 Ford Focus Zetec 2.0L, blue, 3 door, automatic, lots of miles. "If something cannot go on forever, it will stop," Herbert Stein.
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Look Cute In The Cold & Stock Up On Winter Essentials, Just Like Taylor Swift!
While in Europe, the star combats the cold in adorable jackets, hats, and tights! Stock up on some fall and winter essentials this weekend.
The cold weather always seems to sneak up on me this time of year and I always seem to find myself wishing I bought a new coat or wasting time in the morning rummaging around for tights — (which sometimes results in me wearing a colored pair I wasn't necessarily looking for out of a lack of options, like today). I realize it's time to stock up on essentials, especially after seeing Taylor Swift out and about in Europe.
From her penchant for knit hats, lace-up oxfords, and her hooded Topshop coat I'm obsessed with she manages to look adorable when she's out in the cold. I'm not always obsessed with her style, but I love how she manages to bring her signature girly look into her bundled up get-ups. If you're like me and you're still searching for a new coat or maybe you just want a funky pair of patterned tights to add a new twist to your basic LBD I suggest stocking up soon! Check out my picks below if you want to look just as cute as Taylor when you're out in the cold.


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Healdsburg Wedding at Trentadue Winery from Nicole Paulson PhotographyWow, wow, wow. I don't even know how to start this post to prepare you enough for the absolute gorgeousness you are about to see from this wedding by Nicole Paulson Photography. What to start with first? The Trentadue Winery?
View CreditsRead More


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By Sharon Jayson, USA TODAY
Sex, booze or money just can't compare with the jolt young people get from a boost to their self-esteem, says a new study of college students that found the desire for praise trumped other desires or needs.
"We looked at all the things college students love and they love self-esteem more," says lead author Brad Bushman, a professor of communication and psychology at Ohio State University in Columbus.
Using questionnaires that included the Narcissistic Personality Inventory, researchers at Ohio State and Brookhaven National Laboratory in Upton, N.Y., conducted two separate studies of 282 students that gauged the desire for praise, as well as the desire to engage in sex, drink alcohol, get a paycheck, eat a favorite food or see a best friend. They favored experiences that boosted self-esteem, such as receiving a good grade or a compliment
"All these things make people feel good and self-esteem definitely feels good," Bushman says. The study is published online in the Journal of Personality.
Part of what researchers analyzed was the difference between "liking" and "wanting," based on study participants' self-reports. They said they liked the rewards listed in the study more than they actually wanted them, which Bushman says is considered healthy. However, the liking-wanting distinction was smallest for self-esteem, suggesting a stronger desire for it than the other rewards.
For another gauge, participants took a test they were told measured intellectual ability. Afterward, they were told if they waited 10 minutes, their score would be recalculated with a method that usually produced better results. Researchers found that students who highly valued self-esteem were more likely to stay to get the higher scores.
The findings don't surprise Bill Corsaro, a sociologist at Indiana University-Bloomington. He has not seen the study but has taught for 35 years. He says "more and more kids are being raised with high expectations."
"Kids from a very early age are being socialized to do well — to be hard workers. It creates this sense of entitlement. So achieving and being praised for that achievement is what's most important in their view in terms of themselves," Corsaro says. "It doesn't mean they won't engage in these other activities, but they're not held up as (being) as important."
Bushman says self-esteem levels have been rising ever since a widely used standard was developed in 1965. "People seem to value it more highly today than in the past," he says.
Self-esteem is a basic human need, says Robert Reasoner of Port Ludlow, Wash., who has been involved with the movement for 40 years. "All humans want to have positive emotional feelings about themselves," says Reasoner, a founder of the National Association for Self-Esteem.
It's about confidence in your ability to deal with life's challenges and a sense of personal worth, rather than generalized praise and undeserved rewards, he says.
Bushman is worried, however. "In general, I think self-esteem, though it feels good for the individual, is harmful to society, especially if it goes over the top and becomes narcissism."
We've updated the Conversation Guidelines. Changes include a brief review of the moderation process and an explanation on how to use the "Report Abuse" button. Read more.


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Good Deeds
Read Our Full Review & Parent Discussion Questions Here
Good Deeds
Rating & Content Info
Why is Good Deeds rated PG-13? Good Deeds is rated PG-13 by the MPAA for sexual content, language, some violence and thematic material.
Violence: A couple argues. Characters make mean-spirited comments to others. Other characters yell or argue. A man attacks a woman in a homeless shelter. Men engage in a fistfight that includes shoving and punching. One character slaps another on the face. Characters are thrown out of their home and forced to live on the street.
Sexual Content: Characters engage in sexual activity (only their faces and clothed upper bodies are seen), and comment about it the following morning. Characters kiss and embrace. Brief and sometimes crude sexual innuendo is included in the script. References are made about multiple sexual partners. A man is shown in the shower - nudity is obscured. Characters wear suggestive clothing at a club. Characters make verbal and physical sexual advances. A character is shown in lingerie numerous times.
Language: The script includes a couple of strong slang terms for sexual activity, as well as suggestive slang terms for sexual activity. Also used are racial and sexual slurs, frequent moderate and mild profanities, and crude name calling.
Alcohol / Drug Use: A character loses his driver's license after drinking and driving, but still drinks often to deal with stress. A character makes a comment about using cocaine. A character becomes drunk during an evening at a nightclub. Another character is portrayed as being inebriated. Characters drink with dinner or in social settings. Brief tobacco use is shown.
Page last updated July 8, 2016
|Rating Descriptor
|British Columbia
|Not Rated
|Not Rated
|Mature Theme.
|Not Rated
|Canadian Home Video
NEW: Listen to our Parent Previews Podcast and take control of media and technology in your family!
News About "Good Deeds"
Cast and Crew
Good Deeds is directed by Tyler Perry and stars Tyler Perry, Gabrielle Union, Thandie Newton.
Home Video
The most recent home video release of Good Deeds movie is June 12, 2012. Here are some details…
Tyler Perry's Good Deeds releases to home video on June 12, 2012, with the following extras:
- Two featurettes exploring the film's central themes.
Related home video titles:
Tyler Perry is a writer, director and actor (actress too, if you count the roles he's played in drag). You can see his work in Madea's Family Reunion, Diary of a Mad Black Woman and I Can Do Bad All By Myself. He leaves behind the Madea character to play Admiral Richard Barnett in Star Trek.
In The Soloist, a news reporter takes an interest in a homeless man with unusual musical talent. A sports journalist decides to write an article that will revive his career and that of a former boxer in Resurrecting the Champ.
Other movies featuring men with the title character's same last name include Mr. Deeds and Mr. Deeds Goes to Town.


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- Hawaii Police Department - http://www.hawaiipolice.com -
Wanted: Brooks 06-18-08
Hawaii Police Department
Criminal Investigations Section, Area II
Lieutenant Robert Wagner
Phone: 326-4646, Ext. 266
June 18, 2008
Report No. C08016806
Big Island police are asking for the public's help in locating a man wanted in connection with a Kailua-Kona burglary.
Someone entered a home in the Kailua View Estates subdivision sometime between May 27 and May 29 and removed several items, including more than 1,000 pounds of green coffee beans packaged in 100-pound burlap bags.
Police are looking for 24-year-old Donimic Brooks. He is described as an African-American male, 6-foot-3, 165 pounds with brown eyes and brown hair. He has no permanent address but is believed to be staying in Mountain View.
Police ask that you call the police department's non-emergency line at 935-3311 if you have any information about his whereabouts or about anyone trying to sell 100-pound bags of green coffee beans in burlap bags.
Tipsters who prefer to remain anonymous may call Crime Stoppers at 961-8300 in Hilo or 329-8181 in Kona. All Crime Stoppers information is kept confidential.
Article printed from Hawaii Police Department: http://www.hawaiipolice.com
URL to article: http://www.hawaiipolice.com/wanted-brooks-06-18-08
Copyright © 2014 Hawaii Police Department. All rights reserved.


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Paddy Breathnach co-founded Treasure Films with Robert Walpole in 1992. In the intervening time Breathnach (director) and Walpole (producer) have completed numerous projects including shorts, TV series, documentaries and award-winning features.
Breathnach progressed successfully from his first fiction short, A Stone of the Heart which won the Special Jury Prize at the Cork Film Festival 1991, to feature length projects beginning with the documentaries The Road to America and The Charlton Years. Both dealing with the Irish soccer team, they became the most successful Irish sell-through videos ever.
Breathnach and Walpole again worked together on WRH a six-part documentary series for RTE set in the regional hospital in Waterford.
Breathnachs first feature film, Ailsa, produced by Ed Guiney, won the Euskal Media Award for best new director at the San Sebastian Film Festival. Adapted by Joseph OConnor from his own short story, Ailsa is a tale of male obsession and violence set in south Dublin. On its release in 1994 Ailsa was perceived as a European film, both in theme and style and it coincided with a period of unprecedented growth in the Irish film industry.
Perhaps Breathnach is best known for I Went Down, a gangster road movie which employs Hollywood's genre codes and places them in modern day Ireland among small time criminals. Released in 1997 I Went Down won the Best New Director and the Jury Prize at the San Sebastian Film Festival and won Best Director at Thessalonica 1997 and Best Film at Bogota International Film Festival 1998. It was critically acclaimed worldwide and also won numerous awards for screenwriter Conor McPherson.
Breathnach and Walpole co-produced the feature documentary Southpaw - The Francis Barrett Story directed by Liam Mc Grath and selected for the Sundance Film Festival in 1999.
Breathnach also directed 'Blow Dry' from a script by Simon Beaufoy, writer of 'The Full Monty , ' the film starred Josh Hartnett and Alan Rickman and was released by Miramax in 2001.
His most recent feature as director 'Man About Dog', which was produced by Robert Walpole and Simon Channing Williams from a script by Pearse Elliott was released last year, taking over 2.1 million at the Irish Box Office, becoming one of the most successful independently produced Irish films ever at the Irish Box Office. It stars Allen Leech, Sean McGinley, Pat Shortt and Fionnula Flanagan.
Last year he produced 'The Mighty Celt' with Robert Walpole and Paddy McDonald which shot in Belfast in May 2004, starring Robert Carlyle and Gillian Anderson. The film, which is being handled worldwide by The Works, was backed by BBC Films, the Irish Film Board, the NIFTC and TV3, and recently premiered in competition at the Berlinale. It will be released by Metrodome in Ireland and the UK on 26th August, 2005.
Breathnach has served on the board of the Dublin Film Festival and Filmbase, and he was also the Irish Representative of Jury for the European Film Festival, a festival of European Cinema co-ordinated by the European Parliament. He is currently working on a number of projects, including a horror 'Shrooms', which is written by Pearse Elliott, and a self-penned Irish language supernatural Western, 'Swordland'.
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Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)
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DOI: 10.1007/s00262-007-0380-6
- Cite this article as:
- Janetzki, S., Panageas, K.S., Ben-Porat, L. et al. Cancer Immunol Immunother (2008) 57: 303. doi:10.1007/s00262-007-0380-6
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The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.
KeywordsElispot Proficiency panel Validation Harmonization Immune monitoring
Cancer Vaccine Consortium of the Sabin Vaccine Institute
Peripheral blood mononuclear cells
Cytomegalie, Epstein Barr, and influenza virus
Cytomegalie virus
National Institute of Allergy and Infectious Diseases
Coefficient of variation
Standard operating procedure
Elispot is a widely used assay for immune monitoring purposes [6, 17, 25, 39]. Measuring immune responses has been accepted as an important endpoint in early clinical trial settings in order to prioritize further vaccine or other immunotherapy development [11, 22, 24, 26, 38]. Despite the overwhelming use of various immune assays for exactly that purpose, reported results are often met with skepticism, caused mainly by two reasons: (1) high variability among results from the same laboratories and/or among different laboratories, and (2) the lack of demand to report standardization, validation and training strategies as well as assay acceptance criteria by the laboratories conducting immune testing. This is surprising since the reporting of results for other clinical endpoints, e.g., side effects, has to follow strict guidelines and definitions.
- 1.
To provide regulatory and sponsoring agencies with confidence that reported data are generated following necessary standards and rigor that supports product licensure.
- 2.
To provide an external validation tool for individual labs.
- 3.
To provide proof to patients and volunteers that necessary measures have been taken to allow successful study participation [3].
In contrast, immune monitoring approaches in the cancer vaccine field are more heterogeneous, based on the vast variety of vaccine design, type of cancer, and availability of antigen presenting cells. Standardization of the entire Elispot protocol across laboratories is therefore not feasible. We set out to devise a strategy to identify issues and deficiencies in current Elispot practices, and to identify common sources of assay variability within and between laboratories, with the extended goal of standardizing the identified factors in an assay harmonization effort across laboratories.
In 2005, the Cancer CVC/SVI initiated an Elispot proficiency panel program to achieve this goal. In addition to offering an external validation program, the CVC addressed the need for such strategy by comparing assay performance across the field, identifying critical protocol choices and gaining an overview of training and validation practices among participating laboratories.
For this program, predefined PBMCs from four donors with different ranges of reactivity against two peptide pools were sent to participants for Elispot testing. Laboratories had to further provide cell recovery and viability data, as well as respond to surveys describing their protocol choices and training and validation status.
In response to the survey results, the CVC/SVI established requirements for laboratories to participate in future proficiency panels, which included the existence of a Standard Operating Procedure (SOP) prior to joining the program. Further, individualized assay performance assessment was offered to all laboratories, together with suggestions for implementation of protocol optimization steps.
Materials and methods
Participants and organizational setup
All participants were members of the Cancer Vaccine Consortium or its affiliated institutions, the Ludwig Institute for Cancer Research (LICR) and the Association for Immunotherapy of Cancer (C-IMT). Laboratories were located in ten countries (Australia, Belgium, Canada, Germany, Italy, Japan, France, Switzerland, UK, and USA). Each laboratory received an individual lab ID number. Panel leadership was provided by a scientific leader experienced in Elispot, in collaboration with the CVC Executive office. SeraCare BioServices, Gaithersburg, MD, served as central laboratory, providing cells, pretesting and shipping services, as well as logistical services like blinding of panelists. IDs were not revealed to panel leader, CVC or statistician during the panel.
Thirty-six laboratories including the central lab participated in panel 1, 29 including the central lab in panel 2. Twenty-three laboratories participated in both panels. Six new panelists were added to the second testing round. Thirteen dropped out after the first panel. Main reasons for drop out were switch of assay priorities and not meeting criteria for panel participation. Various groups stated that one time participation fulfilled their need for external validation.
PBMCs and peptides
PBMCs from healthy donors were obtained from a commercial donor bank, manufactured and processed under GMP conditions using established Standard Operating Procedures at SeraCare. PBMCs were frozen using a rate controlled freezer and transferred to the vapor phase of liquid nitrogen. A three lot validation study was performed [20] in which the following was validated: cell separation procedure, freezing media, dispensing effect on function, freezing procedure, and shipping on dry ice. It was demonstrated that functionality and viability were maintained throughout the procedure. In addition, Elispot values from fresh and frozen PBMCs, shipped on dry ice, were nearly equivalent.
Each vial of PBMCs contained enough cells to ensure a recovery of 10 million cells or more under Seracare's SOP.
PBMCs were pretested at the central laboratory for reactivity against the CEF [7] and CMV pp65 peptide pools [23]. The CEF peptide pool was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. It contains 32 8-11mers known to elicit CD8-restricted responses, and serves as a widely used control in IFNγ Elispot assays [7]. The CMV pp65 peptide pool was a generous gift of the NIAID and Becton Dickenson. This pool consists of 135 15mers overlapping by 11 amino acids, and has been shown to elicit CD8- and CD4-restricted responses [23]. PBMCs were selected for no, low, medium and strong responses against both peptide pools, and repeatedly Elispot-tested at Seracare in order to confirm responder status. Response definition was set arbitrarily for the spot number/well (200,000 PBMC): no responder: average median/panel 1 + 2 = 1 (CMV and CEF); low responder: average median/panel 1 + 2 = 18 (CMV) and 40 (CEF); medium responder: average median/panel 1 + 2 = 98 (CMV) and 127 (CEF); and high responder: average median/panel 1 + 2 = 396 (CMV) and 398 (CEF).
Peptide pools were resuspended in DMSO and further diluted with PBS to a final concentration of 20 μg/ml. Aliquots of 150 μl of peptide pool were prepared for final shipment to participants. Corresponding PBS/DMSO aliquots for medium controls were also prepared. Participants were blinded to the content of these vials, which were labeled as "Reagent 1, 2 or 3".
All cells and reagents sent to participants in both panels were obtained from the same batches.
Cells and reagent vials were shipped to all participants for overnight delivery on sufficient dry ice for 48 h. Shipping was performed by Seracare under their existing SOPs.
Participants received a plate layout template and instructions for a direct IFNγ Elispot assay which had to be performed in one Elispot plate. Each donor was tested in six replicates against three reagents (medium, CEF and CMV peptide pool). Further, 24 wells were tested for the occurrence of false positive spots by the addition of T cell medium only. About 200,000 PBMC/well were tested against 1 μg/ml peptide pool or the equivalent amount of PBS/DMSO. All other protocol choices were left to the participants, including choices about: Elispot plate, antibodies, spot development, use of DNAse, resting of cells, T cell serum, cell counting and spot counting method. All plates were reevaluated at ZellNet Consulting (Fort Lee, NJ) with a KS Elispot system (Carl Zeiss, Thornwood, NY), software KS Elispot 4.7 (panel 1) and KS Elispot 4.9 (panel 2) in blinded fashion. Each well in each plate was audited.
Since the focus of this study was on assay performance and protocol evaluation and not on the definition of a positive response, we prospectively defined the ability to detect a response (independent of magnitude) by using the following "empirical" method: the antigen-specific spot counts per 2 × 105 PBMCs had to be >10, and at least 3× as high as the background reactivity. Similar approaches have been described elsewhere [5, 8].
Statistical analysis
The following parameters were calculated for the overall panel and the individual participant's performance, using either lab-specific counts or central recounts: the mean, standard deviation, and coefficient of variation (CV), the median, minimum, and maximum spot counts for each donor and reagent and the media only wells. Box plots were used to illustrate the distribution of spot counts across the panel per given test condition. Further, individual results were represented as box plots comparing lab counts with recounts, central lab results and overall panel results. For panel 2, results from repeating laboratories were also compared in box plot format for each donor and condition. The Fisher's Exact test was used to compare the proportions of laboratories which missed to detect responses in each panel. For the comparison of recovery and viability, the Student's t test was applied.
In the first proficiency panel, shipping and Elispot testing among 36 laboratories from 9 countries were conducted without delays. The success of this panel demonstrates the feasibility of such large international studies, the biggest of such format as of today, under the described organizational setup. The second panel with 29 participating laboratories from 6 countries followed the approach of panel 1. However, customs delays of dry ice shipments to some international sites required repeated shipments of cells and antigens to these destinations. Based on this experience, the use of dryshippers with liquid nitrogen is being implemented for international destinations in the third CVC panel round in 2007.
Recovery and viability of PBMCs in panel 1 and 2
Cell recovery and viability in both proficiency panels
Mean P1/P2b
Median P1/P2
Minimum P1/P2
Maximum P1/P2
Cell recovery in 106 cells
Viability (%)
Interestingly, only 4/10 laboratories in panel 1, and 4/7 laboratories in panel 2 with recoveries below 8 million cells were from international locations. Similar, only 1/5 laboratories in panel 1 and 2/5 in panel 2 reporting viabilities less than 70% belonged to international sites. This clearly demonstrates that location for dry ice shipment had no effect on overall cell recovery and viability.
We also investigated whether low (<8 million) or very high (>20 million) cell recovery had an influence on spot counts, assuming that these were potential erroneous cell counts, leading to too low or too high cell dilutions, respectively, what in turn would lead to too high (in case of underestimating cell number) or too low (in case of overestimating cell number) spots counts. However, except for a few sporadic incidents, there was no correlation between cell counts and spot counts (data not shown) in either direction. Only one laboratory with low recovery and low viability was found to have peptide pool-specific spot counts for all donors much below the panel median.
The use of an automated cell counter (Guava) did not reveal any trend in recovery for this group (recovery ranged from 6.7 to 25.1 million cells), nor a difference compared to the overall panel recovery data. In contrast, the mean cell viability per donor measured by Guava counter users was significantly lower than the overall cell viability in users of a hemocytometer (P < 0.01, see table in Fig. 5).
Elispot results in panel 1
All 36 laboratories completed testing of all 4 donors against medium, CEF and CMV peptide pool. Spot appearance and size as well as occurrence of artifacts differed dramatically among laboratories (not shown). Four outlier laboratories were identified, which detected less than half of the responses correctly. In all four cases, detected responses were well below the panel median, and often, there was high background reactivity (up to 270 spots/well) in medium controls. No obvious protocol choices could be identified which could have been responsible for the suboptimal performance. One out of the four laboratories had little experience at the date of the panel. Another group reported a less experienced scientist performing the assay. A third outlier repeated the assay, and was able to perform adequately. No feed back was available from the fourth group.
A similar scenario was found for response detection against the CEF peptide pool. Thirteen laboratories missed to detect the response, one of which due to high reactivity against medium, and one due to inaccurate spot counting.
An interesting observation was that 23 groups reported false positive spots in a range of 1–26 spots/well. Reevaluation revealed that the actual number of groups with false positive spots was lower (12), and the false positive spot number range per well fell between 0 and 8.
Survey results about protocol
During the first panel, participants had to provide information about their protocol choices: plates, potential prewetting of PVDF, antibodies, enzyme, substrate, use of DNAse during PBMC thawing, resting of cells, serum used, cell counting, and plate reader. There was a wide range of protocol choices across the panel participants. The most common choices for the parameters listed above were as follows: use of PVDF plates (64%) prewetted with Ethanol (52%), coated with Mabtech antibodies (67%) at 0.5–0.75 μg/well (33%); spot development with HRP (53%) from Vector Laboratories (33%) using AEC (44%) from Sigma (42%); no use of DNAse when thawing cells (83%), and no resting period for cells prior to the assay (53%); use of human serum (64%); cell counting with trypan blue exclusion using a hemocytometer (78%); and plate evaluation with a Zeiss reader (36%).
There were some clear trends for international sites with preferred use of the AP/BCIP/NBT development system (83 via 29% in the US), the use of nitrocellulose plates (75 via 21% in the US), and the use of Mabtech antibodies (83 via 58% in the US). On the other hand, 7/8 laboratories using an automated cell counter were located in the US.
Survey results about validation and training practices
During the lively discussion of the results of panel 1 and its protocol survey at the Annual CVC meeting in Alexandria in November 2005, it was suggested that the level of experience, standardization and validation of participating laboratories might have been the cause for the variability and performances observed. In response, we conducted a survey among panelists, in which 30 laboratories participated. As expected, the experience and Elispot usage varied significantly. Some laboratories had the Elispot assay established less than one year before panel testing, whereas others used the assay for more than 10 years. The experience of the actual performer of the panel assay also varied widely.
Interestingly, even though 2/3 of participants reported to have specific training guidelines for new Elispot performers in place, more than 50% never or rarely checked on the scientist's performance after the initial training.
Almost all laboratories indicated that they use an SOP that had been at least partially qualified and/or validated. Validation tools and strategies varied widely. Only 12 groups monitored variability, whereas 23 reported the use of external controls of some kind (e.g., T cell lines, predefined PBMC, parallel tetramer testing).
Thirteen groups were found to have some kind of criteria implemented for assay acceptance. Among the 20 different criteria reported, not one was described by more than one lab.
Mirroring these survey results, 20 laboratories believed that they need to implement more validation steps. All except one group expressed their strong interest in published guidelines for validation and training strategies for Elispot.
Elispot results in panel 2
Based on the experience from the first panel, acceptance criteria for participation in the program were redefined. Only laboratories with established SOPs were accepted. The second panel was repeated with identical experimental setup as the first panel, and with the same batches of PBMCs, peptides and control reagents. Twenty-nine laboratories participated including the central lab. This time, there were no outlier performers identified. The number of groups that did not detect the weak responder dropped dramatically (P < 0.01) from 47% (17/36 labs) in panel 1 to 14% (4/29) in panel 2 (Fig. 2). The overall panel median for the CMV-response in the weak responder increased from 14 spots in panel 1 to 21 spots in panel 2, and for the CEF response from 30 to 51, respectively.
Of the 23 labs repeating the panel, 8 had changed their protocol before panel 2, 3 of which as an immediate response to results from the first panel. One outlier lab from panel 1 participated in panel 2, and improved its performance by detecting all responses correctly as per reevaluation counts. Only their lab-specific evaluation did not detect the low CMV responder. Overall, 4/23 panel-repeating labs (17%) did not detect the low CMV-responder, 3 of which also did not detect this response in panel 1. About 10/23 groups missed the low CMV responder in panel 1, but 7 of these laboratories were able to detect it in panel 2. Only one repeater detected the low CMV response in panel 1, but not panel 2. This is a clear performance improvement for that group (47% missed this response in panel 1), and highlights the usefulness of multiple participation in panel testing as an external training program.
We ran an in-depth analysis of the results and previous survey responses, where available, from participants who missed the weak responder, as well as from laboratories with marginal detection of response, including personal communication. We were able to narrow down the possible sources for these performances. Two laboratories missed responses due to inaccurate evaluation, during which they either included artifacts into spot counts or simply did not count the majority of true spots, as central reevaluation revealed. One laboratory did not follow the assay guidelines. The majority of laboratories, however, followed common protocol choices, but had either very low response detection across all donors and antigens, or detected very high background reactivity in some or all donors. This pattern pointed to serum as the possible cause for suppressed reactivity or non-specific stimulation. Three of these laboratories shared with the CVC that retesting their serum choice indicated that they had worked with a suboptimal serum during the panel; and that they now successfully introduced a different serum/medium to their protocol with improved spot counts. Serum choices included human AB serum, FCS, FBS, and various serum-free media. There was no difference in assay performance detectable between these groups.
The CVC conducted two large international Elispot proficiency panels in which participants tested four batches of predefined donor PBMC for CEF and CMV peptide pool reactivity. Common guidelines like number of cells per well and amount of antigen had to be followed, in order to allow result comparison and reduce variability due to the known influence of cell numbers plated [2, 14]. A surprising finding from the first panel was that almost half of the participants were not able to detect the weak responder. The initial protocol survey did not allow the detection of common sources for this sub-optimal performance. In some cases, laboratories with identical protocol choices performed very differently, whereas others with distinct protocols had almost identical spot counts (Fig. 4). This observation supports the premise that many common Elispot materials and reagents (e.g., plates, antibodies, spot development reagents) perform equally or similarly well; and that there are other factors which influence the outcome of the Elispot assay.
Various laboratories missed the response detection by inaccurately evaluating their Elispot plate, independent of the reader used, as reevaluation revealed (Fig. 3). This observation is in contrast to results from the first NIAID proficiency panel, which describes good agreement in spot counts from participants and experienced, independent centers [5]. Operator-dependent variability in Elispot evaluation results is a known phenomenon [15]. Despite the availability of high resolution readers and software features for automated spot gating and other potentially helpful options, it is essential to employ well trained operators for spot counting, to audit all plates, and to implement changes of reading parameter in cases when well and spot appearances differ from the overall assay, typically for technical reasons. For that, SOPs used for plate evaluation might require revision. The use of available certification and training services can be helpful.
Cell counting is a protocol step known for introducing variability. Because of the wide range in PBMC recovery, we investigated whether cell counts could have been potentially erroneous, which would lead to wrong final cell dilutions, and can therefore lead to too high or low spot counts. Despite large differences in cell recovery (from 5.5 to 34.4 million cells per vial in panel 1), we did not find a correlation between cell recovery and spot counts. Very low (<8 million) and very high (>20 million) cell counts were consistently found for all donors among the same few laboratories. Both, hemocytometers and automated counters were used in those laboratories. Various automated cell counters have been introduced to the market, which do not only offer automated spot counting features, but also the discrimination of apoptotic, viable and dead cells. The use of such systems can potentially decrease variability in cell counts, and most importantly increase the accuracy of viable cell counts [4, 21]. Seven laboratories in panel 1 used the Guava counter, which allows the discrimination of apoptotic cells. Even though our panel did not reveal a difference in cell counts between Guava and hemocytometer users, it could be demonstrated that the overall cell viability reported by Guava users was significantly lower. This could likely be attributed to the ability to discriminate apoptotic cells with this method. Smith et al. [35] recently reported the usefulness of apoptosis acceptance criteria that allowed the separation of PBMC samples by their ability to respond to an antigenic stimulus or not.
The introduction of a resting period for thawed cells is known to be advantageous since apoptotic cells die during the resting period, and final dilutions for the assay are based on a more homogenous population of viable cells [16, 18]. In contrast, the addition of a mixture of viable and apoptotic cells, which are prone to die during assay incubation time, directly after thawing leads to lower spot counts. During panel 1, the central laboratory performed a side-by-side comparison of the influence of a 2 and 20 h resting period on final spot counts, and demonstrated that a 20 h resting period yielded significantly higher counts (Fig. 6). Proficiency testing results from the C-IMT also support the introduction of a cell resting period for Elispot assays [2]. Even though almost half of participants in panel 1 let cells rest before addition to the plate, there was no clear correlation to the magnitude of peptide-specific spot counts. This might have been due to the variation of resting times between 0.5 and 20 h, and other protocol variables, which included the actual resting protocol. Factors like serum and serum concentration, cell concentration, and actual storage condition (e.g., tissue culture flasks or plates can lead to cell adherence and therefore loss of professional antigen-presenting cells) are known to influence the success of cell resting.
There are no scientific studies published about the effect of serum choices on immune assay results, one of the best known "secrets" in immunology [16]. It is critical to choose serum that supports low background reactivity, but strong signals. The leading choice in this panel to use human AB serum reflects the historic preference for human immune assays. Each serum batch, however, is unique in its ability to support optimal assay resolution, and may potentially contain mitogenic or immune suppressive factors. There was some anecdotal evidence that the serum choice among our panelists was responsible for suboptimal performance. Interestingly, six laboratories preferred to work with serum-free medium. None of these groups observed high background reactivity, but two failed to detect the weak responder.
A survey conducted among participating laboratories shed light on validation and training practices. The wide experience range of participating laboratories, in combination with various levels and approaches to validation and training, correlated with the overall high variability in panel results. In response, new criteria for panel participation were introduced for panel 2. The outlier lab number decreased from 4 to 0 in the new panel, however 3/4 outliers from panel 1 did not participate in panel 2. The percentage of laboratories that did not detect the weak responder decreased significantly from 47 to 14% (P < 0.01). This improvement might have been partially due to the stricter participant selection in panel 2. However, 7/10 labs repeating the panel improved their performance by correctly detecting the low CMV responder in panel 2, while missed in panel 1. A striking finding was that 2/3 of all laboratories stated that they believed they needed to implement more validation. And all but one group expressed their wish for published guidelines for Elispot assay validation and training.
The outcomes from these two proficiency panels first provide Initial Elispot Harmonization Guidelines for Optimizing Assay Performance (Fig. 1) that can fulfill this need and may provide—if implemented widely—the grounds for substantial improvement of assay utility for research applications and development of immune therapies. This can be implemented in accordance with assay recommendations made for cancer immunotherapy clinical trials by the Cancer Vaccine Clinical Trial Working Group [12]. Further optimization is aimed for through ongoing proficiency panel work conducted by the CVC.
Validation of assays is now a requirement for all endpoint parameters in clinical trials [30]. There are an increasing number of publications available describing validated Elispot assays [1, 19, 25, 34]. These papers contain valuable scientific information, but only limited referral to FDA regulations. The FDA guidelines for validation of analytical procedures [9] describe validation as the process of determining the suitability of a given methodology for providing useful analytical data, which consists of analyzing or verifying the eight or nine assay parameters as described in the US pharmacopeia or the ICH guidelines [13, 37]. Only few publications address validation of bioassays and Elispot in FDA terms [10, 16, 29, 36]. And even these few publications give only limited advice on how to validate the Elispot assay in a given laboratory setting, not to mention specific training guidelines.
Furthermore, acceptance criteria for assay performance were only used by a limited number of laboratories, and each criterion was unique for the laboratory that used it.
These observations should be a wake-up call for the immune monitoring community, which does not only include the cancer vaccine field, but also the infectious disease and autoimmunity field and others. General assay practices for the detection of antigen-specific T cells are comparable across all fields. The CVC as part of the Sabin Vaccine Institute is intending to develop and tighten collaborations with groups from other research and vaccine development areas. Published documents with specific criteria for Elispot assay validation, assay acceptance criteria and training guidelines will be most valuable for the immune monitoring field, and are now being established as CVC guidelines as a result of the described studies. Continuous external validation programs need to be a part of these efforts in order to check upon the success of inter-laboratory harmonization including assay optimization, standardization and validation as well as of laboratory-specific implementation of guidelines and protocol recommendations. These efforts are essential to establish the Elispot assay and other immune assays as standard monitoring tools for clinical trials.
Special thanks to C. Wrotnowski for his work in initializing the proficiency panel program of the CVC. W. M. Kast is the holder of the Walter A. Richter Cancer Research Chair and is supported by the Beckman Center for Immune Monitoring.