general viro plenary Flashcards
virus as obligatory cell parasites mean:
they can not survive or multiply in the environment. They need living cells to do that.
Need of specified laboratories in case of/It is also obligatory in case of:
- Regulations or rules, notifiable diseases e.g: FMD, ASF, BSE
- Suspected zoonosis e.g: rabies
- Eradication programs, or in order to control the eradication and declare the free status of the herd (Bovine Lucosis Virus, Infectious Bovine Rhinotrachetitis virus etc.)
- Certifications: free status, SPF herd, transport, competitions
- Clinical signs, pathological findings are not sufficient to establish the diagnosis
- Herd diagnosis: endemic viruses present in the herd
- In the need of official certifications of free status, specified pathogen free (SPF) herd: before exhibitions/animal shows/competitions, transport, export, travel.
Methods of laboratory diagnosis:
- Direct virus demonstration
- Indirect processes (virus serology)
Direct virus demonstration consists of?
- Whole virus (isolation)
* Its components: proteins, Nucleic acids
Indirect processes (virus serology) consists of?
• Antibody detection from the infected animals
Aim of the examination (i.e diagnosis) determines:
- The type of sample
- Its amount
- Shipping
Aspects of the sample collection and transport
- Sample type an timing of sampling
- Unambiguous mark
- Letter of basic information = accompanying letter
- Period and circumstances of the samples shipping to a diagnostic institute
Necessary onformation for documentation for packaging:
- Location and contact information
- Case information
- Epidermiological information
- Submitted samples
Sampling for virological investigation
- Direct method:
Direct method (in early phase): Virus shedding is the highest in this phase!
- Corpse, organ, tissue (cadaver)
- Secretion: ventricle (thorax), organ (udder)
- Blood anticoagulant treated (leucocyte separation)
Sampling for virological investigation
- Indirect method:
Indirect method (generally in late phase)
- Blood coagulated, serum
- Milk, liquor
- Ventricle and organ secretion
Why do you use samples for serological detection of viral pathogens?
In order to be able to see an increase in antibody titres, whereas an increase of min 4 x is considered significant: sera pair investigation
Types of Laboratory investigations:
- Direct virus demonstration
- Indirect virus demonstration
Direct virus demonstration:
The whole virus or its components (proteins, nucleic acids) can be investigated by propagation of viruses in cell culture (virus isolation) or more specific tests such as:
- Antigen test (ELISA, haemagglutination, peroxidase or immne-fluorescence (IF))
- Protein detection (Western blot)
- Nucleic acid detection (i.e Nucleic acid hybridization, PCR)
Indirect virus demonstration:
Serological methods (ELISA, virus neutralization test, indirect IF, haemagglutnination inhibition (HAI)) by which the virus-induced antibodies from the blood or body fluids of the infected animals can be detected.
Virus isolation:
- period of the diagnosis?
2-3 weeks
Virus isolation
- Prerequisite of in vitro propagation?
infective virion –> early phase (acute stadium) –> virus shedding
Virus isolation:
Sample may be taken from?
- Ante-morten: animal alive (buffy coat, body fluids, faeces)
- Post-mortem: animal dead (organ samples)
what type of sample preparation of virus isolation is used in Direct virus demonstration?
Sample preparation in PBS (Ab-Am supplemented) 1:10 dilution
The method of sample preparation in PBS:
(Ab-Am supplemented) 1:10 dilution
- Organ pieces: cutting and homogenization in potter/seramic mortar
- Swabs: rinsing for 1-2 hours
1st centrifugation: cell debris quartz sand (1000 x g, 10min)
2nd centrifugation: purification (300 x g, 10min) or filtration
How is Buffy coat (WBCs) separated from non-coagulated blood?
by haemolytic resistance or buoyant density
Production of primary monolayer cell culture:
- Organ of origin
- Rich in epithelial cells (- virus multiplication)
- Actively dividing cells – from young animal (a few days old)
- Kidney, testicles, thymus, embryo, etc.
- Aseptic removal, processing within a few hours
Production of primary monolayer cell culture:
- Processing in aseptic circumstances
- Removal of outer membranes, connective tissue
- The tissue is cut into small pieces
- Separation of the cells by digestion with trypsin – EDTA (versen) solution
- Cell-containing suspension is repeatedly removed and replaced with trypsin solution
- Blocking the effect of trypsin in ice bed (0C)
- Sedimentation of the cells by centrifugation, removal of trypsin
- Suspension of cells in culturing medium
- Cell couting (in a Bürker chamber)
- The cell suspension is transferred into a sterile culturing flask (Plate, Roux-flask, Petri-dish, etc)
- Incubation (usually at 37 *C, 5% CO2)
What is used for Suspension of cells in culturing medium which gives Optimal environment?
(Production of primary monolayer cell culture)
MEM: Minimal Essential Medium.
- Isotonic, isoionic, isosmotic (salts, buffer systems)
- Nutritive (amino acids, carbohydrates)
- Antibiotics, antimyotics, indicator
- Foetal (neonatal) calf serum (FCS)
What is true for Foetal (neonatal) calf serum (FCS)?
Protein source + mediators for cellular division
Taken from colostrum-free calves
Growth medium: 5-10%
Maintenance medium: 2% FCS
What happens during incubation?
Production of primary monolayer cell culture
cells settle down, attach and divide
within 3-5 days they cover the bottom of the flask
contact inhibition: when the cytoplasmic membranes meet, the cells stop their division
a primary monolayer culture is developed
When do we use monolayers?
–> inoculation: virus is added into the maintenance fluid
Maintenance of cell cultures:
monolayers
• Aging of the cells – degeneration within 7-14 days
• Cell division – young cells are formed
–> Subculturing (passage)
• Removal of the medium
• Removal of the cells from the wall of the flask (trypsin digestion)
• Dilution (2-3x), putting into fresh culturing flask
–> secondary culture
• Fresh, homogenous, increased amount
• Further 2-3x passages are possible
• The cell content changes in subsequent passages (fibroblast increase), hence sensitivity might decrease
What is cellular cloning?
Cultivation of a single cell, production of permanent cell line
• Dilution of the cell suspension – 1cell/well
• Propagation, selection of quickly dividing clones
Types of cellular cloning:
- Diploidic (i.e: PK-15, MDBK, RK-13)
* Aneuploidic – tumor cells (ie. HeLa, BHK-21, Vero)
Advantages of Cellular cloning:
- Genetically homogenous, standard
- Unlimited number of passages
- Long term storage (-80C, liquid nitrogen)
Disadvantages of cellular cloning:
- Sensitivity for virus infections varies (- presence of cellular receptors)
- Contamination may occur (virus, mycoplasma, leptospira)
- Presence of active oncogene
Adsorption is used to?
avoid CPE caused by toxins
Suspension is used for?
viruses which need dividing cells
What is Co-cultivation?
isolation of cell-associated and latent viruses
- Simultaneous processing and mixing of virus-infected and healthy cells
Suspension cultures:
continuous stirring, no adhesion: vaccine production
Microcarrier cultures:
- Cells adhere on the surface of microcarrier beads (Cytodex), continuous stirring
- Aim: increased surface (vaccine production)
Shell vial assay:
Herpesviruses (CMV, HSV, VZV) Adeno-, entero-, flavi-, orthomyxo-, paramyxoviruses - Inoculation - Centrifugation (700 x g, 45min) - Incubation 16h - Mab staining
What is important characteristics for the egg during inoculation?
- Embryonated
- SPF (specific pathogen free)
- White shelled – easier transillumination
Characteristics for the yolk sac?
- Picorna- (i.e avian encephalomyelitis virus) reo-adenoviruses
- Rickettsia, chlamydia
- 5-7 days old embryo
Characteristics for the Allantoic cavity, Amniotic cavity (embryo)?
- Orthomyxo-, paramyxo-, coronaviruses
- 9-12 days old embryo
Characteristic for Chorio-allantoic membrane (CAM)?
- Pox-, herpesviruses
- 10-13 days old embryo
characteristic for Intravenous inoculation?
- Orbiviruses (i.e Bluetongue virus)
- 16-17 days old embryo
Incubation temp for inoculation of embryonated eggs
33-37 *C
Control after inoculation of embryonated eggs to check?
- Tranillumination
Death within 24h – usually non-specific - Egg necropsy (after 4-5days)
Dwarfism, distorsion, death
CAM: pock (size, inflammation, haemorrhage, necrosis) - Haemagglutination test on the allantoic fluid
If there is no CPE of the embryonated egg:
- Blind-passage (increase of virus titer and CPE
- Auxiliary examinations: EM, HA, IF, IP
Final steps of inoculation of embryonated egg:
isolate –> plaque -isolation –> purification –> virus strain
Daily examination to check:
embryonated egg
- Sample collection: allantoic fluid, CAM, embryo
* Auxillary examinations: HA, EM, histopathology
Aim for concentration and purification of viruses:
virus analytical invesstigations
Prerequisite or concentration and purification of viruses:
Microbiologically clean cultures
- Virus isolate –> plaque purification –> virus strain –> propagation of genetically identical viruses in larger scale
Name different methods for release of viruses from infected cells:
• Mechanical method: freezing-thawing (3x)
• Sonication (heat generation – virus protein may damage)(ultrasound)
• Detergents (for nucleic acid investigations)
–> Opens the cell membrane. Virus can be dead or alive.
Rough purification can be done by?
• Centrifugation
- sedimentation of cells, cell debris (3000-5000 x g)
- the virus stays in the supernatant, NOT in the sediment
• Filtration
- Removal of particles larger than viruses (bacteria, yeasts, moulds, cell components)
- 450nm filter pore size
concentration methods depends on?
The physico-chemical resistance of viruses
Why do we use Precipitation?
- When we don’t need the suspected virus, only the Nucleic Acids etc.
- (NH4)2SO4, PEG 6000, ethanol
- Resolve in buffer
Methods for concentration:
- Precipitation
- Adsorption
- Ultrafiltration
- Dialysis
- Pelletisation
Why do we use Adsorption, and what do we use:
- Can not separate the virus and smaller particles.
- Al(OH)3, Ca3(PO4)2
- Non-specific chromatography
Why do we use ultrafiltration, and what do we use
- Hydrostatic pressure
- Pore size are smaller than the diameter of viruses
Why do we use Dialysis, and what do we use
- Osmotic pressure
- Thorugh a semi permeable membrane
Why do we use Pelletisation, and what do we use
- Virion sedimentation at 25 000-200 000 x g
- In ultracentrifuge (mitochondria and other organells can also be analysed)
- Even 1000 x concentration
What are contaminating elements that also should be removed?
elements similar to viruses in size (eg mitochondria, chromosomes, ribosomes)
Concentration and purification methods:
- Affinity chromatography
- Density gradient ultracentrifugation
- Rate Zonal technique
- Isotopic technique
Affinity chromatography method:
- Virus-specific antibodies are bound to the chromatography column matrix
- Adsorption of viruses
- Rinsing
- Elution with buffer (pH change)
- Filter that binds antibody specific pathogens:
What is the function of filter that binds antibody specific pathogens?
(Affinity chromatography method)
- Virus suspension go through the filter.
- The virus binds specifically.
- Mitochondria etc. only stays on the filter.
- With the buffer, you remove all the cell organelles except the virus itself
What is the principle of Stoke´s Law in Density gradient ultracentrifugation?
The virus particles density divides until it has the same density as the density of the tube.
What is used for sedimentation of viruses in dense solutions?
CsCl, Cs2SO4, sucrose, metrizamide
What is Rate Zonal technique based on?
on sedimentation rate in sequential gradients, which are less dense than the viruses (Stoke 1)
What is Isotopic technique based on?
based on density differences.
- Viruses will sink as deep as their buoyant density is equal to the environemnt´s density (Stoke2)
Preprative ultracentrifugation is used for?
purification
Analytical ultracentrifugation is used for?
measuring buoyant density
What is virion buoyant density?
nucleic acid/protein/lipid ratio (incomplete virions are lighter)
dialysis is used for?
collect and purify virus-specific zones
Physico-chemical test for virus identification:
- Electron-microscopic investigation
- Chloroform resistance
- Halogenic uridin derivates
- Acridin-orange staining
When is Electron microscopic investigation used?
virus identification
–> morphological recognizable virions (complete/incomplete)
Advantage in case of viruses not replicating in cell-cultures
Methods of electron microscopic investigation:
- Ultra thin section
- Negative contrast method
- Immune-electron microscopic method
Explain the method of Ultra thin sections in Electron-microscopic investigation:
- Ultra thin section (from the organs, from the predilection sites)
- fixation (glutaraldehyde)
- Embedding (durcupan resin)
- Tungsten- or urnanium salts treatment (electron absorbent)
Explain the method of Negative contrast method in Electron-microscopic investigation:
- Negative contrast method (diluted sample e.g: fecal)
- Treatment with contrast material (phosphotungsten acid)
- Drying onto the grid (contrast material does not bind)
Explain the method of Immune-electron microscopic method in Electron-microscopic investigation:
- Immune-electron microscopic method (diluted samples)
- centrifugation
- Immune serum added to the sample –> precipitation
- Centrifugation –> precipitated virus in the sediment
How to investigate serotye
species –> antigen investigation, serological methods (VN)
Subtype, variants:
- Alteration in genotype:
NA examination: REA, sequencing
Subtype, variants:
- Alteration structure protein:
MAB, PAGE
what is needed for Virus antigen detection:
with specific antibodies (virus identification too)
- Incomplete virions, non-matures proteins are demonstrated
- Proper serum
What is characteristic for the proper serum needed for Virus antigen detection?
- Hyperimmune
- Monospecific (polyclonal)
- Monoclonal
What test can be used for Virus Antigen Detection?
- Immunofluorescence (IF) test
- Immunoperoxidase (IPA, PLA) test
- Complement fication (CF) test
- Agar Gel Immune electro-foresis (CIEF) test
- Radio Immune Assay (RIA) test
- Enzyme linked Immunosrobent Assay (ELISA) test
When can you use IF test for virus antigen detection:
Immunofluorescence (IF) test: antigen intracellularly
- Direct: rabies, classical swine fever
- Indirect: 10x more sensitive
- Specificity sensitivity optimation
When can you use IPA, PLA test for virus antigen detection:
Immunoperoxidase (IPA, PLA) test: antigen intracellularly
- Histology slide/cell culture
- Spec, antibody labelled with peroxidase enzyme
- Substrate digestion colour change
When can you use CF test for virus antigen detection:
Complement fixation (CF) test: antigen released off the cells - The hemolysin lyses the sheep RBCs in the presence of complement (FMD)
When can you use AGID test for virus antigen detection:
Agar Gel Immune Diffusion test (AGID):
- Adeno, bluetongue, EHD
When can you use CIEF test for virus antigen detection:
Counter current immune electro-forezis (CIEF):
- Rota, parvo, adeno
When can you use ELISA test for virus antigen detection:
Enzyme Linked Immunosorbant Assay (ELISA):
- Direct/indirect: antigen/antibody detection
- Competitive: discriminating/multispecies antobody detection
- Sandwich (FMD antigen detection)
Nucleic acid hybridization
- Theory: heating/cooling down
- dsDNA heating a–> the double helix splits
- Cooling down: the complementary threads rejoin
What is the function of the probe in Nucleic acid hybridization?
Probe: labels oligonucleotide (DNA or mRNA) Complementary to the viral genome Labelling: isotope (32P) or enzyme
Southern Blot:
- Agarose gel lectrophoresis
- Nitrocellulose/nylon filter
- Hybridization
- Electricity first horizontally, then vertically
DNA microarray technique:
• DNA samples are bound to glass slides or membrane filters –> “DNA chip “
• Hybridization with fluorescently labelled probes
• Laser scanning
• Computer analysis
–> Identification, “typing” of viruses
–> Comparison of virus strains, genetic relationships
What are the different Nucleic acid investigation methods:
- Nucleic acid hybridization
- Southern blot
- DNA microarray technique
- Polymerase chain reaction
- Real time PCR
Polymerase chain reaction is used for:
Amplification of specific DNA fragments
What is needed in PCR?
Template + primers + free deoxy-nucleotides + thermos-resistant polymerase
(Taq)
How PCR works:
• Template + primers + free deoxy-nucleotides + thermos-resistant polymerase
(Taq)
• Heating and cooling in cycles (n)
• Polymerase enzyme; moves at 72°C (important number), makes a new
complementary strand –> double amount of nucleic acid
• RNA: reverse transcription
- ssDNA stay ss –> reverse transcription
• Compare sequence strains to see if it is a zoonosis, mutation or epidemic virus
ex.
–> 2n copies of the fragment
• Detection: agarose-gel electrophoresis, NA hybridization etc.
What is the function of real-time PCR?
(detect amplification of NA during the reaction)
• Fluorescent labelling
• Laser detection of amplification products, computer analysis
• Quantification only
What is sequencing
determination of the nucleotide sequence
What is Sangers method?
A type of Sequencing
Sangers method: polymerization
- Template + primer + deoxy nucleotides + labelled dideoxy-nucleotides + polymerase
enzyme –> polymerization of the complementary thread
What is needed for sequencing:
- The complete information about the nucleic acid
- Sangers method
- Dideoxy- nucleotide: chain termination
- Polyacrylamide gel-electrophoresis
What is Next generation sequencing methods:
• High efficacy – 20 million base read within a few hours
• Fragmentation of the DNA sample, binding to carrier surface
• Non-specific amplification
• Separation into wells of a microplate
• Sequencing reactions, detection of signal generated by the incorporating nucleotides
• Automated processing of the reads:
Alignments of repeatedly determined, overlapping sequences, consensus sequence generation
Name some Next generation sequencing methods:
- Oligonucleotide fingerprint technique
- Reverse transcriptase conversion
What is Oligonucleotide fingerprint technique?
- investigation of RNA viruses
- Digestion with ribonuclease A, or T1 rubonuclease
- Two dimensional PAGE –> relation, epidemiology investigation
What is Reverse transcriptase conversion?
- Investigation of RNA viruses
- RNA dependent RNA polymerase –> transcription to dsDNA
- Use of DNA investigation methods.
Aim of titration of viruses:
“standardization” of the virus suspension –> amount of infective viruses –> titration, quantification
Methods for titration of viruses:
- Physical assays: direct particle counting – EM, Haemagglutination
- Biological assays: determination of the infective titre (endpoint method), plaque assay, focus assay, pock formation etc.
When is End point dilution - infective titer used?
in vitro propagation and identification
Method of End point dilution - infective titer:
- Serial tenfold dilution of the virus suspension
- Inoculation of the cell-cultures with each dilution
- Incubation, the period is characteristic to the virus
Diagnostics with Titration of viruses:
Virus neutralization test, Haemagglutination inhibition test
What is infective titer?
CPE in 50% of the inoculated cell-cultures
What is characteristic to the virus suspension?
The highest dilution of the virus, which cause CPE in 50% of the inoculated cell-cultures
What does TCID50/ml mean?
The highest dilution of the virus, which cause CPE in 50% of the inoculated cell-cultures, is characteristic to the virus suspension
–> Its virus concentration unit is; 1 Tissue Culture Infective DOSE TCID50/ml
What does EID50 mean?
- Titration in embryonated eggs – determination of EID50
What does LD50 mean?
- Titration in experimental animals – determination of LD50
Methods of calculation:
• Reed-Muench method: very first, most commonly used. We are looking for
concentration of original virus suspension.
• Spearman-Karber method: easier, no need of lot of numbers, only the data of
dilution which is the next higher dilution with 100% response (CPE).
- A difficult equation, but also looking for the original concentration of the virus suspension.
Method of Plaque counting - infective titer:
• Both stained and unstained
• Serial tenfold dilution of the certain virus suspension
• Inoculation of the cell cultures from each dilution
• Adsorption: 1 hour at 37 degrees
• Covering with semisolid maintenance: agar or CMC
- Incubation characteristic to the virus
- Local CPE in the inoculated cell-cultures
- Staining with vital stain; gentian purple, Evans blue, Janus green
How do you calculate the Infective titre in plaque counting?
at the inoculated cell-cultures containing less than 10 plaques/inoculated cell-cultures, we take the average number (mean) of the plaques, multiply it with the negative reciprocal of the dilution levelàthis gives the infective titre
Evaluation: The concentration of the virus suspension is given in plaque forming units = PFU/ml
Lattice formation =
no infection – only RBC + virions (or hemagglutinating proteins of
virions)
Method of Haemagglutinating tire determination:
• Lattice formation, no infection – only RBC + virions (or hemagglutinating proteins of
virions)
• Often species specific
• On micro-haemagglutination plates (Takatsy plates)
• Serial twofold dilution of the virus suspension
• Washed erythrocytes (1%) of proper species
• Incubation –> at proper temperature for the virus
Haemagglutinating tire =
The highest dilution of the virus suspension, in which we can observe hemagglutination
Virus demonstration Advantage/disadvantage:
- Advantage: isolation of the causative agent within short time (in case of NA/aG detection).
- Disadvantage: can be performed only for few days, and it is expensive.
Serological methods Advantage/disadvantage:
- Advantage: higher chance of demonstration (longer) persistence of antibodies), and it is cheaper. Antibodies present for a much longer time –> higher chance of detection.
- Disadvantage: cannot differentiate maternal, vaccine and seroconversion antibodies
- -> see the age and vaccination history in accompanying letter
Method of Virus neutralization:
• Mix virus with antibodies –> cant infect. Then see CPE on cell. Answer over 2 –>
enough antibodies to neutralize virus
• Heat inactivation of the sera at 56 degrees in 30 min. –> non-specific antibodies are
heat sensitive
Principle of Virus neutralization:
In the presence of blocking antibodies reacting with antireceptors of
viruses, the virus is not able to adsorb to the cell
- Serotype (species) specific
Method of Serial twofold dilution from sera:
- 100 TCID50 (or EID50 or PFU50) viruses
- Incubation 1 hour at 37 degrees –> antibodies neutralize the viruses - Inoculation of the cell-cultures with each dilution
- Incubation at 37 degrees for several days
- -> CPE
What is Serum neutralizing titer:
the highest dilution of the serum where there is 50% CPE
Method of Rapid fluorescent focus inhibition/fluorescent antibody virus neutralization test:
• RFFIT/FAVNT – ex. Rabies
- Virus neutralization – 20 or 48 hours, virus concentration: FFD50/TCID50
- Addition of fluorescent labelled virus-specific antibody: residual virus titer shows the antibody content of the serum sample
Method of
Indirect immunofluorescent test (iIFA):
- Virus neutralization test
- Addition of fluorescent labelled host IG-specific antibody: the fluorescence shows the antibody content of the serum sample
method of Constant serum varying virus dilution method:
• Neutralization index calculation –> for virus indentification
- Two serial tenfold dilution of the given virus suspension - Adding negative serum
- Positive serum
- Incubation at 37 degrees for 1 hour
- Inoculation of the cell-cultures from each dilution - Incubation at 37 degrees for several days
- -> CPE
Plaque reduction test is used to:
- To see if it is enough antibodies in blood
* Fewer plaques compared to the control viruses
What is the plaque reduction titre of the serum:
- The highest dilution of the serum where in 50% of the cell cultures the plaque formation is inhibited (reduction % is characteristic to the given virus)
Method of Hemagglutination inhibition (HAI):
• Which sample does not agglutinate? Opposite of hemagglutination test
• Exhausting the sera 37 degrees for 60 minutes – eliminates the non-specific
hemagglutinating activity: 10-25% RBC or with 10% caolin
• Only for hemagglutinating viruses
- Serial twofold dilution of the serum sample
- 4-8 HAU (standard virus suspension) of virus to each serum dilutions - Incubation for 1 hour at a temperature characteristic to the virus
- Addition of washed erythrocytes (1%)
What is the Serum hemagglutination inhibition titer?
the highest dilution where there is no hemagglutination (enough antibodies to block the HA proteins)
Validity of the HAI test:
- A virus between 4-8 HAU
- Actual titer of the known positive serum did not change
reduction test - More than one dilution level since the previous test
Antibody detecting can be done by:
ELISA (cELISA, iELISA)
Advantage of cELISA?
Competitive ELISA
Advantage:
- “multispecies” kits, discriminative ELISAs (ex. IBR)
- antisbody in the serum binds / no antibody
- Secunder antibody: peroxidase labelled, specific to viral antigen, binding/no binding – competition
What is true in the case of Binding in cELISA?
colour change–sample is negative
What is true in the case of No Binding in cELISA?
No colour change–sample is positive
Advantage of iELISA?
Indirect ELISA
- Antibody in the serum binds/no binding
- Secunder antibody: peroxidase labelled, host Ig-specific
What is true in the case of Binding in iELISA?
colour change–serum sample is positive
What is true in the case of No Binding in iELISA?
No colour change–serum sample is negative
What is true for AGID test?
positive/negative test
Agarose gel immune diffusion test (AGID)
o Positive- precipitation occurs
o Negative – nothing happens
What is true for CF, iCF test?
positive/negative test
(Indirect) complement fixation test (CF, iCF)
o Positive – sedimenting RBC
o Negative–hemolysis
