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231 Cards in this Set

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Organism that causes Syphilis?
Treponema pallidum
About the syphilis organism?
cannot be cultured in artifical media, must rely on serological testing. Anaerobic organism that is long, thin, spiral shaped and unicellular spirochete, has flagella and corkscrew motility. 6-15 micrometers in length, 6-24 coils and sensitive to drying. Can't survive more than 72 hours at 4C
Transmission of syphilis?
sexual contact (primary route or direct inocultion via contact between sores) Congenital via placenta transfer, and very rarely due to transfusion.
Incubation period
lasts 10-90days, average incubation is 21 days and there are NO clinical s/s or symptoms at this stagee and serological tests negative.
Primary Stage of syphilis
Chancre appears at site of inoculation approx. 2-3 weeks after infection, usually around gentals. Heals w/in 4-6 weeks.
Secondary stage of syphilis
usually 1-2 months after appearance of primary chancre. s/s: Headache, sore throat, low-grade fever, nasal discharge, lymphadenopathy, non-itching rash on genitalia and palms of hands and soles of feet, flat lesions in gnital region that are very infectious (Condylomata lata), CNS involvement in up to 40% of pt.
Latent Stage of syphilis
Pt is asymptomatic and noninfectious, except for pregnant women who can still transmit to the fetus. Syphilis can be detected through serologic methods in this stage, 1/3 develop tertiary and 1/3 relapse to secondary.
Tertiary Stage of syphilis
Slowly progressive inflammatory disease that produces clinical illness on average 10-30 yrs after infction and may occur a few months after secondary infection. granulomas called "gummas" form and cardiovascular symptoms occur. CNS involvement occurs including meniingitis, stroke, dementia, and blindness.
Congenital Syphilis
result of placnta transfer during any stage of disease. Can cause abootion, but someteimes viable infant w/ congenital syphilis is born. hutchinson's triad: notched central incisors, interstitial keratitis, deafness. Characteristic saddle-nose deformity, mental retardation, hemolytic anemia, jaundice, and hepatosplenomegaly.
Direct detection of spirochetes
require active lesions and include darkfield microscopy and fluorescent antibody microscopy.
nontreponemal serology
include RPR and VDRL to detect reagin antibody.
Treponemal serology
specific but not very sensitive inn detection of congenital or neurosyphilis. BEST FOR SECONDARY STAGE. includes FTA-ABS (Fluorescent treponemal antibody absorption) and MHA-TP (microhemagglutination for T pallidum).
Venereal disease research laboratory. Flocculation test where reagin antibody combines w/ antigen to form microscopically visible, or flocculent, masses in positive patients.
Rapid Plasma Reagin. most widely used nontreponemal test.
Treponemal Tests
specific for antibodies to T. pallidum.. Used to confirm a positive screening result
Indirect Fluorescent antibody test. complement inactivated in pt serum by heat. Pt serum and Reiter's strain combined to absorb out non-specific treponemal antibodies. Heat-inactivated pt erum verlaid on slide containing t. pallidum and incubated. add fluorescent AHG, wash and observe for fluorescence.
Treatment for syphilis
Historically treated w/ heavy metals like arsenic. Penicillin treatment began in the 1940s and continues to be the predominant form of treatment today.
Rickettsiae and orientia
very small, stain poorly w/ gram stain and much better w/ giemsa stain but rarely dx by staining. Very fragile and transmitted from animals via arthropod vectors. do not grow on artificial media. Usually dx by immunodiagnostic or molecuar methods.
Murine Endemic flea-borne Typhus
Caused by Rickettsia typhi spread by Rat flea. Not common in the US, but occasionally seen in souhern and SE US in summer and fall.
Sx of Murine Endemic flea-borne Typhus
abdominal pain, backache, diarrhea, dull red rash starting on trunk, extremely high fever, hacking/dry cough, headache, join/muscle pain, nausea and vomiting. Rarely it is fatal
Murine Endemic flea-borne Typhus Tx
prompt antibiotic treatment will cure nearly all typhus.
Epidemic Louse-borne typhus
Caused by rickettsia prowazekii spread by human body louse primarily. Not common in the US and associated w/ refugee camps, impoverished areas and regions that recently experienced war or natural disaster
Sx of Epidemic Louse-borne typhus
chills, cough, delirium, high fever, joint pain, light aversion, ow blood pressure, rash over whole body, severe headache, severe muscle pain, stupor More severe than epidemic typhus and death may occur in 10-60% w/o treatment. Brill-Zinser disease: mild, re-activated form, more common in the elderly.
Scrub/mite-borne typhus
caused by Orientia tsutsugamushi and spread by trombiculid mites (chigger). not common in the US, but more than 1 million cases annually, mostly in Asia and Australia. endemic in northern Japan.
Sx of Scrub/mite-borne typhus
skin ulcer w/ "punched out" appearance where mite attached as well as fever, headache, sweating, blood-shot eyes, swollen lymph nodes, rash, vomiting, diarrhea.
Rocky mountain spotted fever
Caused by Rickettsia rickettsii and spread by wood and dog ticks. endemic to the US and especially in children. Mainly seen in warm months in Arkansas, Oklahoma, Georgia, North and South Carolina, and Virginia.
Sx of Rocky mountain spotted fever
fever, headache, rash, nausea vomitting, diarrhea, hx of tick bite.
Dx of Rocky mountain spotted fever
PCR only useful when the disease has progressed to very severe phase. Skin biopsy of rash site used for PCR or immunohistochemical staining which are rapid but not very sensitivve. Indirect Imunofluorescence assay is the gold standard and uses paired samples (withi 1 week of onset and then 2-4 weeks later). Four fold rise in titer between acute and convalescent is diagnostic.
Cat-flea Typhus
Caused by Rickettsia felis and spread by cat fleas via the fleas or the fleas' feces. rare, but reported to occur world-wide. Spotted fever group, but clinically very similar to endemic typhus. OmpA differentiates it from typhus group as well as the vector.
Dx of Cat-flea Typhus
PCR amplification of targeted genes.
caused by Rickettsia akary and spread by house-mouse mites after contact w/ infected rodents, especially during natural die-offs/extermination. Seen globally, mainly in urban areas.
Sx of Rickettsialpox
disseminated rash and eschar at inoculation site. Mild and self-limiting
American Butonneuse Fever / Tidewater spotted fever
Caused by Rickettsia parkeri spread by gulf coast tick in the southern US. only 20 infections since the first one in 2002 have been reported.
Sx of American Butonneuse Fever / Tidewater spotted fever
fever, headache, eschar at inoculation site, and variable rash.
Tx of Rickettsia and Orientia
treat w/ tetracycline antibiotics such as doxycycline
Erlichia and Anaplasma
intraleukocytic pathogens (live within leukocytes, in this case monocytes and granulocytes). Transmitted by arthropod vectors and can also infect animals, especially dogs. cases tend to cluster geographically and it is fairly common in the US.
Tx of Erlichia and Anaplasma
with doxycicline
compact cluster of cells w/in infected cells, range in size from 1-6 micrometers, stain pale blue to dark violet.
Caused by Ehrlichia chaffeensis spread by the lone star tick.
Sx of Ehrlichiosis
high fever, headache, malaise, myalgia, a rash is seen in <60% f pediatric cases and <30% f dult.
Tx of Ehrlichiosis
Doxycycline (healthy individuals typically make a full recovery.
caused by Anaplasma phagocytophilum spread by deer ticks
Sx of Anaplasmosis
high fever, headache, chills, uscle ches, rash is rare.
Dx of Erlichia and Anaplasma
PCR is most effective during first week of illness, sensitivity decreases after Abx administered and not sensitive enough to rule out dx. Periph blood smear shows morulae in about 20% of Pts. IFA is the gold standard.
Weil-Felix Screening test
Not senitive or specific and takes advantage of the shared antigens of Proteus Ox-2, OX-19, and OX-K and Rickettsia/Orientia species. Incubate pt serum w/ commerically prepared proteus antigens and observe agglutination.
Indirect Immunofluorescence in ehrlichia and anaplasma dx methods
gold standard. Patient serum is tested for Ab on paired samples (within 1 week of onset and then 2-4 weeks later.) 4 fold rise in titer between acute and convalescent is diagnostic.
Rickettsia serology
detectable antibody titers observed by 7-10 days after illness (titers obtained in first week after sx appear are usually negative).
Lyme Disease
Recognized in 1975 in Lyme Connecticut. Borrelia burgdorferi is the causative spirochete bacterium and is the most commonly reported vector-borne disease in the US. Most commonly from deer ticks and usually must be attached for 36+ hours to transmit.
Stage 1 Lyme disease
Occurs between 2-30 days after bite. Sx include bulls-eye rash (erythema migrans), flu-like symptoms, right upper quad tenderness.
Stage 2 Lyme disease
after a variable latent period of weeks or months. 10-15% of pt develop borrelia lymphocytomas, muscle/joint pain, nervous involvement (facial palsy, sleep disturbances and possibly can lead to aseptic meningitis). Cardiac manifestations may also occur as atrioventricular block.
Stage 3 Lyme disease
Months to years after infection. Sx include arthritis in 50-60% of individuals often in the knees and late neuroborreliosis (polyneuropathy w/ radiating pain or distal paresthesias, fatigue, headach, hearing loss, verbal memory impairment.
Lyme disease in pregnancy
Can cross the placenta and cause fetal infection that usually leads to still birth if mom is not treated. no negative effects if mother is treated w/ abx after infection.
Lyme disease serology
Ab titers may not be detectable until 3-6 weeks after bite. Two step Dx process recommended: EIA screening followed by western blot confirmation.
Lyme Disease Indirect immunofluorescence
typically titer is 1:256 or more for positive result. may be cross-reactive w/ closely related organisms including T. pallidum.
Tx of Lyme disease
Prevention is key (wear light colored clothing and long pants tucked into socks and be cautious in wooded/grassy areas). Antibiotics are preferred method for treating infection.
Human Ehrlichiosis
can be fatal if not recognized early and properly treated, causesd by lone star tick bite infected w/ tick-borne microscopic parasite that infects human leukocytes.
microscopic tick-borne parasite that infects human RBCs. may be asymptomatic or have flu-like symptms. May result in hemolytic anemia due to RBC destruction. severe in immunocompromised, elderly, or splenectomized individuals.
Dx of babesiosis
gold standard is microscopic visualization of intraerythrocytic Babesia organisms in Giemsa stained peripheral smears. Distinguished by appearance of 4-5 rings per cell that may sometimes form a maltese cross tetrad
Serologic testing of babesiosis
Ab testing by IFA may be indicated in pts w/ low levels of parasitemia and/or discrimination between P falciparum and babesia in inconclusive cases. uses infected RBCs from hamsters.
Tx of babesiosis
Asymptomatic/mild cases resolve spontaneously. severe cases require IV abx/antimalarials. may require exchange transfusion if really severe.
West nile virus
caused by a single-strand virus of the Flaviviridae family.
Clinical presentation of West Nile Virus
incubates 2-10 days. Most who experience symptoms have fever, headache, fatigue,, skin rash on trunk, swollen lymph glands, eye pain. West Nile meningitis can include fever, gastrointestinal symptoms, ataxia, optic neuritis, seizures, weakness, change in mental status, myelitis, polyradiculitis, rash of neck, trunk, arms, or legs, flaccid paralysis.
Non-specific Lab Dx of West Nile Virus
CBCD normal or slightly elevated with lymphocytopenia and anemia. hyponatremia, CSF with increased WBC and protein as well as normal glucose.
Specific Lab Dx of west nile virus
Sandwich ELISA (presence of IgM in serum -14 days after onest or IgM in CSF withing 8 days of onset with 4-fold acute/convalescent titer), PCR has limited usefulness, and viral culture is the gold standard but rarely positive except in autopsy.
Tx of West Nile Virus
no treatment specifically available
Streptococcus pyogenes
Beta-hemolytic, gpc. Most common cause of pharyngitis, scarlet fever, and impetigo. Some surrounded by capsule (anti-phagocytic properties). M protein on cell surface is major virulence factor.
M protein
cell protein on the surface of S. pyogenes that confers virulence. without it, S pyogenes cannot cause infection. 100 serotypes and infection with one strain will not provide protection against another.
M protein shares antigenic epitopes with
proteins in synovium, heart muscle and heart valve. This suggests damage in rheumatic fever is due to an autoimmune response.
Scarlet fever
Post strep sequelae after pharyngeal infection w/ a strain of group A strep that produces pyrogenic exotoxin. Rash usually on the second day of illess that looks like sunburn and feels like sandpaper. Tx w/ Abx. Exposure to pyrogenic exotoxin confers immunity.
Acute Rheumatic Fever
Post-strep sequelae3-4 weeks after upper respiratory tract infections. Causes endocarditis, abnormal involuntary movements called chorea, erytema marginatum which is pink rings on the skin, and plyarthritis. strength of antibody response to streptolysin O is proportional to the severity of the rheumatic fever.
Post-strep sequelae that causes inflammation of the glomerulus and nephron. Sx include proteinuria, hematuria, hypertenssion, impaired renal function, edema. 10 days after pharyngitis or 18-21days after skin infections.
Streptococcal Toxic Shock Syndrome
appears following skin wounds most often. BP drops rapidly and rgans fail. Sx include fever, severe pain, dizziness, flu-like syndrome, confusion, flat red rash
Lab Dx of STSS
4x rise in ASO and DNAse B titers, immature neuts, + blood cultures
Tx of STSS
IV abx, surgical debridement when necessary.
begins w/ skin lesion, may itch at first and then crust over and heal.
subcutaneous infection resulting in warm, red, tender and mildly swollen tissue
a type of cellulitis associated w/ pharyngitis characterized by toxicity and high fever.
Necrotizing Fasciitis
Highly invasive group A strep prduces a pyrogenic exotoxin which produces a tumor necrosis factor which causes rapid destruction of skin tissue
Sx of Necrotizing Fasciitis
severe swelling and pain, fever, redness at wound site.
Tx of Necrotizing Fasciitis
IV fluids, high dose abx, surgical debridement
Acute infection uses lateral flow immunoassay screen (rapid beta kit) and culture confirmation. ASO titer, Anti-DNAse B and Streptozyme (less commonly) for post-strep sequelae.
Rapid Strep Test
Uses lateral fow immunoassay for detection of antigen from a throat swab. If the rapid screen is negative, culture is set up to confirm results.
Anti-Streptolysin O
Body produces antibodies to extracellular products of Group A strep, especially Streptolysin O. Clinical symptoms of rheumatic fever or glomerulnephritis coincides w/ peak of antibody response
Classic ASO titer
Neutralization test shoing antibodies to streptolysin O prevent hemolysis. Serial dilutions of pt serum and streptolysin O and indicator cells.
Automated ASO Titer
Quantitative test using nephelometric method.
Rapid Latex Agglutination
latex particles coated with ASO antigen are mixed w/ pt serum. if presentt, agglutination will occur visibly.
Anti-DNAse B
Appears to be the most relliable measure of S pyogenes skin infection leading to glomerulonephritis. no color change means the patient has antibodies. if all color is lost, then the patient does not have antibodies.
screening slide agglutination test that detects antibodies to streptolysin, streptokinase, hyaluronidase, DNAse, and NADase.
Helicobacter pylori
curved microaerophilic gnb that has strong urease activity.
Virulence factors of H pylori
CagA (insertion into gastric epithelial cell induces changes in the cell's signal transduction pathways and structure of cytskeleton), VacA (vacuolating cytotoxin), or both (increases risk of ulcers and gastric carcinoma.
Motility allows H pylori to escape the acidity of the stomach by
burrowing thrugh the gastric mucosa where it colonies and then fights acid with enzyme called urease which it uses to create alkaline microenvironment.
transmission of H pylori
2 routes: oral-oral and fecal-oral
Epidemiology of H pylori
2/3 of world's population estimated to be infected. Most are asyptomatic. Potential to cause several forms of gastritis, PUD, increased risk of gastric cancer, and MALT lymphoma
Sx of H pylori
gnawing or burning pain in stmach, esp. when empty. Nausea, vomiting, loss of appetite may occur. severe cases can lead to bleeding ulcers that can cause anemia, weakness and fatigue.
Serologic testing of H pylori
Most common serologic test detects H pylori IgG antibodies. Usually non-invasive and rapid and 80-95% sensitive. ELISA most accurate, usually used to confirm following psitive rapid latex test.
Breath Testing of H pylori
Patient drinks carbon-labeled urea labeled w/ 14C which is radioactive or 13C. H pylori rapidly metabolizes the urea and the labeled carbon is absorbed and expired breath is measured to detect the presence of labeled carbon. If the label is detected, H pylori is present. Non-invasive 94-98% sensitivity.
Stool Sample of H pylori
to detect H pylori antigen w/ enzyme labeled monoclonal antibody. Relatively good sensitivity and specificity but nt as good as breath test.
MD inserts flexible viewing tube called endoscope through the moouth into the stomach and duodenum. Small tissue sample can be obtained. Upper esophagogastroduodenal endoscopy is the reference method for Dx of H pylori.
Histologic ID of H pylori
Gold standard of H pylori Dx.
Biopsy Urease Test
CLOtest where tissue sample is placed on special slide inoculated w/ urea. urea break down by urease causes colorimetric reaction if H pylori is present.
Tx of H pylori
10-14 days of abx therapy. acid suppression by an H2 blocker or proton pump inhibitor to alleviate symptoms, abx resistance and noncompliance are two major reasons of treatment failure
Prevention of H pylori
hand washing, eating properly prepared foods and drinking safe, clean water.
Mycoplasma pneumoniae
pleomorphic, variable staining bacteria. 1-3 week incubation leading to sore throat, chills, tracheobronchitis, and headache.
M pneumoniae antibody testing
enzyme immunoassay is most widely used and requires small sample volume and ease of automation. As sensitivve as PCR if tested at apprpriate time and when IgG and IgM both tested.
Parasitic and Fungal Immunology Basics
serological Dx still in somewhat limited use. Ag are cruder than viral and bacterial antigens, decreased specificity. Often, parasitic and fungal infections are seen in immunocompromised pts who dont reliably produce Abs to be detected by serologic tests.
Outcomes of parasite encounter w/ host
No infection, infection, host death, chronic infectio, hypersensitivity reaction
evading detection
invade host cells (temporarily hide from immune system), acquire host antigens (schistosomes acquire RBC antigens and are able to evade detection in blood vessels), antigenic variation (periodic changing of surface antigens due to variant surface glycoprotein), antigen shedding (E. histolytica known to shed its antigens), Molecular mimicry (antigens "designed" to mimic host antigens.
Immunologic response to parasites
entire immune system works to fight the infection. innate immune response critical in early recognition. ig M, G, A, and E are formed with especially high amounts in hlminth infections. Eos chemotactic factor is due a specific mediator released in response to parasites which attract them.
Toxoplasma gondii
prtozoan parasite that causes toxoplasmosis and can be transmitted by ingesting cysts from soil, cat litter, and raw/undercooked meat. 1/4 of pop over age 12 infected. Can cross the placenta
consequences of infection w/ T. gondii
health/non-immunocompromised individuals are asymptomatic or have mild self-limiting flu-like symptoms. Immunocompromised individuals have severe symptoms including fever, confusion, headache, seizures, and nausea. congenital infections differ based on how early infection occurs. Can cause miscarriage or stillbirth.
Congenital toxoplasmosis syndrome
asymptomatic at birth but develop vision loss, mental disability, and seizures over time.
Protozoa other than toxoplasma gondii
ID'd by microscopic exam in most cases. Serology provides alternate means of detection.
Entamoeba histolytica antibody detection
Useful in patients with extraintestinal disease such as amebic liver bscess. EIA uses seroconversion to dmonstrate current infection.
entamoeba histolytica antigen detection
can be used to differentiate pathogenic E. histolytica from E. dispar which is morphlogically identicl and non-pathogenic.
Cryptspridium parvum

Helminth infections
usually not difficult to ID by icroscopy due to size/ease of recovery. Serology useful in complicated cases where the organism has invaded tissue or organs. Most commercial kits use ELISA but CDC accepts samples submitted from state health depts and performs a variety of methods.
Limitations to parasitic serology
no external proficiency testing in parasitic serology except for Toxoplasma and Babesia. FDA must approve kits but weak criteria for approval. Timing f test baed on when Abs are formed and missing that windo may cause false negatives, cannot detect if the organism is sequestered in cells, and low specificity because of crss reactions of antibodies.
fungal infections
opportunistic infections seen especially in immunocompromised, increased use of B-S ABX, and immunosuppressive agents for cancer/transplants. Immune response is not widely studied but innate resistance to infection is high, skin and other barriers are good prtection and cellular immunity is the most important defense.
Serology in fungal infections
allows for more rapid ID. Tests selected based on clinical presentation. Acute and convalescent titers needed. Antigen is tested for in immunocompromised since antibody production is unreliable.
found worldwide in soil, air, decaying vegetation and stored grains. tests for galactomannan antigen in serum using Enzyme immunoassay.
candida albicans is most common cause of all serious fungal diseases. Potential for disseminated infection in immunocompromised due to indwelling cath and long term abx/corticosteroid therapy. Latex agglutination for C. albicans is an antibody test only useful in immunocompetent patients.
Coccidioides immitis is endemic in SW US. Asymptomatic, self-limiting pulmonary infection i most immunocompetent individuals (some exhibit flu-like symptoms).
Cryptococcus neoformans found in pigeon droppings. inhalation of yeast may result in symptomatic pulmonary infection. May progress to meningitis (esp. in immunocompromised). No humoral response, so antigen testing preferred.
What is the name of the substitution nucleic acid alteration that causes a coding for a different amino acid?
Product produced in an amplification is called
an amplicon
What is the target of molecular diagnostics?
the cause of the condition suspected. This could be antigens, bacteria, viral particles, mis-sequenced DNA, and so on.
Which nucleic acid is only in DNA?
Which nucleic acid is only in RNA?
normally very stable and consists of two strands of complimentary base pairs.
passage of hereditary information from parent cell to daughter cell that occurs every time a cell divides
process of generating a messenger RNA strand from DNA to code for proteins
process by which messenger RNA is used to make functional proteins
missing nucleotide or other portion of DNA sequence
extra DNA nucleotide or other prtion of DNA sequence
nucleotie or sequence substitution that codes for a different amino acid
nucleotide substitution that ends in early terminaation of the protein manufacturig process, usually due to a stop codon.
trait/group of traits resulting from transcription and translation of genes
DNA nucleotide sequence responsible for phenotype
sequence change present in at least 1-2% of a population
sequence change present in very small proportion of the population
balanced polymorphism
maintained in a population through a balance of a positive and negative phenotype (Sickle cell anemia and resistance to P. falciparum infection)
Benign polymorphism
provide no selective advantage (ABO blood groups)
stability of mRNA
labile and sensitive to contamination
stability of rRNA
relatively robust
stability of DNA
stable and useful even from nonviable sources
What anticoagulant is not acceptable because it may interfere w/ PCR?
is EDTA an acceptable or unacceptable as an anticoagulant?
EDTA is an acceptable anticoagulant.
What is the preferred specimen collection technique
Collected by routine venipuncture and preferrable collected as fasting to prevent interference by lipids in fluorescence techniques
use the spontaneous and specific pairing of complementary DNA strands known as hybridization that occurs through the use of nucleic acid probes that are labled to facilitate detection.
Factors that affect hybridization
Tremperature (too high denatures, too low anneals), pH (too high denatures, too low anneals), guinine to cytosine ratio (increase delays separation due to strength of GC bond) Salt concentration
Direct testing
use principles that detect RNA or DNA that is currently available in the sample - no multiplication or amplification occurs
amplified methods
use principles that amplify or multiply the target of interest, usually incorporating an enzyme to produce millions or billions of copies in a relatively short time..
Southern Blot
uses a restriction endonuclease enzyme to extract DNA from cells; DNA detection uses agarose gel electrophoresis.
How southern blot is performed
DNA isolated, then cleaved w/ restriction endonucleases (enzymes cleave DNA at specific sites, derived from bacteria, and creates restriction fragment length polymorphisms), separated by size using gel electrophoresis, transferred to a membrane, and dectected w/ a labeled probe complementary to the sequence of interest.
Advantages of southern blot
can analyze large spans of DNA where PCR isn't practical.
disadvantages of southern blot
complex, time-consuming, and requires large sample volume
Fluorescent In Situ Hybridization (FISH)
used to detect protein, RNA and DNA in cells. Capture chromosomes in interphase (can use non-dividing cells) or metaphase (requires dividing cells)
Advantage of interphase FISH
faster because no culture is required and has increased sensitivity because 200-500 cells are analyzed.
disadvantage of interphase FISH
cannot detect chroosomal changes other than those at the specific bindig region of the probe.
Amplified nucleic acid testing
use enzyme to produce millions or billions of copies of the target of interest and can amplify the probe, target, or signal.
Thermal cycling
heating and cooling the sample many times to allow the DNA to denature and anneal several times.
Manual PCR
sample cells mixed w/ reagent containing detergents, enzymes, and salts. Sample is centrifuged resulting in DNA pellet. reagents decanted and pellet re-suspended
Automated PCR
Systems that isolate DNA and can work on multiple samples at a time. requires little itervention and pore pure samples are produced than manual.
reverse transcriptase PCR
RT-PCR modifies PCR for amplification of RNA using reverse transcriptase to convert original RNA to DNA.
conventional PCR
requires PCR product be fully amplified before detection. disadvantages are that it is time consuming, room for interpretation/error when based on size, low res/low sensitivity, qualitative only, and cannot be automated
Real time PCR benefit over conventional
amplicons measured at each cycle allowing for quantification of genetic material present in sample.
Advantages of real-time PCR
measures throughout cyclle, ellimiates need for post-PCR analysis, and reduced potential for contamination.
Multiplex PCR
each probe has different color reporter allowing quantification of several different sequence products at the same time. Probes need to be ensured not to interfere with eachother.
Drawbacks of PCR
potential for false positives due to sample contamination, very few pathogens present in a sample cn result in positive results and does not indicate whether pathogens are viable and/or the cause, inhibiting substances in the specimen may result in false negatives.
Type I Hypersensitivity
Immediate hypersensitivity reaction. Commonlly known as allergies. First exposure, IgE (primarily) attaches to mast cells and basophils through Fc receptors. Second exposure, antigen binds to cell-bound immunoglobulin triggering the release of inflammatory mediators such as histamine, occurs w/in seconds or minutes after contact w/ antigen and can last for prolonged periods
Localized hypersensitivity
Itching, angioedema, watery eyes, runny nose, urticaria
Doesn't occur in all allergic individuals but can be fatal in those that are affected. Even minute quantities entering circulation causes systemic release of mediator substances. Most common triggers are peanuts, seafood, egg albumin, and be stings.
Dx of Type I Hypersensitivity
1. establish that IgE mediated immune reactivity is cause of symptoms (RIST in vitro). 2. then, detect which antigens are responsible (RAST in vitro).
Radioimmunosorbent test. Determine the total amount of IgE. Sandwich immunoassay. treats IgE like an antigen
Radioallergosorbent test used to determine response against specific allergens. Non-competitive immunoassay. Specific allergen bound to a microtiter plate, serum added and allowed to bind w/ allergen, anti-IgE is then added and allwed to bind IgE-allergen complex.
Tx for Type I Hypersensitivity
Immunotherapy/hyposensitization (useful for environmental allergens/bee venom, exposure to small doses of antigen over a period of time elicits maximum anamnestic production of IgG whcih "blocks" antigen attachment to cell bound IgE), Anti-histamines, Hormones (epinephrine causes vasoconstriction and smooth muscle relaxation, corticosteroids are anti-inflammatory and offten used in bronchodilators).
Type II Hypersensitivity
Free antibody mooecules react directly w/ antigen on a cell surface or tissue membrane (activates complement)
examples of Type II Hypersensitivity
incompatible blood transfusions, HDFN, WAIHA/CAIHA
Type III Hypersensitivity
soluble antibody binds w/ soluble antigen and complexes precipitate out of solution which deposit on various sites i the body. Damage from subsequent activation of complement at these sites
examples of Type III hypersensitivity
acute post-streptococcal glomerulonephritis, rheumatoid arthritis, and vasculitis
Type IV Hypersensitivity
delayed hypersensitivity that is cell-mediated. Mediated by T-DTH cells which attack the protein carrying the antigen.
Examples of Type IV hypersensitivity
Tuberculin Reaction (like what is used in the PPD skin test) and Contact sensitivity like those with poison ivy, latex allergies, etc.)
Basic Principle of Flow Cytometry
single cells in fluid suspension are analyzed in terms of intrinsic (light scattering characteristics) and extrinsic properties (Presence of specific surface proteins using a fluorescent probe to label them).
use hydrodynamic focusing to pass one cell at a time through a laser (also known as laminar flow)
Laser light source
produces light scatter to determine size and intracellular complexity.
Light scatter and fluorescence are detected by PMTs and detectors. the light that hits the PMT results in a digital signal proporitonal to the intensity of light detected.
computer for data analysis and management
Uses the digital signal from the PMT to create graphical representation of the data using either single-parameter histogram or dual-parameter dot plot.
Type of specimens used
whole blood, bone marrow, fluid aspirates, and tissue
Sample Prep
Proper specimens are collected. Hemolyzed and clotted specimens are rejected and red cells are lysed for proper analysis.Steps are to wash cells, centrifuge and decant then resuspend in phosphate buffered saline.
Single Parameter histogram
plots flurescence on x-axis vs # of events on the y-axis. Operator can set a marker to differentiate low and high levels of fluorescence for a particular antibody within the group of cells.
Dual-parameter dot plot
two parameters plotted against each other, one on each axis. Operator designates the parameters and the plot is divided into four quadrants and "gates" are drawn around populations of interest.
Absolute Lymph Count
WBC x 1000 x % lymphs (in decimal format)
Differentiate HIV from AIDS based on CD4
HIV >/= 200 or AIDS <200
Major Histocompatibility (MHC)
membrane glycoproteins responsible for antigen presentation. T cells only recognize antigen presented in association w/ MHC.
Class I MHC
encode the info for MHC molecules present on nearly all nucleated cells. Present endogenous antigen so good for CD4 T cells
Class II MHC
expressed by phagocytic cells and other cells capable of phagocytosis and antigen presentation. Present exogenous antigen on surface of antigen-presenting cells.
Class III MhC
encode for a mixture of non-HLA products including the complement proteins and some cytokines.
Tissue Typing
identifing MHC polymorphisms previously was done by antibodies against MHC molecules, but is now done via DNA testing
HLA Typing
Molecular techniques in histocompatibility labs to determine pattern of HLA antigens in each individual
Sequence Specific Primers. lowest resolution, rapid test used to type deceased organ donors.
Sequence Specific Probes. screening to ID potential donors, used for high volume such as in a bone marrow drive.
Sequence Based Typing. highest resolution, used for donors/recipients of stem cell transplants (BMT, PBSC) or in examining disease associations.
Pre-formed HLA antibodies
Pt may develop HLA antibodies due to pregnancy, blood transfusions, or previous transplant. Blood or plasma recipients may have HLA antibodies already formed that cause hyperacute rejection of certain grafts, failure of PLT xfusion, and TRALI.
Panel Reactive Antibody
% portion of the population to which the person bing tested will react via pre-existing antibodies. Use CDC, ELISA or Flow Cytometry. CDC or pooled ELISA must be followed with Flow Cytometry to determine specific HLA antibodies.
Complement-Dependent Cytotoxicity (CDC) Technique
Periph blood lymphs (from potential xplant recipient) and commercially prepared anti-sera incubated together to allow any present antibody to attach. Standardized prep of complement is added and if Abs bound to cell membrane, complement is fixed, cascade s activated to completion and cell is irreversibly damaged. Dye added can only penetrate nonviable cells.
Mixed Lymphocyte culture
when a donor is living to test the degree of donor's cells to stimulate a response in the recipient's T lymphocytes. Blast transformation represents the response of the recipient cell to foreign antigens of the donor cell.
Crossmatch procedure
after donor is selected, recipient serum is tested against donor cells. 'virtual" crossmatch is done at the site where the transplant tissue comes from and actually done by CDC and/or flow.
tissue removed from one area of the body and reintroduced into another area of the same person. no immune rejection.
transfer of tissues /organs between genetically identical individuals such as identical twins. no immune rejection.
tissue/organ transferred from one individual to another individual of the same species but with different genetic characteristics. Possible rejection.
transfer of tissue/organ from one species to another. Common, successful example is pig heart valves
Types of transplanted tissue
cornea, bone, skin, kidney, heart, liver, pancreas, lungs, intestine, bone marrow,
Cornea transplant
avascular, so nonimmunogenic
bone transplant
mechanical is sterilized, nnimmunogenic while regenerative is actively metabolizing bone which is immunogeenic
skin transplant
temporary is allo- or xenograft. Also, permanent autografts possible.
Kidney transplant
Many variables influence success and may be recieved from living or deceased donor.
Heart transplant
major indications are inflammatory or metablic disease of the heart and has over half surviving 5 years.
Liver transplant
donors are difficult to find and it is indicated by tumors and cirrhosis.
Pancreas transplant
May be attempted to "cure" IDDM but prognosis is guarded
lung transplant
donation of a lung lobe from each of two living donors and more successful w/ heart and lungs transplanted together
intestine transplant
most performed along w/ liver transplant from cadaver donor.
Bone Marrow Transplant
ABO not required to be compatible because recipient becomes immunosuppressed so profoundly that the recipient's blood type becomes that of the donor. HLA must match!
Hyperacute rejection
occurs w/iin minutes or hours. previously existing antibodies in recipient cause this reaction. complement is activated in the vessels of transplanted tissues, inflammation with neut accumulation occurs, platelets aggregate, and blood supply is compromised.
accelerated rejection
occurs up to 5 days following transplant. T cells become sensitized to donor antigens over time.
acute rejection
begins w/in 1 week and remains as long as graft persists. most common type of rejection. Can be temporarily revesed by immunosuppression.
chronic rejection
occurs months or year after transplantation.
Graft-versus-host disease
most serious long-term complication of BMT but potentially can occur in any transplant. Result of viable T cells transplanted into a recipient who is profoundly immunodeficient.
methods of immunosuppression may be antigen specific or antigen non-specific.
what loci are coded for on MHC I
HLA-A, B, and C
What loci are coded for on MHC II
HLA-DP, DQ, and DR