Practical Aspects of Platelet Crossmatching

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Practical Aspects of Platelet Crossmatching

• Jody Smalley MT(ASCP)SBB
• Inova Fairfax Hospital
• Falls Church, VA

Technical Workshop MAABB Annual Meeting
April 18, 2002
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Platelet Crossmatching Is

• Not a test to be taken lightly
• Technique Dependent
• Done infrequently in our area
• Done using specialized equipment
• Loads of Fun!

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Practical Aspects of Platelet Crossmatching Will

• Explain Platelet Crossmatch Technique in use
• List Other Research Techniques
• Review Available Commercial Techniques
• Discuss Area Platelet Crossmatch Survey Results

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Immucor Capture-P Most Often Used Technique

• Capture-P is a solid phase antibody detection
  system modified from procedures published by
  Rachel, Juji, and Shibata.

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Capture-P
A specific platelet binding agent is covalently
bound to the wells by the manufacturer.
Winters 94
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Capture-P

• Bring all test reagents to room temperature
  (18-30o C) before use.
• Platelet concentrates from donors must be diluted
  with normal saline before use to prevent
  excessive platelet layering.

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Capture-P

• A rigid U -bottom plastic strip is used for
  testing.
• The wells contain a platelet binding agent.
• Donor platelets are bound to the well by
  centrifugation.

Immucor
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Capture-P
Paternal platelets, donor platelets, or the
patient's platelets are added to the wells.
Winters 94
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Capture-P

• Centrifuge the strip at 45-65 x g for 5
  minutes.

Immucor
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Capture-P

• Using a semi-automated washer,wash the strip
  with phosphate buffered saline to remove excess
  unbound platelets.

Immucor
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Capture-P

• Capture LISS is a low ionic solution containing
  glycine, bromcresol purple, and the preservative
  sodium azide (0.1).
• Capture LISS purple turns to a slate blue in the
  presence of serum or plasma.

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Capture-P

• Add 2 drops of Capture LISS to each well.
• Add one drop of plasma or control serum to the
  appropriate wells.

Immucor
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Capture-P
The patient's serum or antibody of known
specificity is added to the wells.
Winters 94
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Capture-P

• The plate containing strips is then incubated
  in a dry heat incubator at 37o C for 30-60
  minutes.

Immucor
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Capture-P

• Wash again using semi-automated washer to
  remove Capture LISS and unbound platelet
  antibody.

Immucor
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Capture-P

• Add 1 drop of IgG
• coated indicator cells

Immucor
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Capture-P
Indicator cells consisting of red cells
with attached rabbit anti-human IgG are added.
Winters 94
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Capture-P

• Centrifuge the strip at 700-900 x g for 3
  minutes.

Immucor
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Capture-P
In the presence of anti-platelet antibodies,
the red cells coat the well and a hazy pattern is
seen.
Winters 94
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Capture-P
In the absence of anti-platelet antibodies, the
red cells form a button on the bottom of the
well.
Winters 94
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Capture-P

• Place plate containing strips on an illuminated
  surface such as a slide view box.
• Observe for adherence or non-adherence of the
  indicator cells.

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Capture-P
Immucor
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Causes False Positive Using Capture-P(Hazy or
Button not Formed)

• Anti-HLA antibodies are present
• Insufficient centrifugation
• Braking the centrifuge too rapidly
• Insufficient red cell indicator added
• Platelets used are coated with IgG

Winters 94
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Causes False Negative Using Capture-P(Button
Formed)

• Excess centrifugation
• Braking of centrifuge too slowly
• Excess red cell indicator added
• Insufficient platelets added
• Maternal or infant antibody titer too low
• Low platelet antigen expression (i.e. HPA-5b)
• Causative antigen not present on test platelets

Winters 94
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Capture-P Precautions

• Samples should be tested as soon as possible. (If
  not, plasma may be stored at 1-10o C for 48
  hours or frozen).
• Indicator cells outdate 60 days from manufacture
  date.
• Donor platelets used in testing should be ABO
  compatible.
• Droppers should be held at a 45o angle.

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Capture-P Precautions

• Strips must be kept in foil pouch with desiccant
  and used within 30 minutes from removal.
• LISS reagent must be added immediately (within 1
  minute) after washing.
• Indicator red cells must be added immediately
  (within 30 seconds) after washing.

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Capture-P Controls

• A pool of donor platelets is used with control
  serum provided in kit.
• Strong positive, weak positive and negative
  controls are tested.
• All wells are compared to the negative control.
• Test is invalid if positive controls are not
  positive or negative control is not negative.

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Capture-P Ready ScreenTM

• A Capture-P screening test is available
• The screening test consists of double strips
  containing known HLA-A, HLA-B (Class I) and IgG
  platelet antibodies.
• Reaction patterns indicating a specific platelet
  antibody present must be confirmed by another
  method.

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Capture-P Ready ScreenTM

• The Capture-P Ready ScreenTM is intended for
  use only as a screening test.
• Reaction patterns should not be relied upon to
  identify specific platelet antibody.
• Reagents may contain antibody other than those
  recorded.

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Capture-P Ready-ScreenTM Worksheet
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Capture-P Ready-ScreenTM

• Anti-HPA-1a (PLA1) pattern in the screen
  Positive in wells 1-8 and negative in 9-12
• As previously mentionedMaternal Anti-HPA-1a may
  cause NAIT.
• The strong positive control containing
  anti-HPA-1a maybe used as a typing serum to type
  maternal platelets but positives must be
  confirmed by another method.

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Other Platelet Crossmatch Techniques

• Platelet Suspension Immunofluorescence Test
  (PSFIT)
• Flow Cytometry
• Monoclonal Antibody Immobilization of Platelet
  antigens (MAIPA)
• Microcytotoxicity using the Amos modification
• Molecular Techniques such as PCA can confirm
  genotypes of HPA-1 through HPA-6

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Other Commercially Available Platelet Testing
Systems

• GTI -PAK 12 (GTI Inc, Brookfield, WI) is a
  solid phase ELISA assay in which platelet
  glycoproteins obtained from group O donors
  homozygous for HPA-1a, HPA-1b, HPA-3a, HPA-5a,
  HPA-5b, affinity purified platelet glycoproteins
  and HLA Class I are immobilized in microtiter
  wells.

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GTI -PAK 12
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Other Commercially Available Platelet Testing
Systems

• GTI PAKPLUS is an ELISA screening test for the
  detection of antibodies to HLA Class I and
  platelet antigens. It is designed to detect
  weaker expressions of Anti-HPA-1b (Anti-PlA2)
  and Anti-HPA-5b (Anti-Bra ) plus more common
  platelet specific antibodies and HLA antibodies.

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GTI PAKPLUS
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GTI PAKAUTO

• Autoantibodies to certain platelet glycoproteins
  can often be detected in the plasma of patients
  with AITP.
• PAKAUTO is an ELISA screening test for
  autoantibodies to platelet glycoproteins
  IIb/IIIa, Ib/IX and Ia/IIa.

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GTI PAKAUTO
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GTI-MACETM 1

• GTI-MACETM 1 is a solid phase ELISA assay
  designed to detect IgG antibodies to platelet
  glycoproteins IIb/IIIa and HLA Class I
• The assay may be used for platelet crossmatches
  and used instead of Capture P or the MAIPA
  technique.

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GTI-MACETM 1
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GTI-MACETM 2

• GTI-MACETM 2 is a solid phase ELISA assay
  designed to detect IgG antibodies to platelet
  glycoproteins Ia/IIa and Ib/IX and IV.
• The assay may be also used for platelet
  crossmatches and used instead of Capture P or
  the MAIPA technique.

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GTI-MACETM 2
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GTI-PF4TM

• GTI-PF4TM is an ELISA assay for the detection of
  antibodies directed against Platelet Factor 4
  (PF4) when it is complexed with heparin or other
  polyanionic compounds. These antibodies are
  formed in nearly all patients with HIT.

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Laboratory Tests For HAT

• Functional
• Platelet Aggregation
• Serotonin Release
• Flow Cytometry
• Antigenic
• ELISA (H-PF4) (GTI)
• DiaMed (not available in USA)

Triplett
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Inova Fairfax HospitalCrossmatches Platelets
using Immucor Capture-P

• IFH considers ABO when crossmatching when
  possible
• IFH requires a platelet count 1 hour post
  platelet transfusion
• IFH crossmatches platelets. When the patient
  becomes refractory, we use HLA matched platelets
  provided by ARC

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Platelet Crossmatching Facility Survey

• Survey of 14 Facilities
• 12 Hospitals
• 3 large teaching/research hospitals out of area
• 5 area teaching/research hospitals in the area
• 4 other large area hospitals
• 2 Collection Facilities

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Platelet Crossmatching Facility Survey

• 7 facilities crossmatch platelets
• All 7 facilities use Capture-P
• 7 facilities do not crossmatch platelets
• 6 get crossmatched platelets from collection
  facilities
• 1 facility does not use crossmatched platelets

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Platelet Crossmatching Facility Survey

• Of the 7 hospitals who crossmatch platelets
• 5 consider ABO when crossmatching
• and 2 do not consider ABO
• 5 may crossmatch HLA platelets
• 1 does not use HLA matched platelets
• 2 work up heparin induced thrombocytopenia
  patients

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Platelet CrossmatchFacility Survey

• 11 hospitals transfuse HLA matched platelets
• 2 collection facilities and 4 hospitals do HLA
  testing on site and HLA match platelets
• 2 collection facilities and 4 hospitals also have
  large numbers of HLA typed platelet donors
• 1 hospital transfuses random platelets if
  crossmatched or HLA matched platelets become
  refractory

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The End
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Bibliography

• Current Issues in Platelet Transfusion Therapy
  and Platelet Alloimmunity Edited by Thomas S.
  Kickler, MD and Jay H. Herman, MD
• GTI, Inc., Brookfield, WI, Web SiteGTI-incorporat
  ed.com
• Immucor Capture-P Ready-ScreenTM , Immucor Inc.
  Norcross, GA, package insert
• Immucor Capture-P , Immucor Inc. Norcross, GA,
  package insert

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Bibliography

• Detection of Platelet Antibodies by Newly
  Developed Mixed Agglutination With Platelets by
  Y. Shibata, T. Juji, Y. Nishizawa, H. Sakamoto,
  and N. Ozawa
• Mixed Agglutination With Platelets by T. Juji,
  K. Kano, and F. Milgrom
• Use of a Solid Phase Red Blood Cell Adherence
  Method for Pretransfusion Platelet Compatibility
  Testing by J. Rachel, T Summers, L. Sinor and F.
  Plapp