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Accutech provides both engineering and key-stroke support for Arrow backed up the developer, Applied Flow Technology. The annually renewable Support, Update and Maintenance (SUM) agreement ensures that your licence is always up-to-date and that we can provide timely assistance. Send us your design file and we can explain your results, check for errors and feedback suggestions for improving your design. We use our video-chat capability to navigate you through your model, whilst we both concurrently view the flowsheet.
We can provide training on Fathom via our two- or three-day training course.

subset: commoncrawl


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The right to participate in education is protected under state, federal and international legislation. We have a number of resources to help you to promote these rights and succeed in education.
Building relationships is a great way to maximise learning outcomes and success in education.
Getting to know your child's teachers is the best place to start, as well as student support and wellbeing staff. It's important to have a productive relationship with your child's teachers, both for you to feel comfortable to check in on a regular basis and for them to be able to raise any concerns with you.
From knowing your rights in education to thoroughly researching potential schools, it's important to be prepared for any conversations with the school about changes which could be made to improve your child's inclusion.
You and your child (either with your assistance or independently) will be more effective at negotiating education related adjustments if you are both knowledgeable about your child's vision condition and what they might need to succeed.
Teachers and administrators have a lot of competing pressures they need to address at their school, and they won't always understand the need for, or the importance of the things your child may need. By understanding what these limitations might be, you can find some common ground.
Schools will also think about what is best for the majority, so finding ways to express your child's needs in ways that show how they would benefit everyone will help.
Encourage your child to know their own needs and what should be happening in the classroom and at school generally to support their learning. This will enable them to speak up for themselves in the moment and concerns can be addressed immediately.
Your child will be encouraged by seeing you model good advocacy and communication with their school.
Your child may encounter many barriers throughout their education, and realistically, you will not be able to invest energy into every issue you would like to address.
Advocacy can take a lot of time, energy and persistence, so you should make your choice about where to invest your efforts, thinking about which changes are likely to result in achieving the most impact, and what might be 'quick wins' which result in immediate (but not long term) change.
Vision Australia has fact sheets on advocacy in education including:
Complete the form below and a member from Vision Australia's advocacy team will get in touch with your copy of the toolkit.


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Laws of rugby must change to protect players
RUGBY'S laws may have to be modified to reduce the risk of players suffering concussion, the game's chief medical officer has said.
BBC Scotland presenter and former Scotland international John Beattie
Martin Raftery's comments came as it was revealed the incidence of head injuries in Scotland has doubled in the past two years.
As the Rugby World Cup heats up this week, a review to improve safety in the game is continuing, with the emphasis on tackling.
Mr Raftery, of World Rugby, told the BBC that the organisation was reviewing 900 videos of concussions.
He told BBC Scotland presenter and former Scotland international John Beattie: "There's no doubt that the biggest area that we think, that we know, where concussion is going to occur is in the tackle, so that will help us to look at the tackle and see what we can do to make it safer.
"My job is to identify risk and then look for solutions to the law makers to make the changes that will bring about protection of the athlete."
Two seasons ago we had a real push, a real initiative – we educated people that, only by knowing what was happening with injury, could we make a significant difference
During the programme, Rugby And The Brain: Tackling The Truth, Beattie also interviews Peter Robinson whose son, Ben, died in 2011 as a result of sustaining concussion in a school rugby match when he was 14.
Dr James Robson, Scottish Rugby Union's chief medical officer, also said that changes were needed to make the game safer.
He told the programme that injury data from the grassroots game had been gathered over the past couple of years, and the number of concussions had almost doubled in that time.
Dr Robson said: "Two seasons ago we had a real push, a real initiative – we educated people that, only by knowing what was happening with injury, could we make a significant difference.
"I truly believe that the doubling between that season and the following season of the number was down to that education. I strongly believe that we do need to look at the laws of the game and the way that it's played."


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book publishing services; electronic book publishing services; publishing-related services; book distribution services; electronic book distribution services; book marketing services; electronic book marketing services
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Dogs' lives are too short. Their only fault, really. Votes: 3Agnes Sligh Turnbull
Their monument sticks like a fishbone in the city's throat. Votes: 3Robert Lowell
Their free verse was no form at all, yet it made history. Votes: 3John Crowe Ransom
The passions often engender their contraries. Votes: 0Francois de La Rochefoucauld
No one as ever completed their apprenticeship. Votes: 0Johann Wolfgang von Goethe
Women wrap men with their second attention. Votes: 0Frederick Lindemann, 1st Viscount Cherwell
Hate no one; hate their vices, not themselves. Votes: 0John Gardiner Calkins Brainard
Women know not the whole of their coquetry. Votes: 0Francois de La Rochefoucauld
Idiots and lunatics see only their own wit. Votes: 0Francois de La Rochefoucauld
Modern poets add a lot of water to their ink. Votes: 0Johann Wolfgang von Goethe
Men give away nothing so liberally as their advice.Francois de La Rochefoucauld
There are few women whose charm survives their beauty.Francois de La Rochefoucauld
Let me pass, I have to follow them, I am their leader.Alexandre Auguste Ledru-Rollin
Parents have to really talk to their children before they leave home. Votes: 1 Aretha Franklin
The thing that impresses me most about America is the way parents obey their children. Votes: 1 Edward VIII
There's no meaning to life without music. Everybody has their own connect with music. Votes: 1 Kailash Kher
She remembered something she overheard at a dinner party -- Everyone loses their mind at least once in this lifetime. Everyone. Votes: 3 Lawren Leo
It took me a while to warm to the '20s costumes on 'Downton.' I love it when women accentuate their curves, and that era was all about hiding them. The shapes they wore then were in tune with female empowerment. Cutting off their hair and hiding their busts was a way of saying, 'We're equal to men!' Votes: 1 Lily James
Government is a parasite-a cancer that by nature tries to spread deeper into society. Those who want to run others' lives won't give up and start minding their own business. Votes: 3 Harry Browne
It is impossible to preserve my friendship with people who are allegedly leaders when they are attacking their own people, shooting at them, using tanks and other forms of heavy weaponry. Votes: 1 Recep Tayyip Erdogan
I wanted people to see me, to change their minds about me. Votes: 1 Fantasia Barrino
Great storytellers in the past would go to an unknown land and return to tell the stories they've found. Those were also journeys into their inner psyches and that's still true today. An actor, a writer, does that as if saying, 'Here's what I've discovered about myself and about the world I'm in. I would like to share this with you.' Votes: 1 Robert Davi
But of course when people watch morning television, Terry, it's a very different animal. You know, they're running around, they're getting their kids ready for school, they're probably doing eight million things, they're brushing their teeth. Votes: 1 Katie Couric
It took me a while to warm to the '20s costumes on 'Downton.' I love it when women accentuate their curves, and that era was all about hiding them. The shapes they wore then were in tune with female empowerment. Cutting off their hair and hiding their busts was a way of saying, 'We're equal to men!' Votes: 1 Lily James


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Outside the automotive world, the term hybrid basically means a cross-breed � the offspring of two different species or plants or animals � a labradoodle, for example.
In motoring terms, though, hybrid refers to a marriage of two forms of propulsion. In the case of the world�s best-selling hybrid, the Toyota Prius, that is a combination of a petrol engine and an electrical system that includes a motor, generator and battery.
But it doesn�t necessarily have to be the case. While some sort of electric motor is usually part of the equation, the other power source can be anything from an internal combustion diesel, LPG, ethanol or hydrogen engine to a fuel cell stack.
In the case of the Prius - and all hybrids currently on sale - the petrol engine is the main driving force for the car, supplemented by the electric motor.
Both internal combustion engines and electric motors have their strengths and weaknesses; a hybrid engine aims to harness the best of each.
Internal combustion engines are powerful, cost-efficient and have excellent range. But for most of the time, especially in stop-start city traffic, they are simply too big and thirsty for the task at hand.
The typical Aussie family car rarely uses the 200-odd kilowatts of power at its disposal; normally only during overtaking or climbing steep hills.
Once a car is up to speed on a highway, or when it is crawling through city traffic, it really only needs about 20 or 30kW to keep it going. This is where the hybrid system makes sense.
The electric motor can do this task on its own, allowing the petrol unit to switch off completely and save fuel. Today�s electric motors can operate on their own at speeds of up to 40km/h, ideal for heavy traffic situations.
They also can take over from the petrol unit when the car is coasting on a downhill stretch of freeway. When that happens, the car is not burning fuel or emitting harmful substances into the atmosphere.
So why don�t we use the electric motor all the time? Because despite rapid development of the technology in recent years, the size of the batteries required to provide the sort of driving range motorists expect would severely compromise the overall design of the vehicle.
Even today the batteries in a hybrid car eat into boot space and add unwanted weight. The other problem is refuelling. While a petrol engine can be refuelled in minutes, batteries take several hours.
And while petrol engines work as well on the last few litres of fuel, an electric motor loses its effectiveness as the charge runs out. A hybrid system can offer the best of both worlds.
It basically consists of four major parts: a petrol engine, an electric motor, a battery pack to power the electric motor and a generator to recharge the battery.
The petrol engine not only drives the wheels but also runs the generator that charges the battery supplying power to the electric motor. In return, the electric motor can help to drive the wheels, saving fuel and reducing emissions.
On most hybrids, there is also a third power source � regenerative braking. When the car brakes or coasts down a hill, kinetic energy usually lost through heat in the braking system can be captured by the generator and converted into electricity that's stored in the battery.
If a hybrid car is stopped, the petrol engine shuts down. If only mild acceleration is needed, the electric motor provides the power, fed by the battery. If maximum power is needed, both the petrol engine and electric motor kick in.
When the car is up to speed, the petrol engine�s power is then split between driving the wheels and feeding the generator, which in turn supplies the electric motor.
The car�s on-board computer continuously monitors all the systems and distributes the power to maintain the most efficient equilibrium.
The next step in hybrid vehicles is the plug-in hybrid.


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At the end of Castlevania: Lords of Shadow, the origin of Dracula and his legendary connection with the Belmonts was revealed. Castlevania: Lords of Shadow 2 is the sequel to that 2010 reboot of the Castlevania series for play on Next-Gen game consoles. In it fans can expect thrilling new twists, action, adventure and challenges, as Dracula returns, this time to battle ultimate evil. Yet the dilemma is that he himself is greatly weakened and yearning for release from his immortal bonds. To succeed against the powerful threat he stands against, the vampire lord must reacquire his old powers - and only his castle holds the key. However, the famed Belmont clan also seeks his ultimate destruction.
- Become the ultimate anti-hero - Play as Dracula for the first time! Collect all of the vampire Lord's deadly powers and abilities.
- Open world environments – journey through a world of deep exploration in a "modern day" setting and get lost in the sprawling labyrinthine corridors within Dracula's castle, discovering new 'Lords of Shadow' environments and secrets along the way.
- Concludes the immersive and powerful storyline – lose yourself once again, in this final installment of the 'Lords of Shadow' trilogy. A fitting conclusion to the saga!
- Utilize the powers of the great vampire Lord - Master Dracula's vampiric aspects for deep strategic combat and devastating abilities! Develop Dracula's skills and techniques your way.
- Void & Chaos Powers – use these magical capabilities, each with its unique weapon, to change the course of a battle. Master each weapon to unlock devastating skills in which to overshadow your foes.
- Drink blood for eternal strength - Intensify your immortal powers by drinking the blood of your enemies. Blood fuels your Void & Chaos capabilities! Use focus to control the flow of blood and dominate your enemies.
Credit Cards (Mastercard & Visa)
We currently accept Mastercard and Visa credit cards. Your credit card information is secured and safeguarded when shopping at Youbeli.com. All information are encrypted and sent through secured and protected channels. As we are employing Mobile88 (iPay88) as our payment gateway, Mobile88 will be stated in your credit card statement.
Financial Process Exchange (FPX)
We also accepts FPX payment method as it is an ideal payment solution for customers who does not have a credit card. All that is needed to make an online payment is an online banking account. Payment is directly linked and connected to the customer's specified online banking account, transactions are usually instant and fast. Supported banks includes:
Cash Bank-in / Online Fund Transfer / Cheque
You may bank-in cash or cheque into the selected bank account below:
Bank : PUBLIC BANK
Account Name : YOUBUY ONLINE SDN BHD
Account No. : 3194858935
Bank : MAYBANK
Account Name : YOUBUY ONLINE SDN BHD
Account No. : 514048612629
Once you have successfully bank-in the amount, email the bank in slip to firstname.lastname@example.com with the stated Order No.
PayPal lets you send payments quickly and securely online using a credit card or bank account.
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We will provide you with the tracking number via email once we send out your package so that you can track it while it is in transit.
The lead shipping time to West Malaysia is approximately 3 to 4 working days after dispatch. An additional day is required for deliveries to East Malaysia.
You will be happy to know that delivery is free of charge throughout Malaysia!


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Director Davis Guggenheim follows three rock legends in his new documentary about the electric guitar, It Might Get Loud. In what looks to be an awesome documentary, we get to go behind the scenes and learn the tricks of the rock 'n roll trade from The Edge, Jack White, and Jimmy Page. They show us how they craft their sound and in the process we see some pretty awesome music get made. Check out the trailer after the jump.
© 2017 Spike Cable Networks. All Rights Reserved.


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Page: 1 of 1 Items For Sale - Jewelry / Jewelry / Initial
Item #PriceItem Name / Description
$ 5 each
$ 5 each
Large Vintage Single Initial Goldtone PendantsThis is a selection of large retro initial pendants each measuring 2 - 2-1/2" high. They are done in goldtone metal with the front having a cut-in texture design and the back smooth. Each has a loop at the top for hanging.. The following letters are available C - E - F - G - K - L - M - N - P - R and there is a discount available for 3 or more purchases. While they last !... more http://www.tace.com/i/43166.htmlFrom: Jarretts Jade Jewelry & Small Antiques
$ 5 see description
$ 5 see description
Lot Of Initial PinsThis is a lot of various initial pins - Oh, remember when we wore them on everything ! Here are a few and we hope to add more. The smaller goldtone ones run about 1". The one "T" in the center is by Trifari. The others in the picture are not siged. $5.00 each. The large 2-1/2" dark blue "B" is early plastic $3.00. Order by email with vendor # and description... more http://www.tace.com/i/34939.htmlFrom: Jarretts Jade Jewelry & Small Antiques
Sarah Coventry Initial PinsInitial pins by Sarah Coventry all measure about 1-1/2". See individual item below for more description. # 1 Initial J - Matte Goldtone # 2 Initial B - Light Shiney Goldtone Order by Vendor # on the top left and also Item # or letter being ordered. Look for more initial pins in the ... more http://www.tace.com/i/44606.htmlFrom: Jarretts Jade Jewelry & Small Antiques
Page: 1


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Toshiba has announced its super-slim touch-screen handset at the Mobile World Congress 2008 in Barcelona. Able to support both HSDPA and HSUPA, users will be able to surf the web, downloading and uploading pictures and videos at broadband speeds. The handset also boasts built-in GPS and A-GPS functions, a three-megapixel camera, FM radio and Windows Media Player Mobile to play video clips, listen to music and view images.
With its fetching black mirror finish and slim design, the Toshiba Portégé G810 looks great. It's also ideal for working when away from the office, thanks to its range of Microsoft Office Mobile applications. As well as supporting both HSDPA and HSUPA, the Portégé G810 is fully connected with GSM/GPRS/EDGE, 3G, Bluetooth and Wi-Fi.
The Toshiba Portégé G810 is expected to start shipping across Europe.
Mobile Industry Awards 2017 - B2B & Distribution Finalists
Mobile Industry Awards 2017 – Retail Finalists announced
Mobile Industry Awards 2017 - Best Ruggedised Device
Nazish Dossa Joins Brighstar as UK Managing Director.
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Huawei - 'Our 2017 focus is premium products'
Retail Special
S6 to re-ignite the market
Brightstar 20:20 and HTC join forces
Meeting the challenge of in-building coverage


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We help you find a New Malden ukulele teacher. Ukulele lessons are a brilliant method to begin an entertaining new hobby, acquire a new skill and gain confidence in ukulele. Please use our service to find New Malden ukulele teachers and select which matches your requirements so you can begin your ukulele tuition in New Malden today!
This is the best way to discover New Malden ukulele teachers in the UK, helping you find New Malden ukulele lessons close to home and New Malden tutors to assist with any instrument like piano, violin or guitar. We can help you find New Malden ukulele lessons or a New Malden ukulele tutor to prepare for ukulele exams and develop your ukulele skills! We order New Malden ukulele teachers near you according to their lesson charges and proximity to you.
If you are a ukulele teacher wishing to offer your ukulele tuition services in New Malden or anywhere else across the UK, please register with First Tutors: Music or learn about us here. Becoming a New Malden ukulele teacher is a wonderful way to improve your own skills through giving ukulele lessons while assisting someone else, all in the New Malden area!


text: I am toying with the idea of putting a Froyo Rom on my desire, but have several questions that aren't answered in other posts. 1. Will I need to unlock my phone before, during or after changing the Rom? I am not overly bothered by the orange stuff on my phone, but am worried about losing my WiFi connections! 2. Do I need a goldcard? Some instructions say yes, other no. None mention orange handsets specifically. 3. Which is the easiest & safest method to root my phone, if I want to put the Froyo Rom on it? 4. What if I want to change Roms - do I need to do the whole process again? I've seen something about a Rom manager, but where do you get it and how does it work? 5. Which Rom do I use? I've looked at some I'd the modacco forum ROMs, but most don't actually say what you do or don't get with them. 6. What's that difference between a2sd and a2sd+? Does it come as part of the ROM or so I news to get it separately? Sorry if this is in the wrong forum, but thought is ask here dost for guidance - most of the modacco stuff went sailing over my head!


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ALBANY, N.Y. -- Merlin Thomas, 48, was an employee in Albany County Correctional Facility for 18 years. On Saturday, that career ended.
Albany County Sheriff Craig Apple says Thomas smuggled items for inmates.
"We do have a number of surveillance photos indicating that people on the bottom row met the sergeant outside and handed off cash and packages in our parking lot," said Apple.
Those packages allegedly contained marijuana, tobacco and cellphones.
During a lockdown Saturday, those items were found in the jail along with homemade knives. The people who brought the cash and contraband are connected to the inmates who had the items.
But every person involved in this three-month investigation is connected to Thomas.
"You really look for honesty and trust with your employees other than strip-searching your employees prior to coming to work, you just pray to god that you have good stand up employees," said Apple. "People think, 'well the phone, how is that dangerous?' Well they can put hits on people, they can barter with people. Everything that gets smuggled in is dangerous."
Thomas and the other six civilians each face ten charges, including conspiracy and criminal possession of marijuana.
Apple says stricter rules will be put in place to stop something like this from happening again. The "no cell phone" policy in place will continue and people will be searched more thoroughly before entering the facility.
"Anybody that follows the sheriff's office knows we work really hard to try and build in roads into community and gain public trust and, again, it only takes one idiot like this to screw it up and set us back years," said Apple.


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Discussion & Communities
Blamonet Facebook Group
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Classic Blamonet Emojis & Smilies!
Random Chuck Norris Inspiration
July 4th is Independence day. And the day Chuck Norris was born. Coincidence? I think not.
© 1995-2037 - Blamonet &
Aaron Grant


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The College of Business will launch the Young Scholars Programme (YSP) to nurture outstanding students (admitted to the Bachelor of Business Administration programmes in September.) to become future leaders in the region/world.
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Many readers are likely familiar with the audit process that estate planners and high net worth individuals have been using to establish that they are not Minnesota residents for income and estate tax purposes. One element of these audits requires the individual to demonstrate that he or she is present in the state of Minnesota fewer than 183 days per year.
Some of these individuals continue to own a residence or cabin within Minnesota. In some cases the residence can be worth well in excess of $1,000,000. We have become aware that some counties may now be attempting to reclassify such property for real estate tax purposes from "non-homestead residential" to "noncommercial seasonal residential recreational". View the exemplar notice here.
The significance of this change in classification is that the rates for "noncommercial seasonal residential recreational" (sometimes called the "ma and pa resort") will likely be going up, a lot, for properties worth more than $1 million.
Minn Stat. 273.13 is the statute that governs our system of classifying real estate for tax purposes. Minnesota has a comparatively extensive system (some may call it Byzantine) for classifying property. Classification depends on actual use of the property, not its physical character. It is a factual issue. The procedure for disputing a change in classification is the same as that for disputing an increase in assessed value -- that is, file an appeal prior to April 30 of the year following the receipt of the notice.
To retain a "non-homestead residential" classification, it appears that the property needs to be occupied at least 250 days per year. This places many non-resident owners in a Catch-22, because they must in fact be present in the state fewer than 183 days.
It appears this change in classification may be the first step in increasing the tax rates on these properties. Minnesota Revenue publishes a table of tax rates for the various classifications of property. The current table can be found here. The relevant classifications are 4c(12), 4bb, and 1a. As can be seen from the note at the bottom of that page, under current rates seasonal properties are taxed at the same rate as residential non-homestead.
However, tax rates for each classification can be changed on a year-to-year basis, and as indicated in the notice sent by Hennepin County, taxes payable in 2015 will change for properties categorized as seasonal. We have not been able to unearth a rate table that will apply in 2015. However, given the fact that there is already an indication that the rates for high value 4c properties will increase in 2015, we feel it is prudent to resist the change in classification if possible.
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Preludes & elegies
A companion to The Dreambetween Symphony
The essential companion volume for readers of The Dreambetween Symphony…
Get ready for more tales of the Dreambetween as author M.S. Hund expands his historical fantasy saga with four new stories…
- La Chanson du Sang. An orphan boy lives in perpetual fear of his family's legacy of madness.
- Reunion (Kiss Me). A family reunion inspires fear, jealousy…and hope.
- Howl. A man who would rather be a dog is granted his wish.
- Dissolution. A woman tries to save her daughter as her fragile psyche falls apart and her world collapses around her.
But that's not all. Preludes & Elegies also contains a field guide to the world of the Dreambetween, with appendices that include…
- Details about the history of the dreaming blood and a character index for each time period explored in the novels.
- Reference lists and descriptions of the various places that appear in the novels, broken down by time period.
- A glossary detailing the workings of the Dreambetween and translation and pronunciation guides for some of the non-English words that appear in the novels.


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Will Smith – Gettin' Jiggy Wit It (Viceroy "Jet Life" Remix)
Really, I don't have to say much. Viceroy continues the "Jet Life" series remixing the Fresh Prince. Summertime all the time. And it's free. Air Fresh Prince/Jazzy Jeff-shake-it-up-and-snap-while-going-psshh me. You know, like from the show? How hot is Hilary by the way?
2 Comments
Cliff July 25, 2012 at 1:26 am
Oh, and I got shit for you for at least the next two days. So, if you get back tomorrow, I may show up at Harry's Bar, so you know, keep your eyes open.
P.I.A July 27, 2012 at 8:57 pm


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Street magician's elastic band trick upstaged by stunner's cleavage in background
A STREET magician would have been delighted to find the video of a trick he'd performed had gone viral – only to find out the reason why.
The clip begins showing the nifty-fingered conjurer entertaining two volunteers, stretching two interlocked elastic bands between his hands.
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After returning to the middle, he wiggles one against another and it suddenly becomes free – blowing the minds of the watching pair.
The clip went viral after being shared on social media – although users had clearly found a different reason to get excited than the magic.
"I am a heterosexual woman and I cannot stop staring at this woman's lovely breasts long enough to see the actual trick," one viewer wrote.
Another comment read: "Let's just get this over with. That girl's rack is insane."
"The real magic is when her chest comes into focus," agreed another.
The original video had formed part of a kickstarter campaign by a company called Ellusionist, aiming to crowdsource a kit teaching people how to perform 47 different tricks.
Earlier this year, TV magician Dynamo was forced to brush off an awkward botched trick during a show at London's O2 arena.
The Bolton-born performer had attempted to call a volunteer's phone while trapped inside a glass bottle – but failed miserably.


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by Michelle N. Johnson
So, today is the last day of Endometrosis Awareness Month. I hope you guys have have been educated enlightened and empowered by all the posts, info, and dialogue I've been sharing with you all month. Hugs and Thank Yous to EVERYBODY who pitched in and helped me to make this happen! When I started this, I was asked to complete a challenge by answering a series of questions about my personal experience to help others learn more about what it is actually like to live w/ this disease. I decided to share my challenge with you as my last 'official' post for awareness month. I hope you'll take a few moments to read this and continue to help me Fight Fiercely, not just this day but EVERY DAY until we finally put an END to Endo!! Peace & Love Y'all!!!
Endometriosis Awareness - 31 Day Challenge
Day 1. For me, living with Endometriosis is: EXTREMELY UNPREDICTABLE
Day 2. I was diagnosed with Endometriosis in the year: 2008
Day 3. But I had symptoms since: Looking back, probably around 2000
Day 4. The biggest adjustment I've had to make is: sacrificing a certain quality of life and learning how to function optimally while being in some sort of semi - medicated state a great majority of the time.
Day 5. Most people assume: #1 It must not be 'that bad' because I don't 'look sick' and #2 that because I had surgery, 'everything is okay now…right?'
Day 6. These are the treatments I have tried so far: surgery, continuous birth control pills (multiple/various types), Hormone/'Pain Management' Therapies (Lupron, Danazol, Neurontin, Elavil, Norco, Vocodin, Ibuporphen 800 up to 4x's a day)
Day 7. The hardest part about mornings are: getting out of bed, getting out of bed on time, getting out of bed with a clear focused mind and train of thought.
Day 8. My Favorite medical TV show is: House
Day 9. A gadget I couldn't live without is: my mp3 player
Day 10. The hardest part about nights is: GETTING ENOUGH SLEEP/REST, Getting a truly restful, peaceful, undisturbed sleep, Insomnia, Being able to get sleep and stay asleep when the pain is bad, Having the meds disrupt your sleep natural cycle, having only a short period of time to be functional and/or get things done before your pain meds kick in and zombie you out!!!{usus60-90 mins}
Day 11. Each day I take 4-7 pills & vitamins: I take one bc pill, 2 Elavil, up to 4 IB 800s and up to 3 Norco/Vicodin depending on the pain/symptoms, and 1 multivitamin or Prenatal ( how 'bout THAT for irony – Endo is on of the top three causes of infertility and yet Pre-Natal vitamins are often given to help give needed vitamins/minerals to the reproductive system)
Day 12. Regarding alternative treatments I use: massage therapy self are techniques (obviously, sine I'm a Massage Therapist), Pelvic Floor Physical Therapy, and aromatherapy w/ my hot packs/compresses/baths/showers and massage PRAYER
Day 13. If I had to choose between invisible illness or visible I would choose: NOT TO BE SICK at all! W/an invisible illness you often get ridiculed b/c you don't 'look sick', so its hard for people to understand and /or empathize. With a visible illness, you may get more understanding, but still get ridiculed and/or shunned b/c people CAN see your symptoms. It's a lose-lose
Day 14. Regarding working and career: It has been SEVERELY affected by having Endo. As a business
owner, I not only am the lead therapist, but I'm the president, administrator, the HR person, the bookkeeper, etc. There are days when I can't meet deadlines or meetings or when I have to postpone projects, or simply can't go to work. That bothers me b/c I tend to be a perfectionist and I pride myself on giving my clients and business partners the ultimate in excellence of service. I hate it when I can't give 100% of myself to my clients, my circle, or myself. When I'm in crisis mode, I often struggle w/ guilt of being 'idle' or 'lazy' b/c even when I'm lying there doubled over in pain crying my eyes out, or so doped up I literally can't see straight – all I can think about is 'what needs to be done', 'what I need to be doing', all the projects/meetings/networking oops that are falling by the wayside. When I'm able to I push through, I do, and when I can't I feel absolutely wretched about it!!!!
Day 15. People would be surprised to know: The seriousness of the degree and the severity of my condition. Because it can't be seen outwardly, and b/c (until now), I haven't been vocal about it, the few people that (did) now assume that because I've had surgery for it – that everything is 'ok now'. I often don't let it show on the outside all that's going on inside, I've become the MASTER of the 'smile and nod.'
Day 16. The hardest thing to accept about my new reality has been: My new reality! Having to RE-adjust nearly EVERYTHING about the way I live.
Day 17. Something I never thought I could do with my illness that I did was: 'Come out' publicly and allow myself to be a spokes-person AND a FACE for this disease so that hopefully my trials with this can lead to someone else's triumph
Day 18. I feel that Endometriosis Awareness is: EXTREMELY IMPORTANT! Before being diagnosed I had never even heard of this disease. After living with it for four years and STILL learning new things about it, I became really pressed, and inspired to educate enlighten and empower other women, and their families and loved ones to truly learn how to be survivors and not just sufferers of this disease
Day 19. Something I really miss doing since I was diagnosed is: TMI Alert: sleeping with no panties on!!!
Day 20. It was really hard to have to give up: #1 My left ovary and fallopian tube in order to preserve the rest of me so that I can still be able to have children if/when I'm ready, #2 that super sexy size 14 that I used to be and lost b/c of all the drugs and hormones and their side effects wreaking having on my body! #3 sexy/cute/'girlie' underwear, matching bra and panty sets!
Day 21. A new hobby I have taken up since my diagnosis is: researching reading and learning as much as I can and educating as many people as I can that will let me talk their ears off about it.
Day 22. If I could have one day of feeling normal again I would: start kickboxing again, go out w/ my girls and juke my lil heart away until I sweat my perm out!!! Go panty-less whenever I felt like it!!!
Day 23. Endometriosis has taught me: that although it came close, that what didn't kill me will DEF made me stronger; it taught me that real life is what HAPPENS to you while you're making PLANS for it; it has taught me that the ONLY thing constant in life is CHANGE; It taught me that I can face some of the darkest nights that life can throw at me, but the Diamond in me we will SOMEHOW find a way to shine through, it taught me that you can still be in the midst of YOUR wilderness and STILL help someone else find a way out of theirs.
Day 24. Want to know a secret? One thing people say that gets under my skin is: #1 You need to hurry up and get pregnant – you KNOW you ain't got that much time left {THIS coming from DOCTORS!!! IKR!!!!} #2 'oooh girl, you go through all that - I would just get they hysterectomy and be done with it!! {THIS coming from chicks w/ a litter of kids runnin' around}
Day 25. But I love it when people: Are genuinely concerned about me. Not feeling 'sorry for me', but sincerely being compassionate and trying to understand. Gimme hugs when I need them, let me vent when I need too w/o judgement
Day 26. My favorite motto, scripture, quote that gets me through tough times is: #1 For I know the plans I have for you," declares the LORD, "plans to prosper you and not to harm yo
u, plans to give you hope and a future."
~Jeremiah 29:11#2 - I need you, you meed me, we all need each other - ~Pastor Danjuma Gibson
Day 27. When someone is diagnosed I'd like to tell them: If you need me, I am here. Few people can say this and mean it, but I KNOW what you are going through.
Day 28. Something that has surprised me about living with an illness is: That I (unknowingly) went so long and lived so many years with this w/o ever being diagnosed
Day 29. The nicest thing someone did for me when I wasn't feeling well was:{different people at different times} Bring me food when I wasn't able to stand/walk/cook for myself; took me to the doctor and stayed with me; Ask me if I was okay and if they could do anything to help; Held me and rubbed my tummy and my lower back when it was killing me; Helped me throw up so I could rid of that nauseated 'blah' feeling!
Day 30. I'm involved with Endometriosis awareness month because: OVER 176 MILLION WOMEN, TEENS, AND GIRLS suffer from this disease and only a fraction of people have ever even heard the word!
Day 31. The fact that you read this list makes me feel: like you cared enough about me, that I meant enough to you, that you would take the time to see through my eyes, what it's like to live with endometriosis.
copyright March 31, 2012
Contact Michelle N. Johnson


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Study of the pathophysiology and treatment of anemia of prematurity is facilitated by direct measurement of red cell volume (RCV) utilizing microliter quantities of blood samples. Our objective was to compare concurrent measurements of multiple direct RCV methods in infants.
Eighteen preterm infants receiving clinically-indicated transfusions had concurrent flow cytometric determinations of RCV and 24 h red blood cell (RBC) recovery based on donor-recipient differences of biotin–labeled RBCs (BioRBCs), Kidd antigen mismatched RBCs, and HbF positive (HbF+) RBCs. HPLC was also used to measure HbF and HbA protein concentrations for RCV determination.
Concurrent RCV measurements using BioRBCs (18 and 54 μg/ml), Kidd antigen, and HbF flow cytometry were not statistically different compared to RCVs measured using the reference BioRBC density (6 μg/ml). In contrast, the HbF HPLC method over estimated RCV by 45% compared to the reference method. All methods demonstrated 100% 24 h post-transfusion RBC recovery (PTR24).
Because BioRBC, Kidd antigen, and HbF flow cytometry are safe and accurate methods requiring <10 μl of patient blood to determine RCV and PTR24 in preterm infants, they can be useful in clinical and research studies of anemia and other conditions.
Anemia is a common and serious clinical problem among critically ill premature infants. Important contributors to anemia of prematurity include low Hb at birth, blood loss due to phlebotomy for laboratory testing, and inability of the infant to produce sufficient red blood cells (RBCs) to overcome blood loss and postnatal expansion of blood volume with rapid growth. Currently, the two most common treatments for anemia in premature infants are allogeneic RBC transfusion and recombinant erythropoietin (rEPO) (1). To assess the effectiveness of these and other therapies, safe, accurate, and versatile methods for determining circulating red cell volume (RCV), blood volume (BV), and 24 h post-transfusion recovery (PTR24) of transfused blood are needed. Among ill premature infants, RCV is deemed a better indicator of the need for RBC transfusion than either whole blood Hb or hematocrit (HCT) levels (2, 3).
Flow cytometric enumeration of RBCs can be used in determining RCV and BV and requires only a few microliters of blood. This method relies on its ability to discriminate transfused from endogenously produced RBC populations. Flow cytometry has been used to detect fetomaternal hemorrhage (4, 5), determine RBC phenotype following bone marrow transplant (6), measure RCV (7-9), detect illicit blood transfusions in athletes (10), and determine RBC survival (11, 12). Recently, our group has for the first time demonstrated that RCV can be accurately determined in adult humans and sheep using multiple distinct populations of biotin–labeled RBCs (BioRBCs) enumerated by flow cytometry (8, 13).
In the present study, we extend this previous work with the objective of comparing concurrent RCV determinations using four different methods in very low birth weight (VLBW) premature infants weighing < 1.5 kg at birth: flow cytometric methods, 1) multi-density BioRBC; 2) Kidd antigen (Jka and Jkb) mismatches between adult donor and infant RBCs; 3) dilution of infant RBCs containing primarily HbF by donor RBCs containing primarily HbA; and 4) a non-flow cytometric method, change in proportion of HbA and HbF proteins measured by HPLC. None of these three methods requires labeling of donor RBCs prior to transfusion. Because of extremely limited recovery data for stored donor blood in infants, we also determined the PTR24 of transfused RBCs using the same methods.
Based on our prior findings in adults (8, 11), we hypothesized that 1) RCV determined using RBCs biotinylated at three high biotin densities (18, 54 and 162 μg of biotinylating reagent per ml RBC), Kidd antigen mismatch, and Hb type differences methods would not differ significantly from RCV determined using a previously validated low BioRBC density (6 μg/ml) as the reference method; 2) allogeneic RBCs would completely equilibrate within first 20 min post-transfusion (i.e., there would be no mixing or spleen effect); and 3) PTR24 assessed by all the methods would not be significantly different than 100%.
Eighteen premature infants with gestational ages at birth between 26 and 30 wks were studied (Table 1). Mean (± SD) birth weight was 0.96 ± 0.24 kg (range 0.39 to 1.40 kg). On the day the study transfusion was administrated, infants were 18 ± 14 d old (range 1 to 45 d) with body weights of 1.21 ± 0.45 kg (range 0.37 to 2.21 kg).
The four discrete BioRBC densities and the unlabeled RBCs demonstrated complete separation, permitting accurate enumeration of each of the four BioRBC populations (Figure 1a). Pre- and post-transfusion histograms based on Kidd Jka and Jkb antigens also exhibited no peak overlap (Figure 1b and c). Because the study transfusion was the first transfusion for this specific infant, there was only a single peak (Jkb-RBCs) originating from the infant in the pre-transfusion sample (Figure 1b). After transfusion of Jkb mismatched RBCs, the expected two peaks were present. In contrast, both pre- and post-transfusion samples from an infant who had already received Kidd antigen mismatched blood prior to the study showed two distinct peaks (Figure 1c). After the study transfusion, the proportion of Jka− donor RBCs increased while the proportion of Jka+ infant RBCs decreased. The histograms for HbF+RBCs in subjects without and with previous transfusions demonstrated similar peak patterns to the two Kidd antigens (Figure 1d and e).
RCV measured at 20 min post-transfusion using the reference BioRBC density agreed well with RCV measured using the higher BioRBC densities, Kidd antigen, and HbF flow cytometry methods, but not with the HbF HPLC method (Figure 2). The agreement of the reference BioRBC density and densities 18, 54, 162 μg/ml were greater than for the Kidd antigen and HbF flow cytometry methods as judged by range of the 95% CI for both the slope and y intercept (Figure 2a-c; Table 2). Among all the methods, the HbF HPLC method showed the weakest correlation with the reference BioRBC density (R2 = 0.869 and the widest 95% CIs) (Figure 2f; Table 2).
There was a single notable outlier RCV determined using the Kidd antigen and both HbF methods (Subject 13 shown as a triangle in Figure 2d-f). RCV values for this subject were greater than mean ± 2 SD relative to the other RCV values. We have no explanation for this outlier. This point was excluded from the regression analyses.
In our previous adult sheep studies, we observed a slowly equilibrating pool of intravascular RBCs that accounted for about one-third of the total circulating RBCs (13). This was likely due to pool of RBCs sequestered in the ovine spleen that equilibrates slowly with the circulating RBCs and BioRBCs. To determine whether a similar phenomenon was present in VLBW infants, we compared paired RCV values at 20 and 90 min post-transfusion using a linear mixed model analysis for repeated measures (Table 3). Differences between mean RCV values at 20 and 90 min were not significant, thus indicating no reservoir of slowly mixing RBCs in the spleen of VLBW infants.
Two statistical comparisons of the RCV results from the individual methods were performed: linear mixed model analysis with repeated measures and Bland-Altman plot analysis. In both comparisons RCV determined by individual methods was expressed as a ratio relative to the reference BioRBC density RCV. Because mean post-transfusion RCV values at 20 and 90 min were not significantly different, the linear mixed model analysis of the methods was based on the RCV values averaged for these two post-transfusion times. In this analysis results were deemed not different if the ± 95% CI ratio included 1.0. Mean RCV ratios were not significantly different among the methods with two exceptions: 1) 162 μg/ml BioRBC density overestimated the RCV by 4.4%, a difference likely to be unimportant for most clinical and research applications; and 2) HbF HPLC overestimated RCV by 44.9%, a large difference (Table 4).
In the second statistical comparison, agreement among the methods for measuring RCV at 20 min post-transfusion was assessed by Bland-Altman plots (Figure 3). Using the reference BioRBC density as the denominator, the ratios of the RCV for each method demonstrated agreement among all methods except for HbF HPLC. For the three higher BioRBC densities, the 95% limits of agreement with the reference method were as follows: −3.3% to +6.5% for 18 μg/ml; −4.4% to +7.9% for 54 μg/ml; and −7.6% to +15.6% for 162 μg/ml. Agreement with the reference BioRBC method was not as close for the Kidd antigen (Jkb) or the HbF flow cytometry methods. The limits of agreement were −6.4% to +19.2% for Jkb, and −9.6% to +20.1% for HbF flow cytometry. The HbF HPLC method significantly overestimated RCV; the limits of agreement were +6.8% to +56.5%. As with the regression analyses, Subject 13's outlier data point was excluded from the Kidd antigen and both HbF analyses. Due to the small sample number (n = 4), comparison of RCV values by the Kidd Jka method against the reference BioRBC density was not performed.
To determine PTR24, we assessed the percentage of transfused RBCs remaining 24 h post transfusion using the four BioRBC densities, Kidd antigen, and both HbF methods. Differences in means between 20 min and 24 h for each method were tested for significance by mixed model analysis for repeated measures. Mean PTR24 measured by BioRBC for the 14 study subjects were not different between 20 min and 24 h (Table 5). Indeed, PTR24was not significantly different from 100% for any of the methods (Bonferroni adjusted P-values: 0.14 to >0.99), thus providing evidence that the reticuloendothelial system of VLBW infants does not remove any of the transfused RBCs — whether labeled with biotin or differentiated from recipient RBCs by minor antigen or HbF — within the first 24 h post-transfusion. The mean percent change from 20 min ranged from a decrease of –3.7% (95% CI: –9.8%, 2.8%) for HbF flow cytometry, to an increase of 3.7% (95% CI: –2.4%, 10.2%) for Kidd Jkb. Due to the small sample number, Kidd Jka method (2 data points) data were not analyzed. Because four subjects did not have an appropriate 24 h post-transfusion sample, they were also excluded.
Because RCV and BV values at 20 min post-transfusion normalized by body weight are of clinical interest, we calculated the mean pre- and post-transfusion RCV (ml/kg) and BV (ml/kg) using each of the four BioRBC densities, Kidd antigen, and both HbF methods. Mean pre-transfusion RCV ranged between 18.9 and 23.6 ml/kg for the flow cytometric methods, but was 30.8 ml/kg for HbF HPLC method (Figure 4a). Following RBC transfusion, infant mean RCV ml/kg increased about 50% for all methods except for RCV determined by HbF HPLC method where the mean increase was only 37%. Mean post-transfusion BV ranged between 73.7 and 85.1 ml/kg when measured using the flow cytometric methods. In contrast, mean post-transfusion BV measured by the HbF HPLC method was 104.2 ml/kg (Figure 4b).
The present study determined concurrent RCV and PTR24 measured in VLBW infants undergoing clinically indicated allogeneic RBC transfusions using multiple methods of analysis. These methods included flow cytometric analysis of multi-density-labeled BioRBCs, Kidd antigen and HbF positive RBCs, and HbF protein by HPLC. Our study provide evidence that RCV measured using lowest density reference BioRBC (6 μg/ml) agreed well with RCV measured using BioRBC densities 18 and 54 μg/ml, Kidd antigen, and HbF flow cytometry. PTR24 also agreed well among all methods. All methods required only microliter volumes of blood and none were associated with identifiable safety issues.
Since the 1970s, accurate measurement of RCV in infants has not been reported, primarily for ethical concerns related to unnecessary radiation exposure of infants enrolled in research studies. Simultaneous determinations of multiple distinct RBC populations are not possible with 51Cr or with most other RBC labeling methods. Accordingly, we contend that the multi-density BioRBC method utilizing <10 μl sample volumes offers substantial potential for advancing our understanding of hematological and cardiorespiratory conditions and disease processes in the smallest, least mature anemic premature infants. Although a large majority of such infants develop significant anemia requiring RBC transfusion, RBC kinetic studies have never before been performed in the smallest, most fragile, high risk patients being cared for today.
Like the multi-density BioRBC method, the Kidd antigen and both HbF methods utilize microliter volumes of blood. Although the latter methods can only be performed at the time an allogeneic RBC transfusion is administrated, neither requires ex vivo pre-transfusion RBC manipulation (i.e., labeling). This feature of the Kidd antigen and HbF methods negates all safety issues other than those inherently associated with clinically ordered RBC transfusions.
In the present study, we first demonstrated the agreement among RCV measurements by the methods mentioned above using regression analysis (Figure 2; Table 2). For these comparisons the lowest BioRBC density was selected as the reference method. This was based of our prior data-supported hypothesis that labeling with the lowest biotin density would perturb RBCs the least (11, 12). RCV determined by the four BioRBC densities demonstrated a very close agreement with one another (R2 = 0.973 to 0.997). This observation is similar to our previous RCV results in adults using the same four BioRBC densities (8). Similar agreement was also observed in our adult sheep multi-density BioRBC RCV study with the exception that the highest BioRBC density studied (96 μg/ml for sheep) slightly overestimated RCV (7.6%) compared to the lowest BioRBC density (12 μg/ml) (13). Although in the present study, the non-BioRBC RCV determinations also agreed with the reference BioRBC density, level of agreement tended not to be as strong. When the inexplicable outlier was excluded from the Kidd antigen and HbF flow cytometry analyses, RCV results of these two methods demonstrated stronger agreement with the reference BioRBC density. The HPLC method of HbF determination demonstrated the weakest correlation with the reference BioRBC density, but HbF HPLC method also had the fewest number of usable data pairs (n = 6).
In addition, we demonstrated that 20 min was sufficiently long for complete equilibration of transfused RBCs with the infant's RBCs. RCV determinations can be perturbed by changes in the enrichment of labeled or mismatched RBCs in the immediate (minutes) post-transfusion period as a result of incomplete mixing or equilibration with body distribution pools. As an example of the latter, in sheep we reported an apparent 37% increase in RCV during the first 60 min post-transfusion as the circulating transfused RBCs equilibrated with the non-circulating pool of RBCs in the ovine (13). In the present study, we observed no significant change in RCV determinations for any of the RCV methods between 20 and 90 min post-transfusion (Table 3). These RCV observations are consistent with our prior observations of no change in RCV after 20 min in adult humans (8). They are also consistent with results reported by Bratteby in term infants in which 51Cr-labeled autologous RBCs reached steady-state levels after an initial 10 min mixing phase (14).
We also showed that different BioRBC densities, Kidd antigen mismatch and HbF flow cytometric methods can be interchangeably used to determine RCV in infants undergoing RBC transfusions. Although statistical analyses indicated a significant (P = 0.02), but clinically unimportant, 4.4% over estimate of mean RCV (95% CI 0.7%to 8.2%) for the 162 μg/ml BioRBC density (Table 4), we concluded that 162 μg/ml BioRBC density can be used for RCV determination. In contrast, the HbF HPLC method overestimated RCV by 45%, clinically important discrepancy makes the HPLC method unsuitable for RCV determination.
As hypothesized based on our previous report in infants (15), PTR24 of stored allogeneic RBCs with all methods was not significantly different from 100%, i.e., relative to the 20 min post-transfusion sample. In addition, PTR24 values of 100% as determined by methods that do not require ex vivo RBC modification, i.e., the Kidd antigen and both HbF methods, supports our hypothesis that biotinylation of RBCs does not cause a change in PTR24 in infants. These findings stand in contrast to stored RBC PTR24 data in adults in which PTR24 data show a significant decline relative to immediate post-transfusion enrichment (16).
Using the RCV data determined in this study BV per kg was derived by dividing RCV by simultaneously determined hematocrit measurements. Mean post-transfusion BV derived in this manner ranged from 73.7 to 85.1 ml/kg for the three flow cytometric methods (Figure 4). Because accurate hematocrit values immediately prior to transfusion were not available, it was not possible to determine pre-transfusion BV. The close agreement among the post-transfusion BV determined among the flow cytometry methods and the lack of close agreement in the measurement of RCV using HbF protein by HPLC method (104.2 ml/kg) indicates that the latter method is associated with one or more unidentified systematic errors. While the derived BV values observed are within the ranges previously reported (17-20), reports of BV are highly variable because some — as in the present study — are derived from only RCV and HCT, while others are based on direct measurement of both RCV and plasma volume, or plasma volume alone. Studies using the latter approach report larger BVs because of the larger distribution space of the plasma (18, 21). In considering infant BV measurements derived using only RCV and HCT values must also be recognized that HCT determinations in infants are not as reproducible as in older children and adults. This is because of discrepancies in HCT with capillary versus venous sampling (22).
In conclusion, the present study demonstrates that RCV can be safely measured in premature VLBW infants using <10 μl of blood without exposure to radioactivity. Evidence of strong agreement in the concurrent RCV determinations among the flow cytometric methods suggests that these methods provide true and accurate direct measurement of infant RCV. Because of the high sensitivity, safety, and reproducibility of the multi-density BioRBC method and because BioRBC studies can be performed using either autologous or allogeneic RBCs (or both simultaneously), we contend that RCV measurements using biotin-labeled RBCs is the most useful and versatile in infants. Kidd antigen and HbF flow cytometry methods also perform well for evaluating the kinetics of allogeneic donor blood and have the advantage of not requiring ex-vivo labeling. The application of these newer flow cytometric methods in measuring RCV in the smallest, least mature, and critically ill infants are useful tools in advancing our understanding of their hematological and cardiorespiratory conditions and disease processes.
Institutional Review Boards at the University of Iowa (performance site) and the University of Arkansas for Medical Sciences (analysis site) approved the study. This included approval for administering Kidd mismatched RBCs. Informed written parental consent was obtained.
A prospective, convenience sample of 18 newborn infants eligible for study included those being treated with the expectation of survival who were <31 wks gestation and expected to receive a clinically indicated RBC transfusion. Infants with congenital anomalies were excluded. Infants who had received prior RBC transfusions were not excluded. Eight of 18 subjects had received one or more previous allogeneic RBC transfusions.
When a clinical RBC transfusion (15 ml/kg packed RBCs with HCT of ~75%) was ordered, equal aliquots of allogeneic RBC (1.0 ml/kg packed RBCs for each density) were labeled at four discrete biotin densities as described below. Each BioRBC population transfused was calculated to produce a final enrichment of approximately 2% of the total number of circulating RBCs. Immediately following the infusion of the 11 ml/kg of the unlabeled clinical transfusion, the remaining 4 ml/kg containing the four BioRBC densities were infused over 10 min.
Potential donor blood units were screened to identify Kidd antigen mismatches with infant study subjects when possible. Donor blood units identified as mismatches were then used when a clinical transfusion was ordered. Some infants received mismatched RBCs by chance i.e. the donor was not pre-screened and selected for that infant.
Pre- and post-RBC transfusion blood samples were collected. Post-transfusion infant samples included 150 μl volumes drawn within 20 to 90 min following the completion of the BioRBC transfusion. Two of the 18 infants did not have 20 min post-transfusion samples, so we used 70 and 90 min samples for those two infants as earliest post-transfusion samples. A complete blood count analysis was performed using an automated hematology analyzer (Sysmex XE-2100, Sysmex Corp., Kobe, Japan) to determine RBC count and HCT on the first post-transfusion sample.
The method for biotin labeling of RBCs at distinct densities in adult humans was used and aliquots of post-transfusion blood samples were processed for flow cytometric analysis of BioRBCs as described previously (8). Each distinct BioRBC population was detected as a separate peak on the post-transfusion histogram and the number of events counted under each peak used to determine the enrichment of each BioRBC population.
For Jka+ or Jkb+ RBC flow cytometry analysis, triplicate 3 μl aliquots of the pre- and post-transfusion samples were washed to remove plasma proteins following the same procedures as for BioRBCs (13, 23). The RBCs were resuspended in 0.3 ml of wash buffer with 2% BSA (A30075, RPI Corp., Mount Prospect, IL) and incubated with 5 μl of anti-Jka or anti-Jkb primary antibody (Immucor Inc., Norcross, GA) overnight at room temperature with continuous mixing on a rotating wheel. These RBCs were washed twice and were incubated at 37 °C for 1 h with 5 μl of Alexa Fluor 488 conjugated secondary antibody (H10120, Invitrogen, Carlsbad, CA). RBCs were then washed three times and resuspended in wash buffer with 2% BSA (RPI Corp.) that had been filtered through a 0.2 micron filter to a final volume of 0.5 ml. Jka+ or Jkb+ RBCs were enumerated as described above for BioRBCs.
The method for flow cytometric analysis of HbF+RBCs used to detect HbF+ cells following feto-maternal hemorrhage (4) was adapted to determine the RCV in premature infants undergoing adult donor RBC transfusion. The percent HbF+RBCs was determined using Invitrogen Fetal Hemoglobin Test (Invitrogen) with FITC conjugated monoclonal antibody directed against HbF. The manufacturer's manual was followed with the following minor modifications. Duplicate 1.5 μl whole blood samples (~ 5×106 cells) were fixed in 0.25 ml of 0.05% glutaraldehyde. RBCs were permeabilized in 0.2 ml of 0.1% Triton X-100. Washed and pelleted RBCs were resuspended in 80 μl of PBS/BSA (Invitrogen/RPI Corp.) and incubated with FITC conjugated HbF monoclonal antibody for 30 min at room temperature. RBCs were washed and resuspended in 0.4 ml of 2% paraformaldehyde prior to flow cytometry analysis. HbF+RBCs were enumerated as described above for BioRBCs.
Analysis of HbA and HbF proteins by HPLC was performed using the method described by Witzhandler et al. (24). Briefly, analysis of stored blood samples maintained at −80°C was performed using a Dionex ProPac SCX-10 column (Thermo Scientific, Sunnyvale, CA) interfaced with a Waters 600E pump controller (GenTech Scientific, Arcade, NY), a SPD-10AV UV detector (Shimadzu Scientific Instruments, Columbia, MD), and an auto sampler (Perkin Elmer Inc., San Jose, CA). Replicate hemolysate samples were prepared by mixing 5 μl of blood in 1 ml of eluent solution (50 mM sodium phosphate and 2 mM potassium cyanide, pH 6.0). Ten μl of sample hemolysate was injected and the relative percentages of HbF and HbA proteins derived by using area under the peak analysis using Shimadzu data management software. With this method, acetylated HbF elutes as a separate peak that is added to the larger non-acetylated HbF to obtain the total percentage of HbF peak protein in the sample.
Post-transfusion RCV was calculated for all methods using the dilution principle as follows:
where the total number of RBC (Tx)= Σ [volume of individual infusate × RBC concentration]. The volume of infusate for individual BioRBC densities was determined gravimetrically (11) using a specific gravity for transfused RBCs of 1.05 (g/ml); %A = percentage of individual BioRBC densities (6, 18, 54, or 162 μg/ml), Jka+, Jkb+, or HbF+RBCs (for HPLC, %HbF protein); Tx = transfusion; pre-Tx = pre-transfusion sample; post-Tx = post-transfusion sample; and MCV = mean corpuscular volume.
Although all study subjects had RCV measurements by BioRBC performed at all four densities, only 14 had Kidd antigen mismatches. For two infants, technical difficulties precluded inclusion of the Kidd antigen results, leaving 12 RCV determinations. For the HbF flow cytometry and HbF HPLC determinations, these methods had not been fully developed in our laboratory at the time the study was initiated: only 11 had RCV determined by HbF flow cytometry, and only 7 had RCV determined by HbF HPLC.
The post-transfusion BV ml/kg of each study subject was calculated using the following formula:
BV (ml/kg) = RCV(ml/kg)/ post-transfusion HCT.
Because pre-transfusion HCT was not measured, pre-transfusion RCV was determined by subtracting the total volume of RBCs transfused from post-transfusion RCV.
Test for differences in mean RCV among the methods was performed by linear mixed model analysis for repeated measures. The natural log transformation of RCV was used to normalize the data distribution. The mixed model included method, time, and method by time interactions. In addition to estimating the fixed effects in the mixed model, this method of analysis allows for selection of the covariance structure that best fits the variance-covariance of the RCVs for the different methods and times in the same subject. From the linear mixed model analysis, the test for method by time interaction and the test for the post-transfusion time effect were examined. To report mean RCV in the original scale, the method ln(RCV) means from the fitted linear mixed model were back transformed, and the standard error computed using the delta method The differences in ln(RCV) between the BioRBC densities were also back-transformed. This provided estimates for the mean RCV ratio relative to reference BioRBC density.
Bland-Altman plots of the observed RCV ratio for a given density, Kidd antigen (Jkb), or both HbF methods relative to reference BioRBC method were included to display the distribution of ratios. The plots also show the mean ratio and the 95% limits of agreement for the mean ratio (i.e., mean ± 1.96 SD).
Descriptive data were expressed as the mean ± SD. Bonferroni correction for multiple comparisons was used. All the statistical analyses were performed with computer software (SAS, Version 9.3, 2002-2008, SAS Institute, Inc., Cary, NC). A P-value <0.05 was considered significant.
We acknowledge helpful discussions with Robert S. Franco and Peter Veng-Pedersen, regarding technical and theoretical aspects of the present study. We appreciate the many contributions of the clinical laboratory staff led by Mitchell J. Owen and overseen by Matthew D. Krasowski, M.D., Ph.D., without which this work would not have been possible. We acknowledge the many outstanding clinical research contributions of Iowa's neonatal nurse research team (Karen Johnson, Laura Knosp, Nancy Krutzfield, Ruthann Schrock Sara Scott, and Jin Zhou) and the research laboratory team (Earl Gingerich and Jessica Goehring). We thank S. Isil Cevik for her assistance with preparation of figures and Mark Hart for valuable secretarial assistance. The Sysmex XE-2100 automatic hematology analyzer used in this study was generously provided on an on-loan basis from Sysmex Corporation, Kobe, Japan. Last, we appreciate the willingness of study subject families in allowing their infants to participate.
STATEMENT OF FINANCIAL SUPPORT: This publication was supported in part by U.S. National Institutes of Health (NIH) grant P01 HL046925, by the Thrasher Research Fund 0285-3 (Salt Lake City, UT), and by grant UL1RR024979 from the National Center for Research Resources (NCRR). The paper's contents are solely the responsibility of the authors and do not represent the official views of the NIH, the Thrasher Research Fund, or the NCRR.
Note: This trial has been registered at www.clinicaltrials.gov (identifier NCT 00731588).
DISCLOSURE: The authors have nothing to disclose.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Date Posted: Thursday, August 18th, 2016
Categories: Department of Justice DOJ Press Releases
With Delaware's primary elections coming up in less than a month and the general election shortly thereafter, the Attorney General wishes to notify the public that the Delaware Department of Justice will be strictly enforcing the voter intimidation provisions of the Delaware Constitution. Those provisions make it a crime in Delaware for a person to "by force, threat, menace or intimidation, prevent or hinder, or attempt to prevent or hinder, any person…qualified to vote from voting according to said person's choice at any such general, special or municipal election."
The DOJ Office of Civil Rights and Public Trust has provided written guidance to the state Department of Elections that will be provided to poll workers that requests that any observed cases of voter intimidation be reported to the Office for investigation. The guidance states that "among the activities that have been identified by a federal court as potential election-day voter intimidation are the following: persons other than duly-appointed election officers questioning voters about their credentials; persons impeding or delaying voters by asking for identification, videotaping, photographing, or otherwise making visual records of voters or their vehicles; or persons distributing literature at the polls outlining the fact that voter fraud is a crime and/or detailing the legal penalties for impermissibly casting ballots."
DOJ notes that the Delaware Code has detailed provisions, which are administered by elections officials, allowing each political party to ensure the absence of election day fraud by challenging the eligibility of persons to vote within polling places in a proper fashion. In the upcoming primary and general elections, no citizens should take it upon themselves to interfere with people attempting to vote, but should report any concerns to state or county election officials. The Office of Civil Rights and Public Trust will be prepared to respond on the primary and general election days to reports of possible voter intimidation.Related Topics: Attorney General Matt Denn • Delaware • Delaware Department of Justice • Office of Civil Rights and Public Trust • Voter Intimidation
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Senate Minority Leader Charles E. Schumer traveled to the Niagara Falls Air Reserve Station on Monday to try to jump-start the push to get the military to put a flight simulator into the building it built for one here. Eight months after officials cut the ribbon on a new $5.2 million simulator building, Schumer came to the base to say that the push for a simulator to fill the building continues. Read More
Schumer Pushes For Flight Simulator at Niagara Falls Base
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Discussion in 'Lifestyle' started by Jcolman, Sep 30, 2005.
I found this truck near my buddys cabin. Thought you guys would enjoy the pics.
I think I've got you beat in the oldness department. I found this thing in a walk in the woods.
i like the first shot the best
Third one is my favorite.
I like the third one but the clouds are over exposed and bug me
I like the first pic but also coma's is pretty cool too!
wow...cool pix.
It's very pretty in CO.. Wish I could visit sometime.
third one is the best.
I wish Fall looked like that around here.
Nice pics.
I also like the third picture the best. Where in Colorado was this taken?
Just outside of Grand Lake, about 1 1/2 hours from Denver.
Kodak gold 100.