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Porselvi Chockalingam, MD Ronald A. Sacher, MD

Management of Patients Refractory to Platelet Transfusion

Prevention strategies include efforts to limit

Abstract

recipients exposure to human leukocyte antigen specicities by using single-donor platelets, ltration to reduce the number of human leukocyte antigenbearing leukocytes, and pretransfusion ultraviolet B irradiation to decrease their immunogenicity. For appropriate management of patients refractory to platelets, close cooperation and good communication are necessary between clinicians and blood centers.

This article discusses the causes and management of platelet refractoriness. Improvements in the quality of platelets and leukoreduction have reduced the morbidity and mortality related to alloimmunization and refractoriness of patients to platelet transfusion. Alloimmunization can be distinguished from other causes of poor posttransfusion platelet increments by the measurement of platelet alloantibodies. Options for managing platelet refractoriness caused by alloimmunization include platelet transfusion from human leukocyte antigenmatched or donor-recipient cross-matched platelets.

he rst successful attempt to raise the platelet count in thrombocytopenic patients by transfusing whole blood was described by Duke in 1910.1 In the current era, intensive therapies producing severe and sustained thrombocytopenia are used routinely in patients with hematologic malignancies and many patients with solid tumors.2 Advances in platelet collection, storage, and transfusion have decreased the morbidity of such therapies, and death from

Porselvi Chockalingam is currently doing her fellowship in transfusion medicine at Hoxworth Blood Center, University of Cincinnati, OH. She is a medical graduate from Kasturba Medical College, Karnataka, India. Ronald A. Sacher is Director of Hoxworth Blood Center, and Professor of Internal Medicine and Pathology, University of Cincinnati Medical Center, OH. Address correspondence to: Ronald A. Sacher at the University of Cincinnati Academic Health Center, 3130 Highland Avenue, P.O. Box 670055, Cincinnati, OH 45267-0055 (e-mail: Ronald.Sacher@uc.edu).

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hemorrhage is now an unusual occurrence, despite the large number of patients being treated aggressively. These patients undergo either prophylactic transfusion to prevent bleeding or therapeutic transfusion in the case of bleeding.3 Most platelets are transfused prophylactically, which is generally accepted as reducing the risk of bleeding in thrombocytopenic patients.4 Recent guidelines for the use of prophylactic transfusion adopted the threshold of 10 109/L, which is as safe as higher levels for patients without additional risk factors such as sepsis, concurrent use of antibiotics, or other abnormalities of hemostasis.5-7 Strict compliance with the transfusion trigger may save costs and unnecessary exposure to the risks of transfusionrelated complications, such as allergic or febrile transfusion reactions, transmission of infections, and alloimmunization. Platelets for transfusion can be prepared either by separation of units of platelet concentrates from whole blood, which are pooled before administration, or by apheresis from single donors. A single-unit platelet concentrate contains more than 5.5 1010 platelets that are suspended in 40 to 70 cc of plasma to maintain the pH higher than 6.2 throughout storage. Apheresis products derived from one donor contain 3 1011 platelets in a single unit (average volume 300 mL) collected by cytapheresis procedure. These platelets are then stored at 20 to 24C for 5 days (maximum storage time) using continuous, gentle, horizontal agitation in storage bags specically designed to permit O2 and CO2 exchange to optimize platelet quality. Comparative studies have shown that the post-transfusion increments, hemostatic benet, and side effects are similar with either product.5

A sample of the pre- and postcalculation using the CCI is inserted as an example. For example, if 4 1011 platelets were administered to an individual with a body surface area of 2 m2 and an increment of 40,000/L platelet count is obtained, this individual has a CCI of 20,000. Although different values of the CCI have been used to dene an adequate transfusion response, the recently completed Trial to Reduce Alloimmunization to Platelets (TRAP) study10 used a CCI of 5,000 to dene a satisfactory response. The normal CCI 24-hour post-transfusion is approximately 64% of the CCI at 1 hour.

CAUSES OF REFRACTORINESS
Major factors causing platelet refractoriness include clinical condition of the patient, quality of the platelet concentrate, handling, transportation,11 and alloimmunization. During platelet concentrate preparation and storage, platelets are exposed to several mechanical and chemical factors, which results in several alterations. An increase in lactic dehydrogenase and reduction in pH are expressions of biochemical changes, whereas platelet activation may be detected by increased expression of Pselectin and glycocalcin as the cleavage product of the surface ligand, glycoprotein 1b.12 These changes may affect platelet survival and can inuence refractoriness to transfusions. Nonimmune causes for refractoriness may occur with clinical factors such as fever, sepsis, bleeding, disseminated intravascular coagulation (DIC), drugs, viral infections, splenomegaly, and graft-versus-host disease in up to 80% of patients. Platelet recovery/survival is impaired in only 50% of patients with these conditions, however. With nonimmune causes of platelet destruction, the platelet count increases initially (normal platelet recovery) and then drops considerably in 18 to 24 hours (decreased platelet survival).13 Studies by van Marwijk Kooy et al14 and Sniecinski et al15 estimated that 25% to 50% of all refractoriness are caused by immune-mediated processes. Alloimmunization against human leukocyte antigen (HLA) class I antigens is a major cause of permanent refractoriness to random platelet transfusions, although platelet-specic antibodies and ABO incompatibility also may contribute. The predisposing factors for alloimmunization include a history of multiple transfusions (>20), transfusion of nonleukoreduced blood products, and history of pregnancies in female patients.8 Platelet alloimmunization may develop within a few days to several months of the initial transfusion. Patients who have been immunized by transfusion, pregnancy, or organ transplantation may develop platelet antibodies as early as 4 days after transfusion. During pregnancy, fetal cells can enter the maternal circulation, leading to primary alloimmunization. The HLA system includes 2 classes of molecules; class I molecules, the HLA-A, -B and -C antigens, are pre-

PLATELET REFRACTORINESS
Platelet transfusion refractoriness is dened as an inability to achieve an expected incremental response to a platelet transfusion. By denition, a diagnosis of refractoriness should be made only after at least 2 ABO-compatible transfusions, stored less than 72 hours, resulting in poor platelet increments.8 The platelet increment is determined by subtracting the pretransfusion platelet count from the count determined 1 hour after transfusion. Studies have shown, however, that a 10-minute post-transfusion count correlates closely with the 1-hour post-transfusion value. This 10-minute platelet count is convenient to obtain and is very useful before surgical procedures.9 The percentage of platelet recovery or the corrected count increment (CCI) is determined using the following formula: Post-transfusion Platelet Count L Pretransfusion Platelet Count L Body surface area CCI = No. of platelets 1011

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sent on leukocytes and platelets. On platelets, however, the amount of HLA-A and -B antigens is at least 10-fold higher than that of HLA-C antigen, which makes it less common to match the HLA-C antigen for donor-recipient compatibility in platelet transfusions.16 The class II HLA-D antigens, including DR, DP, and DQ, are present only on leukocytes. The class II antigens present on the lymphocytes and antigen-presenting cells of the donor are needed to stimulate the recipient B cells to produce a primary HLA class I alloimmunization, resulting in platelet refractoriness. This mechanism has been demonstrated by recent studies, indicating that the antigens presented by leukocytes (HLA class II antigens) are the primary immunogens rather than platelets (HLA class I antigens), and platelets should be considered innocent bystanders.

DIAGNOSIS OF REFRACTORINESS
Once the criterion for refractoriness is fullled, it is critical to document that the patient is, in fact, alloimmunized, because such patients are managed differently from patients with other causes of refractoriness to platelet transfusion. The use of techniques to identify platelet alloantibodies in patients suspected of becoming refractory to platelet therapy has increased signicantly from 73.6% to 94.3%. The lymphocytotoxicity test modied by the addition of human antiglobulin sera is useful for identifying complement-dependent platelet-associated antibodies that react with most public and private epitopes representing a wide variety of HLA specicities. Antibodies to private epitopes include HLA-A2 and B12, whereas antibodies to public epitopes include HLA-Bw4 and Bw6. This test detects antibodies that are capable of killing lymphocytes in the presence of complement.17 HLA antibodies in sera bind to corresponding antigens, and the resulting patterns of reactivity establish the panel reactive antibody (PRA) levels. A PRA value of more than 20% indicates signicant alloimmunization to HLA. Several other serologic assays have been used, including those listed in Table 1. A solid-phase HLA-specic enzyme-linked immunosorbent assay (ELISA) that detects

the presence of IgG anti-HLA antibodies has been used in many laboratories to document alloimmunization as the cause of refractoriness. This test distinguishes HLA from non-HLA antibodies and correlates well with standard lymphocytotoxicity testing. Although this assay cannot be used to identify a specic HLA, the strength of reactivity does correlate with the extent of sensitization. A modication of ELISA is the monoclonal antibody-specic immobilization of platelet antigens assay (MAIPA). The MAIPA assay is more sensitive than the standard lymphocytotoxicity test in detecting platelet-reactive HLA antibodies. This sensitive technique detects antibodies directed to different platelet glycoproteins separately, which allows the identication of mixtures of platelet-specic antibodies in the presence of HLA antibodies. Mouse monoclonal antibodies are used to immobilize the platelet glycoprotein required to an ELISA plate, and an ELISA system then identies the presence of platelet antibody bound to the selected platelet glycoprotein. An effective alternative to both methods is ow cytometric detection of platelet-reactive antibodies. The ow cytometric platelet immunouorescent cross-matching test has a negative predictive value when nonimmunologic factors are absent. Platelet cross-matching by the solid-phase red cell adherence method detects antibodies directed against ABO antigens and platelet-specic antigens, in addition to antibodies directed against HLA.

ANTIBODIES TO HUMAN
PLATELET ANTIGENS
Despite HLA matching of platelets for patients who are refractory to platelet transfusions, unexplained transfusion failures can still occur in approximately 12% to 39% of patients.18,19 These failures can sometimes be partly explained by antibodies to human platelet antigens. The platelet antigens to which antibodies are formed include glycoprotein Ia/IIa and IIb/IIIa, which are measured by MAIPA assay. In this assay the patients serum is incubated with a panel of 3 or 4 HPA-typed platelets. The results are given as number of platelets from the panel showing positive reactions using a particular monoclonal antibody to immobilize the platelet glycoprotein. Antibody specicity is reported if appropriate. The most common antiplatelet allospecicities are anti-HPA Ia and anti-HPA 5b.20

TABLE 1

Test Used to Detect Platelet Alloantibodies

Standard lymphocytotoxicity test Monoclonal antibody-specic immobilization of platelet antigens assay Platelet immunouorescent tests Flow cytometry Adhesion Enzyme-linked immunosorbent assay Solid-phase red cell agglutination test Platelet agglutination test

MANAGEMENT OF
PLATELET REFRACTORINESS
Strategies to prevent platelet transfusion refractoriness in the prevention of bleeding in refractory patients should be the main target of platelet transfusion therapy.18 If no incremental response to platelet therapy occurs, nonstored

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ABO-identical platelets should be transfused to exclude a role of ABO antibodies or impaired quality of the platelets transfused.21 In the presence of factors associated with increased platelet consumption, such as fever, sepsis, or DIC, frequent transfusion of platelets rather than increased dosages per transfusion is recommended because of the impaired platelet survival. In case of splenomegaly, when recovery of transfused platelets is very low but platelet survival is normal, one can consider increasing the platelet transfusion dose to increase post-transfusion platelet counts. If autoantibodies are present, treatment with steroids, immunosuppressive agents, or intravenous immunoglobulin sometimes improves post-transfusion platelet recovery.1,22,23 Whenever a patient develops consistently poor responses to platelet transfusions without a clear cause, the possibility of drug-induced antibodies should be considered. Correlating the onset of thrombocytopenia and administration of a certain drug may indicate possible involvement of that drug. Even if the presence of druginduced antibodies by means of laboratory testing cannot be conrmed, discontinuation of the drug and replacement by alternative drugs should be considered. Once it is determined that a patient is alloimmunized to HLA, compatible platelets are required for transfusion. When only HPA antibodies are present, the best chances to nd compatible donors are within the family. For HLAalloimmunized patients, HLA-matched platelet transfusions are required. Selection of HLA-compatible donors can be based on platelet cross-match techniques or on HLA type selection. The predictability of transfusion efcacy with a donor selected by cross-matching varies between 60% and 90%, whereas donor selection by HLA matching is superior in cases of multispecic HLA antibodies. The guidelines that are used in selecting HLAmatched patients are dened in Table 2.24
TABLE 2

PLATELET CROSS-MATCHING
Numerous investigators have evaluated the usefulness of cross-matching recipients serum with that of potential platelet donors. Platelet cross-matching can be done on available apheresis platelets, making a compatible transfusion available in a few hours rather than the several days it takes to schedule, draw, and test an HLA-typed donor (or longer if the patient was not previously HLA typed). The value of using of a platelet cross-match test is avoiding the administration of cross-matchpositive donor platelets, which are almost uniformly unsuccessful in producing a platelet increment in alloimmunized patient. An additional reason for using platelet cross-match is selecting not only for HLA but also for platelet-specic antigencompatible donors.17,19

USE OF FREQUENT SMALL

DOSES OF PLATELETS
Platelets are involved in an endothelial supportive function, and anecdotal clinical experience suggests that for some patients, platelet transfusionseven if they do not increase the platelet countmay help to control bleeding. Although an increment may not occur, transfused platelets may be able to support the endothelium to maintain vascular integrity. In such patients, frequent small doses of platelets (ie, 2 to 4 units of platelet concentrates every 6-8 hours) rather than large bolus transfusions may be more efcacious.

USE OF E-AMINOCAPROIC ACID

(AMICAR) OR TRANEXAMIC ACID
Some data suggest that the administration of brinolytic inhibitors, such as aminocaproic acid (Amicar) or tranexamic acid, may be associated with stabilization of the platelet plug and improved hemostasis in platelet refractory patients.

Traditional Selection Guidelines

Matching of donor HLA-A and -B antigens Providing platelets that lack the antigens against which the patient has antibodies If HLA-identical platelets are unavailable, platelets from donors whose mismatched HLA types are serologically cross-reactive with the recipients may be substituted Matching for antigens of the HLA-C locus is not necessary Mismatching for some HLA-B locus antigens (eg, HLAB44, HLA-B45) that are weakly expressed on platelets is acceptable for some donor-recipient pairs If cross-reactive platelets are ineffective for some patients, attention to linked HLA specicities may be important Although ABO is expressed on platelets, ABO matching is usually not critical, but should be considered if HLAmatched platelets are not effective
HLA, human leukocyte antigen.

USE OF RECOMBINANT
HUMAN THROMBOPOIETIN
The use of recombinant human thrombopoietin increases platelet levels and delays the time interval between platelet transfusions for patients undergoing intensive chemotherapy.25-27

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PREVENTION OF PLATELET
REFRACTORINESS
The use of single-donor platelets limits a recipients exposure to HLA specicities, as compared with pooled platelet concentrates derived from multiple random donors. The use of single-donor apheresis platelets may delay or decrease the development of alloantibodies to HLA. Hence, restricting the use of platelets obtained by apheresis to those requiring HLA-matched platelet support results in the most efcient and efcacious use of single-donor platelets by patients who require them.28 There is considerable evidence that HLA class II antigen-bearing leukocytes, which contaminate the platelet concentrate, are necessary for sensitization to class I HLA present on platelets that themselves do not express HLA class II specicities. The use of leukocyte-reduced platelet concentrates decreases the incidence of platelet alloimmunization attributable to antibodies against class I HLA.29 Ultraviolet-B (UV-B) irradiation of platelet concentrates inhibits immunogenicity at dosages that do not severely affect platelet function. The TRAP study10 stated that ltration and UV-B irradiation of platelet concentrates are equally effective in preventing HLA alloimmunization. Clearly, in instances in which there are secondary or nonimmune causes of platelet refractoriness, management of these conditions may be associated with an improvement in platelet recovery and survival. Attention should be paid to potential medications that may affect this, such as quinine derivatives, cephalosporin antibiotics, and other medications that may have an impact on platelet production or survival. Further attention should be paid to a patients history of having received blood transfusions and past pregnancies; however, these patients are more likely to be alloimmunized.

immunization has occurred, particularly in individuals who received previous platelet transfusions and persons who have had multiple pregnancies, identication of the antibody responsible or cross-matching may help to improve responses. Some of these strategies have been reviewed in this article. Attention also must be paid to the patients history and concomitant drug therapies.
R E F E R E N C E S 1. Novotny VM. Prevention and management of platelet transfusion refractoriness. Vox Sang. 1999;76:1-13. 2. Pisciotto PT, Benson K, Hume H, et al. Prophylactic versus therapeutic platelet transfusion practices in hematology and/or oncology patients. Transfusion. 1995;35:498-502. 3. Eikenboom JC, van Wordragen R, Brand A. Compliance with prophylactic platelet transfusion trigger in haematological patients. Transfus Med. 2005;15:45-48. 4. Slichter SJ. Relationship between platelet count and bleeding risk in thrombocytopenic patients. Transfus Med Rev. 2004;18:153-167. 5. Schiffer CA, Anderson KC, Bennett CL, et al. Platelet transfusion for patients with cancer: Clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol. 2001;19:1519-1538. 6. Strauss RG. Pretransfusion trigger platelet counts and dose for prophylactic platelet transfusions. Curr Opin Hematol. 2005;12: 499-502. 7. Heal JM, Blumberg N. Optimizing platelet transfusion therapy. Blood Rev. 2004;18:149-165. 8. Kekomaki R. Use of HLA- and HPA-matched platelets in alloimmunized patients. Vox Sang. 1998;74(Suppl 2):359-363. 9. OConnell B, Lee EJ, Schiffer CA. The value of 10-minute posttransfusion platelet counts. Transfusion. 1988;28:66-67. 10. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions: The trial to reduce alloimmunization to platelets study group. N Engl J Med. 1997;337:1861-1869. 11. Friedberg RC, Donnelly SF, Boyd JC, Gray LS, Mintz PD. Clinical and blood bank factors in the management of platelet refractoriness and alloimmunization. Blood. 1993;81:3428-3434. 12. Fabris F, Soini B, Sartori R, Randi ML, Luzzatto G, Girolami A. Clinical and laboratory factors that affect the posttransfusion platelet increment. Transfus Sci. 2000;23:63-68. 13. Rebulla P. Refractoriness to platelet transfusion. Curr Opin Hematol. 2002;9:516-520. 14. van Marwijk Kooy M, van Prooijen HC, Moes M, Bosma-Stants I, Akkerman JW. Use of leukocyte-depleted platelet concentrates for the prevention of refractoriness and primary HLA alloimmunization: a prospective, randomized trial. Blood. 1991;77:201-205. 15. Sniecinski I, Jean JS, Nowicki B. Preparation of leukocyte-poor platelets by ltration. J Clin Apher. 1989;5:7-11. 16. Santoso S, Kiefel V. Human platelet alloantigens. Wien Klin Wochenschr. 2001;113:806-813. 17. Sandler SG. Management of patients with hematologic malignancies and aplastic anemia who are refractory to platelet transfusions. Haematologia (Budap). 1998;29:1-11. 18. Engelfriet CP, Reesink HW, Aster RH, et al. Management of alloimmunized, refractory patients in need of platelet transfusions. Vox Sang. 1997;73:191-198. 19. Sanz C, Freire C, Alcorta I, Ordinas A, Pereira A. Platelet-specic antibodies in HLA-immunized patients receiving chronic platelet support. Transfusion. 2001;41:762-765.

SUMMARY
Platelet transfusions are an integral component of myelosuppressive chemotherapy regimens and are often given repetitively. In general, more than 30% of individuals who receive multiple platelet transfusions become alloimmunized. These patients may not respond to platelet transfusions with the expected increments and may become refractory to platelet transfusions. Alloimmunization is one cause of platelet refractoriness, as has been reviewed. There are also nonimmune causes of a failure to achieve an expected response to platelet transfusions. Clearly, in instances in which secondary or nonimmune mechanisms of platelet refractoriness are the cause, management of these conditions may be associated with improvement in platelet recovery and survival. In instances in which allo-

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20. Dan ME, Schiffer CA. Strategies for managing refractoriness to platelet transfusions. Curr Hematol Rep. 2003;2:158-164. 21. Slichter SJ. Algorithm for managing the platelet refractory patient. J Clin Apher. 1997;12:4-9. 22. Delaor-Weiss E, Mintz PD. The evaluation and management of platelet refractoriness and alloimmunization. Transfus Med Rev. 2000;14:180-196. 23. Choudhry VP. Platelet therapy. Indian J Pediatr. 2002;69:779-783. 24. Sacher RA, Kickler TS, Schiffer CA, et al. Management of patients refractory to platelet transfusion. Arch Pathol Lab Med. 2003;127: 409-414. 25. Bai CM, Zou XY, Zhao YQ, Han SM, Shan YD, Thrombopoietin Clinical Trial Cooperation Group. The clinical study of recombinant human thrombopoietin in the treatment of chemotherapy-induced

26.

27. 28. 29.

severe thrombocytopenia. Zhonghua Yi Xue Za Zhi. 2004;84: 397-400. Vadhan-Raj S, Patel S, Bueso-Ramos C, et al. Importance of predosing of recombinant human thrombopoietin to reduce chemotherapy-induced early thrombocytopenia. J Clin Oncol. 2003;21: 3158-3167. Zhao Y, Jiang J, Jiao L. Clinical tolerance test of recombinant human thrombopoietin. Zhonghua Yi Xue Za Zhi. 2001;81:1508-1511. Kickler TS. The challenge of platelet alloimmunization: Management and prevention. Transfus Med Rev. 1990;4:8-18. Vamvakas EC. Meta-analysis of randomized controlled trials of the efcacy of white cell reduction in preventing HLA-alloimmunization and refractoriness to random-donor platelet transfusions. Transfus Med Rev. 1998;12:258-270.

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