Improved flow cytometric detection of HLA alloantibodies using pronase: Potential implications in renal transplantation

Smita Vaidya, Todd Y. Cooper, Jeanne Avandsalehi, Titus Barnes, Karl Brooks, Phoumymala Hymel, Maryam Noor, Rachel Sellers, Alice Thomas, Dod Stewart, John Daller, Jay C. Fish, Kristene Gugliuzza, Robert A. Bray

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

Background. Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the "standard of practice" in many programs. Although FCXM is the most sensitive method for detecting alloantibody, the B cell FCXM has been problematic. Difficulties with the B cell FCXMs have been centered around high nonspecific fluorescence background owing to Fc-receptors present on the B cells and autoantibodies. To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc receptors from lymphocytes before their use in FCXM. Methods. Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pronase and then used in a three-color FCXM. A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were performed. Testing used serial dilutions of HLA allosera (22 class I and 6 class II), with the titer of each antibody at one dilution past the titer at which the complement-mediated cytotoxicity anti-human globulin crossmatch became negative. Results. After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 78±10 to 57±4 (P<0.05) and 107±11 to 49±3 (P<0.0001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting class I antibody by 20% and 80%, respectively. In no instance was a false-positive reaction observed. In this study, pronase treatment improved the specificity of B cell FCXM for detecting class II antibodies from 75% to 100% (P=0.03). In no instance was a false-negative reaction recorded. Lastly, on the basis of these observations we re-evaluated three primary transplant recipients who lost their allografts because of accelerated rejection. One of the patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negative B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identified in each case. Conclusion. Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.

Original languageEnglish (US)
Pages (from-to)422-428
Number of pages7
JournalTransplantation
Volume71
Issue number3
StatePublished - Feb 15 2001

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Isoantibodies
Pronase
Kidney Transplantation
B-Lymphocytes
T-Lymphocytes
Immunoglobulin Isotypes
Fc Receptors
Lymphocytes
False Negative Reactions
False Positive Reactions
Sensitivity and Specificity
Globulins
Therapeutics
Autoantibodies
Allografts
Peptide Hydrolases
Spleen
Color
Fluorescence
Lymph Nodes

ASJC Scopus subject areas

  • Transplantation
  • Immunology

Cite this

Vaidya, S., Cooper, T. Y., Avandsalehi, J., Barnes, T., Brooks, K., Hymel, P., ... Bray, R. A. (2001). Improved flow cytometric detection of HLA alloantibodies using pronase: Potential implications in renal transplantation. Transplantation, 71(3), 422-428.

Improved flow cytometric detection of HLA alloantibodies using pronase : Potential implications in renal transplantation. / Vaidya, Smita; Cooper, Todd Y.; Avandsalehi, Jeanne; Barnes, Titus; Brooks, Karl; Hymel, Phoumymala; Noor, Maryam; Sellers, Rachel; Thomas, Alice; Stewart, Dod; Daller, John; Fish, Jay C.; Gugliuzza, Kristene; Bray, Robert A.

In: Transplantation, Vol. 71, No. 3, 15.02.2001, p. 422-428.

Research output: Contribution to journalArticle

Vaidya, S, Cooper, TY, Avandsalehi, J, Barnes, T, Brooks, K, Hymel, P, Noor, M, Sellers, R, Thomas, A, Stewart, D, Daller, J, Fish, JC, Gugliuzza, K & Bray, RA 2001, 'Improved flow cytometric detection of HLA alloantibodies using pronase: Potential implications in renal transplantation', Transplantation, vol. 71, no. 3, pp. 422-428.
Vaidya S, Cooper TY, Avandsalehi J, Barnes T, Brooks K, Hymel P et al. Improved flow cytometric detection of HLA alloantibodies using pronase: Potential implications in renal transplantation. Transplantation. 2001 Feb 15;71(3):422-428.
Vaidya, Smita ; Cooper, Todd Y. ; Avandsalehi, Jeanne ; Barnes, Titus ; Brooks, Karl ; Hymel, Phoumymala ; Noor, Maryam ; Sellers, Rachel ; Thomas, Alice ; Stewart, Dod ; Daller, John ; Fish, Jay C. ; Gugliuzza, Kristene ; Bray, Robert A. / Improved flow cytometric detection of HLA alloantibodies using pronase : Potential implications in renal transplantation. In: Transplantation. 2001 ; Vol. 71, No. 3. pp. 422-428.
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author = "Smita Vaidya and Cooper, {Todd Y.} and Jeanne Avandsalehi and Titus Barnes and Karl Brooks and Phoumymala Hymel and Maryam Noor and Rachel Sellers and Alice Thomas and Dod Stewart and John Daller and Fish, {Jay C.} and Kristene Gugliuzza and Bray, {Robert A.}",
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AU - Vaidya, Smita

AU - Cooper, Todd Y.

AU - Avandsalehi, Jeanne

AU - Barnes, Titus

AU - Brooks, Karl

AU - Hymel, Phoumymala

AU - Noor, Maryam

AU - Sellers, Rachel

AU - Thomas, Alice

AU - Stewart, Dod

AU - Daller, John

AU - Fish, Jay C.

AU - Gugliuzza, Kristene

AU - Bray, Robert A.

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N2 - Background. Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the "standard of practice" in many programs. Although FCXM is the most sensitive method for detecting alloantibody, the B cell FCXM has been problematic. Difficulties with the B cell FCXMs have been centered around high nonspecific fluorescence background owing to Fc-receptors present on the B cells and autoantibodies. To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc receptors from lymphocytes before their use in FCXM. Methods. Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pronase and then used in a three-color FCXM. A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were performed. Testing used serial dilutions of HLA allosera (22 class I and 6 class II), with the titer of each antibody at one dilution past the titer at which the complement-mediated cytotoxicity anti-human globulin crossmatch became negative. Results. After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 78±10 to 57±4 (P<0.05) and 107±11 to 49±3 (P<0.0001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting class I antibody by 20% and 80%, respectively. In no instance was a false-positive reaction observed. In this study, pronase treatment improved the specificity of B cell FCXM for detecting class II antibodies from 75% to 100% (P=0.03). In no instance was a false-negative reaction recorded. Lastly, on the basis of these observations we re-evaluated three primary transplant recipients who lost their allografts because of accelerated rejection. One of the patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negative B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identified in each case. Conclusion. Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.

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