血小板輸注無效之供血配對策略探討

Abstract

罹患血小板減少症的病人，常需要藉著血小板的輸注，以避免發生嚴重的出血。然而，在長期輸注血小板後，病人會發生血小板輸注無效(Platelet Transfusion Refractoriness; PTR)，使得體內血小板的數量，在輸注後無法達到預期。由人類白血球抗原(Human Leukocyte Antigen; HLA)所引起的同種異體免疫反應(Allo- immunization)，是造成血小板輸注無效的主要原因之一，病人一旦確認為HLA- alloimmunized PTR，臨床上會以HLA相合之血小板，來解決其血小板輸注無效的問題。傳統上，係以血清學交叉反應群組(Cross-Reactivity Groups; CREGs) 進行受血者與捐血者間HLA抗原相合程度的配對，提供HLA相合之血小板。然而，有些屬於相同CREGs的HLA抗原，因具有一個以上不同的共有epitope，導致彼此間產生對抗不同epitope的特異性抗體，使得以CREGs方式進行HLA相合血小板的配對，在臨床血小板輸注效果上，並不如預期的理想。 在本研究中，我們評估新的HLA配對方式，以HLA epitopes分析為基礎，進行HLA配對(稱作Epitopes-Based Matching ; EBM配對方法)，以提供合適的血小板給HLA-alloimmunized PTR病人使用。EBM配對方法係利用Bead-based single -antigen HLA 抗體的檢驗結果，以及HLAMatchmaker software進行HLA epitopes位點分析；再將HLA epitopes位點分析結果，應用在HLA相合血小板的供應上面。我們以前瞻型研究設計方法，從台大醫院血液科病人隨機抽樣9位HLA-alloimmunized PTR病人，進行長期追蹤並觀察其血小板輸注結果，共收集142筆血小板輸注後的臨床數據，進行不同配對方式的臨床效果分析，並長時間觀察病人體內HLA抗體產生的變化。從研究結果顯示，以EBM配對方法供應HLA-alloimmunized PTR病人的血小板，其血小板輸注效益與A/BU 配對(受血者與捐血者間HLA分型完全相同或是不含相異之HLA抗原)相當，且在追蹤病人使用EBM配對血小板後，並沒有病人產生對抗EBM相合但非自身HLA的抗體。總而言之，可利用EBM配對方法，選擇適當之HLA 相合血小板給HLA-allo- immunized PTR病人使用。 此外，血小板特異性抗原(Human Platelet-specific Antigen; HPA)的多態性，會引發異體免疫反應，造成一些血小板疾病的發生，例如：新生兒血小板減少症 (NAIT)、輸血後紫斑症(PTP)，以及血小板輸注無效(PTR)等。HPA基因分型的檢測及其頻率調查，對族群或是提供HPA相合之血小板給PTR病人使用，是相當重要的。我們利用sequence specific primers (PCR-SSP)方法，分析998位捐血人HPA基因分型，包括：HPA-1, -2, -3, -4, -5, -6 及 -15等7型的建檔資料，並計算HPA基因型頻率。根據HPA基因型頻率調查結果顯示，台灣族群在血小板輸注上，以HPA-3 及-15 分型不相合風險比率較高，需要進行大量捐血人HPA分型建檔作業。然而，傳統HPA基因分型技術有許多缺點，不適合捐血中心進行大量檢體的檢測，因此，本研究利用 Pyrosequencing 原理，建立一套高通量的HPA-1, -3, -15的基因分型檢測技術，並與Sanger定序方法進行結果比較，以驗證新方法的正確性。為節省試劑成本及操作時間，我們進一步發展單管同步檢測HPA-1及HPA-3的HPA pyrosequencing基因分型技術。經與Sanger定序方法比較，一致性達100%。這些結果顯示，以Pyrosequencing進行HPA基因分型檢測，是一項簡單、高正確性、高靈敏度，且高通量檢測技術，適合國內捐血中心進行大量捐血者建檔作業，以提供HPA相合血小板給PTR病人使用。

Patients with thrombocytopenia require frequent platelet (PLT) transfusions to prevent bleeding complications. Unfortunately, long-term PLT transfusions are complicated by refractoriness, which is defined as an insufficient PLT count increment after repeated PLT transfusions. HLA alloimmunization is the major factor for platelet transfusion refractoriness (PTR) and HLA-matched PLTs are widely used to improve the PLT transfusion outcome in HLA-alloimmunized PTR patients. Traditionally, HLA-matched PLTs are selected via serological cross-reactive groups (CREGs) method. CREGs method is based on the comparison of the HLA public epitopes between patient and donor’s HLA antigens. However, many HLA antigens contain more than one public epitope within the same CREGs that consequently develop different epitope-specific antibodies. As a result, the CREGs method was not effective enough to predict transfusion outcomes for PTR patients. In our studies, we conducted a prospective trial with a novel HLA matching approach (called epitope-based matching; EBM method) to provide suitable platelet concentrates for HLA-alloimmunized PTR patients. The EBM method used bead-based single antigen HLA antibody detection and HLAMatchmaker software to define the epitopes recognized by HLA-specific antibodies. We evaluated 142 platelet transfusion outcomes with different matching strategies (A/BU, CREGS, and EBM method) in 9 HLA-alloimmunized PTR patients and monitored the alteration of HLA antibody patterns in patients in a follow-up study. The results indicated that the selection of platelets for HLA-alloimmunized PTR patients by EBM method is as effective as that by A/BU matching, and follow-up antibody analysis confirmed that no specific antibodies were detected against EBM-matched antigens. In conclusion, we suggested that the EBM strategy could be used for proper selection of PLT concentrates for PTR patients. In addition, the polymorphism of human platelet antigens (HPA) leads to alloimmunizations and immune-mediated platelet disorders including neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), and platelet transfusion refractoriness (PTR). HPA typing and knowledge of antigen frequency in a population is important in particular for the provision of HPA-matched blood components for patients with PTR. We have performed allele genotyping for HPA-1 through -6 and -15 among 998 platelet donors from 6 blood centers in Taiwan using sequence-specific-primer polymerase chain reaction. Based on allele frequency study, the HPA-3 and -15 might play an important role in the HPA imcompatibility platelet transfusion and need to set up a large scale HPA-typed donor file in Taiwan. However, there are several disadvantages in the conventional methods of HPA genotyping for large-scale-sample laboratory. Therfore, we developed a new high throughput method for detecting genetic polymorphism of HPA-1, -3 and -15 systems using the pyrosequencing technology and validated this method with Sanger sequencing. Beside, we also developed a simultaneously detecting HPA-1 and HPA-3 genotypes in a single-tube (one test) for cost and time saving. Repeat test and reliability test indicated that the agreement between the pyrosequencing and the Sanger sequencing methods was 100%. Our results indicated that HPA pyrosequencing genotyping is a simple, accuracy, sensitive and high-throughput method for blood donor centers.