INSTRUCTIONS FOR LABORATORY TRAINING - · PDF file1 INSTRUCTIONS FOR LABORATORY TRAINING HUMAN...

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INSTRUCTIONS FOR LABORATORY TRAINING

HUMAN IMMUNODEFICIENCY VIRUS 1 (HIV-1)

FLUOROGNOST HIV-1 IFA

A Qualitative Immunofluorescence Assay for the Detection of Antibodies to Human Immunodeficiency

Type 1 (HIV-1) Virus in Human Serum and Plasma

TABLE OF CONTENTS SECTION A : TRAINING AND READER QUALIFICATION ................................................................................................2

SECTION B: INTERPRETATION GUIDE.............................................................................................................................7

SECTION C: PROFICIENCY TEST ...................................................................................................................................20

11-21-08 Version 3.0

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SECTION A : TRAINING AND READER QUALIFICATION

I. PURPOSE AND NATURE OF THE QUALIFICATION PROCEDURE

Prior to the use of FLUOROGNOST HIV-1 IFA by any laboratory, the laboratory is strongly advised to

demonstrate that at least one technician, researcher or other individual has been adequately trained to

perform and interpret the IFA.

Sanochemia qualification of adequate training to conduct, evaluate and interpret FLUOROGNOST

HIV-1 IFA is awarded to a specific individual in a specific work environment.

Becoming proficient in the Assay requires:

proper conduct of the Assay Protocol

suitable equipment and

sufficient skills to evaluate and interpret the various fluorescence patterns accurately.

Individuals who are qualified by Sanochemia criteria to perform the IFA at a single site will be deemed

qualified to perform the Assay at all sites, provided that the Immunofluorescence microscopes used for

evaluation are technically equivalent with respect to all relevant technical parameters:

- Overall magnification

- Numerical aperture (often referred to as “speed” of the lens)

- Field of view

- UV light source

- Filter system

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II. THE FLUOROGNOST HIV-1 IFA TRAINING SET FOR NOVICE USERS

In order to train and qualify individuals to perform the FLUOROGNOST HIV-1 IFA, Sanochemia will provide

to each laboratory a Training Set, with their initial order.

TRAINING SET:

1 FLUOROGNOST HIV-1 IFA KITS as ordered

2 A copy of the Instructions for Laboratory Training and Qualification brochure

3 A coded Proficiency Panel

The Training Set will be shipped to the attention of the Laboratory Director. Upon receipt of the Training Set,

the Coded Proficiency Panel should be removed from the shipment container. Store the Coded Proficiency

Panel in its original box at 2 to 8°C. It is also recommended that the Laboratory Director removes and files

separately the Proficiency Test Section from the Instructions for Laboratory Training and Qualification.

The qualification of an individual in the use and interpretation of FLUOROGNOST HIV-1 IFA results is

based on satisfactory completion of the following:

1. It is expected that the qualified readers correctly identify all samples in the preparatory written Proficiency

Test or “Paper Exam.”

2. It is also expected that qualified readers will correctly and independently interpret all of the Proficiency

Panel members using the FLUOROGNOST HIV-1 IFA.

The Director should select at least one employee to be trained in the assay. [Several individuals may be

selected for simultaneous qualification with the same Coded Proficiency Panel.] It is not necessary that the

employees have prior experience with indirect immunofluorescence assays.

It is recommended that every selected trainee studies the Instructions for Laboratory Training and

Qualification, and passes the “Paper Exam” in private with the Laboratory Director, before proceeding to the

evaluation of the coded Proficiency Panel.

The designated trainee(s) should be provided with the Instructions for Laboratory Training and Qualification,

excluding the Proficiency Test Section. Instruct the trainee(s) to read the Instructions for Laboratory Training

and Qualification carefully and review all of the sections, paying particular attention to the interpretation guide

and the illustrations provided. The trainee(s) should be instructed to notify the Laboratory Director when he or

she thoroughly understands the instructions and interpretations and is prepared to take the Proficiency Test.

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III. PREPARATORY “PAPER EXAM “PROFICIENCY TEST

For the written Proficiency Test, the Laboratory Director should first provide the trainee(s) with a Test

Worksheet and the Proficiency Test materials, including color photos of the 15 sets of test samples. It is

important to note that there is only one correct interpretation for each test specimen. The trainee(s) should be

instructed to independently evaluate the photographs of the uninfected and infected cell wells for each

specimen, interpret the respective IFA result, and place his/her results on the Worksheet. When finished, the

trainee(s) are asked to return the completed Worksheet to the Laboratory Director. He/she then grades the

completed Worksheet(s) using the “Proficiency Test Worksheet Scorecard” found in Annex 2 at end of this

book.

If the trainee(s) correctly interprets all test samples (15 of 15), he/she is ready for the laboratory testing of the

18 coded Proficiency Panel samples using FLUOROGNOST HIV-1 IFA. However, if the trainee incorrectly

interprets any of the coded Proficiency Panel samples, he/she should be required to study again the

Instruction for Laboratory Training and Qualification and take the written Proficiency Test a second time. If on

the second test the trainee fails to correctly interpret any of the Proficiency Panel samples, the Laboratory

Director should attempt to determine the reason for the failure.

The Laboratory Director is encouraged to consult with Sanochemia support:

PHONE (USA): 203-564-1580

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IV. LABORATORY TESTING OF THE PROFICIENCY PANEL

After correctly completing the written preparatory test, the trainee(s) is ready for the laboratory testing of the

coded Proficiency Panel using the FLUOROGNOST HIV-1 IFA KIT.

The Laboratory Director should prepare the necessary materials for testing the Coded Proficiency Panel

members. For each trainee, 60 µl of the Concentrated FITC-Conjugate should be diluted with 540 µl in a

suitable vial with PBS Working Diluent. (Concentrated Diluent is provided in kit).

5 µl each of the FLUOROGNOST HIV-1 IFA Positive and Negative Serum Controls should be diluted with

145 µl of PBS Diluent in appropriately labeled vials. The quantities prepared need to be adjusted for the

number of trainees testing the Proficiency Panel.

Testing of the Coded Proficiency Panel will consume 4 IFA slides per trainee. The remainder of the Kit may

then be stored for regular use as indicated on the Kit label.

The Laboratory Director should then provide the trainee(s) with the Coded Proficiency Panel and all of the

materials required for carrying out the Assay. A list of these materials is outlined in the Product Insert under

“Additional Materials Required, Not Supplied.”

Instruct the trainee(s) to open one FLUOROGNOST HIV-1 IFA KIT and to follow the instructions in the

Package Insert. The trainee(s) should dilute 5 µl aliquots of each of the 18 Coded Proficiency Panel members

according to instructions for the Kit Controls. The assay should be conducted in strict adherence to the

“Assay Procedures” provided in the Package Insert. The results should be filled into the dedicated worksheet

found in Annex 3.

Note to the Laboratory Director The amount of undiluted serum remaining from the Coded Proficiency Panel members should be stored at -20°C; the

serum may be thawed once and used for qualification of other employees until the expiry date indicated on the Coded

Proficiency Panel Kit label. The Coded Proficiency Panel is not intended as a set of standards for the laboratory, and

should be reserved for the sole purpose of user qualification.

The trainee(s) should, independently and unaided, evaluate the mounted IFA Slides the same day. Ask the trainee(s) to

enter their IFA results for each Panel member onto the Proficiency Test Worksheet (see Annex 3). When the sheets are

completed, have the trainee immediately forward the Worksheet to the Laboratory Director.

It is important that trainee(s) report their interpretations without conferring with anyone else. Other employees should be

encouraged to examine the IFA Slides under the microscope as a means of becoming acquainted with the various IFA

fluorescence patterns. Each individual, however, should independently qualify before being allowed to perform, evaluate,

and interpret the FLUOROGNOST HIV-1 IFA.

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V. NOTIFICATION OF LABORATORY QUALIFICATION

On completion of the laboratory testing, the Laboratory Director should check the Coded Proficiency Panel

Worksheet for completeness and send it directly to Sanochemia. In turn, Sanochemia will notify the

Laboratory Director within two working days whether or not the results are in 100% agreement with the

expected values. On this basis, the laboratory and the individual employee will be provided with, or refused, a

FLUOROGNOST HIV-1 NOTICE OF QUALIFICATION (example, refer to Annex 4) to perform

FLUOROGNOST HIV-1 assays with donor and/or patient sera.

It is strongly recommended that no laboratory employee perform a FLUOROGNOST HIV-1 IFA without

passing both parts of the Proficiency Test (written and laboratory).

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SECTION B: INTERPRETATION GUIDE

TABLE OF CONTENTS

I. INTRODUCTION ...............................................................................................................................................................7

II. SUMMARY OF FLUOROGNOST HIV-1 IFA INTERPRETATION....................................................................................8

III. PHOTOGRAPHIC PORTFOLIO OF IFA INTERPRETATIONS.......................................................................................9

1. Negative Serum Control (Exhibit 1) ..............................................................................................................................9

2. Positive Serum Control (Exhibit 2) ................................................................................................................................9

3. Positive IFA Interpretations (Exhibits 3 - 9)...................................................................................................................9

4. Negative IFA Interpretation (Exhibit 11)......................................................................................................................10

5. Indeterminate IFA Interpretations (Exhibits 12 - 18) ...................................................................................................11

6. Positive IFA interpretations in the presence of non-specific staining (Exhibit 19, 20) .................................................12

I. INTRODUCTION

The FLUOROGNOST HIV-1 IFA Interpretation Guide Section is designed to provide both the new and

experienced IFA reader with a comprehensive tutorial on the evaluation and interpretation of

FLUOROGNOST HIV-1 IFA results. The Guide contains a portfolio of typical microscopic immunofluorescent

photographs of the infected and uninfected control cells of an HIV-1 IFA slide for clinical specimens

representing Positive, Negative or Indeterminate IFA interpretations. An attempt was made to obtain

photographs which are identical in fluorescent color to those a user will see under the microscope. In order to

provide the best possible microscopic field, all of the photographs were taken at a magnification of 400x.

For quick reference, a summary page of the FLUOROGNOST HIV-1 IFA interpretation criteria is provided in

the package insert. The discussion and portfolio of immunofluorescent photographs of the clinical samples

representing the various IFA results are organized into six separate sections and are presented in the

following order:

(1) Negative Control, Exhibit 1

(2) Positive Control, Exhibit 2

(3) Positive IFA interpretations, Exhibits 3-9, and a photographic comparison of three different positive HIV-1

specimens with decreasing fluorescence intensity, Exhibit 10

(4) Negative IFA interpretations, Exhibit 11,

(5) Indeterminate IFA interpretations, Exhibits 12 through 18, and

(6) Positive IFA interpretations in the presence of non-specific staining, Exhibit 19 and 20 (page 19).

Study the photographs and explanations in this section of the Instructions for Laboratory Training and

Qualification until you are satisfied with your understanding of IFA interpretive criteria and your ability to

interpret IFA test results. Notify the Laboratory Director that you are prepared to take a written Proficiency

Test to measure your comprehension of the material.

If you have any questions regarding the interpretation of the IFA, criteria or results, please call the

Sanochemia Technical Support Department Hot Line: 203-564-1580 .

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II. SUMMARY OF FLUOROGNOST HIV-1 IFA INTERPRETATION

Negative Result Interpretation Criteria A test specimen is interpreted as NEGATIVE when there is no specific fluorescent staining of the infected

cells and there is no credible difference in the intensity of fluorescent staining and the pattern of fluorescence

between the HIV-1 infected and uninfected cells. The test specimen is reported as negative and no follow-up

testing is required.

Positive Result Interpretation Criteria A test specimen is interpreted as POSITIVE when there is a specific cytoplasmic staining pattern in the HIV-1

infected cells and there is a credible difference in the intensity of fluorescent staining and the pattern of

fluorescence between the HIV-1 infected and uninfected cells. The test specimen is reported as positive and

no follow-up testing is required.

Indeterminate Result Interpretation Criteria A test specimen is interpreted as INDETERMINATE when there is fluorescent staining present IN BOTH the

HIV-1 infected and uninfected cells, and it is NOT possible to differentiate the intensity of fluorescent staining

and the pattern of fluorescence between the HIV-1 infected and uninfected cells. The test specimen is

reported as INDETERMINATE and repeat testing of the original specimen should be carried out. If an

indeterminate result persists, it may be necessary to obtain a fresh test specimen for follow up testing. Non-

specific staining can be categorized as cellular and/or extracellular and can occur as a result of a variety of

conditions and from a number of sources. Cellular and extracellular staining can also occur in the same

sample. The following is a brief description of typical non-specific staining reactions:

Non-specific Cellular Staining Non-specific cellular staining can occur when antibodies from the test sera bind with non-HIV-1 protein in

both the HIV-1 infected and uninfected control cells. For example, sera from patients with Systemic Lupus

Erythematosus (SLE) can result in intense cell membrane staining without cytoplasmic staining. Sera that

possess antinuclear antibody (ANA) react with the nucleus, but not the cytoplasm of the cells. Some sera

react with non-specific antigens in the cytoplasm of the cells and often display an appearance of a “polar

cap”. Non-specific staining would be evident in infected as well as uninfected cell wells of the slide.

Non-specific Extracellular Staining Non-specific extracellular staining can appear in a wide variety of patterns, including an amorphous film,

droplets, particulate matter, bacterial/fungal contamination and dead cells. This type of staining generally

does not interfere with the interpretation of the IFA result as long as the positive or negative result criteria are

fulfilled.

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III. PHOTOGRAPHIC PORTFOLIO OF IFA INTERPRETATIONS

The following is a presentation of immunofluorescent photographs displaying typical negative and positive

control samples, as well as, samples interpreted to be Positive, Negative, and Indeterminate. A brief

description of each category of IFA interpretation precedes the representative photograph(s). This collection

of illustrations can serve as a useful reference for the categorization and interpretation of FLUOROGNOST

HIV-1 IFA Assay results.

1. Negative Serum Control (Exhibit 1) The FLUOROGNOST HIV-1 IFA Negative Serum Control is composed of heat-inactivated normal human

serum which is negative for antibodies to HIV-1, Hepatitis B, and C. The HIV-1 infected and uninfected cells

of the Negative Serum Control should not exhibit any specific fluorescence and should appear to be

essentially indistinguishable. Please see Exhibit 1.

2. Positive Serum Control (Exhibit 2) The FLUOROGNOST HIV-1 IFA Positive Serum Control is composed of heat-inactivated normal human

serum which is positive for antibodies to HIV-1 and negative for antibodies to hepatitis B and C. The HIV-1

infected cells of the Positive Serum Control should demonstrate an intense apple-green staining of the

cytoplasm, while the uninfected control cells should not display any specific fluorescence. Depending on the

spatial orientation of the infected cell, the fluorescent staining pattern may appear as a “polar cap” or a

crescent shaped structure (half-moon). Validation of both the Negative and Positive Serum Controls is

required to validate each run of the IFA test. Please see Exhibit 2.

3. Positive IFA Interpretations (Exhibits 3 - 9) The immunofluorescent pictures in this section were taken from a series of HIV-1 positive serum samples

obtained from domestic and international sources. All of the specimens display the required cytoplasmic

staining pattern in combination with the contrast in the fluorescence between the infected and uninfected

cells. By studying each sample closely, it is possible to recognize a full scope of the various staining patterns

which result from different orientations of the HIV-1 infected cell.

The positive IFA interpretation criteria require that there is

(1) a specific staining pattern of the cell cytoplasm in the HIV-1 infected cells and that

(2) there is a credible difference in both the intensity of fluorescent staining and the pattern of fluorescence

between the HIV-1 infected and uninfected control cells.

Positive HIV-1 infected cells will exhibit a concentrated fluorescence in the cytoplasm of the cell. Because the

PALL T-cell nucleus occupies a large portion of the cell volume, the cytoplasm tends to be compressed into a

dense acentric structure which is localized at one end of the cell. A positive cytoplasmic pattern can vary in

appearance from an acentric “half moon” to a “polar cap”, depending on the orientation of the T-cell fixed on

the slide well. A positive pattern can range from diffuse to finely reticulated and the intensity can vary from a

very intense brilliant apple-green color to a less intense apple-green color (see Exhibit 10). Some sera also

have non-specific staining of the uninfected cells in combination with specific HIV-1 staining of the infected

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cell and should be interpreted as positive as long as there is a credible contrast in fluorescence (see

Exhibits). The infected cell well contains 40-70% infected cells, often termed a “mixed cell” environment.

Therefore, a positive specimen will exhibit specific fluorescence in at most 40-70% of the cells. The remaining

cells are uninfected, serve as a built in, internal control, and should have an appearance similar to the

Negative Serum Control.

CAUTION: It is ALWAYS NECESSARY to evaluate both the uninfected cell well and the infected cell well

before interpreting the final IFA result. Be sure to scan a range of 3-5 microscopic fields within each cell well

before completing the evaluation.

3a. Photographic Comparison of the Variation in Fluorescent Staining Intensity among HIV-1 Positive Specimens (Exhibit 10) For sera containing antibodies to HIV-1, the intensity of fluorescent staining can vary from a very intense

apple green fluorescence to a dull green fluorescence. In Exhibit 10, the infected and uninfected cells of three

positive HIV-1 specimens, with varying degrees of fluorescence, are displayed together to demonstrate the

color contrast. Each of the samples shows a credible difference in both the intensity and the pattern of

fluorescence between the respective infected and uninfected control cells, thereby, conforming to a positive

HIV-1 interpretation. Note the decreasing intensity of fluorescence between the infected cells of specimens 1,

2 and 3. Even though specimen 3 has a much weaker fluorescence, it is still possible to easily distinguish

between the intensity of the infected and uninfected cells of that sample.

By carefully studying the photograph of the infected cells of each specimen, it is also possible to see

uninfected cells in the same microscopic field which have no fluorescent staining.

The typical pattern of cytoplasmic staining is very evident in each specimen. Note that in the bulk of the

infected cells, the concentration of fluorescent staining is localized at one end of the cell. As mentioned

previously, the nucleus of the cell tends to compress the cytoplasm, and depending on the orientation of the

cell when it was fixed to the HIV-1 slide, the concentration of staining can have the appearance of a polar cap

or an acentric half-moon structure. Most of the infected cells in these samples demonstrate the usual and

distinctive polar cap staining.

4. Negative IFA Interpretation (Exhibit 11) The negative IFA Test result interpretation criteria require that (1) there is no specific cell staining in either the

HIV-1 infected cells or in the uninfected cells and that (2) it is not possible to differentiate the intensity of

fluorescent staining or the pattern of fluorescence between the HIV-1 infected and uninfected cells. Note that

the HIV-1 infected and uninfected cells of the negative specimen have an appearance similar to those of the

Negative Serum Control and the uninfected cells of the Positive Serum Control.

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5. Indeterminate IFA Interpretations (Exhibits 12 - 18) A test specimen is interpreted as INDETERMINATE when there is fluorescent staining present IN BOTH the

HIV-1 infected and uninfected cells, and it is NOT possible to differentiate the intensity of fluorescent staining

and the pattern of fluorescence between the HIV-1 infected and uninfected cells. The test specimen is

reported as INDETERMINATE and repeat testing of the original specimen should be carried out.

The INDETERMINATE IFA interpretation does not imply that HIV-1 antibodies are, or are not, present in the

test specimen. It simply means that the HIV-1 status of the blood specimen cannot be resolved by the results

of that particular Fluorognost HIV-1 IFA test run.

INDETERMINATE ASSAY RESULTS MUST NOT BE CONSIDERED POSITIVE OR NEGATIVE !!!

The correct evaluation in such situations must be based on subsequent repeat testing and/or immunoblot

testing and clinical evaluation.

In most cases, INDETERMINATE IFA results are due to the presence of non-specific staining. In specimens

with non-specific staining reactions, the fluorescence intensity can vary from very weak to very intense, and

staining will be exhibited in BOTH the infected and uninfected cells. When the specimen reacts equally with

both the infected and uninfected cells, the IFA interpretation result must be regarded as inconclusive and

reported as INDETERMINATE. Non-specific staining can be categorized as cellular and/or extracellular and

can occur as a result of a variety of conditions or be introduced from a number of sources. Cellular and

extracellular staining can also occur in the same sample.

Non-specific cellular staining can occur when antibodies from the test sera bind with non - HIV-1 protein in

both the HIV-1 infected cells and uninfected control cells. Examples include the cell membrane staining found

in sera from patients with Systemic Lupus Erythematosus (SLE), the nuclear staining found in sera containing

the antinuclear antibody (ANA), and the “polar cap” cytoplasmic staining occasionally found in non-specific

sera. While there is a myriad of possible patterns, non-specific cellular staining will always occur in both the

HIV-1 infected cells and uninfected control cells.

It is also possible for a test specimen to present non-specific staining in the presence of specific HIV-1

staining. In those specimens where the positive interpretation criteria are clearly fulfilled, the sample is

reported as positive. In some specimens, the nonspecific staining may mask the presence of specific HIV-1

staining and hence, those samples are reported as INDETERMINATE.

Non-specific extracellular staining patterns can vary significantly, and can appear as an amorphous film

with no structure, droplets, particulate matter, dead cells and microbial contamination. Extracellular

fluorescence generally does not interfere with the interpretation of an IFA result, since interpretation of the

test is based on the presence, or absence, of typical fluorescence within the cytoplasm of the HIV-1 infected

cells.

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6. Positive IFA interpretations in the presence of non-specific staining (Exhibit 19, 20) It is important to note that it is also possible for a test specimen to present specific HIV-1 staining in the

presence of non-specific staining (see Exhibit 19). In those specimens where the positive interpretation

criteria are clearly fulfilled, the sample is reported as POSITIVE. In some specimens, the non-specific staining

may mask the presence of specific HIV-1 staining and hence, those samples should be reported as

INDETERMINATE. If the IFA reader does not have confidence in the IFA interpretation, the test result should

be considered to be INDETERMINATE and follow-up testing with the original specimen should be carried out

as outlined above. Caution must be exercised with sera containing microbial contamination since

bacteria/fungus may reduce or eliminate antibody binding to HIV-1 antigens and lead to a false negative

interpretation. Exhibit 1: Negative Serum Control. Note that both the HIV-1 infected and uninfected control cells do not have any specific fluorescence and are essentially indistinguishable.

HIV-1 Infected Cells Uninfected Control Cells

Exhibit 2: Positive Serum Control. Note the typical apple-green fluorescent staining of the cytoplasm in the infected cells and the absence of fluorescent staining in the uninfected control cells.


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Exhibit 3: Positive IFA staining reaction






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Exhibit 10: A comparison of fluorescence intensity in three positive HIV-1 specimens. Note the variation in pattern and intensity of the fluorescent staining among samples. Note the presence of unstained, uninfected cells in the same field with stained, infected cells and the typical pattern of cytoplasmic staining in HIV-1 cells (mostly polar cap staining in these samples).



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Exhibit 11: Negative IFA staining reaction. Note that there is an absence of fluorescence staining in both the infected and uninfected control cells.


Exhibit 12: Non-specific IFA staining reaction. This is an example of a fairly common nonspecific staining pattern involving the cytoplasm of both the infected and uninfected cells. This staining pattern precludes interpretation of a positive or negative IFA result. The cells often display a “polar cap” staining due to the concentration of cytoplasm at one end of the cell.


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Exhibit 13: Non-specific IFA staining reaction. This is another example of a nonspecific “polar cap” of the cytoplasm which occurs both in the infected cells and control cells.


Exhibit 14: Non-Specific IFA staining reaction. This is an SLE serum sample displaying a cytoplasmic membrane staining pattern in both the infected and uninfected control cells. The staining in both cells precludes interpretation of a positive or negative IFA result.


Exhibit 15: Non-specific IFA staining reaction. Sera from patients with SLE can produce non-specific reactions. This SLE specimen illustrates intense cytoplasmic membrane staining of both the infected cells and uninfected control cells making it impossible to interpret the IFA result. Some extracellular staining of “film like” materials also present in both cell wells.


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Exhibit 17: Non-specific extracellular staining reaction. Some sera possess material that sticks to the slide and fluoresces as shown in this picture. Since no cellular structures can be discerned the presence of this material should be ignored because it does not interfere with the interpretation of the presence of absence of fluorescence within the cytoplasm of the cells.


Exhibit 18: Non-specific extracellular staining reaction. This serum does not possess HIV-1 antibody but is reacting with dead cells which are present in both the infected and the uninfected cell wells. Dead cells are smaller than live cells and stain completely hence typical cytoplasmic and nuclear morphology cannot be discerned. This staining does not interfere with the interpretation of a positive or negative IFA result.


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Exhibit 19: Positive IFA staining reaction in the presence of non-specific polar staining.


Exhibit 20: Positive IFA staining reaction plus non-specific polar staining extracellular staining of bacterial and/or fungal contamination. Unless a sample is grossly contaminated this type of non-specific staining does not interfere with the interpretation of specific HIV-1 cytoplasmic staining within the infected cells.


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SECTION C: PROFICIENCY TEST

TABLE OF CONTENTS I. INTRODUCTION .............................................................................................................................................................20

II. INSTRUCTIONS FOR TAKING THE PROFICIENCY TEST ..........................................................................................20

III. INSTRUCTIONS AFTER COMPLETING THE PROFICIENCY TEST...........................................................................20

I. INTRODUCTION

The FLUOROGNOST HIV-1 IFA Proficiency Test is designed to test the ability of an IFA reader to recognize

and correctly interpret HIV-1 specific fluorescence patterns as POSITIVE, absence of specific is staining as

NEGATIVE and non-specific fluorescent staining patterns as INDETERMINATE.

The Proficiency Test consists of 15 sets of two immunofluorescent photographs showing Fluorognost HIV-1

infected cells paired with uninfected control cells. The photographs were all taken at a magnification of 400x

and are reproductions of the actual microscopic views of samples tested by Fluorognost HIV-1 IFA. Each set

represents a single clinical specimen selected from a panel of domestic and international blood samples.

II. INSTRUCTIONS FOR TAKING THE PROFICIENCY TEST

In the following pages, the fluorescent staining of Fluorognost HIV-1 IFA infected cells and uninfected control

cells tested with clinical specimens are presented for your evaluation and interpretation. The Proficiency Test

Worksheet provides answer columns for your interpretation of each test specimen. [Four Proficiency Test

Worksheets are provided in Annex 1]

Remove a Proficiency Test Worksheet. Fill your name and the date in your worksheet.

Study the photographs of each sample carefully. For each test specimen, evaluate the immunofluorescent

patterns in the photographs of BOTH the HIV-1 infected cells (left panel) and the uninfected control cells

(right panel). Select a POSITIVE, NEGATIVE or INDETERMINATE interpretation for each test sample and

place an “X” in the appropriate column of the Proficiency Test Worksheet. Please note that there is only one

correct interpretation for each specimen.

III. INSTRUCTIONS AFTER COMPLETING THE PROFICIENCY TEST

After completing the Proficiency Test, give the completed Test Worksheet to your laboratory supervisor for

review.

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TEST SPECIMEN 1


TEST SPECIMEN 2


TEST SPECIMEN 3


TEST SPECIMEN 4


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TEST SPECIMEN 5


TEST SPECIMEN 6


TEST SPECIMEN 7


TEST SPECIMEN 8


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TEST SPECIMEN 9


TEST SPECIMEN 10


TEST SPECIMEN 11


TEST SPECIMEN 12


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TEST SPECIMEN 13


TEST SPECIMEN 14


TEST SPECIMEN 15


Annex 1:

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Proficiency Test Worksheet

The Fluorognost HIV-1 IFA Proficiency Test is designed to test your ability to correctly interpret the results of

a mixed panel of Fluorognost HIV-1 IFA samples. Evaluate the uninfected and infected photograph of each

serum sample, record your interpretation in the appropriate result box with an “X”, and give the test to your

supervisor for review.

TECHNICIAN NAME: _______________________ DATE: ______________

TEST ADMINISTERED BY: ___________________ SCORE: ______________

TEST SPECIMEN NO. INTERPRETATION RESULTS Positive Negative Non-Specific

1

2

3

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5

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Annex 1:

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1

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Annex 1:

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1

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Annex 1:

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1

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Annex 2:

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Proficiency Test Scorecard:


1 X

2 X

3 X

4 X

5 X

6 X

7 X

8 X

9 X

10 X

11 X

12 X

13 X

14 X

15 X

Annex 3:

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Please enter the results of the PROFICIENCY PANEL TEST (18 serum samples) in the table below by

checking the appropriate box.

NAME & ADDRESS OF INSTITUTION: _________________________________________________

_________________________________________________


TEST ADMINISTERED BY: ___________________ LOT #: ______________

PROFICIENCY PANEL BATCH: _______________


Results should be faxed or emailed to: Sanochemia Tech Support 203-564-1580 [email protected]

Annex 4:

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NOTICE OF QUALIFICATION

This is to certify that the laboratory and the investigators listed below have received qualification to perform

the FLUOROGNOST HIV-1 Indirect Immunofluorescence Assay and to report the presence and absence of

antibodies to human immunodeficiency virus type 1 in human serum, plasma and DBS:

Testing site: XXX Public Health Lab

Investigator`s Name: Name

Address: XXXX

This non-transferable qualification is based on the results you obtained with the coded Proficiency Panel

(series XXXXX) on DD/MM/YYYY and reported to Sanochemia on DD/MM/YYYY, which conform with

expected results.

Your FLUOROGNOST HIV-1 User Reference Number is: KOXXXX

Congratulation!

On behalf of Sanochemia Pharmazeutika AG:

DI Wolfgang Stocker DD/MM/YYYY

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SANOCHEMIA PHARMAZEUTIKA AG

Manufactured for: Sanochemia Corporation

One Stamford Plaza 263 Tresser Blvd.

Stamford, CT 06901

Phone: 203 564 1580

Fax: 203 564 1402

Cell: 203 305 8086

Email: [email protected]

by: Sanochemia Pharmazeutika AG

Boltzmanngasse 11

A-1090 Vienna, Austria

US license #1631

For customer orders or technical service call: 203 564 1580

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