# SEROLOGIC ASSAYS FOR HUMAN 

# 

# IMMUNODEFICIENCY VIRUS ANTIBODY

#  

# IN DRIED‑BLOOD SPECIMENS COLLECTED

# 

#  ON FILTER PAPER

**CONTENTS**

Page

PREFACE
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1

HOW TO USE THIS MANUAL
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SAFETY...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....2

Guidelines to Prevent Transmission of

HIV in the Laboratory
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In Case of a Spill
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SPECIMEN COLLECTION AND STORAGE
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Sample Collection
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Collection from finger sticks:

Collection from vacutainer of blood

Packaging Blood
Spots....................................................................6

Storage of Specimens
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Shipping Stored Specimens to Another Site
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#  Logging and Tracking DBS Specimens................................................7

Punching Disks From Specimens for Testing
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ENZYME IMMUNOASSAY MODIFICATIONS FOR DBS ANALYSIS
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Eluting Filter Paper Blood Specimens
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General Instructions for Preparation of Eluates
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Manufacturers\' Instructions for Preparation of Eluates
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Classification of Results
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WESTERN BLOT MODIFICATIONS FOR BLOOD-SPOT ANANLYSIS
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QUALITY CONTROL OF DBS SPECIMENS FOR ENZYME IMMUNOASSAY AND IMMUNOBLOT
ASSAY\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...21

Quality Control for Elution Procedure
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> Quality Control of Enzyme Immunoassays for DBS Specimens
> ................22
>
> Quality Control for the Western Blot
> Assay\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...22

Interpretation of Quality Control Results
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Number range CHAPTERQUALITY CONTROL (QC) CRITERIA FOR A VALID RUN:
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Figure 1. Dried‑Blood‑Spot Quality Control

Appendix
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**PREFACE**

Antibodies to the human immunodeficiency virus (HIV) can be detected in
whole blood that has been collected and dried in filter paper. The DBS
(DBS) can used to study the prevalence of HIV infection in populations
and can be used to screen and confirm the HIV status of individuals.

Collection of blood samples on absorbent paper has unique advantages for
large‑scale screening programs and epidemiologic surveys. Sufficient
blood to saturate the collection paper can be obtained easily by
sticking the heel, finger, or ear, thereby eliminating the need for
venipuncture. The DBS do not require refrigeration, and they can be
mailed conveniently and inexpensively to a central laboratory for
analysis. Specially formulated absorbent papers for blood collection
(Schleicher & Schuell 903 or Whatman BFC 180) are commercially available
and are registered as in vitro medical devices subject to Federal Food
and Drug Administration regulations.

This manual was prepared to guide laboratorians in screening whole blood
specimens collected on filter paper (to be referred to as blood spots)
for HIV antibody using commercially available enzyme immunoassay (EIA)
and enzyme-linked immunosorbent blot technique procedures. Since these
procedures were developed for clear, non‑hemolyzed serum or plasma
samples, testing the hemolyzed specimens obtained by eluting requires
considerable modification of existing assay procedures. Methods for
eluting the blood from the paper and for testing microvolumes of serum
by EIA and Western Blot are described in this manual. In addition, a
quality assurance program to ensure reliable results when testing this
type of specimen is described.

**HOW TO USE THIS MANUAL**

This manual provides the laboratorian with information necessary to test
dried-bloodspot specimens (DBS) for human immunodeficiency virus (HIV)
antibody by enzyme immunoassay (EIA) and Western Blot assay.

Read all sections of the manual before performing any of the laboratory
procedures. **Pay special attention to the sections on specimen storage,
specimen processing, and quality control, since these procedures differ
from those recommended by the manufacturers for the routine analysis of
serum and plasma specimens.**

This manual complements the instructions provided by manufacturers of
HIV‑EIA antibody kits. The standard assays for serum and plasma
specimens were changed as little as possible, and these changes are
noted.

\*Use of trade names is for identification only and does not constitute
endorsement by the Public Health Service or by the U.S. Department of
Health and Human Services

**SAFETY**

Working with DBS for human immunodeficiency virus (HIV) testing
introduces no new biohazards to laboratorians working in either neonatal
screening or HIV testing laboratories. In fact, the viability of HIV
seems to be reduced in the dried state (1,2). Despite these findings,
universal blood and body fluid precautions should be observed for ALL
blood‑spot specimens. These precautions are described in the August 21,
1987, supplement to the [Morbidity and Mortality Weekly
Report]{.underline} (3) and amplified in the April 1, 1988, supplement
and June 24, 1988, issue of the same publication (4,5), and updated in
the July 12, 1991 recommendations (6). Every laboratory should have a
copy of these guidelines and observe the recommendations.

**Guidelines to Prevent Transmission of HIV in the Laboratory (7)**

1\. Employ appropriate barrier precautions to prevent skin and mucous
membrane exposure when contact with blood or other body fluids of any
person is anticipated:

> ‑ Wear gloves when performing venipuncture and other vascular access
> procedures.
>
> ‑ Use gloves for performing finger sticks on teens and adults and/or
> heel sticks on infants and children (5).
>
> ‑ Change gloves and wash hands after contact with each patient.
>
> ‑ Place all specimens of blood and body fluids in containers that will
> prevent leakage during transport. Avoid contaminating the outside of
> the container and the laboratory form accompanying the specimen.
>
> NOTE: Whole blood dried on filter paper has not been shown to present
> a hazard when mailed in paper envelopes. See the Specimen Collection
> and Storage section for more information.
>
> ‑ Wear gloves when processing blood and body fluid specimens. Remove
> gloves and wash hands with soap and water upon completion of specimen
> processing.

2\. If hands or other skin surfaces become contaminated with blood or
other body fluids, wash them immediately and thoroughly with soap and
water.

3\. Employ a biological safety cabinet for procedures that have a high
potential for generating droplets (blending, sonicating, vigorous
mixing).

4\. Use mechanical pipetting devices to manipulate all liquids in the
laboratory. **DO NOT PIPETTE BY MOUTH.**

5\. Take precautions to prevent injuries caused by needles, scalpels,
and other sharp instruments.

> ‑ Do not recap needles, bend or break needles by hand, or remove
> needles from disposable syringes.
>
> ‑ Discard all sharp instruments in puncture‑ resistant containers
> located close to work area.
>
> ‑ Limit use of needles and syringes to situations in which there is no
> alternative.

6\. Decontaminate laboratory work surfaces at least daily with a freshly
prepared chemical germicide such as a 1:10 dilution of household bleach
(this dilution has a final concentration of 0.5% sodium hypochlorite).
If bleach is to be used dilutions should be mixed daily as bleach looses
its effectiveness within 24 hours. Other commercially available
disinfectants can also be used (dilute as indicated by manufacturer).

7\. To decontaminate equipment that may come in contact with blood or
body fluids:

‑ Disinfect refrigerators by cleaning thoroughly and then by wiping with
1:10 dilution of household bleach.

> ‑ Disinfect centrifuge components by swabbing head, bowl trunion, and
> carriers with 80% ethanol.
>
> ‑ Autoclave or soak specimen racks in 1:10 dilution of household
> bleach for 5 minutes and then rinse thoroughly with water.
>
> ‑ Discard as hazardous waste any disposable components of instrument
> systems that come in contact with patient specimens. Clean
> non-disposable components with 80% ethanol.
>
> ‑ Allow disinfectant to remain in contact with surfaces for at least 5
> minutes at ambient temperature for optimal effectiveness against dried
> blood or serum.
>
> ‑ If equipment needs maintenance, clean and decontaminate it in the
> laboratory before transporting it to the manufacturer for repair.

8\. Use special precautions in handling microbiology laboratory waste,
pathology waste, and blood specimens or blood products.

> ‑ Incinerate or autoclave all waste before disposal in a sanitary
> landfill. Solutions containing bleach may corrode the autoclave;
> therefore, these solutions may be poured down a drain connected to a
> sanitary sewer.

‑ After decontaminating, carefully pour down a drain connected to a
sanitary sewer bulk blood, suctioned fluids, excretions, and secretions.

9\. Wash hands thoroughly after completing laboratory activities. Remove
protective clothing before leaving the laboratory.

**In Case of a Spill**

Decontaminate spills of blood and body fluids:

> ‑ Wear disposable gloves.
>
> ‑ Cover visible blood or body fluids with paper towels and soak with a
> 1:10 dilution of household bleach. Allow to stand for at least 5
> minutes.
>
> ‑ Discard contaminated towels in infective waste containers.
>
> ‑ Wipe down the area with clean towels soaked in a 1:10 dilution of
> household bleach.

References

1\. Resnick L, Veren K, Salahuddin SZ, Tondreau S, Markham PD. Stability
and inactivation of HTLV‑III/LAV under clinical and laboratory
environments. JAMA 1986;255:1887‑91.

2\. McDougal JS, Martin LS, Cort SP, Mozen M, Heldebrant CM, Evatt BL.
Thermal inactivation of acquired immunodeficiency syndrome virus, human
T‑lymphotropic virus type‑III/lymphadenopathy‑associated virus, with
special reference to antihemophilic factor. J Clin Invest 1985;76:875‑7.

3\. Centers for Disease Control. Recommendations for prevention of HIV
transmission in health‑care settings. [MMWR]{.underline} 1987;36(suppl.
no. 2S):3S‑18S.

4\. Centers for Disease Control. Agent summary statement for human
immunodeficiency virus and report on laboratory‑aquired infection with
human immunodeficiency virus. [MMWR]{.underline} 1988;37(suppl. no.
S‑4):1S‑17S.

5\. Centers for Disease Control. Update: Universal precautions for
prevention of transmission of human immunodeficiency virus, hepatitis B
virus, and other bloodborne pathogens in health‑care settings. MMWR
1988;37:377‑88.

6\. Recommendations for Preventing Transmission of Human
Immunodeficiency Virus and Hepatitis B Virus to Patients During
Exposure-Prone Invasive Procedures. MMWR 1991;40:RR08;1-9.

7\. American Public Health Association. Prevention of HIV transmission
in laboratory settings. Laboratory Section Newsletter, October 1987:1‑2.

**SPECIMEN COLLECTION AND STORAGE**

The accuracy of any laboratory procedure used to detect HIV antibody in
DBS on filter paper depends on the manner in which the specimen is
obtained and handled. Dried‑blood‑spot specimens have been used to
screen newborns for congenital metabolic diseases for many years, and
have been successfully used to assess the prevalence of HIV infection in
childbearing women and other adult populations . The following
recommendations reflect completed studies on stability and optimal
storage conditions.

Best test results are obtained when the DBS have not been stored for
prolonged periods at room temperature or exposed to conditions of high
humidity or high temperatures.

**Sample Collection**

In the United States, the National Committee for Clinical Laboratory
Standards (NCCLS) has published a standard for collecting samples
entitled \"Blood Collection on Filter Paper for Neonatal Screening
Programs\" (LA4‑A; 1997). The NCCLS recommendations are followed when
samples are collected for HIV screening program.

Use only No. 903, Schleicher and Schuell, or No. BFC 180, Whatman,
cotton‑fiber‑based paper product designed and used nationally within the
United States for the collection of body fluids. Validate with
Schleicher and Schuell or Whatman that the lot number of the paper is
that currently in use See Appendix A for a list of supplies and
manufacturers for blood spot collection and storage.

**Collection from finger sticks:**

Add identification information for the client/patient to each filter
paper card.

Select one of the 2 middle fingers.

Thoroughly cleanse the finger with 70% isopropanol.

Allow to air dry a few seconds.

Use a sterile, disposable lancet to puncture the skin off to the side of
the finger tip. We recommend a single-use lancet such as BD Genie
Lancet. These are spring loaded and once they have been used the
blade/needle retracts into a plastic housing to prevent reuse. BD
lancets come in a variety of needle or blade widths and depths. (See
Appendix A)

Wipe away the first small blood drop with a gauze pad.

Place the card close to the lanced area but do not touch it. Apply
gentle pressure to the base of the finger and allow the second LARGE
blood drop to fall from the tip of the finger onto surface of the filter
paper.

The filter paper cards may come with printed circles, apply blood to the
inside of the circles. Attempt to fill the circle completely with a
single drop before moving to the next empty circle.

Apply blood to only one side of the filter paper (the side with
printing).

When all circles are filled (or client no longer bleeds) apply cotton to
the puncture site until blood flow stops.

NOTE: Avoid using capillary tubes to collect blood specimens.
Considerable danger of infection exists for lab workers from puncture
wounds resulting from accidental breakage of the capillary tubes.

**Collection from vacutainer of blood:**

Write on the filter paper card identification information for the
client/patient.

Mix blood completely.

Use a pipet (with disposable tip) to aspirate 110 μl of whole blood and
apply to the center of each pre-printed circle.

Avoid touching the part of the card with the blood spot

. Dry all specimens at least 3 hours in a suspended horizontal position.
Depending on the climate it might be necessary to allow spots to dry
over night. Schleicher and Schuell sell cardboard drying racks which
have slots to hold about 12 cards. (See Appendix A)

# Packaging Blood Spots

Once DBS are completely dry, stack them between sheets of glassine paper
so that blood spot cards from different patients are not touching each
other. Pack 10-15 blood spot cards in Low Gas Permeable zip-lock bags.
The following items should also be placed in this bag: 5-10 desiccant
packs (this will remove any residual moisture form the cards), humidity
indicator cards (this will tell you the relative humidity inside the
bag) and QC material. Press as much air out of the bag as possible and
seal it shut. The humidity indicator cards and desiccant packs have a
color indicator which changes from blue to pink as humidity increases.
All cards and packs should be replaced with fresh material before they
have all changed to a pink color.

Moist humidity cards and desiccant packs can be re-used. Simply place in
a 65°C oven over night until the color indicator returns to a blue
color. Remove from the oven and store in a sealed bag until reuse.

> NOTE: **Plastic bags used for storage [must]{.underline} be gas‑
> impermeable. Bags available from grocery stores are inadequate. Use
> bags such as Bitran Saranex Series S multipurpose bags available from
> Number range CHAPTERVWR Scientific, Fisher Scientific, or Number range
> CHAPTERS&S (See Appendix A.) These bags come in variety of sizes**.

**Storage of Specimens**

For short term storage, DBS should be kept in zip-lock bags with
desiccant and stored at 4^o^C. DBS should only be taken out of cold
storage when they are needed for testing.

For long term storage (over 90 days), keep specimens in a freezer
(‑20^o^C).

Specimens stored differently from those just described are considered
compromised and should not be tested. It is better to reject such
specimens than to include potentially unreliable results that might bias
the survey data.

**Shipping Stored Specimens to Another Site**

Use special care when shipping to another location any DBS that have
been stored at 4 ^o^C or ‑20 ^o^C. Remove the bagged samples from the
refrigerator and allow them to reach room temperature
[before]{.underline} opening the bag. Once the sealed bag has
equilibrated, open it and remove the old desiccants. Add fresh
desiccants and reseal the bag. Ship the bag by the fastest means. If a
cooler is available for transport this will protect samples from short
periods of high temperature. Upon receiving such specimens, store them
immediately in a refrigerator (4^o^C) or freezer (‑20^o^C).

# Logging and Tracking DBS Specimens

DBS arriving from other locations should be examined for the quality of
the spots and packaging:

> All spots should be in appropriate low gas permeable zip-lock bags
> described elsewhere in this document.
>
> Each blood card should be examined for collection quality and possible
> damage and a note should be made of any poor quality samples.

Samples should be separated by glassine paper.

> NOTE**: Desiccant packs and humidity indicator cards which have
> changed to a pink color should be replaced with fresh material.**

All DBS specimens arrived in your lab should be logged into your
existing specimen inventory system (whether it is a notebook or a
computer software package).

**Punching Disks From Specimens for Testing**

Dried‑bloodspot specimens or controls must be acclimated to room
temperature before punching disks. If they have been stored in sealed
bags with desiccant at 4 ^o^C or ‑20 ^o^C, allow the sealed bags to
reach room temperature before opening them (a minimum of 30 minutes).

Examine DBS before testing. Do not test spots that:

> ‑ Are obviously contaminated by some foreign substance
>
> ‑ Contain blood clots
>
> ‑ Are not saturated with blood or appear spotted
>
> ‑ For any other reason appear to be of poor quality

In general, these guidelines for rejecting specimens are the same as
those used in all newborn screening programs.

Punch the disk in an area that is saturated with blood. Do not take the
punch from the edge of the circle where the degree of saturation may be
uneven. Avoid blood clots.

Whether using an automated punching machine or a manual punch, remove
paper fiber residue as it accumulates on the instruments. Although no
cases of false‑positive results have been associated with transfer of
paper dust, periodic cleaning reduces this possibility.

**ENZYME IMMUNOASSAY MODIFICATIONS FOR DBS ANALYSIS**

**Eluting Filter Paper Blood Specimens**

The procedure for eluting filter paper specimens is dependent upon the
dilution required by the EIA kit used to test for HIV antibody. Use an
EIA kit licensed by the U.S. Food and Drug Administration or one that
has been previously validated for DBS to detect HIV antibody. Since
these HIV kits were licensed to detect HIV antibody in serum or plasma,
it is important that the performance of the kit for detecting HIV
antibody in serum eluted from DBS has been evaluated and that adequate
specificity and sensitivity studies have been conducted. For more
information on kits and protocols see the list of contacts at the end of
this document.

Elution of the DBS results in a dilution of the specimen (1:40; see
below). Prepare the eluates and perform the tests exactly as directed to
obtain consistent and reliable results.

**General Instructions for Preparation of Eluates**

1\. Elute specimens in a \"blank,\" 96‑well, flat‑bottomed microplate
with lid. (The top well diameter must be 7 mm to accommodate the 6mm DBS
punch.) Arrange samples in the elution plates in a way that leaves room
for the appropriate serum and filter paper controls. Leave empty the
wells designated for serum controls. By configuring the elution plate
exactly like the test plate, you can transfer eluates easily to the test
plate with a multichannel pipetter. Each elution and test plate contains
duplicate determinations for high‑positive, low‑positive, and negative
blood‑spot controls.

2\. Record the placement of each control and specimen disk before
punching the disks. See example 96-well plate sample form (See Figure
1). Place filter paper disks (controls and samples) in their designated
microplate wells.

A 1/4‑inch (6.3 mm) disk collected as prescribed is estimated to contain
10 ul of blood or approximately 5 ul of serum. Subsequent dilutions are
made on the assumption that complete elution of the specimen yields 5 ul
of serum.

3\. Elute the disks by adding 200 ul of PBS with 0.05% Tween 20 to each
well. Check each well to ensure the disk is completely submerged in the
diluent and not floating on top. If necessary, use a separate applicator
stick to submerge each disk and removed any air bubbles. Elute overnight
at 4°C.

4\. Make the appropriate dilution of the eluates according to the
protocol modifications then proceed with the EIA test procedure.

5\. DO NOT store DBS elutions for longer than 5 days at 4^o^C. The
specimen diluents do not contain bacteriostatic agents and DBS are
nonsterile specimens, so bacterial contamination of stored eluates is
possible.

> CAUTION: Bacterial growth can destroy antibody, producing
> false‑negative results, or introduce bacterial metabolites that may
> give false‑positive results.

6\. It is possible to store eluted DBS in the freezer for future
testing, a single freeze‑thaw cycle will have little effect on assays
currently in use. It might be necessary to do this for some blood spot
specimens which have been depleted. The entire 96-well plate with it's
lid should be labeled, placed in a zip-lock bag and stored upright in
the freezer. However this practice is not encouraged since it can result
in false test results due to cross contamination of samples from one
well to the next. Care should be taken to completely label the plate and
avoid bumping or spilling the eluates

**Manufacturers\' Instructions for Preparation of Eluates**

A universal procedure for the elution of DBS has been developed. The use
of these elutions has been incorporated into modified EIA protocols.
These have been reproduced below. Follow the procedure exactly for
consistent and reliable results.

**Genetic Systems rLAV EIA ([This is an HIV-1 only detection
kit]{.underline})**

**BioRad Laboraroties , Redmond WA 98052**

USDA licensed product for DBS testing.

Genetic Systems\' HIV antibody assay requires 100 ul of a 1:400 dilution
of serum. Elution yields a 1:40 dilution.

> 1\. Add 200 ul of supplemented specimen diluent to each well
> containing a filter paper disk (1:40 dilution).
>
> 2\. Cover plate with a clean plate sealer.
>
> 3\. Elute overnight (14‑18 hours) at 4 ^o^C, warm to room temperature
> before testing.
>
> 4\. Remove an antigen‑coated Genetic Systems plate (at room
> temperature) from its protective wrapper. If less than a full plate is
> needed to test all controls and samples, return the unused wells to
> the wrapper and reseal with desiccant.
>
> 5\. Add 60 ul of specimen diluent to each well and 40uL of dried blood
> spot eluate directly to the appropriate well. This represents an
> additional dilution of 1:2.5 or a final dilution of 1:100. Do not add
> diluent to the wells designated for serum controls.
>
> 6\. Mix eluates thoroughly by stirring them gently with clean tips on
> a multichannel pipetter. Cover the elution plate with a clean plate
> sealer and return it to 4 ^o^C pending the results of screening.
>
> 7\. Prepare Genetic Systems serum controls as directed in the product
> insert and add them to the designated wells.
>
> 8\. Cover the test plate with a clean plate sealer.

PROCEED TO STEP 7 (INCUBATION) IN THE GENETIC SYSTEMS PRODUCT INSERT TO
CONTINUE THE ASSAY.

NOTE: WASH THE MICROWELL PLATE OR STRIP A MINIMUM OF FIVE TIMES.

**Vironostika HIV Microelisa System ([This is an HIV-1 only detection
kit]{.underline})**

**bioMerieux, Inc., Durham NC 27704**

USDA licensed product for DBS testing.

bioMerieux's HIV antibody assay modified for DBS requires 25 ul of a
1:30 dilution of serum.

> 1\. Add 150 ul of Dilsim II to each microtiter well containing a
> filter paper disk.
>
> 2\. Cover plate with a clean plate sealer.
>
> 3\. Elute overnight (14‑18 hours) at 2-8 ^o^C.
>
> 4\. After incubation, fit a stripholder with the required number of
> HIV-1 Microelisa strips. Return unused strips to the wrapper and
> reseal with desiccant.
>
> 5\. Add 125 ul of Dilsim II to each well of the test plate that
> corresponds to a well in the elution plate containing a blood‑spot
> sample or control. Do not add Dilsim II to the wells designated for
> serum controls.
>
> 6\. Mix eluates thoroughly by stirring them gently with clean tips on
> a multichannel pipetter or plate shaker. Change tips, then transfer 25
> ul of each eluate to its respective well in the test plate. Cover the
> elution plate with a clean plate sealer and return it to 4 ^o^C
> pending the results of screening.
>
> 7\. Prepare Vironostika serum controls as directed in the product
> insert and add them to the designated wells.
>
> 8\. Cover the test plate with a clean plate sealer.

PROCEED TO STEP 3 (INCUBATION) IN THE VIRONOSTIKA PRODUCT INSERT TO
CONTINUE THE ASSAY.

> NOTE: Modify the microtiter plate WASH PROCEDURE (page 7 in the
> product insert) as follows:
>
> a\. Aspirate the well contents.
>
> b\. Dispense wash buffer and soak wells for 10 seconds.
>
> c\. Repeat steps a. and b. for a total of four wash cycles.
>
> Failure to incorporate these soak periods into the wash procedure may
> result in increased numbers of falsely reactive samples.

## Vironostika HIV Uni-Form II plus O

**Organon Teknika, Boxtel, The Netherlands**

The Uni-Form II plus O assay requires a 1:3 dilution of serum. For dried
blood spot (DBS) testing, this concentration is unattainable as elution
of a 6mm punch in 200 ul of PBS-T yields a 1:40 dilution.

Procedure:

1.  Bring all regaents to room temperature.

2.  Remove any strips from the microwell plate that are not needed for
    the assay and replace with null strips.

3.  Add 75 ul of well-mixed specimen diluent to each well of the test
    tray that

> corresponds to to a well in the elution tray containing a blood spot
> control or sample. Add 100 ul of diluent to the wells designated for
> the serum controls.

4.  Using a multichannel pipette, mix the DBS elutions 4-5 times and
    transfer 75ul to the test plate and mix well. Discard the pipette
    tips and continue the diltuions until all DBS elutions have been
    transferred. Final dilution of the serum contained in the DBS is
    1:80. Cover the elution tray and stroe appropriately.

5.  Add 75ul of each eluate to its respective well in the test tray. Add
    50ul of the kit controls to the designated wells.

6.  If all wells of a strip are not used, fill the unused wells with
    specimen diluent to dissolve the conjugate sphere in order to
    prevent problems with the wash system.

7.  Proceed to Step 4 of the Uni-form II ;plus O kit insert and follow
    the instructions to complete the assay.

## Select-HIV

**BioChem ImmunoSystemes Inc**.

The Select HIV immunoassay requires a 1:50 dilution of serum. The DBS
modified procedure requires 50 ul of the 1:40 DBS elution diluted in 50
ul of the specimen diltuent for a final dilution of 1:80.

Procedure

1.  Bring all regaents to room temperature.

2.  Remove any strips from the microwell plate that are not needed for
    the assay and replace with null strips.

> 3\. Dispense 100 ul of specimen diluent into a well A1 to serve as the
> substrate
>
> blank.

4.  Dispense 50 ul of specimen diluent to each well of the test tray
    that

> corresponds to to a well in the elution tray containing a blood spot
> control or
>
> sample.

5.  Using a multichannel pipette, mix the DBS elutions 4-5 times and
    transfer 50ul to the test plate and mix well. Discard the pipette
    tips and continue the diltuions until all DBS elutions have been
    transferred. Final dilution of the serum contained in the DBS is
    1:80. Cover the elution tray and store appropriately.

6.  Kit controls are pre-diluted and should be added directly without
    further

> dilution. Proceed to Step 5 of the Select-HIV product insert and
> follow the
>
> instructions to complete the assay.

**Detect-HIV (version 2)**

**BioChem Immunosystemes, Inc.**

The Delect HIV immunoassay requires a 1:1.33 dilution of serum. The DBS
modified procedure requires 50 ul of the 1:40 DBS elution diluted in 50
ul of the specimen diltuent for a final dilution of 1:80.

Procedure

> 1\. Bring all regaents to room temperature.

2.  Remove any strips from the microwell plate that are not needed for
    the assay and replace with null strips.

3.  Prepare solution of conjugate 1 according to the product insert so
    that reconstitution is assured.

4.  Dispense 50 ul of specimen diluent to each well of the test tray
    that

> corresponds to a well in the elution tray containing a blood spot
> control, kit control, or sample.

5.  Using a multichannel pipette, mix the DBS elutions 4-5 times and
    transfer 50ul to the test plate and mix well. Discard the pipette
    tips and continue the diltuions until all DBS elutions have been
    transferred. Final dilution of the serum contained in the DBS is
    1:80. Cover the elution tray and store appropriately.

6.  Add 150 ul of kit controls to the designated wells according to kit
    insert steps

    4.  . Cover the microplate with an adhesive cover and proceed to
        Step 6 of

> the product insert.

## Genetic Systems HIV-1/HIV-2 Peptide EIA

**Biorad Laboratories, Hercules, CA**

The Genetic Systems HIV-1/HIV-2 peptide EIA requires a 1:10 dilution of
serum. The DBS modified procedure requires 50 ul of the 1:40 DBS elution
diluted in 50 ul of the specimen diluent for a final dilution of 1:80.

Procedure

1.  Bring all regaents to room temperature except for the conjugate
    concentrate.

> 2\. Remove any strips from the microwell plate that are not needed for
> the assay
>
> and replace with null strips.

3.  Dilute kit controls 1:10 in specimen diluent (e.g. 15 ul into 135 ul
    of specimen diluent).

4.  Dispense 50 ul of specimen diluent to each well of the test tray
    that

> corresponds to a well in the elution tray containing a blood spot
> control or
>
> sample.

5.  Using a multichannel pipette, mix the DBS elutions 4-5 times and
    transfer

> 50ul to the test plate and mix well. Discard the pipette tips and
> continue until
>
> all DBS elutions have been transferred. Final dilution of the serum
> contained
>
> in the DBS is 1:80. Cover the elution tray and store appropriately.

6.  Add 100 ul of kit controls to designated wells.

> 7\. Proceed to Step 7 of the kit insert and follow the instructions to
> complete the
>
> assay.

## Inno-test HIV-1/HIV-2 Ab

**Innogenetics, Ghent, Belgium**

The Inno-test HIV-1/HIV-2 Ab requires a 1:4 dilution of serum. The DBS
modified procedure requires 75 ul of the 1:40 DBS elution diluted in 25
ul of the specimen diluent for a final dilution of 1:53.

Procedure

1.  Bring all regaents to room temperature.

2.  Remove any strips from the microwell plate that are not needed for
    the assay and replace with null strips.

3.  Dispense 25 ul of specimen diluent to each well of the test tray
    that

> corresponds to a well in the elution tray containing a blood spot
> control, kit
>
> control, or sample. Add 200 ul of specimen diluent to kit control
> wells.
>
> 4\. Using a multichannel pipette, mix the DBS elutions 4-5 times and
> transfer
>
> 75ul to the test plate and mix well. Discard the pipette tips and
> continue until
>
> all DBS elutions have been transferred. Final dilution of the serum
> contained
>
> in the DBS is 1:53. Cover the elution tray and store appropriately.

5\. Add 10 ul of controls to designated wells.

> 6\. Proceed to Step 5 of the kit insert and follow the instructions to
> complete the
>
> assay.

**Murex 1.2.O**

**Abbott Murex, Dartford, UK**

The Murex 1.2.O requires a 1:2 dilution of serum. The DBS modified
procedure requires 75 ul of the 1:40 DBS elution diluted in 25 ul of the
specimen diluent for a final dilution of 1:53.

Procedure

1.  Bring all regaents to room temperature.

2.  Remove any strips from the microwell plate that are not needed for
    the assay and replace with null strips.

3.  Dispense 25 ul of specimen diluent to each well of the test tray
    that

> corresponds to a well in the elution tray containing a blood spot
> control, kit
>
> control, or sample. Add 50 ul of specimen diluent to kit control
> wells.
>
> 4\. Using a multichannel pipette, mix the DBS elutions 4-5 times and
> transfer
>
> 75ul to the test plate and mix well. Discard the pipette tips and
> continue until
>
> all DBS elutions have been transferred. Final dilution of the serum
> contained
>
> in the DBS is 1:53. Cover the elution tray and store appropriately.

5\. Add 50 ul of controls to designated wells.

> 6\. Proceed to Step 5 of the kit insert and follow the instructions to
> complete the
>
> assay.
>
> **Classification of Results**

The presence or absence of HIV antibody is determined by relating the
absorbance value of the specimen to the cutoff value calculated from the
serum controls as prescribed by the enzyme immunoassay (EIA) kit being
used. Each EIA kit has its own parameters for calculating the cutoff
value. The detection of HIV antibody in serum eluted from DBS is also
determined by relating the absorbance value of the specimens to the kit
serum cutoff value.

Note: Some EIA kit manufacturers may recommend a cutoff value for
dried‑blood‑spot specimens that is different from the serum calculated
cutoff value. Consult the manufacturer\'s supplemental instructions for
calculating the cutoff value for dried‑blood‑spot specimens.

Specimens are classified as reactive or nonreactive by comparing them to
the cutoff value.

> NONREACTIVE SPECIMENS have absorbance values less than the cutoff
> value. Nonreactive specimens do not require further testing.
>
> REACTIVE SPECIMENS have absorbance values equal to or greater than the
> cutoff value for the particular EIA.

**WESTERN BLOT MODIFICATIONS**

**FOR BLOOD-SPOT ANANLYSIS**

## Calypte Biomedical Corporation

Rockville, Maryland

Phone: 877-225-9783

Human Imunodeficiency Virus Type 1 (HIV-1) Cambridge Biotech HIV-1
Western Blot kit (for detection of antibodies to HIV-1)

Follow the manufacturer's instructions for the **overnight protocol**
only for DBS specimens.

Kit control sera (high-positive, low-positive and negative) should be
included in each Western blot run along with Quality Control DBS.

> 1\. Bring all blood-spot specimens and kit reagents to room
> temperature (approximately 30 minutes).
>
> 2\. Each blot strip is printed with a number unique within that kit.
> Make a note of which strips are tested with which specimens or
> controls. Each tray has wells for 9 strips.
>
> 3\. Follow the manufacture's directions for wetting each strip in the
> tray with [diluted wash buffer]{.underline}.
>
> 4\. Add the [working buffer]{.underline} to each strip.
>
> 5\. Punch one ¼ inch (6.3 mm) DBS directly into the appropriate well.
> For kit serum controls add 20ul as directed.
>
> 6\. Cover the tray and incubated on a rocker overnight (14-20 hours)
> at 20° to 28°C.

7.  Follow the manufacturer's directions for completing the assay.
    Carefully remove the paper punches from each well during the first
    set of washes.

8.  Follow the manufacturer's directions for interpretation of the
    results.

Due to the small amount of serum within a ¼ inch DBS punch (5 ul of
serum from the single blood-spot punch versus the 20 ul of serum used
for the kit controls) the virus-specific bands will appear fainter than
the kit sera controls. Use the results from the DBS quality control
material to assist with interpretation of results from DBS specimens.

**QUALITY CONTROL OF DBS SPECIMENS FOR ENZYME IMMUNOASSAY AND IMMUNOBLOT
ASSAY**

Quality assurance is the dynamic and ongoing process of monitoring a
system for reproducibility and reliability that permits corrective
action when established criteria are not met. Maintaining these
acceptable levels of performance is quality control. Laboratory aspects
of quality assurance include the numerous steps from specimen collection
through confirmation of positive specimens. Good laboratory practice for
repeat and confirmatory testing requires that the original specimen, not
an intermediate dilution, be used as the sample source.

The overall quality control scheme allows serum controls to monitor the
effectiveness of each commercial kit\'s reagents throughout the analysis
and permits linkage of the dried‑blood‑ spot controls throughout the
system, thus controlling the effectiveness of each analytical system.

The criteria for accepting or rejecting an analysis are based on the
performance of the quality control materials (serum and dried‑ blood
spots) and must be predetermined as part of the laboratory protocol.
Quality assurance and quality control steps must be outlined in writing
in the laboratory operations manual. Quality control specimens must be
handled in a manner identical to the manner in which unknown specimens
are handled. All quality control events must be documented, from
specimen processing to reporting. Good quality assurance for the HIV
screening laboratory involves setting criteria of performance for
[all]{.underline} steps in the specimen logistics, from collecting
specimens to reporting data.

Each enzyme immunoassay (EIA) plate is considered a separate run and
must have its own serum and DBS controls. Data from EIA plates and
Western blots are accepted or rejected on the basis of the performance
of the serum controls supplied in the kits and on the DBS controls in an
interactive process. The analytical system presented in Figure 1
illustrates one possible scheme for incorporating quality control
material in a testing algorithm. This scenario uses an initial EIA, a
repeat duplicate EIA, and a Western blot.

The serum kit controls (negative and positive) verify that the kit
reagents are performing correctly and are used to calculate the positive
and negative cutoff. The dried‑blood‑spot controls (low‑ positive,
high‑positive, and negative) monitor the stability of the
dried‑blood‑spot specimens from collection through the testing process.

**Quality Control for Elution Procedure**

Establish the placement of the dried‑blood‑spot disks in the elution
plate so that the blank wells for the kit serum controls follow the
configuration of the plate as directed by the manufacturer\'s protocol.
DBS of all specimens and controls must be at room temperature before
they are punched. Specimens or controls stored cold or frozen in sealed
bags with desiccant must be allowed to equilibrate at room temperature
before the bags are opened. See the chapter on specimen collection and
storage for details. Duplicate dried‑blood‑spot control disks
(low‑positive, high‑positive, and negative) are eluted along with the
dried‑blood‑spot specimens.

Before performing the initial EIA, examine the eluted DBS to visually
ensure complete elution. The filter paper punch should be white. If it
is not, complete elution has not occurred. Continue incubation and do
not analyze the specimen until elution is effective. If complete elution
is not accomplished within 24 hours, discontinue the analysis of these
specimens and document the observation. Several factors, such as
specimen age and heat exposure, will affect the elution of blood dried
on filter paper.

**Quality Control of**

**Enzyme Immunoassays for DBS Specimens**

Examine the results of serum controls as described in the
manufacturer\'s product insert. Accept or reject assay results on the
basis of the performance criteria for the serum controls as described by
each manufacturer. Reject any analytical run (EIA plate) that violates
these standards, regardless of results obtained for other control
materials.

If the serum controls are within specifications, examine the blood‑spot
controls. Accept the assay results if [all]{.underline} blood‑spot
controls are correctly classified as positive or negative. If any
blood‑spot control is incorrectly classified (positive / negative)
reject the entire plate and repeat the EIA on the original eluates
produced for this EIA plate or create fresh elutes from blood‑spot
controls and specimens. If blood‑spot controls are still incorrectly
classified, troubleshoot the method before proceeding.

Record in the quality control log the absorbance values of the
dried‑blood‑spot controls, along with the calculated serum kit cutoff
for each run. Plot these values as quality control charts, to identify
trends in data and single control values that differ significantly from
previous observations. See QC criteria for a valid run below.

**Quality Control for the Western Blot Assay**

The Western blot assay employs both serum and DBS control material. The
serum controls should consist of a high‑positive serum, displaying all
of the significant viral bands; a low‑positive serum of defined
reactivity; and a negative serum.

If particular bands are missing from the high‑positive DBS control, a
stability or elution problem must be considered in the interpretation of
the specimens.

**Interpretation of Quality Control Results**

Examine the serum controls first. The high‑positive serum control must
consistently demonstrate [all]{.underline} significant viral specific
protein bands at an intensity equal to that usually observed in the test
system. These bands are identified, numbered, and used as a reference
for identifying bands present in the unknown specimens. If bands usually
present in the high‑positive control are absent in a particular run, the
run must be rejected. The system should be examined to determine the
source of the performance problem before repeating the assay.

The low‑positive serum control is used as a control for assay
sensitivity. The serum selection for this purpose should have
[all]{.underline} the viral specific protein bands critical in
identifying a positive specimen. The intensity of these bands should
make them definitely visible but weak in comparison to the high‑positive
control. If a serum having these properties is not available, a serum
containing only key viral protein bands (p24, gp41) can be substituted.
Again, the bands present in a particular run must always be of the
intensity usually observed for that control specimen. To compensate for
run‑to‑run variations in the assay conditions, the low‑positive serum
control is used to adjust the substrate incubation period to ensure a
specified reactivity for each run. The test is incubated until a
selected band (or bands) present in the low‑positive control reach an
expected intensity, based on previous experience. If the expected
intensity is not achieved within a 30‑minute incubation with substrate,
the run should be rejected and the method should be examined for the
source of the malfunction.

The negative serum control should not demonstrate any viral protein
bands.

Number range CHAPTER**QUALITY CONTROL (QC) CRITERIA FOR A VALID RUN**:

**HIV Antibodies in DBS**

**A. Monitoring Control Values with QC Charts**

QC charts must be made using the OD values for negative control (NC),
low positive control (LP) and high positive control (HP). QC limits
(either ±95% and 99% confidence intervals or ±2 or 3 standard
deviations) should be calculated using data from the first 10 analytical
runs. These limits should be recalculated and held after 20 runs. The
mean values from each run, the overall mean of the 10 (or 20) mean
values, and upper and lower 95 and 99% confidence intervals or 2 and 3
standard deviations must be plotted on one chart. For each subsequent
analytical run, the mean OD for the control materials must fall within
the defined control limits for each material.

***An analytical run is considered out of control if any of the
following events occur:***

1\. The mean OD for NC, LP or HP falls outside the upper or lower 99%
control limits (or 3 SD)**.** The 99% control limits (or ± 3 SD) are
considered action limits. The run must be repeated if these limits are
exceeded.

2\. Two successive mean ODs for NC, LP or HP fall outside of the upper
or lower 95% control limits (or ± 2 SD). The second run must be
repeated. The 95% control limits (or ± 2 SD) are considered warning
limits.

3\. Eight successive mean ODs (for NC, LP or HP) are above or below the
mean value line. The eighth run must be repeated.

**B. Troubleshooting Guidelines**

The list below should be used to troubleshoot the assay if control
values fall outside of the established ranges.

1.  Check to be sure all pipettes or instruments used for making
    dilutions are calibrated and capable of making accurate dilutions.

2.  Check all instrumentation used to wash and read plates.

3.  Review kit lot information to be sure kits are not outdated.
    Reviewing kit lot information may also reveal a kit lot bias.
    Perform 5 runs and recalculate control limits for new kit lots.

4.  Check storage and handling conditions of control materials.

5\. Check quality of substrate solution. Substrate solution that has
been reconstituted and stored for a period of time may increase
background.

Number range CHAPTER

APPENDIX A

SUPPLIES AND PREPARATION OF DRIED BLOOD SPOTS FROM BLOOD COLLECTED BY
VARIOUS METHODS

**1. Supplies**

a\. FDA-approved Filter Paper Collection Device

1.73310\--3\" x 4.25\" card with 5 circles/card.

Order \# 10538414

Job \# A01520

Schleicher & Schuell (S&S) 903™ paper

Keene, NH 03431

Contact: Judy Peter 800- 437-7003

2\. Whatman BFC 180^TM^ paper

Whatman, Inc.

6 Just Road

Fairfield, NJ 07004

Contact: Mark Fry 800-343-5853 ext 132

Fry@whatman.co

> b\. Drying Racks
>
> 105-395-21, Dry Rak™
>
> Schleicher & Schuell (S&S)

Keene, NH 03431

\(800\) 437-7003

> c\. Double-sided Carpet Tape
>
> ST501, DF Paper Tape ½\" x 36 yds
>
> Minimum order: 1 case of 72 rolls
>
> Spectape of Atlanta
>
> 1661 Roadhaven Drive
>
> Stone Mountain, GA 30083
>
> \(770\) 934-4053

d\. Low-gas permeable plastic bags for card storage

VWR Scientific #11217-106

\(800\) 932-5000

Fisher Scientific #19240127

\(800\) 766-7000

7 x 8\" bags are marketed by the above companies but manufactured by

Com-Pac International, (800) 824-0817, manufacturer #4743S)

S&S #79692

These are low gas perm. bags from S&S

> e\. Desiccant Packs--1 gram desiccant packs with blue indicator that
> turns pink with high humidity

#02-00040-AG37 Minipax Indicating Silica Gel Tyvek Bag

Multisorb Technologies

325 Harlem Road, Buffalo, NY 14224

\(800\) 445-9890

f\. Weigh Paper

#09-898-12C

Fisher Scientific

PO Box 829

Norcross, GA 30091

\(800\) 766-7000

g\. Hole Puncher

1/4\" stainless hole punch

Available from any office or school supply store

h\. BD Genie Lancet (single use, permanently retractable lancet with 1.5
mm blade)

#02-683-105

Fisher Scientific

PO Box 829

Norcross, GA 30091

\(800\) 766-7000

i.  96-well microtiter plate, flat bottom, with lid

> #07-200-90
>
> Fisher Scientific

PO Box 829

Norcross, GA 30091

\(800\) 766-7000

j.  PBS pH 7.4 with TWEEN 20 0.05% powder

> #P3563
>
> Sigma Aldrich
>
> \(800\) 325-5052

*The use of trade names is for identification only and does not imply
endorsement by the Public Health Service or the U.S. Department of
Health and Human Services.*

**2. Description of collection device**

> DBS can be collected from a finger stick or venous blood and dried
> into FDA-approved filter paper. The paper is preprinted with circles
> that contain approximately 100 μL blood when fully filled. Patient
> identifiers can be written in pen or pencil directly on the paper.

**3. Collecting blood on filter paper**

a\. *From a finger stick*: Lightly touch the filter paper to a LARGE
blood drop. Allow the blood to soak through and completely fill the
circle with a single application of blood. To enhance blood flow, gently
apply intermittent pressure to the area surrounding the puncture site.
Avoid "milking" the finger. Apply blood to only one side of the paper.
Fill remaining circles in the same manner. Dry the filter paper
horizontally for a minimum of 3 hours at room temperature (see "Drying
dried blood spots" below)

> b\. *From blood collected into vacuum tubes*: Use blood from
> anticoagulant tubes (EDTA-lavender top\*\* or Heparin-green top). Fill
> the blood collection tube to the recommended volume so anticoagulant
> is at proper dilution. Before making spots allow the blood to come to
> room temperature if it has been refrigerated. Gently and thoroughly
> mix the tubes to resuspend the red cells, either by inverting the tube
> by hand (25x) or by placing the tube on a mechanical hematology mixer
> for approximately 5 min. After the blood is mixed, remove 100 μL
> aliquots of blood and apply the blood to the filter paper that has
> been secured (see "Drying dried blood spots" below).
>
> c\. *From blood collected from a finger stick into EDTA\*\* or heparin
> capillary tubes*: Gently touch the capillary tube to the surface of
> filter paper that has been secured (see "Drying dried blood spots"
> below). The blood will flow from the tube into the filter paper. Do
> not scratch, poke, or otherwise score the filter paper with the
> capillary tube.

\*\*EDTA can interfere with some analytical tests. Consult the testing
protocol or package insert from a commercially available kit to
determine which blood collection tubes to use.

**4. Drying Dried Blood Spots**

> a\. Use S&S #903™ Dry Rak™ to dry blood spot cards. If these are not
> available, follow procedure b.
>
> b\. Place double-sided carpet tape on two books, boards, boxes, or
> other suitable solid support to suspend the filter paper used to
> collect blood. Remove cover strip to expose sticky side of tape.
> Carefully place the paper containing DBS between the supports so as to
> avoid contact between the table and wet blood spots. Nothing should
> touch the wet paper. DBS should be dried at room temperature for a
> minimum of 3 hours. They may also be dried overnight at room
> temperature.
>
> *Note:* Desiccant packs can be regenerated by heating overnight in a
> 60˚C oven.

**5. Storing Dried Blood Spots**

> After spots are dried, place DBS between 2 sheets of glassine paper
> (weighing paper), or wrap sheets of weighing paper around spots. Store
> spots at -20˚C, in low gas permeable zip-closure bags with several
> desiccant packs. The desiccant packs should be checked often and
> changed when the indicator turns pink. Initially, desiccant packs may
> need to be changed frequently. After a few changes the cards will be
> drier and the desiccant packs will need less changing. The buildup of
> humidity can damage the quality of the sample.

**6. Shipping**

> Transport low gas-permeable bags containing dried DBS by the fastest
> means possible. Enclose the bags inside a foam or plastic cooler for
> transport. This will provide a double-layer barrier that protects
> casual handlers from accidental exposure, and protects the specimens
> from the environment during transport.

**7. References:**

National Committee for Clinical Laboratory Standards. NCCLS Approved
Standard LA4_A3. Blood collection on filter paper for neonatal screening
programs. Villanova, PA: National Committee for Laboratory Standards,
1997.

Knudson RC, Slazyk WE, Richmond JY, Hannon WH. 1993. Guidelines for the
shipment of dried blood spot specimens. Infant Screening. Volume 16.
Document can also be found at:

http://www.cdc.gov/od/ohs/biosfty/driblood.htm

**For more information please contact any of the following
individuals:**

**Joanne V. Mei, Ph.D.**

Lead Research Chemist

Newborn Screening Quality Assurance Laboratory

Division of Laboratory Science

National Center of Environmental Health

Centers for Disease Control and Prevention

Mail Stop F-19, 4770 Buford Hwy NE

Atlanta, GA 30341-3724

Phone: 770-488-7945

Fax: 770-488-7459

Email: [[jmei@cdc.gov]{.underline}](mailto:jvm0@cdc.gov)

## Timothy C. Granade 

Division of AIDS, STD, and TB Laboratory Research

Nation Center for Infectious Disease

Centers for Disease Control and Prevention

Mailstop A-25, 1600 Clifton Road

Atlanta, GA 30333

Phone: 404-639-3850

Fax: 404-639-2660

Email: [[txg1@cdc.gov]{.underline}](mailto:jvm0@cdc.gov)

## Kyle B. Bond

Global AIDS Program, Laboratory Support

Nation Center for Infectious Disease

Centers for Disease Control and Prevention

Mailstop: A-12, 1600 Clifton Road

Atlanta, GA 30333

Phone: 404-639-2643

Fax: 404-639-2475

Email: <kbb5@cdc.gov>

## Marie Downer

Global AIDS Program, Laboratory Support

Nation Center for Infectious Disease

Centers for Disease Control and Prevention

Mailstop: A-12, 1600 Clifton Road

Atlanta, GA 30333

Phone: 404-639-3050

Fax: 404-639-1286

Email: [mld8@cdc.gov]{.underline}

**U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES**

Public Health Service

Centers for Disease Control

Atlanta, Georgia 30333