This is Principles of Capture®, an Immucor products course.

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Immucor is approved as a provider of continuing education programs in the Clinical

Laboratory Sciences by the ASCLS P.A.C.E. ® Program.

This course provides one P.A.C.E. Contact Hour. The program number is 437-905-13.

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Disclaimer: Graphics in this course are for information and illustration purposes only.

Immucor makes no representation or warranties about the accuracy or reliability of the

information presented in the graphics, and this information is not to be used for clinical or

maintenance evaluations.

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Through this course, you will learn about Immucor Capture assays.

With this information, you will be able to identify Capture assay principles and interpret

assay results.

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After completing this course, you will be able to:

• Describe the principles and procedure for Capture Assays

•List the process steps for both traditional tube and solid phase technologies

• Identify the similarities and differences between traditional tube and solid phase

technologies

• Identify types of Capture assays and their assay processes and results

To understand Capture principles, you will first need to know about traditional tube

antiglobulin testing and solid phase technology, as well as the differences between them.

Let’s look at these testing types.

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Traditional Tube Testing uses red blood cells (RBCs) suspended in liquid.

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Click each of the buttons below to view the traditional antiglobulin tube testing process, which uses agglutination as the method of antibody/antigen detection.

Traditional tube testing uses a drop of a 3-5% saline suspension of either reagent RBCs or sample RBCs.

Depending on what is being tested, either plasma or an antisera is added to the reaction mixture in the tube.

If a sample plasma is being tested, a potentiator may also be added to the reaction mixture to enhance the antibody/antigen reaction during incubation.

The reaction mixture in the tube is then incubated at 37 degrees for a prescribed period of time, during which the antibody/antigen reaction takes place. If there is an antigen on the RBC to which an antibody in the plasma is directed, the antibody will attach to the RBC in the reaction mixture.

The tube is then washed with saline to remove any unbound globulin. Any antibody attached to the RBC will remain on the RBC.

Next, Anti-IgG (also referred to as AHG, anti-human globulin, Coombs serum or antiglobulinreagent) is added. If IgG antibody binds to the RBC during incubation, the anti-IgG will attach to the bound IgG.

The assay mixture is centrifuged to move the RBCs closer together, enhancing lattice formation and thus visible agglutination.

The RBCs in each tube are gently resuspended and RBC agglutination reactions are read and graded.

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In Traditional Tube Testing, a tech uses a light source and a magnifying mirror to rate,

grade, and record agglutination reactions from negative (no RBC agglutination) through 4+

(maximum RBC agglutination).

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Shown here is a negative reaction followed by the four grades of positive agglutination.

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Now, let’s review the Capture antibody/antigen test process, which uses the principles of

solid phase technology.

Capture assays are manufactured for the detection of RBC, platelet, or virus

antibody/antigen reactions. For this part of the presentation, we will limit the discussion to

Capture-R® used for RBC antibody/antigen testing and expand it to platelets and viruses

later in the presentation.

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The Capture® process is similar to tube testing; however, there are some differences. Click

on each of the buttons below to view the Capture testing process, which uses solid phase

RBC adherence, or SPRCA, as the method of antibody/antigen detection.

LISS and plasma or serum are added to a microtiter well. The microtiter wells already have

the RBCs to be tested attached to the well bottom.

The wells are then incubated at 37 degrees to allow for the antibody/antigen reaction to

occur.

After incubation, the wells are washed with saline to remove the LISS and the plasma with

unbound antibody. Any bound antibody remains attached to the RBCs at the bottom of the

well.

Indicator cells are added to the wells.

The wells are centrifuged for two minutes.

Finally, the results are read and interpreted.

To understand the reactions, you must understand the reagents used for Capture® testing.

Let’s start with the microwell itself.

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An individual Capture microwell resembles a short test tube.

Its size and shape enable pre-coating of substances onto the smooth surface of its well

bottom.

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Capture microwells are provided in a plate of detachable single strips. Each strip has eight

microwells.

Panels are also provided in plates; however, instead of single strips, they are supplied as

double strips to accommodate the additional cells. We will discuss the cell configurations of

the strips later in the course.

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Now let’s talk about the “solid phase” of our test, the RBCs bound to the bottom of each

well. During the manufacturing of our Capture plates, reagent RBCs are applied to the

bottom of each well, hemolyzed, and then dried.

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Because only RBC membrane is present in the well, Capture RBC reagents are very stable. The

plates are packaged in foil pouches that may be stored at room temperature and have an extended

outdate of 120 days from the date of manufacture. Either screening cells or panel cells may be

used as reagents.

Capture Screening cells include these three products:

•Capture-R® Ready-Screen® (Pooled Cells)

•Capture-R® Ready-Screen® I and II

•Capture-R® Ready-Screen® (3)

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Here is a picture of the Capture-R Ready-Screen (Pooled) package. This reagent is approved only

for antibody screening of blood donors and is similar to a liquid pooled screening cell ,such as

Hemantigen®, used in tube antibody screening. The configuration on an 8-well strip would look like

this, with pooled cells coating each individual well. One well would represent one test on one

donor. In addition to the wells used for antibody screening, positive and negative control sera are

tested to ensure the validity of the test. If the controls do not perform as expected, the test is

invalid.

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Next is the Capture-R Ready-Screen (I and II), which is similar to a liquid 2-cell set of screening cells

such as the Panoscreen® I and II used for tube testing. The configuration on the strips would look

like this , with cells I and II coated on every other well of the strip. Each set of I and II would be

used for one antibody screen on one patient. In addition to the wells used for antibody screening,

positive and negative control sera are tested to ensure the validity of the test. Again, if the controls

do not perform as expected, the test is invalid.

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Finally, we have the Capture-R Ready-Screen (3), which is similar to a liquid 3-cell set of screening

cells such as the Panoscreen® I, II and III used for tube testing. The configuration on the strips

would look like this, with cells I,II and III and a positive control cell coating a sequence of 4 wells on

each 8-well strip. Each set of 4 wells would be used for one antibody screen on one patient. The

positive control well functions as a sort of “check cell” to ensure that the test was acceptable. If the

positive control is not positive upon completion of the test, the test is invalid.

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Immucor also manufactures three different panels:

•Capture-R® Ready-ID®

•Capture-R® Ready-ID® EXTEND I

and

•Capture-R® Ready-ID® EXTEND II

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The panels are made up similarly to liquid RBC panels, but are manufactured by applying

the RBCs to the wells of a double microwell strip as shown in the configuration here. Each

panel includes a positive and a negative control.

The three panels contain donor cells selected with varying criteria to fulfill different needs

in the blood bank.

Capture-R Ready-ID is created to be used as a primary panel. Each of the microwells

contain RBCs, 14 of which are from reagent RBC donors. The two remaining wells contain a

positive and negative control respectively.

Capture-R EXTEND I also contains RBCs from 14 reagent RBC donors and microwells with

positive and negative controls. However, all of the cells on this panel are D positive and

include 7 little c and 7 little e negative cells to help resolve patients with those antibodies.

Capture-R EXTEND II is formatted in the same way as the other two panels with 14 reagent

RBC donors and the positive and negative controls. However, all but one of the cells on this

panel are D negative. In the case of a sample with a suspected anti-D, the one D positive

cell on this panel can serve as the 3rd required positive to the two positive cells from the

screening cells. The remaining 13 D negative cells can determine the presence and, in

some cases, identify any additional antibody that may be present in the sample.

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Capture LISS® is used to enhance antibody/antigen reactions during the incubation period.

Its purpose is similar to tube-LISS reagents such as ImmuAdd™, Gamma LO-ION™ and

Gamma N-HANCE®.

However, Capture LISS is only approved for Capture testing and cannot be used for tube

testing. Likewise, tube-LISS reagents are only approved for tube testing and cannot be used

for Capture testing.

The other difference is that Capture LISS contains bromcresol purple dye which changes to

blue in the presence of protein. This is a great tool for Capture testing, as the color change

acts as an indicator that plasma has indeed been added to the well.

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Slide 22

ND2 I removed the bottle of ImmuAdd that was in the pictureNichols, Dickie, 3/14/2013

Lastly, let’s review how the indicator cells work in the test. Indicator cells are added to

each well after washing out the LISS , plasma, and any unbound antibody. If there is an

antibody/antigen reaction during the incubation period, bound antibody will remain

attached to the RBCs at the bottom of the well as shown here. The attachment of the

indicator cells is also referred to as “RBC adherence.”

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Indicator cells are RBCs with Anti-IgG attached to their surfaces. If any IgG antibody is

attached to the cells at the bottom of the well during incubation, the Anti-IgG on the

indicator cells will bind to those cells.

When the wells are then centrifuged, the IgG/Anti-IgG binding will prevent the indicator

cells from migrating to the bottom of the well – if antibody was bound during the

incubation phase. Therefore, a positive reaction is due to “RBC adherence” of the indicator

cells at the bottom of the well.

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If no antibody was bound during the incubation phase, the anti-IgG on the indicator cells

has nothing to bind to or to keep the cells from migrating when centrifuged. In this case,

the indicator cells would form a button at the bottom of the well, because there is no RBC

adherence.

Essentially, the indicator cells perform the same function as the anti-IgG in traditional tube

testing.

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Let’s review and compare traditional tube technology to Capture solid phase technology.

In the ADD process for both procedures, samples and potentiator are added to the reaction container—tube or microwell. In the tube procedure, liquid reagent RBCs are also added to the test mixture. With Capture, the RBC reagents are already in the reaction container (the microwell) stuck to the bottom of the well, hemolyzed and dry.

In the INCUBATE process for both procedures, the test mixture is given the correct time and temperature to enable any antibody present to react with antigen.

In the WASH process for both procedures, saline is added and removed to eliminate unbound antibody, leaving the bound antibody stuck to the RBC.In the second ADD process for tube technology, anti-IgG is added and will bind to any antibody that was bound to the reagent RBC during the incubation phase of the test.In the second ADD process for Capture, reagent indicator RBCs coated with anti-IgG are added for the same purpose.

In the CENTRIFUGE process for tube technology, the cells are brought closer together into a button at the bottom of the tube to allow for the anti-IgG to agglutinate the sensitized RBCs. In the CENTRIFUGE process for Capture, anti-IgG on the indicator RBCs bind to antibody that is bound to the reagent RBC, which in turn are bound to the bottom of the microwell. This preventsthe RBC from migrating to the bottom of the well to form a button. This is also called adherence.

In the READ process for tube technology, the user shakes the tube to disperse the RBC button, analyzes the cells as they shake off of the button to look for agglutinated RBCs and grades the resulting reactions. In the READ process for Capture, the user or the instrument analyzes the well, looks for RBC adherence indicating a positive reaction, and grades the reaction.

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Because the end point of the test for tube testing is agglutination while for solid phase

technology the end point is adherence, the results are read differently.

A strong positive reaction in a tube test produces a solid RBC button in the bottom of the

test tube, whereas a strong positive reaction in a Capture test produces a completely

adhered layer of indicator RBCs in the bottom of the microwell.

A negative reaction in a tube test produces freely dispersed RBCs, but a negative reaction in

a Capture test produces a defined button of collected indicator RBCs in the bottom of the

microwell.

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Reading Capture results is actually similar to reading gel columns. In gel, a button at the

bottom of the column indicates a negative reaction just as with Capture. Similarly, more

disbursement of the RBCs through the column or across the top indicates a positive

reaction in gel just as the broader disbursement of RBCs in the microwell caused by RBC

adherence in Capture indicates a positive reaction.

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This image provides a clear comparison of negative results in Capture forming a button at the

bottom of the microwell alongside strong positive reactions resulting in adherence.

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We’ve now discussed the basic principles of Capture and how Capture-R compares to tube testing.

We’ve also discussed the different Capture-R products that may be used for antibody screening and

identification as shown here.

There is also a second type of plate called Capture-R® Select. The Select plate allows the user to

select any cell to be used for testing and bind it to the bottom of the well.

The Capture-R Select plates allow the user the opportunity to run such tests as IgG crossmatches,

IgG DATs (which are automated only), and weak D testing.

Capture-R Select microwells contain only a RBC binding agent, which allows the user to

create RBC monolayers from any sample chosen.

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The Capture-R Select Assay process includes two parts: monolayer preparation and assay

processing.

First the RBC sample to be tested is added to the Select well along with a drop of saline.

The wells are centrifuged to enhance the binding of the cells to the bottom of the well.

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The supernatant and extra cells are washed out of the well leaving behind a monolayer of

the cells to be tested.

The rest of the test may now be completed either as:

• An IgG crossmatch, if using donor cells and testing with patient plasma

• A weak D test using either patient or donor cells and incubating with anti-D

• An IgG DAT using patient or donor cells and simply adding indicator cells (automated

testing only)

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For instance, an IgG crossmatch could be performed by laying down a monolayer of donor

cells on the bottom of the well and testing the cells with the patient’s plasma.

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Weak D testing may be performed by using a monolayer of patient’s or donor’s RBCs and

incubating them with anti-D, washing and adding indicator cells to see if anti-D was

attached to the cell.

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On the Echo®, Galileo® and NEO® instruments, an IgG DAT may be performed using Select

strips by creating a monolayer on the bottom of the well of the RBC to be tested. Indicator

cells are then added to the well to detect any IgG on the cells being tested.

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That brings us to the available equipment that may be used for Capture testing. Some

hospital and clinical laboratories perform Capture testing on small numbers of samples

using manual methods of testing.

With manual testing, sample and reagents are applied to the wells using the reagent

dropper or a regular blood bank pipette; no calibrated pipettor is required.

The following equipment is used:

• Manual Capture wash station

• Capture incubator

• Immuspin centrifuge

• Lighted reaction viewer.

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Automated platforms provide the benefits of walk-away testing and the ability to interface

to an LIS. These benefits may also be applied to other tests not available on the manual

format such as ABO/Rh testing, Rh phenotyping and IgG DAT testing.

There are two instruments that perform automated Capture testing.

The Galileo Echo is a table-top instrument scaled for use in small to medium-sized labs.

The NEO is a larger floor instrument, designed to manage much larger volumes such as for

blood centers and larger transfusion services.

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We’ve now reviewed solid phase technology as it applies to detecting RBC antigens and antibodies.

These are also known as the Capture-R products. Now let’s discuss Capture-P products, which test

for platelet antibodies, and Capture-CMV®, which test for cytomegalovirus infection.

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Capture-P® Ready-Screen® is a test for detecting antibodies in patient or donor plasma and

serum samples.

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Capture-P Ready-Screen utilizes a double strip similar to the double strip used for Capture-

R Ready-ID. However, instead of being coated with RBCs, the wells are coated with

platelets from a variety of Group O donors, to facilitate the detection of the most

commonly encountered HLA or platelet-specific antibodies. The double-strip set of

microwells contain platelet antigens from 13 reagent platelet donors, a blank microwell,

and microwells for positive and negative controls.

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The Capture-P Ready-Screen platelet antibody screen process is very similar to the Capture-

R process.

First, LISS and plasma or serum are added to the platelet-coated microwells.

The strip is incubated to allow for any antibody to react with its platelet antigen.

The wells are washed with saline to remove the unbound antibody and protein, leaving

behind any bound antibody.

Indicator RBCs are added.

The strip is centrifuged to promote the adherence of indicator RBCs to any bound antibody.

The tech or instrument analyzes the wells to look for indicator cell adherence and grades

reactions.

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Capture-P is intended for use in the detection of antibodies to platelets. Test processing

includes two parts: monolayer preparation, and assay processing.

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Capture-P plates are manufactured with only the platelet binding agent at the bottom of

the well. Platelets added to the wells are bound by this binding agent.

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The first step is to prepare the platelet monolayer from selected platelet samples.

The user may select several platelet samples to be screened for reactivity to

antibody. Each platelet sample would be added to a different well for binding.

After the Platelet samples are added to the microwells, the microwell strips

are centrifuged to move the platelets close to the binding agent, allowing

them to attach to the microwells.

The microwell strips are washed with saline to remove unbound platelets.

Microwells with monolayers are now ready for assay processing.

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After the monolayers are created, the platelet antibody testing may be done by the user.

This testing may be done using a patient’s plasma with platelets from selected units to

determine serological compatibility before transfusion.

Testing continues by adding Capture LISS and sample plasma or serum to each of the

platelet coated microwells.

The strip is incubated to allow any sample antibody to react with platelet antigen.

The wells are washed to remove the unbound antibody and protein, leaving behind the

antibody bound to the platelets.

Reagent indicator cells are added.

The strips are then centrifuged to enhance the adherence of the indicator RBCs to any

bound antibody.

The user analyzes each well to look for any indicator RBC adherence and grades reactions.

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Capture-CMV is a solid phase RBC adherence test system for the detection of IgG and IgM

antibodies to cytomegalovirus, or CMV, in serum or plasma. Capture-CMV may be used in

screening of donors or patients for serological evidence of previous infection by CMV.

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Capture-CMV microwells contain CMV antigen, which is inactivated CMV virus.

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The CMV antigen-coated wells are tested similarly to other Capture products as shown

here:

In the ADD process, potentiator and samples are mixed in the microwells.

In the INCUBATE process, time and temperature allow any CMV antibody to react with the

CMV antigen.

In the WASH process, the wells are washed to remove LISS, plasma, and any unbound

antibody.

In the ADD process, reagent indicator RBCs are added.

In the CENTRIFUGE process, indicator RBCs adhere to any bound antibody.

In the READ process, the user uses a light source to analyze indicator RBC adherence.

Results are interpreted as “negative” or “positive” without a reaction grade.

The unique elements in this test are, of course, the CMV antigen coated wells and the indicator

cells, which are very different from the other Capture products. Because we want to detect both

IgG and IgM CMV antibodies, the indicator cells are coated with both anti-IgM and anti-IgG.

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