TY - JOUR
AU - Coppage,, Myra
AB - Abstract Objectives To provide an overview of the complexities associated with the human leukocyte antigen (HLA)-mediated platelet refractoriness. HLA antibody detection technologies and limitations associated with methodologies are discussed. Methods A case scenario and review of relevant literature describing platelet refractoriness are presented, followed by a discussion of HLA antibody testing. Results Following diagnosis of HLA-mediated refractoriness, a decision is made regarding the approach to obtain the appropriate platelets. The panel reactive antibodies (PRA) % of the patient, HLA typing, and limitations of the HLA testing should be taken into account when deciding which type of product would be the best option for a given patient. Conclusions Following confirmation and review of HLA antibody testing, platelets are ordered based upon the PRA% and approach employed, HLA-matched platelets, antigen restricted platelets, or cross-matched platelets. The platelets are transfused and a posttransfusion increment count is monitored to determine transfusion success. Consult An 80-year-old woman had a history of myelodysplastic syndrome that progressed to acute myelogenous leukemia; her status after cycle 2 with decitabine was found to be pancytopenic during her regular hematology/oncology follow-up visit. Her medical history was significant for arthritis, hypertension, chronic kidney disease, and depression. Her current blood work-up revealed a WBC count of 0.7 × 103/μL (reference range: 4.0-10.0 × 103 /μL), hemoglobin of 7.0 g/dL (11.2-15.7 g/dL), and platelet count of 5 × 109/L (160-370 × 109/L). Two units of RBCs and one unit of platelets were ordered and transfused. Approximately 6 hours after transfusion, her platelet count was 4 × 109/L. She received another unit of platelets and 2 hours later, her platelet count dropped further to 3 × 109/L. The patient was deemed to be refractory to platelet transfusion and the blood bank and human leukocyte antigen (HLA) laboratory were consulted. Platelet serum antibody test, Pak Plus (Immucor, Peachtree Corners, GA) was performed by the blood bank and found to be positive for antibodies against the HLA. Using phenotypic beads (LABScreen, One Lambda, Canoga Park, CA), she was found to have a panel reactive antibodies (PRA) of 100%. Her HLA work-up showed Class 1 HLA typing of A2, 24; B35, 60 Figure 1. This was then reflexed to single-antigen bead testing (SAB) where the patient was found to have numerous HLA antibodies for a PRA of 94% Table 1 . The mean fluorescence intensities (MFIs) ranged from 3,084 to 18,665. To evaluate for prozone effect, a molecular weight (MW) spin column (Pall Nanosep Centrifugal Devices, 300K Omega, Westborough, MA) was used and the SAB class I assay was repeated. A prozone effect was detected and the HLA antibody specificities identified are shown in Table 1. The MFIs ranged from 3,164 to 26,009. The PRA with these results was 95%. Using the results with the MW spin column to eliminate prozone effect, several additional HLA-A antibodies were detected and some differences in the HLA-B antibodies were seen. Finally, to determine if the HLA antibodies were able to fix complement, the complement-dependent cytotoxicity (CDC) assay (GEN TRAK FCT 60 Frozen T cell tray, Gen Trak, Liberty, NC) was performed. In this assay, the patient’s HLA antibodies were found to be able to fix complement and mediate killing of 93% of the various cells of known HLA phenotype (Figure 1). Table 1 Patient’s Single-Antigen Bead (SAB) Results HLA-A Antibodies HLA-B Antibodies SAB results 24, 34, 68, 69 7, 13, 18, 27, 37, 38, 39, 41, 42, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 67, 71, 72, 73, 75, 76, 77, 81 SAB results with MW column to eliminate prozone effect 1, 3, 11, 25, 26, 29, 30, 31, 32, 33, 34, 36, 43, 66, 74, 80 7, 8, 13, 18, 27, 37, 38, 39, 42, 44, 45, 46, 47, 54, 55, 56, 57, 58, 59, 64, 65, 67, 76, 82 HLA-A Antibodies HLA-B Antibodies SAB results 24, 34, 68, 69 7, 13, 18, 27, 37, 38, 39, 41, 42, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 67, 71, 72, 73, 75, 76, 77, 81 SAB results with MW column to eliminate prozone effect 1, 3, 11, 25, 26, 29, 30, 31, 32, 33, 34, 36, 43, 66, 74, 80 7, 8, 13, 18, 27, 37, 38, 39, 42, 44, 45, 46, 47, 54, 55, 56, 57, 58, 59, 64, 65, 67, 76, 82 HLA, human leukocyte antigen; MW, molecular weight. View Large Table 1 Patient’s Single-Antigen Bead (SAB) Results HLA-A Antibodies HLA-B Antibodies SAB results 24, 34, 68, 69 7, 13, 18, 27, 37, 38, 39, 41, 42, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 67, 71, 72, 73, 75, 76, 77, 81 SAB results with MW column to eliminate prozone effect 1, 3, 11, 25, 26, 29, 30, 31, 32, 33, 34, 36, 43, 66, 74, 80 7, 8, 13, 18, 27, 37, 38, 39, 42, 44, 45, 46, 47, 54, 55, 56, 57, 58, 59, 64, 65, 67, 76, 82 HLA-A Antibodies HLA-B Antibodies SAB results 24, 34, 68, 69 7, 13, 18, 27, 37, 38, 39, 41, 42, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 67, 71, 72, 73, 75, 76, 77, 81 SAB results with MW column to eliminate prozone effect 1, 3, 11, 25, 26, 29, 30, 31, 32, 33, 34, 36, 43, 66, 74, 80 7, 8, 13, 18, 27, 37, 38, 39, 42, 44, 45, 46, 47, 54, 55, 56, 57, 58, 59, 64, 65, 67, 76, 82 HLA, human leukocyte antigen; MW, molecular weight. View Large Figure 1 View largeDownload slide Human leukocyte antigen (HLA) laboratory report. The patient’s HLA typing is indicated by the double arrows. The asterisk by the A and B indicates that the typing was done using molecular methods. HLA typing results are given as the allele group followed by a colon and then a given number or letter code that is indicative of a series of specific HLA proteins or a specific HLA protein. For example, in the case of HLA-B, the allele group is 35 and the specific protein is 8. For the other B allele, it is given as 40(60):AFHYS. HLA-B40 is comprised of the splits B60 and B61. In this case, the patient has the B60 split. The specific protein is given by a code, AFHYS, which describes a group of proteins. This code is then described lower in the report and it lists the specific proteins that are included. To further elucidate which specific protein of B40(60) that she has, sequence-based typing (SBT) or higher-resolution sequence-specific oligonucleotide (SSO) is needed. In the case of HLA-A, the allele is 02 and the specific protein is given as a code, AEGCT. The antibody screen results are indicated with a single arrow. The test listed is TSTAT, which is a reference to the complement-dependent cytotoxicity (CDC) test. The patient’s panel reactive antibodies (PRA)% is 93%. Our center uses the CDC in HLA antibody-mediated platelet refractoriness evaluations. We find it helpful in elucidating which HLA antibodies are likely to cause platelet destruction. LMX-ID, Luminex identity; LMX-SAB, Luminex single-antigen bead; POS, positive; SSP, sequence-specific primer. Figure 1 View largeDownload slide Human leukocyte antigen (HLA) laboratory report. The patient’s HLA typing is indicated by the double arrows. The asterisk by the A and B indicates that the typing was done using molecular methods. HLA typing results are given as the allele group followed by a colon and then a given number or letter code that is indicative of a series of specific HLA proteins or a specific HLA protein. For example, in the case of HLA-B, the allele group is 35 and the specific protein is 8. For the other B allele, it is given as 40(60):AFHYS. HLA-B40 is comprised of the splits B60 and B61. In this case, the patient has the B60 split. The specific protein is given by a code, AFHYS, which describes a group of proteins. This code is then described lower in the report and it lists the specific proteins that are included. To further elucidate which specific protein of B40(60) that she has, sequence-based typing (SBT) or higher-resolution sequence-specific oligonucleotide (SSO) is needed. In the case of HLA-A, the allele is 02 and the specific protein is given as a code, AEGCT. The antibody screen results are indicated with a single arrow. The test listed is TSTAT, which is a reference to the complement-dependent cytotoxicity (CDC) test. The patient’s panel reactive antibodies (PRA)% is 93%. Our center uses the CDC in HLA antibody-mediated platelet refractoriness evaluations. We find it helpful in elucidating which HLA antibodies are likely to cause platelet destruction. LMX-ID, Luminex identity; LMX-SAB, Luminex single-antigen bead; POS, positive; SSP, sequence-specific primer. Background Platelet refractoriness represents a serious complication in platelet-transfusion-dependent patients. It is associated with adverse clinical outcomes, such as increased bleeding risk, and decreased survival and increased health care costs due to increased length of stay.1-3 Following platelet transfusion, the effectiveness of the transfusion is most commonly evaluated by using the posttransfusion increment, percentage of platelet recovery, and/or the corrected count increment (CCI). The posttransfusion increment is the most frequently used method as it uses readily available information from the patient’s chart, namely the platelet counts before and after transfusion. A postinfusion increment increase of greater than 10 × 109/L anytime up to 24 hours posttransfusion is considered an adequate response.4 The percentage of platelet recovery is calculated from platelet increment increase, blood volume (BV) in liters, and estimated number of transfused platelets. A platelet count increase of 30%, up to 1 hour posttransfusion, or greater than 20% at 20 to 24 hours is considered a successful transfusion.5 % platelet recovery = platelet count increment increase × BV# platelets transfused × 1011 To calculate the CCI, the platelet increment increase, body surface area (BSA) of the patient (m2), and the estimated number of platelets transfused must be known. A successful transfusion at 1 hour is typically assumed to be a CCI greater than 7.5 × 109/L or greater than 4.5 × 109/L at 20 to 24 hours.6 Notably, the Trial to Reduce Alloimmunization to Platelets study (TRAP) defined platelet refractoriness as a 1-hour CCI of less than 5 × 109/L on two sequential occasions.6 CCI  =  platelet count increment increase × BSA# platelets transfused × 1011 Once a patient has been determined to be platelet refractory, the etiology of the refractoriness should be evaluated. The main causes of platelet refractoriness can be grouped into two categories, nonimmune and immune Table 2 , with the former being more common. Following platelet transfusion, patients with nonimmune causes will typically have a normal response in platelet count when measured within 1 hour. Platelet count will often return to baseline within 24 hours.6 This is indicative of decreased platelet survival. Patients with immune causes, however, typically show little to no increase in platelet count at 1 hour posttransfusion.7,8 Once the platelet refractoriness has been established as immune or nonimmune mediated, the underlying cause can be investigated. A thorough patient medical history and physical is extremely valuable. Additional tests that can further clarify nonimmune causes include d-dimer, blood/urine culture, and liver function tests. Immune causes of platelet refractoriness can be distinguished via HLA testing, platelet antibody testing, and drug-dependent platelet antibody testing. To avoid the high cost of these tests, a careful review of patient medical history should be used to decide the most likely etiology. This article will mainly focus on HLA-mediated platelet refractoriness. Table 2 Causes of Platelet Refractoriness Nonimmune  Infection/sepsis  Antibiotics  Disseminated intravascular coagulation  Fever  Splenomegaly  Hepatic venoocclusive disease  Graft-versus-host disease  Medications (eg, vancomycin, amphotericin B, heparin) Immune  HLA antibodies  Platelet auto and alloantibodies  ABO antibodies  Drug-dependent platelet antibodies  Immune complexes Nonimmune  Infection/sepsis  Antibiotics  Disseminated intravascular coagulation  Fever  Splenomegaly  Hepatic venoocclusive disease  Graft-versus-host disease  Medications (eg, vancomycin, amphotericin B, heparin) Immune  HLA antibodies  Platelet auto and alloantibodies  ABO antibodies  Drug-dependent platelet antibodies  Immune complexes View Large Table 2 Causes of Platelet Refractoriness Nonimmune  Infection/sepsis  Antibiotics  Disseminated intravascular coagulation  Fever  Splenomegaly  Hepatic venoocclusive disease  Graft-versus-host disease  Medications (eg, vancomycin, amphotericin B, heparin) Immune  HLA antibodies  Platelet auto and alloantibodies  ABO antibodies  Drug-dependent platelet antibodies  Immune complexes Nonimmune  Infection/sepsis  Antibiotics  Disseminated intravascular coagulation  Fever  Splenomegaly  Hepatic venoocclusive disease  Graft-versus-host disease  Medications (eg, vancomycin, amphotericin B, heparin) Immune  HLA antibodies  Platelet auto and alloantibodies  ABO antibodies  Drug-dependent platelet antibodies  Immune complexes View Large Platelets only express class 1 HLA.9 HLA-A, -B, and -C comprise the class 1 HLA antigens. Platelets predominantly express HLA-A and -B.9,10 HLA-C is not well expressed on platelets and therefore is typically not regarded as a significant contributing factor in HLA-mediated platelet refractoriness.10,11 However, HLA antibodies to C antigens have been implicated in some cases of platelet refractoriness.11,12 Platelet donors typed for HLA-C are not readily available in the United States. Exposure to foreign HLA antigens via pregnancy, blood component transfusion, and/or organ transplantation is the primary mechanism of HLA antibody induction.8 Almost 50% reduction (from 16% to 7%-8%) in HLA alloimmunization from blood product transfusions was seen following introduction of leokoreduction.13 Most hospitals and transfusion centers in the United States have adopted the use of leukoreduced blood products. Several tools are available to evaluate immune causes of platelet refractoriness. The most general screening method is an enzyme-linked immunosorbent assay (ELISA) based method that evaluates the patient’s plasma or serum for a variety of antibodies directed against platelet antigens and HLA antigens. In this assay, a variety of class I HLA antigens are tested but not categorized. This method is useful in distinguishing between patients with idiopathic thrombocytopenic purpura, who typically have antibodies against glycoprotein IIbIIIa, and patients with HLA-mediated platelet refractoriness.14 Additionally, this method may be particularly helpful when an immune cause is suspected but suspicion for HLA antibodies is low. This moderate complexity test takes approximately 4 hours to perform and is commonly used in large medical centers. It is also frequently available as a send out test from most local blood suppliers (eg, American Red Cross). Another commonly used test employs Luminex-based phenotypic beads to screen for HLA antibodies of either class I or II antigens.15,16 For class I beads, each bead will have a set of HLA antigens from a single individual (two A antigens, two B antigens, and two C antigens). These beads will distinguish whether a patient has class I HLA antibodies and in some cases of limited antibodies, a specificity can be determined. However, in most cases, Luminex-based HLA class I SABs will be utilized Figure 2.16 In this assay, each individual bead is coated with a single HLA antigen for better determination of HLA antibodies. A calculated panel-reactive antibody percentage (cPRA) or PRA is then generated. The cPRA takes into account the population frequency of the identified HLA antigens.17 There are several websites to facilitate cPRA calculation, such as that of the Organ Procurement and Transplantation Network (https://optn.transplant.hrsa.gov/resources/allocation-calculators/cpra-calculator). Figure 2 View largeDownload slide Single-antigen bead (SAB) assay. Luminex beads are coated with a single human leukocyte antigen (HLA) on their surface and incubated with patient serum. If the patient has HLA antibodies, they will bind to the bead that is coated with the respective antigen. The beads are then washed and incubated with phycoerythrin (PE)-labeled anti-human IgG antibodies. This antibody will bind to the Fc region of antibodies bound to beads. The mixture is then washed and analyzed using a Luminex instrument. Each bead is identified through a unique combination of two fluorescent dyes (red and infrared) impregnated into the bead. The anti-human IgG complex will emit fluorescence upon exposure to laser light. This fluorescence is detected on a Luminex platform and antibody reactivity is recorded as mean fluorescence intensity. Figure 2 View largeDownload slide Single-antigen bead (SAB) assay. Luminex beads are coated with a single human leukocyte antigen (HLA) on their surface and incubated with patient serum. If the patient has HLA antibodies, they will bind to the bead that is coated with the respective antigen. The beads are then washed and incubated with phycoerythrin (PE)-labeled anti-human IgG antibodies. This antibody will bind to the Fc region of antibodies bound to beads. The mixture is then washed and analyzed using a Luminex instrument. Each bead is identified through a unique combination of two fluorescent dyes (red and infrared) impregnated into the bead. The anti-human IgG complex will emit fluorescence upon exposure to laser light. This fluorescence is detected on a Luminex platform and antibody reactivity is recorded as mean fluorescence intensity. Bead-based assays using Luminex technology are very sensitive. In these assays, the patient’s serum is incubated with a given Luminex bead type such as class I phenotype bead or class I SAB.18 The beads are then washed and anti-human IgG (AHG) with a fluorochrome is added to each well. The Luminex beads are dyed with two additional fluorochromes to allow for discrimination of individual beads. If the patient has an HLA antibody to the antigen present on a given bead, the AHG fluorochrome will provide a signal linked to the bead. The strength or amount of antibody binding is expressed as the MFI. The MFI cutoff values vary between different laboratories. Experts disagree as to what is a significant MFI value. Some laboratories use 1,000, 1,500, 2,000, 3,000, or even 5,000 as a cutoff.16 Notably, not all detected HLA antibodies are clinically significant.19 The Luminex bead-based assays described above detect all subclasses of IgG regardless of their ability to fix complement, although there are kits available to specifically detect C1q binding.20 The C1q assay is depicted in Figure 3. HLA antibodies (IgG1 and IgG3) that are capable of fixing complement are more likely to contribute to platelet refractoriness. Thus, a CDC may be performed to determine HLA antibodies ability to fix complement. The CDC assay requires coincubation of patient serum with a panel of HLA-typed lymphocytes. Rabbit serum as a source of complement is then added with a vital dye and the amount of cell killing is determined microscopically.21 The CDC assay is depicted in Figure 4. Figure 3 View largeDownload slide C1q assay. Luminex beads are coated with a single human leukocyte antigen (HLA) antigen on their surface and incubated with patient serum. If the patient has HLA antibodies, they will bind to the bead that is coated with the respective antigen. The beads are then washed and incubated with C1q. The coated beads are washed again and incubated with phycoerythrin (PE)-labeled anti-C1q antibodies. The mixture is then washed and analyzed using a Luminex instrument. Each bead is identified through a unique combination of two fluorescent dyes (red and infrared) impregnated into the bead. The anti-C1q complex will emit fluorescence upon exposure to laser light. This fluorescence is detected on a Luminex platform and antibody reactivity is recorded as mean fluorescence intensity. Figure 3 View largeDownload slide C1q assay. Luminex beads are coated with a single human leukocyte antigen (HLA) antigen on their surface and incubated with patient serum. If the patient has HLA antibodies, they will bind to the bead that is coated with the respective antigen. The beads are then washed and incubated with C1q. The coated beads are washed again and incubated with phycoerythrin (PE)-labeled anti-C1q antibodies. The mixture is then washed and analyzed using a Luminex instrument. Each bead is identified through a unique combination of two fluorescent dyes (red and infrared) impregnated into the bead. The anti-C1q complex will emit fluorescence upon exposure to laser light. This fluorescence is detected on a Luminex platform and antibody reactivity is recorded as mean fluorescence intensity. Figure 4 View largeDownload slide Complement-dependent cytotoxicity assay. A positive reaction is shown above. Lymphocytes with known human leukocyte antigen (HLA) typing are incubated with patient serum containing antibodies. If the patient has an HLA antibody against any HLA antigens expressed on the lymphocytes, it will bind. Rabbit complement is then added. The complement will fix at the sites of bound patient antibodies. The membrane attack complex then forms followed by lysis and cell death. A dye is added and is taken up by cells that are dead. The amount of dead cells in each well is scored. A panel of reactive antibodies is calculated based upon the number of wells with dead cells as a percent of all wells. Figure 4 View largeDownload slide Complement-dependent cytotoxicity assay. A positive reaction is shown above. Lymphocytes with known human leukocyte antigen (HLA) typing are incubated with patient serum containing antibodies. If the patient has an HLA antibody against any HLA antigens expressed on the lymphocytes, it will bind. Rabbit complement is then added. The complement will fix at the sites of bound patient antibodies. The membrane attack complex then forms followed by lysis and cell death. A dye is added and is taken up by cells that are dead. The amount of dead cells in each well is scored. A panel of reactive antibodies is calculated based upon the number of wells with dead cells as a percent of all wells. The SAB, C1q, and CDC assay all detect patient HLA antibodies; however, they are different as depicted in Figure 2, Figure 3, and Figure 4. The SAB assay is the most sensitive and can detect HLA antibodies that may not be clinically relevant. The C1q assay attempts to determine if the patient’s HLA antibodies are capable of C1q complement binding. If they do bind C1q, the assumption is that they are more clinically relevant HLA antibodies. Lastly is the CDC assay, which is a more clinically relevant assay as the HLA antigens are on lymphocytes. This assay looks at the ability of the patient’s HLA antibodies to bind and fix complement that mediates cell death. Different HLA laboratories rely on various combinations of these tests to help ascertain which HLA antibodies are clinically relevant. Management Depending upon availability of HLA testing (in-house vs send out), HLA typing may take several days as will HLA antibody identification. Some larger in-house HLA laboratories can offer same-day results. Once HLA antibodies are identified, there are several different ways to order platelets. The first option is to order HLA-matched platelets. To do this, the patient’s Class 1 HLA typing must be known. Platelets are only matched at the A and B locus. The degree of match is determined based upon a classification system described in Table 3 .22 Platelets with a higher degree of match have improved survival following transfusion. For example, grade A, B1U, and B2U matches have been shown to provide the best increases in platelet counts. Similarly, platelets from donors that have HLA antigen typings that are in the same cross-reactive groups (CREGs; Table 423,24) as the patient’s antigens have also been shown to successfully produce better count increments.25,26 This is thought to be due to inability of the patient’s immune system to recognize the CREGs as foreign proteins. Notably, most HLA antibodies are directed against public epitopes.23 Public epitopes are shared by two or more antigens and private epitopes are only found on one antigen.27 Many HLA antibodies are against public epitopes, thus they cross-react with numerous HLA alleles. Lastly, B2X, C, and D matches have been shown to provide platelet responses similar to transfusion of random donor platelets. Unfortunately, HLA-matched platelets may be difficult to obtain. This is frequently the case for patients with uncommon or rare HLA antigens as well as for patients of various ethnic groups that are not well represented in the blood donor pool. In these cases, partially matched platelets are provided as they have been shown in several studies to provide adequate platelet increments.28,29 Table 3 Human Leukocyte Antigen (HLA)-Matched Platelet Classification Grade Description A All 4 donor HLA antigens are identical to the recipient B1U Only 3 antigens are detected in the donor (homozygous at 1 HLA allele); all 3 detected antigens are identical to the recipient B1X 2 HLA antigens are identical to the recipient and 1 HLA antigen is cross-reactive B2U Only 2 antigens are detected in the donor (homozygous at 2 HLA alleles); both detected antigens are identical to the recipient B2UX Only 3 HLA antigens are detected in the donor (homozygous at 1 allele); 2 antigens are identical with the recipient and 1 antigen is cross-reactive B2X 2 antigens are identical to the recipient and 2 antigens are cross-reactive C 1 antigen of donor is not present in the recipient and are not cross-reactive with the recipient D 2 antigens of the donor are not present in the recipient and are not cross-reactive with the recipient R Random donor Grade Description A All 4 donor HLA antigens are identical to the recipient B1U Only 3 antigens are detected in the donor (homozygous at 1 HLA allele); all 3 detected antigens are identical to the recipient B1X 2 HLA antigens are identical to the recipient and 1 HLA antigen is cross-reactive B2U Only 2 antigens are detected in the donor (homozygous at 2 HLA alleles); both detected antigens are identical to the recipient B2UX Only 3 HLA antigens are detected in the donor (homozygous at 1 allele); 2 antigens are identical with the recipient and 1 antigen is cross-reactive B2X 2 antigens are identical to the recipient and 2 antigens are cross-reactive C 1 antigen of donor is not present in the recipient and are not cross-reactive with the recipient D 2 antigens of the donor are not present in the recipient and are not cross-reactive with the recipient R Random donor View Large Table 3 Human Leukocyte Antigen (HLA)-Matched Platelet Classification Grade Description A All 4 donor HLA antigens are identical to the recipient B1U Only 3 antigens are detected in the donor (homozygous at 1 HLA allele); all 3 detected antigens are identical to the recipient B1X 2 HLA antigens are identical to the recipient and 1 HLA antigen is cross-reactive B2U Only 2 antigens are detected in the donor (homozygous at 2 HLA alleles); both detected antigens are identical to the recipient B2UX Only 3 HLA antigens are detected in the donor (homozygous at 1 allele); 2 antigens are identical with the recipient and 1 antigen is cross-reactive B2X 2 antigens are identical to the recipient and 2 antigens are cross-reactive C 1 antigen of donor is not present in the recipient and are not cross-reactive with the recipient D 2 antigens of the donor are not present in the recipient and are not cross-reactive with the recipient R Random donor Grade Description A All 4 donor HLA antigens are identical to the recipient B1U Only 3 antigens are detected in the donor (homozygous at 1 HLA allele); all 3 detected antigens are identical to the recipient B1X 2 HLA antigens are identical to the recipient and 1 HLA antigen is cross-reactive B2U Only 2 antigens are detected in the donor (homozygous at 2 HLA alleles); both detected antigens are identical to the recipient B2UX Only 3 HLA antigens are detected in the donor (homozygous at 1 allele); 2 antigens are identical with the recipient and 1 antigen is cross-reactive B2X 2 antigens are identical to the recipient and 2 antigens are cross-reactive C 1 antigen of donor is not present in the recipient and are not cross-reactive with the recipient D 2 antigens of the donor are not present in the recipient and are not cross-reactive with the recipient R Random donor View Large Table 4 Cross-Reactive Groups (CREGs); Human Leukocyte Antigens (HLA) in CREGs Share a Common Public Epitope CREGa HLA Antigens A1 CREG A1, 3, 9 (23, 24), 11, 29, 30, 31, 36, 80 A2 CREG A2, 9 (23, 24), 28 (68, 69), B17 (57, 58) A10 CREG A10 (25, 26, 34, 66), 11, 28 (68, 69), 32, 33, 43, 74 B5 CREG B5 (51, 52), 15 (62, 63, 75, 76, 77), 17 (57, 58), 18, 21 (49, 50), 35, 46, 53, 70 (71, 72), 73, 78 B7 CREG B7, 8, 13, 22 (54, 55, 56), 27, 40 (60, 61), 41, 42, 47, 48, 59, 67,81, 82 B8 CREG B8, 14 (64, 65), 16 (38, 39), 18, 59, 67 B12 CREG B12 (44, 45), 13, 21 (49, 50), 37, 40 (60, 61), 41, 47 Bw4 CREG A23, A24, A25, A32, B13, 27, 37, 38, 44, 47, 49, 51, 52, 53, 57, 58, 59, 63, 77 Bw6 CREG B7, 8, 18, 35, 39, 41, 42, 45, 46, 48, 50, 54, 55, 56, 60, 61, 62, 64, 65, 67, 71, 72, 73, 75, 76, 78, 81, 82 CREGa HLA Antigens A1 CREG A1, 3, 9 (23, 24), 11, 29, 30, 31, 36, 80 A2 CREG A2, 9 (23, 24), 28 (68, 69), B17 (57, 58) A10 CREG A10 (25, 26, 34, 66), 11, 28 (68, 69), 32, 33, 43, 74 B5 CREG B5 (51, 52), 15 (62, 63, 75, 76, 77), 17 (57, 58), 18, 21 (49, 50), 35, 46, 53, 70 (71, 72), 73, 78 B7 CREG B7, 8, 13, 22 (54, 55, 56), 27, 40 (60, 61), 41, 42, 47, 48, 59, 67,81, 82 B8 CREG B8, 14 (64, 65), 16 (38, 39), 18, 59, 67 B12 CREG B12 (44, 45), 13, 21 (49, 50), 37, 40 (60, 61), 41, 47 Bw4 CREG A23, A24, A25, A32, B13, 27, 37, 38, 44, 47, 49, 51, 52, 53, 57, 58, 59, 63, 77 Bw6 CREG B7, 8, 18, 35, 39, 41, 42, 45, 46, 48, 50, 54, 55, 56, 60, 61, 62, 64, 65, 67, 71, 72, 73, 75, 76, 78, 81, 82 aAs defined by Rodey et al23 and Wade et al24 View Large Table 4 Cross-Reactive Groups (CREGs); Human Leukocyte Antigens (HLA) in CREGs Share a Common Public Epitope CREGa HLA Antigens A1 CREG A1, 3, 9 (23, 24), 11, 29, 30, 31, 36, 80 A2 CREG A2, 9 (23, 24), 28 (68, 69), B17 (57, 58) A10 CREG A10 (25, 26, 34, 66), 11, 28 (68, 69), 32, 33, 43, 74 B5 CREG B5 (51, 52), 15 (62, 63, 75, 76, 77), 17 (57, 58), 18, 21 (49, 50), 35, 46, 53, 70 (71, 72), 73, 78 B7 CREG B7, 8, 13, 22 (54, 55, 56), 27, 40 (60, 61), 41, 42, 47, 48, 59, 67,81, 82 B8 CREG B8, 14 (64, 65), 16 (38, 39), 18, 59, 67 B12 CREG B12 (44, 45), 13, 21 (49, 50), 37, 40 (60, 61), 41, 47 Bw4 CREG A23, A24, A25, A32, B13, 27, 37, 38, 44, 47, 49, 51, 52, 53, 57, 58, 59, 63, 77 Bw6 CREG B7, 8, 18, 35, 39, 41, 42, 45, 46, 48, 50, 54, 55, 56, 60, 61, 62, 64, 65, 67, 71, 72, 73, 75, 76, 78, 81, 82 CREGa HLA Antigens A1 CREG A1, 3, 9 (23, 24), 11, 29, 30, 31, 36, 80 A2 CREG A2, 9 (23, 24), 28 (68, 69), B17 (57, 58) A10 CREG A10 (25, 26, 34, 66), 11, 28 (68, 69), 32, 33, 43, 74 B5 CREG B5 (51, 52), 15 (62, 63, 75, 76, 77), 17 (57, 58), 18, 21 (49, 50), 35, 46, 53, 70 (71, 72), 73, 78 B7 CREG B7, 8, 13, 22 (54, 55, 56), 27, 40 (60, 61), 41, 42, 47, 48, 59, 67,81, 82 B8 CREG B8, 14 (64, 65), 16 (38, 39), 18, 59, 67 B12 CREG B12 (44, 45), 13, 21 (49, 50), 37, 40 (60, 61), 41, 47 Bw4 CREG A23, A24, A25, A32, B13, 27, 37, 38, 44, 47, 49, 51, 52, 53, 57, 58, 59, 63, 77 Bw6 CREG B7, 8, 18, 35, 39, 41, 42, 45, 46, 48, 50, 54, 55, 56, 60, 61, 62, 64, 65, 67, 71, 72, 73, 75, 76, 78, 81, 82 aAs defined by Rodey et al23 and Wade et al24 View Large A second option is to order HLA antigen-restricted platelets that avoid the HLA antibodies that the patient has in a “virtual” crossmatch. In this case, only the patient’s HLA antibodies need to be known rather than the HLA typing. This approach expands the likelihood of finding compatible platelets as compared to using HLA-matched platelets. Petz et al30 looked at the availability of platelet donors when patients were matched with an A grade match, BU grade match, or antigen-negative matched. In their study, they had 7,247 donors that were HLA-typed and 29 patients that had HLA antibodies. They identified a mean number of six grade A matched donors, 33 BU matched donors, and 1,426 antigen-negative donors.30 Figure 5 depicts the evaluation and approach that we use for platelet refractoriness at our medical center. As shown, depending upon the PRA score, we decide to use antigen restricted or HLA-matched platelets. Following transfusion, we reevaluate the platelet count increment. If antigen-restricted platelets fail, we will move to HLA-matched platelets. However, if the patient fails to receive platelet count increments with HLA-matched platelets, we discontinue HLA-matched platelets and transfuse standard random donor apheresis platelets. Figure 5 View largeDownload slide Flow chart for evaluation and work-up of platelet refractoriness, describing the protocol we use. The patient is deemed refractory to platelet transfusions following at least one platelet count with suboptimal corrected count increment, count increment, or % platelet recovery that is obtained within 1 hour posttransfusion of a unit of random donor platelets. HLA, human leukocyte antigen; PRA, panel reactive antibodies. Figure 5 View largeDownload slide Flow chart for evaluation and work-up of platelet refractoriness, describing the protocol we use. The patient is deemed refractory to platelet transfusions following at least one platelet count with suboptimal corrected count increment, count increment, or % platelet recovery that is obtained within 1 hour posttransfusion of a unit of random donor platelets. HLA, human leukocyte antigen; PRA, panel reactive antibodies. A third option is to order crossmatch compatible platelets. For this, neither the patient’s HLA type nor HLA antibody specificity needs to be known. This method can be used while waiting for HLA typing and HLA antibody results. This approach can also be used for patients that have a lower PRA percentage as testing of 10 to 20 apheresis platelet units will yield a compatible match. However, in highly sensitized patients, this method is unlikely to find compatible platelets without testing numerous units. Wiita and Nambiar31 found that patients with initial crossmatches with PRA greater than 66% were more likely to have at least one panreactive crossmatch assay. Recently, Wang et al32 administered 480 crossmatched platelets to 82 of their 114 alloimmunized patients and found that the mean CCI was 7.8 ± 5.2 × 109/L, which was statistically significantly different as compared to random donor platelets (P < .001). The commonly crossmatched methods used are solid-phase red cell adherence (SPRCA), modified antigen capture ELISA, and flow cytometry.33 In the SPRCA method, the most popular, platelets from different random apheresis units are bound to the bottom of a microtiter plate. Patient’s serum is added to each well, incubated, and washed before the addition of the AHG-coated RBCs. This test is moderate complexity and can be performed in hospital blood banks. However, due to the need for large numbers of apheresis platelets of a given blood group, this test is most frequently performed by the local blood supplier (eg, American Red Cross). Typically, the patient’s serum is tested against 10 different apheresis platelets of the same blood group. Due to the short shelf life of platelets, most hospital blood banks typically do not have enough apheresis units of a given blood group to set aside for testing. A newer method of HLA matching that has been incorporated at some blood centers is epitope-based matching. Epitopes are conformational arrangements of amino acids that are targeted by antibodies. As described above, epitopes can be private, on a single HLA antigen, or public, shared by two or more HLA antigens. CREGs are defined by sharing of a public epitope and can be grouped by serologic cross-reactivity patterns. A study by Moroff et al34 showed that about 40% of CREG-matched platelet transfusions, in patients who had intra-CREG antibodies, resulted in unsuccessful platelet count increments. To address this, Pai et al35 evaluated transfusion success rates using 1-hour CCIs for perfectly matched platelets, CREG-matched platelets, and epitope-based matching platelets. A higher successful rate was found with epitope-based matching (83.7%) as compared to CREG matching (63.2%).35 To perform epitope-based matching, the Luminex HLA class I SAB assay can be coupled with the HLAMatchmaker program (www.hlamatchmaker.net). This software program takes into account the patient’s HLA antibodies and generates epitopes that are possibly recognized by these antibodies.26,36,37 It identifies immunogenic epitopes represented by amino acids located within approximately 3 to 3.5 Å radius of a polymorphic residue in antibody accessible regions of HLA antigens.26 This software also uses the patient’s HLA typing at higher resolution levels than the serological level. A limitation to this approach is the cost of the required high-resolution HLA typing of both the patient and platelet donors. Additionally, epitope prediction can be difficult and there may be no suitable epitopes for some patients. In summary, regardless of which type of platelet matching is used, a large pool of donors is needed. Additionally, there is no guarantee of a successful platelet transfusion increment, and matching of platelets this way typically takes at least a day or more. For urgent platelet transfusion, a random donor platelet off-the-shelf should be provided. HLA antibody production can be transitory; therefore, it may be beneficial to reevaluate the HLA platelet refractory patient over time. The Luminex Class 1 SAB assay can be used to detect antibodies specific to class 1 HLA antigens and to determine their strength using MFI. A CDC assay can also be repeated to verify that the antibodies are able to fix complement. Repeat testing is helpful to ensure that HLA-matched platelets, which are quite expensive, are still necessary and no additional HLA antibodies have been produced. HLA Antibody Testing Limitations The Luminex SAB assay is a very powerful assay and can detect very low levels of antibody. Many laboratories use different MFI cutoffs when deciding to “call” antibodies for patients. Some laboratories use a higher MFI cutoff and will report less antibodies and possibly leave out a low MFI antibody that is relevant; while others may use a low MFI cutoff and call many low-level antibodies that are not important or unlikely to be biologically relevant. There is ongoing debate as to whether the bead-based Luminex assays are too sensitive for use in HLA platelet refractoriness testing. Jackman et al38 showed that HLA antibodies detected by cytotoxic methods predicted platelet refractoriness but that antibodies detected by bead-based assays or microarrays did not. This indicates that weak or low-level HLA antibodies may not participate in platelet refractoriness. Furthermore, it should be noted that antibody cross-linking is required for complement-mediated destruction and weak or low-level HLA antibodies are unlikely to facilitate cross-linking.39,40 Hence, some HLA laboratories have increased the MFI that they use to “call” antibodies in cases of HLA-mediated platelet refractoriness. Bead-based Luminex assays have additional limitations. The antigens on the phenotypic beads are removed from cells and placed on the beads. The antigens on the SAB beads are recombinant proteins. Thus, the antigens may not be in their true native form, resulting in both false-positive and false-negative reactions. Attachment of the HLA antigen to the beads can result in a conformational change in the antigen that exposes a cryptic binding site that is not normally accessible.41 Additionally, individuals have been found to make antibodies to cross-reactive epitopes found in microorganisms, ingested proteins, and allergens that are reactive with HLA antigen epitopes.42 Another limitation is that the HLA protein binds and presents specific peptides; in some cases these peptides are not present or have fallen out, which exposes cryptic sites in the HLA protein.27 The patient may have antibodies that recognize this denatured or altered antigen, causing a positive reaction and MFI for a particular bead. However, this would be a false-positive HLA antibody identification. There are several ways to try to prevent false-positive HLA antibody identification. One way is to treat the SABs with acid to denature all HLA antigens and then incubate with the patient’s serum. Any positive reactions would be false positive. Approximately 21% to 39% of patients are believed to have at least one false-positive antibody identified against denatured HLA antigens.41,43 The bead-based Luminex assay is also subject to interference by IgM antibodies, autoantibodies, antithymocyte globulin, intravenous immune globulin, immune complexes, complement, and nonspecific antibody binding to the beads themselves.33,44-47 The SAB assay also has problems with prozone effect where high-titer anti-HLA antibodies saturate the beads and/or interfere with alloantibody binding and lead to false-negative results or lower MFI values for other HLA antibodies. Treatment of the patient’s serum with ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), heat, dialysis, or MW spin columns can decrease the prozone effect.48,49 High-titer antibodies that fix complement can also yield negative results due to prozone effect created by C1qrs complexes on the beads, which inhibits binding of AHG. This can be mitigated by use of EDTA or dilution.50 Taylor et al51 looked at serum from 25 highly sensitized patients using the bead-based Luminex HLA SAB and C1q-SAB assays. They found that undiluted samples had poor correlation between the two assays (r2 = 0.42), and treatment with EDTA and dilution improved the correlation to 0.57 and 0.77, respectively.51 Serum treatment with EDTA, DTT, heat, dialysis, or MW columns can dilute out the sample and/or alter the sample and yield lower MFI results as well as less reproducible MFI results. In addition to the limitations described, the SAB assay has been shown to have intra- and interlaboratory variability, which is thought to be attributable to background fluorescence in the assay. Variation in MFI is higher at lower values of 1,000 to 3,000 MFI and has been reported to be as high as 62%.52 Other sources of variability can be attributed to differences in bead manufacturer, batch, and lot as different manufacturers have different sources of antigen and varied antigen density on beads.52-55 Notably, even with the same kit, variation in results can be seen with different testing personnel, reagents, equipment, and other conditions.54,55 Last but not least, not all HLA antigens are represented in phenotypic or SAB bead-based assays. Thus, a patient may have additional HLA antibodies that are not being detected. This is unlikely to contribute to a patient’s continued platelet refractoriness as most common HLA antigens are represented in the HLA antibody detection assays. Conclusions Platelet refractoriness can be attributed to nonimmune or immune causes. Notably, nonimmune causes comprise the largest proportion. Immune-mediated platelet refractoriness is indicated by a 1-hour CCI of less than 5 × 109/L on two sequential occasions.6 HLA antibody testing and/or HLA typing can then be initiated. This testing typically takes several days. In the interim, random donor platelets can be given to the patient or crossmatch compatible platelets can be ordered. Crossmatched platelets do not require any HLA information to be known; however, if the patient has a large amount of antibody or high cPRA, crossmatched compatible platelets may be hard to find. When the HLA results come back, it is important to understand the limitations of the bead-based Luminex SAB assays and C1q assay (if performed). Additionally, the SAB assay is very sensitive and may detect HLA antibodies of unknown significance. In some centers, a CDC assay is done to help determine which HLA antibodies fix complement and enhance platelet destruction; however, many HLA laboratories do not perform the CDC assay as it uses older technology. Yet another limitation to be aware of are the various MFI cutoffs used in determining which HLA antibodies a patient has. Following determination of the patient’s HLA antibodies and/or HLA typing, a decision can be made based upon the cPRA as to whether to order HLA-matched or antigen-restricted platelets. Crossmatch platelets can also still be ordered. For patients with high cPRA, there will be difficulty obtaining platelets that avoid the antibodies. Similarly, for patients with uncommon or rare HLA A and B antigens, there will also be difficulty obtaining grade A matched HLA platelets. Typically, a compromise on avoided antigen and grade of match will be needed and every patient should be considered individually. Case Summary The second set of HLA antibody results using the MW columns to remove miscellaneous substances contributing to an interfering prozone effect were deemed accurate and representative of the patient’s true HLA antibodies. The patient had prior pregnancies and blood product transfusion as sensitizing events. HLA-matched platelets were then ordered for the patient from the local blood supplier with an A or B match listed as acceptable. The patient’s HLA antibodies were not listed as antigens to avoid. This choice was made due to the patient’s high PRA and perceived increased difficulty in finding compatible platelets. The first product received was a B2X grade (Donor A2, 2; B7, 62) and the patient’s platelet count went from 10 × 109/L pretransfusion to 24 × 109/L posttransfusion. Approximately 2 weeks later, the patient received another HLA-matched platelet transfusion, graded as a B2X (donor A2, 24; B 7, 18), and her platelet count went from 13 × 109/L to 21 × 109/L. 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TI - HLA-Mediated Platelet RefractorinessAn ACLPS Critical Review
JF - American Journal of Clinical Pathology
DO - 10.1093/ajcp/aqy121
DA - 2019-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/hla-mediated-platelet-refractorinessan-aclps-critical-review-bHqjWgvJsL
SP - 353
VL - 151
IS - 4
DP - DeepDyve
ER -