Aplastic anemia falls into the category of "anemias-in-which-the-cells-don't-look-weird" category. Anemias
in this category can sometimes be difficult to diagnose (for example,
in anemia of chronic disease, the cells are of normal size and
shape...so it's dang hard to find anything weird to point you in a
diagnostic direction). In aplastic anemia, however, there's a giant clue staring you
straight in the face: pancytopenia. Not only are the red cells decreased
in number, but so are the white cells and platelets. That's because the
marrow is basically empty. Check out the image above of a bone marrow
biopsy from a patient with aplastic anemia. There's supposed to be
hematopoietic tissue in there - but basically all you see are mature red
cells and lymphocytes. All precursors are decreased: erythroblasts,
granulocyte precursors, and megakaryocytes. So when you look at the
blood, the mature cells in the blood are all decreased too. Clinically, patients present with just what you'd expect: findings
related to their cytopenias. Patients will usually be pale and fatigued
(from the anemia), with bleeding and bruising (from the
thrombocytopenia) and recurrent infections (from the leukopenia). Lots
of things can cause aplastic anemia (drugs, viruses, pregnancy, Fanconi
anemia), but many times, no cause is found. Patients are usually treated with blood products as needed, and if
the aplasia doesn't resolve, then drugs such as G-CSF and prednisone are
given. Bone marrow transplant is successful in many patients, but
because of the high morbidity and mortality of the procedure, it's used
only as a last resort. For a serious hematologic disorder, the prognosis
is relatively good: 3 year survival is on the order of 70%.
Here are some troubleshooting hints that we have gathered regarding non-specific bands after PCR reactions:
Non-optimal Mg++ concentration
Nucleotide concentration is too high or unbalanced
DNA contamination / carryover
Primer annealing temperature is too low
Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers
Primers are degraded or sequence is not optimal
Primer concentration is too high
Cycle number is too high
Incorrect template to enzyme ratio
Nucleotide concentration is too high or unbalanced
The standard concentration is 20-200 µM of each nucleotide Suggestions:
Check the concentration of stock solutions of all nucleotides
Double check the final concentrations of all nucleotides
DNA contamination / carry-over
Suggestions:
Test for carryover by performing PCR without adding target DNA.
Avoid carryover
To prevent carryover, good lab practices should be used such as:
Physically isolating PCR preps and products
Autoclaving solutions, tips, and tubes
Aliquoting reagents to minimize repeated sampling (no more than 20 reactions per aliquote)
Eliminating aerosols by using positive displacement pipettes
Premixing reagents
Adding DNA to reaction last
Choosing (+) and (-) controls carefully
Soaking gel box and combs in 1M HCl to depurinate DNA
Using new razor blades to exise bands
Using new razor blades to exise bands
Covering UV box with fresh plastic wrap
Always using oil overlay
To eliminate contamination / carryover:
UV irradiation: Mix all components, except template DNA, irradiate in clear 0.5 ml polypropylene tubes in direct contact with glass transilluminator (254 and 300 nm UV bulbs) for 5 minutes
UNG Digestion: Incorporate d-UTP nucleotides into reaction and do subsequent uracyl DNA glycosylase digestion
Primer annealing temperature is too low
Primer annealing temperature is typically 50-60°C (may be higher or lower based on primer sequence and buffer components).
Suggestion: Determine Tm/annealing temperature based on one of the following equations:
If primers are 20-35 bases If primers are 14 - 70 bases
Tp = 22 + 1.46(Ln)
Ln = 2(# G or C) + (# A or T)
TP = Effective annealing
temperature ± 2 - 5 Tm = 81.5 + 16.6 (log10 [J+]) + 0.41
(% G + C) - (600/l) - 0.063
(% Formamide) + 3 to 12
[J+] = concentration of Monovalent cations
l = length of oligo
Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers
Suggestions:
Increase initial denaturation temperature to 95-97°C
Denature DNA minus enzyme & buffer for 4-6 minutes
Increase cycling denaturation time 15-30 seconds
Try "Hot Start" technique
Add T4 Gene 32 protein 3-5 µl/ml
When designing primers, make sure there is no more than 2 bases of homology at the 3' end. Use a primer design program if available
Consider addition of cosolvent to reaction buffer:
3-15% DMSO
1-10% Formamide
5-15% Polyethylene glycol
10-15% glycerol
Primers are degraded or sequence is not optimal
Primers should have same number A & T's versus G & C's, and they should be at least 14 bases for specificity. Suggestions:
If primers are short and A-T rich, add 0.9 - 2.0%(v/v) DMSO
If primers are G-C rich, add 1-10% (v/v) Formamide
Double check priming sequence, use primer design program if available
Check aliquot of primers on a gel to ensure they are not degraded
Primer concentration is too high
Suggestion: Adjust the primer concentration (0.1 - 1.0 µM of each primer is optimal)
Cycle number is too high
Most templates require 25-30 cycles. Suggestion: Cycle number should be based on starting concentration of template DNA.
If the number of target molecules in your sample is...
Then we recommend the following number of Cycles...
3 x 105 25-30
1.5 x 104 30-35
1.0 x 103 35-40
50 40-45
Incorrect template to enzyme ratio
The necessary amount of template varies from reaction to reaction. As a guideline, 100 - 750 ng human DNA (105 - 106 copies) per 100 µl reaction. The amount of enzyme should be optimized for each template. Suggestions:
Titrate the amount of template in the reaction
Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range (0.5 to 5.0 units)
Talaro/Chess: Foundations in Microbiology is an allied health microbiology text for non-science majors with a taxonomic approach to the disease chapters. It offers an engaging and accessible writing style through the use of tools such as case studies and analogies to thoroughly explain difficult microbiology concepts. We are so excited to offer a robust learning program with student-focused learning activities, allowing the student to manage their learning while you easily manage their assessment. Detailed reports show how your assignments measure various learning objectives from the book (or input your own!), levels of Bloom’s Taxonomy or other categories, and how your students are doing.
The Talaro Learning program will save you time while improving your students success in this course.
Big Advance Against Cystic Fibrosis: Stem Cell Researchers Create Lung Surface Tissue in a Dish
Harvard stem cell researchers at Massachusetts General Hospital (MGH) have taken a critical step in making possible the discovery in the relatively near future of a drug to control cystic fibrosis (CF), a fatal lung disease that claims about 500 lives each year, with 1,000 new cases diagnosed annually.
Beginning with the skin cells of patients with CF, Jayaraj Rajagopal, MD, and colleagues first created induced pluripotent stem (iPS) cells, and then used those cells to create human disease-specific functioning lung epithelium, the tissue that lines the airways and is the site of the most lethal aspect of CF, where the genes cause irreversible lung disease and inexorable respiratory failure.
That tissue, which researchers now can grow in unlimited quantities in the laboratory, contains the delta-508 mutation, the gene responsible for about 70 percent of all CF cases and 90 percent of the ones in the United States. The tissue also contains the G551D mutation, a gene that is involved in about 2 percent of CF cases and the one cause of the disease for which there is now a drug.
The work is featured on the cover of this month's Cell Stem Cell journal. Postdoctoral fellow Hongmei Mou, PhD, is first author on the paper, and Rajagopal is the senior author.
Mou credits learning the underlying developmental biology in mice as the key to making tremendous progress in only two years. "I was able to apply these lessons to the iPS cell systems," she said. "I was pleasantly surprised the research went so fast, and it makes me excited to think important things are within reach. It opens up the door to identifying new small molecules [drugs] to treat lung disease."
Doug Melton, PhD, co-director of the Harvard Stem Cell Institute, said, "This work makes it possible to produce millions of cells for drug screening, and for the first time human patients' cells can be used as the target." Melton, who is also co-chair of Harvard's inter-School Department of Stem Cell and Regenerative Biology and is the Xander University Professor, added, "I would expect to see rapid progress in this area now that human cells, the very cells that are defective in the disease, can be used for screening."
Rajagopal said, "The key to our success was the ecosystem of the Harvard Stem Cell Institute and MGH. HSCI investigators pioneered the strategies we used, helped us at the bench, and gave us advice on how to combine our knowledge of lung development with their exciting new platforms. Indeed, we also enjoyed a wonderful collaboration with Darrell Kotton's lab at Boston University that was able to convert mouse cells into lung tissue. These interactions really helped fuel us ahead."
The epithelial tissue created by Rajagopal and his colleagues at the MGH Center for Regenerative Medicine also provides researchers with the same cells that are involved in a number of common lung conditions, including asthma, lung cancer, and chronic bronchitis, and may hasten the development of new insights and treatments into those conditions as well.
"We're not talking about a cure for CF; we're talking about a drug that hits the major problem in the disease. This is the enabling technology that will allow that to happen in a matter of years," said Rajagopal, a Harvard Medical School assistant professor of Medicine.
Also a physician trained as a pulmonologist, the specialty that treats CF patients, Rajagopal said, "When we talk about research and advances, donors and patients ask: 'When? How soon?' And we usually hesitate to answer. But we now have every single piece we need for the final push. So I have every hope that we'll have a therapy in a matter of years."
Cystic fibrosis, which used to claim its victims in infancy or early childhood, has evolved into a killer of those in their 30s because treatments of the infections that characterize the disease have improved. But despite those advances, there has been little progress in treating the underlying condition that affects the vast majority of patients: a defect in a single gene that interferes with the fluid balance in the surface layers of the airways and leads to a thickening of mucus, difficulty breathing and repeated infections and hospitalizations.
The discovery and recent FDA approval of the drug Ivacaftor, which corrects the G551D defect seen in about 2 percent of CF patients, has served as a proof of concept to demonstrate that the disease can be attacked with a conventional molecular treatment. In fact, Ivacaftor was found by screening thousands of drugs on a far less than ideal cell line. In the end, many drugs that functioned well on this cell line proved ineffective when used on genuine human airway tissue.
Genuine human airway tissue is the gold standard prior to drugs being tested clinically, but it has been extremely difficult to obtain the tissue from patients, and when it could be obtained, the tissue rarely survived long in the lab -- all of which created a major bottleneck in screening for a therapy. But by creating iPS cells that contain the entire genome of a CF patient and directing those cells to develop into lung progenitor cells, which then develop into epithelium, the group appears to have solved this key problem.
Rajagopal, who did his own postdoctoral fellowship in Melton's laboratory during the first half of the past decade after completing his training in pulmonary medicine, said that having both the G551D and 508 genes in the epithelial tissue provides a way to prove that the tissue will be effective in testing drugs against CF.
"We've created the perfect cell line to show that the drug out there that works against G551D mutation works in this system, and then we're in business to screen for a drug against delta 508," he said. "We'll know soon that the cell line works. We know it makes bonafide airway epithelium, and we'll have the proof of principle that the tissue responds properly to the only known drug. We think this is the near-ideal tissue platform to find a drug for the majority of CF."
Rajagopal's lab has created numerous other cell lines to further show that a CF drug that works in one patient should work in others and to see whether this will be an area that allows a more personalized approach to medicine.
