MALDI-TOF Working Protocol

Process of rapid identification of pathogenic bacterial species using MALDI-TOF MS (Matrix-assisted laser desorption/ionization – time of flight mass spectrometry)

The MALDI-TOF system can be used to identify clinically significant pathogens (bacteria, fungi) isolated from vaginal, cervical secretions, blood cultures (of pregnant or infertile patients, hospitalized in the Department of Obstetrics), as well as from hypopharyngeal aspirates, cerebrospinal fluid, blood cultures, etc. (of newborns hospitalized in the Department of Neonatology).

MALDI-TOF MS is a technique used in mass spectrometry that allows the analysis of biomolecules (such as DNA, ribosomal proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), and can be used to analyze the protein composition of a bacterial cell. The identification of bacterial proteins is based on their molecular weight.

MALDI-TOF MS involves a laser that acts on a matrix of small molecules to transform the analyte molecules into the gaseous phase without fragmenting or breaking them down. A mass spectrum is generated and automatically compared to a database of mass spectra by the software, resulting in the identification of the organism.

This technique has been shown to be effective due to its reproducibility, speed and sensitivity, with the advantage of MALDI-TOF MS over other methods of identification being that results are available in minutes to a few hours rather than days.

Equipment and consumables:

• Matrix HCCA (alpha-Cyano-4-hydroxycinnamic acid), ported
• Standard solvent (acetonitrile 50%, water 5% and trifluoroacetic acid 2.5%)
• Wooden chopsticks for transferring biological material or plastic inoculation loops
• Pipette tips 5-10 μL, 2-200 μL, 50–1000 μL and corresponding pipettes
• MALDI target plate
• Tubes Shaker (vortexor)
• Formic acid 70%

Preparation of HCCA matrix solution:

• add 250 μL of standard solvent prepared in advance in a HCCA tube
• dissolve HCCA by shaking/vortexing at room temperature until the solution is clear/transparent

Principle:

• ideally, cultures not older than 24 hours should be used for identification, except for slow-growing germs, in which case older cultures (a few days) can also be
• transfer biological material (e.g. an isolated bacterial colony) from a bacterial culture and apply as a thin film directly to a free position on the MALDI target plate
• using the pipette and the 5-10 μL tips, add 1.0 (± 0.1) μL of the Matrix solution
• leave to dry at room temperature (a homogeneous preparation must be obtained)
• the MALDI-TOF-MS measurement is

Note: Although most bacteria will be easily identified by direct application to the Maldi target plate, some organisms possess the capsule that prevents effective lysis of the bacterial cells and obtaining identification. In these situations (e.g. in the case of fungi) a protein extraction procedure is required to identify them correctly. So when initial attempts at direct identification fail, the material can be superimposed with formic acid before the matrix is added.

References

1. UK Standards for Microbiology Investigations Matrix-assisted laser desorption/ionisation – time of flight mass spectrometry (MALDI-TOF MS) test procedure; Issued by the Standards Unit, Microbiology Services, PHE; Bacteriology – Test Procedures | TP 40 | Issue no: 1 | Issue date: 27.11.19

MALDI Biotyper Bruker Standard Operating Procedure

WORKING PROTOCOL CHEMILUMINESCENTA

Chemiluminescence determinations (Anti-HAV IgM , Anti-HCV Ac, Anti-HIV Ac, HBsAg,
Toxo-IGG, Toxo-IGM, Rub-IGG, Rub-IGM, CMV-IGG, CMV-IGM, EPB-IGG,EPB-IGM)

Viral and parasitic infectious pathology occupy an important place in the management
of pregnancy at risk, as well as neonatal infections. In this regard, viral hepatitis, HIV infection,
toxoplasmosis, rubella, CMV and EBV infection are some of the most common infections
diagnosed.

Anti-HAV IgM

a) Purpose of the examination: qualitative determination of IgM antibodies against
hepatitis A virus (anti-HAV IgM) using the chemiluminescent method; the
determination is used as a support for the diagnosis of an acute or recent (usually
less than 6 months) infection with hepatitis A virus
b) Principle and method of procedure used for examinations: the method is based
on two-site immunometry; the light signals emitted by the system are directly
proportional to the amount of anti-HAV IgM in the sample
c) Performance characteristics: linearity: 0.02 – 7.0 S/CO; specificity = 100%,
sensitivity = 100%, CV = 7.5%
d) Sample type:
• serum, heparinized plasma or EDTA plasma
• test as soon as possible after harvesting.
• primary samples can be stored closed in the refrigerator until 7 days after
collection
• serum/plasma samples can be frozen up to 180 days after collection
e) Type of container and additives: red stopper container without additive or green
stopper container with heparin or purple stopper container with EDTA
f) Calibration procedures
• Calibration is performed when changing the lot of reagent and calibrator,
once every 28 days,after major service interventions, in case of failure of
internal quality control

g) Interference
• Haemoglobin > 500 mg/dL
• Triglycerides >3000 mg/dL
• Bilirubin > 60 mg/dL
• Proteinaemia: > 12 g/dL
h) Reference biological ranges: Non-reactive

Translation from Romanian

i) Clinical Laboratory Interpretation:
• Samples with value < 0.8 S/CO (signal to cutoff) are considered non-reactive
• Samples with a value of ≥ 0.8 S/CO but less than 1.20 S/CO are
considered inconclusive and must be repeated; it is recommended that the
repeat be done in duplicate and the result released be based on the results of
the repeat; if the result of the repeat is still inconclusive, it is recommended to
obtain a new specimen and test it
• Samples ≥ 1.20 S/CO are considered reactive
j) Potential sources of variation:
a) Heterophilic antibodies present in serum or plasma may interfere with
immunological tests
b) The determination is not validated for other biological products
c) igM anti-HAV are usually detectable between 3 and 6 months after the onset of the
disease (anti-HAV IgG persists throughout life)

Anti-HCV needle

a) Purpose of examination: qualitative detection of IgG antibodies against hepatitis C
virus (anti-HCV) using the chemiluminescent method;determination may be used in
conjunction with other serological tests and clinical information for diagnostic
purposes in patients with symptoms of hepatitis and in individuals at risk of HCV
infection; the product is not intended for screening or testing of serum pools, other
blood types or plasma from more than one individual
b) Principle and method of the procedure used for examinations: the method is based
on two-site immunometry (sandwich technique); the light signals emitted by the
system are directly proportional to the anti-HCV in the sample
c) Performance characteristics: linearity: 0.0 – 11.0 index value; specificity = 99.90%,
sensitivity = 100%, CV = 6.6%
d) Sample type:
• serum, heparinized plasma or EDTA plasma
• samples are handled as potentially infectious
• the samples are tested as soon as possible after collection (they can be kept
uncentrifuged for up to 24 hours at room temperature)
• primary samples can be kept for up to 7 days in the refrigerator
• serum/plasma samples can be frozen
e) Type of container and additives: red stopper container without additive or green
stopper container with heparin or purple stopper container with EDTA
f) Calibration procedures
• Calibration is performed when changing the lot of reagent and calibrator, once
every 28 days,
after major service interventions, in case of failure of internal quality control

g) Interference
• Haemoglobin > 500 mg/dL
• Triglycerides > 1000 mg/dL
• Bilirubin > 60 mg/dL
• Protein level 3-12 g/dL
h) Reference biological ranges: Index value < 0.8 (Non-reactive)
i) Clinical Laboratory Interpretation:
• Samples with index value less than 0.8 are considered non-reactive
• Samples with index value ≥ 0.8 but less than 1.0 are considered inconclusive;
testing is repeated in duplicate; if 2 of the 3 are with index value less than 0.8,
the sample is considered non-reactive; if 2 of the 3 have index value ≥1.0, the
sample is considered reactive and confirmatory tests are recommended; if 2 of
the 3 are with index value ≥ 0.8 and <1 it is recommended to perform additional
tests
• Samples with index value ≥ 1.0 are considered reactive; testing is repeated in

duplicate; if 2 of the 3 are with index value < 0.8, the sample is considered non-
reactive; if 2 of the 3 have index value ≥1.0, the sample is considered reactive

and confirmatory tests are recommended; if 2 of the 3 are with index value ≥ 0.8
and <1.0, additional tests are recommended
• The results are considered invalid and must be repeated if the controls are
outside the allowed limits
j) Potential sources of variation:
• Heterophilic antibodies present in serum or plasma may interfere with
immunological tests
• The determination is not validated for other biological products
• If the result is used for diagnostic purposes, its interpretation should be made in
conjunction with the clinical examination, the patient’s history or other types of
tests
• A negative result does not exclude the possibility of exposure or infection with
HCV; HCV antibodies may be undetectable at some stages of infection and in
some clinical situations
• Test performance has not been studied in immunocompromised populations and
patients

Anti-HIV needle

a) Purpose of the examination: Qualitative detection of antibodies against human
immunodeficiency virus type 1, including subtype O and/or type 2 (anti-HIV) using the
chemiluminescent method

b) Principle and method of the procedure used for examinations: the method is
based on two-site immunometry (sandwich technique); the light signals emitted by the
system are directly proportional to the anti-HIV in the sample
c) Performance characteristics: linearity: 0.05 – 50 index value; specificity = 99.87%,
sensitivity = 100%, CV = 8.8 – 11.6%
d) Sample type:
• serum, heparinized plasma or EDTA plasma
• samples are handled as potentially infectious
• the samples are tested as soon as possible after collection (primary samples can be
kept uncentrifuged for up to 24 hours at room temperature)
• primary samples can be kept for up to 7 days in the refrigerator
• serum/plasma samples can be frozen
e) Type of container and additives: red stopper container without additive or green
stopper container with heparin or purple stopper container with EDTA
f) Calibration procedures
• Calibration is performed when changing the lot of reagent and calibrator, once every
28 days, after major service interventions, in case of failure of internal quality
control.
g) Interference
• Haemoglobin > 500 mg/dL
• Triglycerides >3000 mg/dL
• Bilirubin > 30 mg/dL
• Protein level 3-12 g/dL
h) Reference biological ranges: Index value < 1.0 (Non-reactive)
i) Clinical Laboratory Interpretation:
• Samples with index value < 1.0 are considered non-reactive
• Samples with index value ≥ 1.0 are initially considered reactive and are rewritten
in duplicates after centrifugation; if one or both duplicates are reactive, the
sample is considered repeatedly reactive and confirmatory tests are
recommended; if confirmatory tests are positive, then the sample is considered
to be positive
• Initially reactive samples where both duplicates are non-reactive (index value
<1.0) are considered negative
• The results are considered invalid and must be repeated if the controls are
outside the allowed limits
j) Potential sources of variation:
• Heterophilic antibodies present in serum or plasma may interfere with
immunological tests

• The determination is not validated for other biological products
• It is not recommended to test blood pools or products derived from such
pools
• Test performance has not been studied in immunocompromised populations
and patients
• The test may not detect anti-HIV antibodies in all infected individuals; a
negative result does not exclude the possibility of exposure or infection with
HIV; anti-HIV antibodies may be undetectable at some stages of infection or
in some clinical situations

AgHBs

a) Purpose of examination: qualitative detection of hepatitis B virus surface antigen
(HBsAg) using the chemiluminescent method; determination may be used in
conjunction with other serological tests and clinical information for the diagnosis of
acute and chronic hepatitis B virus infection; determination may also be used for
screening for hepatitis B virus infection in pregnant women to identify newborns at risk
of infection in the neonatal period
b) Principle and method of the procedure used for examinations: the method is based on
two-site immunometry (sandwich technique); the light signals emitted by the system
are directly proportional to the AgHBs in the sample
c) Performance characteristics: linearity: 0.1 – 1000 index value; initial specificity =
99.51%, specificity after retesting = 99.91%, sensitivity = 100%, CV = 3.6%
d) Sample type:
• serum, heparinized plasma or EDTA plasma
• samples are handled as potentially infectious
• the samples are tested as soon as possible after collection (they can be kept
uncentrifuged for up to 24 hours at room temperature)
• primary samples can be stored closed in the refrigerator until 7 days after
collection
• serum/plasma samples can be stored in the refrigerator for up to 14 days or can
be frozen

e) Type of container and additives: red stopper container without additive or green stopper
container with heparin or purple stopper container with EDTA
f) Calibration procedures
• Calibration is performed when changing the lot of reagent and calibrator, once every
21 days, after major service interventions, in case of failure of internal quality control
g) Interference
• Haemoglobin > 500 mg/dL
• Triglycerides > 1000 mg/dL
• Bilirubin > 40 mg/dL
• Cholesterol > 400 mg/dL

• Protein level 3-12 g/dL
h) Reference biological ranges: Index value < 1.0 (Negative)
i) Clinical Laboratory Interpretation:
• Samples with an index value of less than 1.0 are considered non-reactive (negative)
• Samples with index value ≥ 1.0 but less than or equal to 50 are considered reactive
(positive); the test is repeated in duplicate; if 2 of the 3 results are non-reactive
(negative) the result is negative for HBsAg; if 2 of the 3 results are reactive (positive)
the result is considered to be repeated reactive (positive) for HBsAg and confirmatory
tests are recommended, other markers of HBB infection
• Samples with index value > 50 or signaled by the device as “>Index Range” are
considered reactive (positive) for HBsAg and confirmatory tests are recommended
• The results are considered invalid and must be repeated if the controls are outside
the allowed limits
j) Potential sources of variation:
• Heterophilic antibodies present in serum or plasma may interfere with
immunological tests
• The determination is not validated for other biological products
• If the result is used for diagnostic purposes, its interpretation should be made in
conjunction with the clinical examination, the patient’s history or other types of tests
• A negative result does not exclude the possibility of exposure or infection with HBV; a
negative result in a patient exposed to HBV may be generated by HBsAg levels below
the limit of detection of the HBsAg method or lack of reactivity with antibodies from
the reaction

TOXO IgG

a) Purpose of the examination: ToxoplasmaG (Toxo G) is an IgG antibody that captures
microparticles directly chemiluminometric for the in vitro diagnosis of the quantity and
quality of Toxoplasma gondii IgG antigens in serum or plasma (EDTA heparin). The test
can also be used to screen for Toxoplasma G infection in pregnant women to identify
newborns at risk of infection during the neonatal period.
b) Principle and method of the procedure used for examinations: the method is based on
direct immunometry with two sites (sandwich technique); human monoclonal antibodies
are covalently coupled with paramagnetic particles in the solid phase.
c) Performance characteristics: linearity: 0.5 – 700 index value; initial specificity = 96.5%,
specificity after retesting = 99.91%, sensitivity = 96.5%,
d) Sample type:

• Serum, heparinized plasma or EDTA plasma
• Samples are handled as potentially infectious
• Store samples at 2–8°C for up to 7 days
• Freeze samples, free of red blood cells, at or below -20°C for longer storage

• Samples stored at room temperature for up to 7 days or refrigerated for up to
14 days

e) Type of container and additives: red stopper container without additive or green
stopper container with heparin or purple stopper container with EDTA
f) CALIBRATION PROCEDURES
• Calibration is performed when changing the lot of reagent and calibrator, once every
14 days, after major service interventions, in case of failure of internal quality control
g) Interference:
• Haemoglobin > 500 mg/dL
• Triglycerides > 1000 mg/dL
• Bilirubin > 40 mg/dL
• Protein level 3-12 g/dL
h) Reference biological ranges: Index value < 10.0 (Negative)
i) Clinical Laboratory Interpretation:

• Samples with a calculated value of less than 6.4 IU/mL are considered non-
reactive

• Samples with a calculated value between 6.4 and 9.9 IU/mL are equivocal
• Samples with a calculated value greater than or equal to 10.0 IU/mL are
reactive
• The results are considered invalid and must be repeated if the controls are
outside the allowed limits
•

j) Potential sources of variation:
• Heterophilic, antinuclear (AAN), and antimytochondrial (AMA) antibodies present
in serum or plasma may interfere with immunological tests.
• The determination is not validated for other biological products
• If the result is used for diagnostic purposes, its interpretation should be made in
conjunction with the clinical examination, the patient’s history or other types of
tests
• Pay attention to the interpretation of the results for patients who have been
transfused recently or for several months.

TOXO-IgM

a) Purpose of the examination: ToxoplasmaM (Toxo M) is an IgM antibody that captures
microparticles directly chemiluminometric for the in vitro diagnosis of the quantity and
quality of IgM antigens of Toxoplasma gondii in serum or plasma (EDTA heparin). The
determination can also be used to screen for Toxoplasma M infection in pregnant
women to identify newborns at risk of infection during the neonatal period.

b) Principle and method of the procedure used for examinations: the method is based on
direct immunometry with two sites (sandwich technique); human monoclonal antibodies
are covalently coupled with paramagnetic particles in the solid phase.
c) Performance characteristics: linearity: 0.5 – 700 index value; initial specificity = 99%,
specificity after retesting = 99.91%, sensitivity = 99.2%,
d) Sample type:
• Serum, heparinized plasma or EDTA plasma
• Samples are handled as potentially infectious
• Store samples at 2–8°C for up to 7 days
• Freeze samples, free of red blood cells, at or below -20°C for longer storage
• Samples stored at room temperature for up to 7 days or refrigerated for up to 14
days

e) Type of container and additives: red stopper container without additive or green
stopper container with heparin or purple stopper container with EDTA
f) Calibration procedures:
• Calibration is performed when changing the lot of reagent and calibrator, once
every 14 days, after major service interventions, in case of failure of internal
quality control
g) Interference:
• Haemoglobin > 500 mg/dL
• Triglycerides > 1000 mg/dL
• Bilirubin > 40 mg/dL
• Protein level 3-12 g/dL
h) Reference biological ranges: Index value < 1.0 (Negative)
i) Clinical Laboratory Interpretation:

• Samples with a calculated value less than <0.9 IU/mL are considered non-
reactive

• Samples with a calculated value between 0.9 and 0.99 IU/mL are equivocal
• Samples with a calculated value greater than or equal to 1.0 IU/mL are reactive
• The results are considered invalid and must be repeated if the controls are
outside the allowed limits
j) Potential sources of variation:
• Heterophilic antibodies present in serum or plasma may interfere with
immunological tests
• The determination is not validated for other biological products
• If the result is used for diagnostic purposes, its interpretation should be made in
conjunction with the clinical examination, the patient’s history or other types of
tests

• Pay attention to the interpretation of the results for patients who have been
transfused recently or for several months.

RUB-IgG

a) Purpose of examination: In vitro diagnosis for the quantitative and qualitative detection
of Rubella virus IgG antibodies in serum or plasma (EDTA heparin)
b) Principle and method of the procedure used for examinations: the method is based on
direct immunometry with two sites (sandwich technique); human monoclonal
antibodies are covalently coupled with paramagnetic particles in the solid phase.
c) Performance characteristics: linearity: 0.2 – 500 index value; initial specificity = 98.8%,
specificity after retesting = 99.91%, sensitivity = 99.4%,
d) Sample type:

• Serum, heparinized plasma or EDTA plasma
• Samples are handled as potentially infectious
• Freeze samples, free of red blood cells, at or below -20°C for longer storage
• Samples stored at room temperature for up to 7 days or refrigerated for up to
14 days

e) Type of container and additives: red stopper container without additive or green
stopper container with heparin or purple stopper container with EDTA
f) CALIBRATION PROCEDURES
• Calibration is performed when changing the lot of reagent and calibrator, once
every 14 days, after major service interventions, in case of failure of internal
quality control
g) Interference:
• Haemoglobin > 500 mg/dL
• Triglycerides > 1000 mg/dL
• Bilirubin > 40 mg/dL
• Protein level 3-12 g/dL
h) Reference biological ranges: Index value < 5.0 IU/mL (Negative)
i) Clinical Laboratory Interpretation:

• Samples with a calculated value less than < 5.0 IU/mL are considered non-
reactive

• Samples with a calculated value between 5 and 9.9 IU/mL are equivocal
• Samples with a calculated value greater than or equal to 10.0 IU/mL are
reactive
• The results are considered invalid and must be repeated if the controls are
outside the allowed limits
j) Potential sources of variation:

• The determination is not validated for other biological products
• If the result is used for diagnostic purposes, its interpretation should be made
in conjunction with the clinical examination, the patient’s history or other
types of tests

RUB-IgM

a) Purpose of the examination: In vitro diagnosis for the quantitative and qualitative
detection of IgM antibodies of Rubella virus, in serum or plasma (EDTA heparin)
b) Principle and method of the procedure used for examinations: the method is based on
direct immunometry with two sites (sandwich technique); human monoclonal
antibodies are covalently coupled with paramagnetic particles in the solid phase.
c) Performance characteristics: linearity: 0.2 – 500 index value; initial specificity = 99.1%,
specificity after retesting = 99.91%, sensitivity = 91.0%,
d) Sample type:
• Serum, heparinized plasma or EDTA plasma
• Samples are handled as potentially infectious
• Freeze samples at or below -20°C for longer storage
• Samples stored at room temperature for up to 7 days or refrigerated for up to 14
days

e) Type of container and additives: red stopper container without additive or green
stopper container with heparin or purple stopper container with EDTA
f) CALIBRATION PROCEDURES

• Calibration is performed when changing the lot of reagent and calibrator,
once every 14 days, after major service interventions, in case of failure of
internal quality control

g) Interference:

• Haemoglobin > 500 mg/dL
• Triglycerides > 1000 mg/dL
• Bilirubin > 40 mg/dL
• Protein level 3-12 g/dL
• Hypergamaglobulinaemia >3 mg/mL
h) Reference biological ranges: Index value < 0.80 IU/mL(Negative)
i) Clinical Laboratory Interpretation:

• Samples with a calculated value less than <0.80 IU/mL are considered
non-reactive
• Samples with a calculated value between 0.80 and 0.99 IU/mL are
equivocal

• Samples with a calculated value greater than or equal to 1.0 IU/mL are
reactive
• The results are considered invalid and must be repeated if the controls
are outside the allowed limits
j) Potential sources of variation:
• The determination is not validated for other biological products
• If the result is used for diagnostic purposes, its interpretation should be made in
conjunction with the clinical examination, the patient’s history or other types of
tests
• Specimens taken early during the acute phase of infection may not contain
detectable levels of Rubella IgM antibodies, this does not rule out a primary
infection.

CMV IgG

a) Purpose of the examination: qualitative determination of IgG antibodies for
Cytomegalovirus (CMV) in human serum in order to determine the immune status to
Cytomegalovirus.
b) Principle and method of procedure used for examinations: Chemiluminescence.
c) Performance characteristics:.
• Sensitivity : 100%
• Specificity: 100%
d) Sample type:
• human serum or plasma (heparinised or EDTA).
• in case of lipemic serums, ultracentrifugation is recommended
• The primary samples can be stored closed in the refrigerator until 3 days after
collection, at 2–8°C,
• Serum samples can be stored in the freezer for up to 6 months at –20°C.
g) Interference
• Bilirubin: The presence of direct or indirect bilirubin in concentrations up to 200 mg/L
does not affect the result.
• Hemolysis: the presence of hemoglobin in concentrations up to 539 mg/dL does not
affect the result.
• Lipemia: The presence of triglycerides in concentrations up to 3000 mg/dL does not
affect the result .
h) Clinical Laboratory Interpretation:

• Reagent: A ratio of ≥ 1.1 S/CO indicates that IgG antibodies have been detected in
the patient’s serum.
• Non-reactive: a ratio < 0.9 S/CO indicates that IgG antibodies have not been
detected in the patient’s serum
• Inconclusive: a ratio of 0.9 to 1.1 S/CO, requires retesting.
i) Potential sources of variation:
• Heterophilic antibodies in human serum may interfere with immunological tests.

• An increased level of IgG antibodies can be found in the case of measles, varicella-
zoster virus, Epstein-Barr.

CMV IgM

a) Purpose of examination: qualitative determination of IgM antibodies against
Cytomegalovirus(CMV) for the purpose of diagnosis of acute cytomegalovirus infection.
b) Principle and method of procedure used for examinations: Chemiluminescence .
c) Type of sample:
• serum or plasma (EDTA or heparinized)
• The primary samples can be stored closed in the refrigerator until 3 days after
collection, at 2–8°C,
• Serum samples can be stored in the freezer for up to 6 months at –20°C.
•
d) Interference:
Bilirubin: The presence of bilirubin in concentrations up to 200 mg/L does not affect the
result.
Hemolysis: The presence of hemoglobin in concentrations up to 522 mg/dL does not affect
the result
Lipemia: The presence of triglycerides in concentrations up to 3000 mg/dL does not affect
the result.
e) Reference biological ranges: Non-reactive
f) Clinical Laboratory Interpretation:
• Reagent: A ratio of ≥ 1.1 S/CO indicates that IgM antibodies have been detected in
the patient’s serum. The presence of IgM antibodies indicates that the patient had a
recent exposure to Cytomegalovirus.
• Non-reactive: a ratio < 0.9 S/CO indicates that IgM antibodies have not been
detected in the patient’s serum
• Inconclusive: a ratio of 0.9 to 1.1 S/CO, requires retesting.
g) Potential sources of variation:

• An increased level of antibodies can be found in the case of measles, varicella-zoster
virus, Epstein-Barr.
• Samples containing antinuclear antibodies may give false reactive results

EBV-EBNA IgG

a) Purpose of the examination: Qualitative detection of IgG antibodies to the Epstein-Barr
virus (EBNA) nuclear antigen in human serum or plasma. This test can also be used to help
diagnose EBV-related diseases.
(b) Principle and method of the procedure used for examinations: two-stage solid-state
chemiluminescent enzyme immunoassay.
d) Sample type:
• human serum or plasma (heparinised or EDTA)
• In the case of lipemic serums it is recommended to use an ultracentrifuge.
• Hemolysed samples may indicate maltreatment of a sample prior to receipt by the
laboratory; therefore results should be interpreted with caution.
• The primary samples can be stored closed in the refrigerator until 3 days after
collection, at 2–8°C,
• Serum samples can be stored in the freezer for up to 6 months at –20°C.
g) Interference
• Bilirubin: The presence of conjugated and unconjugated bilirubin in concentrations
up to 200 mg/L has no effect on the results.
• Haemolysis: The presence of haemoglobin in concentrations up to 537 mg/dL has no
effect on the results.
• Lipemia: The presence of triglycerides in concentrations up to 3000 mg/dL has no
effect on the results
h) Reference biological ranges
• Reagent : a ratio of ≥ 1.1 S/CO . The report indicates that EBV-EBNA IgG antibodies
were detected in the patient sample.
The presence of IgG antibodies to EBV-EBNA is an indication of prior exposure to the
virus.
• Non-reactive: a ratio of < 0.9. Indicates that EBV-EBNA IgG antibodies were not
detected in the patient sample.
• Inconclusive: a ratio of 0.9 to 1.1 S/CO, requires retesting. Alternative testing is
recommended, or a second sample taken, within a reasonable period of time , e.g.
one week.
j) Potential sources of variation:
• Samples containing antinuclear antibodies or other anti-cellular antibodies may give
false positive results.

EBV-EBNA IgM

a) Purpose of the examination: for the qualitative detection of IgM antibodies to
EpsteinBarr virus viral capsid antigen in human serum or plasma.
b) Principle and method of the procedure used for examinations: chemiluminescent IgM
capture immunoassay, solid phase.
c) Performance characteristics: total concordance: 91.1%, sensitivity: 89.4%, specificity:
96.4%.
d) Sample type:
• serum or plasma (with EDTA and heparinized) In the case of lipemic serums it is
recommended to use an ultracentrifuge.
• Hemolysed samples may indicate maltreatment of a sample prior to receipt by the
laboratory; therefore results should be interpreted with caution.
• The primary samples can be stored closed in the refrigerator until 3 days after
collection, at 2–8°C,
• Serum samples can be stored in the freezer for up to 6 months at –20°C.
f) Interference:
• Biotin: Specimens containing biotin at a concentration of 1500 ng/ml demonstrate a
change of less than or equal to 10% in the results. Biotin concentrations higher than
this may result in incorrect results for patient samples.
• Bilirubin: The presence of conjugated and unconjugated bilirubin in concentrations
up to 200 mg/L has no effect on the results.
• Haemolysis: The presence of haemoglobin in concentrations up to 269 mg/dl has no
effect on the results.
• Lipemia: The presence of triglycerides in concentrations up to 3000 mg/dL has no
effect on the results.
g) Biological reference ranges:
• Reagent : a ratio of ≥ 1.1 S/CO . The report indicates that IgM EBV-EBNA antibodies
were detected in the patient sample.
• Non-reactive: a ratio of < 0.9. Indicates that IgM EBV-EBNA antibodies were not
detected in the patient sample.
• Inconclusive: a ratio of 0.9 to 1.1 S/CO, requires retesting. Alternative testing is
recommended, or a second sample taken, within a reasonable period of time , e.g.
one week.
h) Potential sources of variation:
• Samples containing antinuclear antibodies or other anti-cellular antibodies may give
false positive results.

Vitek 2C System Work Protocol

The Vitek 2 Compact System can be used both to identify and to test the sensitivity to antibiotics of pathogens of clinical significance (bacteria, fungi) isolated from vaginal, cervical secretions, blood cultures (of pregnant or infertile patients, hospitalized in the Department of Obstetrics), as well as from hypopharyngeal aspirates, cerebrospinal fluid, blood cultures, etc. (of newborns hospitalized in the Department of Neonatology).

The Vitek 2 Compact System uses a fluorogenic methodology for the identification of microorganisms and a turbidimetric method for antibiotic sensitivity testing (AST) using appropriate 64-well cards.

1.      Identification process on the Vitek 2 system

Aseptically transfer 3 ml of sterile saline (0.45% – 0.50% NaCl, pH 4.5 – 7.0) into a transparent plastic tube. With a sterile loop or swab, the isolated colonies are suspended in the saline solution tube, from the 24-hour culture on blood-geloza. Homogenise the bacterial suspension until a density equivalent to 0,5 – 0,63 McFarland is obtained for unpretentious bacteria, 1,8-2,2 McFarland for fungi and 2,7-3,3 McFarland for the identification of neisserias, species of the genera Haemophilus and Corynebacterium or anaerobes. The tube with the suspension is placed in the cassette, then the Vitek card is inserted. Transferring the inoculum into the card is done automatically in the filling chamber of the device, after which it is transferred to the reader / incubator room.

Identification cards are read based on changes in bacterial growth and metabolism during incubation; they are read every 15 minutes with an optical reader. The results of the biochemical tests are automatically compared with the information in the database, selecting the bacterial species corresponding to the obtained biochemical profile.

2.      The process of establishing antibiotic sensitivity on the Vitek 2C system

Vitek AST cards are used to automatically test the sensitivity to antibiotics with the determination of the minimum inhibitory concentration (mic) for pathogens with clinical significance isolated from vaginal secretions, cervical secretions, hemocultures, from patients hospitalized in the department of Obstetrics or hypopharyngeal aspirates, CSF cultures, hemocultures from

newborns hospitalized in the department of Neonatology of SCJUPBT. The tested germs are: Gram negative bacilli, Staphylococcus species, Enterococcus, Streptococcus agalactiae, Streptococcus pneumoniae, fungi, etc.

Principle: Determination of CMI by the microdilution method. Each AST card contains 64 microcells with antibiotics in different concentrations, as well as the control cell.

Technique: a quantity of 145 µl (for gram-positive bacteria) or 280 µl (for gram-negative bacteria) of the suspension of 0.5-0.63 Mc Farland from the bacterial strain, to be tested, is resuspended in 0.45% saline. Insert the antibiogram card into the tube and insert it into the apparatus (incubator/reader).

The growing conditions are optimized to a microaerophilic atmosphere.

The instrument monitors the growth in each card cell over a defined period of time (up to 18 hours). Throughout the incubation period, CMI values are determined every 15 minutes for each antibiotic in the card.

Once the CMI result is obtained, the valid interpretation standards (breakpoint) specified in the Vitek2 software (CLSI-Clinicaland Laboratory Standards Institute) are applied to provide the clinical categories of sensitive, intermediate and resistant.

For testing the sensitivity of gram-negative bacteria, AST-N204, ASTN-222 and AST N-397

cards are used.

The AST-N204 card contains: ampicillin, amoxicillin/clavulanic acid, ceftazidime, cefotaxime, cefepim, piperacillin/tazobactam, ESBL screening (extended spectrum beta- lactamases) imipenem, meropenem, ertapenem, colistin, phosphomycin, ciprofloxacin, norfloxacin, gentamicin, amikacin.

The AST-N222 card contains: ticarcillin, ticarcillin /clavulanic acid, piperacillin, piperacillin

/clavulanic acid, ceftazidim, cefepim, aztreonam, imipenem, meropenem, amikacin, gentamicin, tobramycin, minocycline, colistin, ciprofloxacin, trimethoprim/sulfamethoxazole.

AST card N-397 contains: amoxicillin, cefotaxime, ceftazidime, ceftazidime/avibactam, piperacillin/tazobactam, colistin, ciprofloxacin, gentamicin, tobramycin, amikacin, imipenem, meropenem, trimethoprim/sulfamethoxazole.

For testing the antibiotic sensitivity of gram-positive bacteria (Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp and Streptococcus agalactiae, the AST-GP67 card is used (card content in the table):

For the anthribiogram of various streptococcal species, the AST-ST03 card is used (table below):

For antifungigram, the AST-YS08 card is used, table below:

The reference strains (ATCC American Type Culture Collection) recommended by the card maker will be used as test control witnesses: Escherichia coli ATCC 25922, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ssp pneumoniae ATCC 700603, Enterococcus faecalis ATCC 29212, Enterococcus faecalis ATCC 51299, Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC BAA-1026, Staphylococcus aureus ATCC BAA-976, Staphylococcus aureus ATCC BAA-977.

To identify fungi

Bibliography: Vitek 2. User Manuals.Biomerieux tool. France, 2009

PGS, preimplantaon genec screening, refers to removing one or more cells from an in vitro ferlized embryo to test for chromosomal normalcy.

An abnormal number of chromosomes is a major cause of in vitro ferlizaon (IVF) failure as most embryos with aneuploidy will not implant or will miscarry during the first trimester of pregnancy. PGS is performed for selecng those embryos with a normal number of chromosomes, increases the chance that a viable embryo will be selected for transfer and the likelihood of successful implantaon and pregnancy.

Our lab has the possibility to use next-generaon sequencing (NGS) technology to provide comprehensive, accurate screening of all 24 chromosomes for selecng euploid embryos. Illumina plaorm that we use enables massively parallel sequencing of millions of DNA fragments, detecng single bases as they are incorporated into growing DNA strands.  

General workflow

DNA extracon and amplificaon

DNA sample -provided by IVF clinics must be suspended in molecular grade 1x PBS (Phosphate Buffered Saline) and, if used, with a maximum concentraon of 0.5% PVP (Polyvinylpyrrolidone) in a volume of 2.5 μl. Samples must be stored at -65 – 85°C following biopsy to preserve integrity and quality. Cell lysis and amplificaon must be completed within 14 days from the harvesng.  

Materials required for sample preparation: SurePlex DNA amplificaon system, 20x PBS (dilute to 1x with nuclease-free water), genomic control DNA, PCR tubes (0.2 ml) or 96-well PCR plate and adhesive plate seals, microcentrifuge tube (1.5 ml), genomic DNA 100ng/ µl. Sample and control required  

Control Tubes for Sample Preparaon

PCR Tubes for Sample Preparaon

Store the control PCR tubes in a 96-well rack on ice unl required. Cell Lysis/Extracon steps 

1.Collect samples in 2.5 µl of 1x PBS and 2.5 µl of collecon buffer control. Centrifuge the tubes/plate at 200 × g for 3 minutes at 4°C

2.Add 2.5 µl of Cell Extracon Buffer to each sample (including controls) and store at 2°C to 8°C.

3. For every sample or control, add 5 µl of the freshly prepared Extracon Cocktail (4.8 µl Extracon

Enzyme Diluon Buffer + 0.2 µl Cell Extracon Enzyme/ sample)  

4. Briefly centrifuge samples to get all contents to the botom of the tube and incubate samples in a PCR thermal cycler for the following program:

Preamplificaon  

For each 10 µl sample prepared in Cell Lysis/Extracon, add 5 µl of SurePlex Pre-amp Cocktail (4.8 µl SurePlex Pre-amp Buffer + 0.2 µl SurePlex Pre-amp Enzyme). Briefly centrifuge and incubate samples according to the following thermal cycler program:

Following preamplificaon the products are maintained on ice  Amplificaon steps

1. Combine the Amplificaon Cocktail components

2.Add 60 μl of the freshly prepared Amplificaon Cocktail to the 15 μl pre-amplificaon reacon product, cap the tube, and invert to mix. Centrifuge briefly.

3. Amplify samples according to the thermal cycler program.

4 Keep amplified SurePlex products on ice before proceeding with the VeriSeq PGS assay protocol.

VeriSeq PGS Library Prep

The VeriSeq PGS Library Prep Kit protocol is opmized for 1 ng of input SurePlex amplified DNA. Illumina® strongly recommends quanfying the starng SurePlex amplified dsDNA. Steps for quanficaon are included in this protocol.  DNA Input Quantification

To obtain an accurate quanficaon of the DNA library, quanfy the starng DNA library using a fluorometric based method specific for duplex DNA.  

Consumables and Equipment 

96-well PCR plate, molecular grade water, Qubit dsDNA HS Assay Kit, Qubit Assay Tubes (1 tube per sample) Qubit 2.0 or greater Fluorometer, Adhesive PCR seal, 

Procedure  

Prepare 1/10 Diluons of SurePlex Sample and Controls  1 Vortex each sample and control.  

• Centrifuge at 280 × g for 1 minute.
• In a new PCR plate, add 45 μl molecular-grade water to the required wells.
• Add 5 μl sample or control to the wells containing molecular-grade water.
• Seal the plate and briefly vortex to mix.
• Centrifuge at 280 × g for 1 minute.
• Set aside on wet ice.

Qubit Method  

• Prepare the working soluon according to the manufacturer instrucons.
• To calibrate the Qubit fluorometer, add 10 μl of each standard to 190 μl of working soluon.
• Add 10 μl of the 1/10 diluted SurePlex sample and 190 μl working soluon to each assay tube. Briefly vortex to mix.
• For opmal fluorescence, incubate the assay tubes for 2 minutes.
• Calculate the concentraon of each 1/10 diluted SurePlex sample as described by the Qubit dsDNA HS Assay Kit user guide. Convert the units to ng/μl.

Tagment Input DNA  

In this step, the SurePlex amplificaon product is tagmented (tagged and fragmented) by the VeriSeq PGS transposome.  

Consumables ATM (Amplicon Tagment Mix), TD (Tagment DNA Buffer), NT (Neutralize Tagment Buffer), SurePlex amplificaon product (diluted at 0.2 ng/µl), 96-well PCR plate, Adhesive PCR seal, PCR 8-tube strips

Tagmentation of SurePlex WGA Product 

• Label a new PCR plate VTA (VeriSeq Tagment Amplicon Plate).
• Calculate the total volume of TD for all reacons. Using a mulchannel pipete, divide the volume equally among the wells of a PCR 8-tube strip, or use a reservoir.
• Add 10 µl TD Buffer to each well. 4 Add 5 µl ATM to the wells containing TD Buffer.
• Add 5 µl SurePlex amplificaon product (diluted at 0.2 ng/µl) to each sample well.
• Mix at 1,800 rpm for 1 minute centrifuge at 280 × g for 1 minute.

7. Make sure that each well contains a volume of 20 µl. Record any nonuniform volumes.

8 Immediately place on a thermal cycler with a heated lid and run the following program: 55°C for 5 minutes/ Hold at 10°C

Neutralization of the Tagmented SurePlex DNA 

• Calculate the total volume of NT buffer required for all reacons. Using a mulchannel pipete, divide the volume equally among the wells of a PCR 8-tube strip.
• Add 5 µl NT Buffer to each well. 3 Mix at 1800 rpm for 1 minute.
• Centrifuge at 280 × g for 1 minute.
• Make sure that each well contains a volume of 25 µl. Record any nonuniform volumes.
• Incubate at room temperature for 5 minutes.

Amplify Tagmented DNA

Consumables and Equipment NPM (Nextera® PCR Master Mix), index 1 primers (N701 to N712), index 2 primers (S503 and S504), TruSeq® Index Plate Fixture, Adhesive PCR seal, Plate sealer

Procedure

• Print the sample assay plate layout using the BlueFuse Workflow Manager.
• Arrange the index primers in the TruSeq Index Plate Fixture, as follows:

-Index 1 (i7) adapters: N701–N712 in columns 1–12  -Index 2 (i5) adapters: S503 in row A, S504 in row C

• Place the plate on the TruSeq Index Plate Fixture.
• Add index adapters according to the sample assay plate layout.

-Add 5 μl of each Index 1 (i7) adapter to each column.  

-Add 5 μl of each Index 2 (i5) adapter to each row.

• Add 15 μl NPM to each well.
• Mix at 1800 rpm for 1 minute.
• Centrifuge at 280 × g for 1 minute.
• Make sure that each well contains a volume of 50 µl. Record any nonuniform volumes.
• Place on the thermal cycler and run the following program on a thermal cycler with a heated lid:

Clean Up PCR -steps

Step that removes both the short library fragments and Primers from the populaon.  

• Centrifuge the VTA plate at 280 × g for 1 minute to collect condensaon.
• Add an appropriate volume of beads to a trough.
• Add 45 µl AMPure XP beads to each required well of a clean deep well plate.
• Transfer 45 µl PCR product from the VTA plate to the plate containing beads.
• Mix at 1800 rpm for 1 minute.
• Incubate at room temperature for 5 minutes. Do not shake the plate.
• Pulse centrifuge. To prevent magnec bead aggregaon, do not centrifuge longer than a pulse. 8 Place on a magnec stand and wait unl the liquid is clear (~2 minutes). Keep the plate on the stand during the following steps.
• Discard the supernatant from each well.
• Wash 2 mes, as follows. Using a mulchannel pipete, add 200 µl freshly prepared 80% EtOH to each row on the opposite side of the aggregated beads, being careful not to disturb the bead pellet at the botom of the well. Do not resuspend the beads. Incubate on the magnec stand for ≤ 30 seconds. Start the mer aer dispensing 80% EtOH into the first well being careful to not exceed 30 seconds for each row. Immediately remove and discard all supernatant from each well and discard to appropriate waste
• Using a mulchannel pipete and fine pipete ps, remove residual EtOH from each well.
• Air-dry on the magnec stand for 15 minutes, or unl beads are completely dry.
• Add 50 µl RSB to each well.
• Remove the plate from the magnec stand.
• Mix at 1800 rpm for 1 minute.
• Centrifuge at 280 × g for 1 minute.
• Place on a magnec stand and wait unl the liquid is clear (~2 minutes).
• Transfer 45 µl of each supernatant from each well to a new PCR plate.
• Run a Quality Control check to determine the success of the library preparaon.

Normalize Libraries – steps

1 In a new 15 ml conical tube, prepare the LNA1/LNB1 mix according to the number of reacons. 2 Vortex thoroughly unl LNA1/LNB1 mix is homogenized.

• Label a new deep-well plate LNP (Library Normalizaon Plate).
• Pour the LNA1/LNB1 mix into a reservoir.
• Transfer 45 µl LNA1/LNB1 mix to each well.
• Add 20 µl dsDNA from the Clean-Up PCR procedure to each well.
• Mix at 1800 rpm for 30 minutes.
• Pulse centrifuge to collect any droplets. To prevent magnec bead aggregaon, do not centrifuge longer than a pulse.
• Place on a magnec stand and wait unl the liquid is clear (~2 minutes). Keep the plate on the stand during the following steps.
• Remove and discard all supernatant from each well.
• Discard the ps in an appropriate hazardous waste container.
• Wash beads 2 mes as follows. Keep on the magnec stand and add 45 µl LNW1 to each well. Seal and shake at 1800 rpm for 5 minutes. To prevent magnec bead aggregaon, pulse centrifuge to collect any droplets. Place on a magnec stand and wait unl the liquid is clear (~2 minutes). Remove and discard all supernatant from each well.
• Add 30 µl 0.1 N NaOH to each well.
• Remove from the magnec stand.
• Mix at 1800 rpm for 5 minutes.
• Centrifuge at 280 × g for 1 minute.
• Place on a magnec stand and wait unl the liquid is clear (~2 minutes).
• Add 25 µl of LNS1 to each well of a new PCR plate.
• Transfer 25 µl of supernatant from the LNP plate to the new PCR plate containing LNS1.
• Vortex, and then centrifuge the PCR plate containing LNS1 and supernatant at 280 × g for 1 minute.

Pool Libraries for the MiSeq System

Equal volumes of normalized libraries are combined, diluted in HT1, and heat-denatured before being transferred to the flow cell for cluster generaon and sequencing.

• Centrifuge the plate at 280 × g for 1 minute.
• Transfer 5 µl of each normalized library to pool into a LoBind tube.
• Vortex and centrifuge the pooled library.
• Transfer 15 μl library pool to a new PCR tube or PCR 8-tube strip. The recommended cluster density of the MiSeq VeriSeq PGS workflow ranges from 1,100 K/mm2 to 1,600 K/mm2. Adjust the volume of the library pool to keep the cluster density in the recommended cluster density range.
• Add 85 µl HT1 record the volumes of library pool and HT1.
• Gently vortex and centrifuge the pool/HT1 mixture.
• Immediately place on the preprogrammed thermal cycler and run the DENATURE program.
• Transfer 600 μl of HT1 into a second clean LoBind tube. Set aside in an ice-water bath.
• When the denaturaon is complete, immediately transfer 100 µl of denatured pool/HT1 mixture to the LoBind tube with HT1. Set aside on wet ice.
• Sequence your library

MiSeq Sequencing VeriSeq PGS workflow

Thaw Reagent Cartridge Inspect the Reagent Cartridge 1 Remove the reagent cartridge from -25°C to -15°C storage.

• Place the reagent cartridge in a water bath containing enough room temperature deionized water to submerge the base of the reagent cartridge. Do not allow the water to exceed the maximum water line printed on the reagent cartridge.
• Allow the reagent cartridge to thaw in the room temperature water bath unl it is thawed completely. 4 Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge. Dry the base of the cartridge.

5.Inspect the reagents in posions 1, 2, and 4 to make sure that they are fully mixed and free of precipitates.

6 Place the reagent cartridge on ice for up to six hours, or set aside at 2°C to 8°C unl ready to set up the run. For best results, proceed directly to loading the sample and seng up the run.

Load Sample Libraries

• Clean the foil seal covering the reservoir labeled Load Samples with a low-lint lab ssue.
• Pierce the foil seal with a clean 1 ml pipete.
• Pipete 600 µl prepared libraries into the reservoir Load Samples. Avoid touching the foil seal
• Proceed directly to the run setup steps using the MiSeq Control Soware (MCS) interface.

Clean/ Load the Flow Cell

• Using plasc forceps, grip the flow cell by the base of the plasc cartridge and remove it from the flow cell container
• Lightly rinse the flow cell with laboratory-grade water unl both the glass and plasc cartridge are thoroughly rinsed of excess salts.
• Using care around the black flow cell port gasket, thoroughly dry the flow cell and cartridge with a linree lens cleaning ssue. Gently pat dry in the area of the gasket and adjacent glass.
• Clean the flow cell glass with an alcohol wipe. Make sure that the glass is free of streaks, fingerprints, and lint or ssue fibers.
• Dry excess alcohol with a lint-free lens cleaning ssue.
• Make sure that the flow cell ports are free of obstrucons and that the gasket is well-seated around the flow cell ports

7.Raise the flow cell compartment door, and then press the release buton to the right of the flow cell clamp.

8.Make sure that the flow cell stage is free of lint. If lint or other debris is present, clean the flow cell stage using an alcohol wipe or a lint-free ssue moistened with ethanol or isopropanol. Carefully wipe the surface of the flow cell stage unl it is clean and dry.

9.Holding the flow cell by the edges, place it on the flow cell stage, gently press down on the flow cell clamp to close it over the flow cell. Close the flow cell compartment door.

Load Reagents and Start the Run

• Remove the botle of PR2 from 2° to 8°C storage. Invert to mix, and then remove the lid.
• Open the reagent compartment door.
• Raise the sipper handle unl it locks into place.
• Remove the wash botle and load the PR2 botle.
• Empty the contents of the waste botle into the appropriate waste container.
• Slowly lower the sipper handle. Make sure that the sippers lower into the PR2 and waste botles.

7. Open the reagent chiller door.
8. Hold the reagent cartridge on the end with the Illumina label, and slide the reagent cartridge into the reagent chiller unl the cartridge stops.
9. Close the reagent chiller door.
10. Aer loading the flow cell and reagents, review the run parameters and perform a pre-run check before starng the run. When all items successfully pass the pre-run check, select Start Run. During a run, monitor run detail using the Sequencing screen on the instrument.

Data Analysis

Aneuploidy screening embryos prior to transfer has been shown to deliver significant improvements IVF success. Preimplantaon genec diagnosis (PGD) enables screening of embryos for specific genec condions prior to transfer. Following sequencing sample informaon is uploaded directly from the MiSeq System, saving me and allowing sample tracking. BlueFuse Mul is a sophiscated soware for analyzing and interpreng results from molecular cytogenomic studies.

Sample informaon includes sample type (e.g., blood, CVS, amnioc fluid), date of birth, pedigree number, and phenotype. Related samples can be overlaid for data comparisons and regions of change compared to phenotype. Data are permanently available and can be revisited and interrogated at any me ensuring that any follow-up quesons can be readily answered. NGS provides reliable, comprehensive screening of 24-chromosome aneuploidy, achieving results with a high degree of concordance to those obtained using established array-based PGS techniques. NGS offers a readily available, high-throughput method for PGS in the clinic with the potenal benefits of reduced costs and enhanced precision.