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Absolute platelet refractoriness associated with HLA antibodies: A case report.


The term "platelet refractorinesses" used to describe a post-transfusion increment in a patient's platelet count that is less than expected. Inadequate responses to platelet transfusion are not uncommon particularly amongst multiply transfused patients. (1) While the incidence of refractoriness has decreased with the use of leukoreduced products; it is still a concern to clinicians and transfusion services as a less than adequate response leads to an increased risk of morbidity and mortality. Platelet transfusions are necessary to prevent hemorrhagic complications after myeloablative therapy. (2) When platelet transfusions first became available in 1959 there was a significant decrease in the number of leukemic patients dying of hemorrhage. (3) Improvements in medical therapy, including prophylactic platelet transfusions, have further reduced the incidence of fatal hemorrhage in patients with acute leukemia. (3) However, some patients fail to benefit from transfusions because they are unable to achieve an appropriate rise in their platelet count following transfusion.

In patients treated for hematological malignancies as in this case with AML, fever, infection, and medications are the most common causes of platelet refractoriness. (4) The association between sepsis and thrombocytopenia is well-established. (5) The mechanism behind this association is not completely understood. Disseminated intravascular coagulation plays a role in some cases, but other mechanisms are also involved. (6) It has been reported that up to 30 percent of intensive care unit patients that are thrombocytopenic have platelet-associated antibodies, a subset of which recognize GPIIb/IIIa or GPIb/IX, which suggests that the immune system may contribute to sepsis-induced thrombocytopenia. (7) Non-antibody-mediated mechanisms, such as haematophagocytosis, which can occur in the bone marrow of septic patients, may also be a contributing factor. (8,9) In addition to sepsis, medications are a relatively common cause of thrombocytopenia and many are known to produce this adverse effect. (10,11)

Thrombocytopenia produced by antibody-mediated destruction must also be considered a mechanism that may underlie refractoriness to platelet transfusions. Alloimmunization to human leukocyte antigen (HLA) and/or platelet-specific antigens (HPA) is included in this category. The HLA system is comprised of polymorphic cell-surface proteins important for antigen presentation. Immunization against HLA-antigens can be caused by contaminating leukocytes in platelet products. (12) HPA polymorphisms occur in the glycoproteins present on the surface of platelets (i.e. GPIIb/IIIa and GPIa/IIa). The prevalence of these polymorphisms varies amongst populations, and patients may be immunized to platelet surface antigens through transfusion. The incidence of refractoriness in patients seen by hematology/ oncology services varies between seven to thirty-four percent based upon the specific study population and definition of refractoriness. (13,14,15) In this report we describe a patient with newly diagnosed AML that demonstrated no response to multiple sequential platelet transfusions that were given during the course of induction chemotherapy to prevent onset of bleeding complications. In this case the patient had a number of potential underlying causes, both immune and non-immune, likely contributing to her response to transfusions, and here we discuss the challenges associated with the diagnostic approach to platelet refractoriness.


An elderly female presented to her primary care physician with chest pain, fatigue, and progressive shortness of breath. Laboratory evaluation revealed severe anemia (Hgb 6 g/dL, reference range 10.9-14.3 g/dL), and the patient was transfused and referred for further evaluation. A bone marrow biopsy was performed which revealed acute myelogenous leukemia. The patient subsequently underwent induction chemotherapy (Cytarabine and Idarubicin (7 + 3)). Thereafter she developed neutropenic fever and progressive thrombocytopenia. Empiric antibiotic coverage was started, and as her platelet count decreased below 10,000/[micro]L, she was transfused platelets. Despite three successive platelet transfusions, her platelet count continued to decline and reached a nadir of 3,000/[micro]L to 4,000/ [micro]L (Figure 1). All units issued were single donor, leukoreduced, irradiated and type specific, except the first unit issued on the first hospital day (Table 1). She displayed no signs of transfusion reaction (i.e. rigors, fever, and respiratory distress) during transfusion of any units. Due to an inadequate response to platelet transfusions, as shown by corrected count increments (CCI) at 24 hours of 760 platelets x [m.sup.2]/[micro]L on days 3 and 4, and a CCI of <1000 platelets x [m.sup.2]/[micro]L at 1 hour on day 5, a platelet refractory panel was requested to evaluate antibody mediated alloimmunity as a cause of platelet refractoriness. Antibody identification by antigen capture enzyme-linked immunosorbent assay (ELISA) revealed alloantibody to Class I human leukocyte antigen (HLA). No antibodies to the human platelet antigen (HPA) system detected by this screen. After a one week period the patient's platelet count began to recover without further need for platelet transfusion and the CCI on hospital day 6 was 5,700 platelets x [m.sup.2]/[micro]L at 24 hours. She successfully completed induction chemotherapy and was later readmitted to begin consolidation phase of chemotherapy.


Platelet refractoriness is manifested by a failure to achieve a rise in the circulating platelet count one hour after infusion of adequate numbers of platelets upon two or more sequential platelet transfusions. The corrected count increment (CCI) can be used to measure the response to platelet transfusion. (16) A platelet refractory state can defined by a post transfusion corrected count increment at 18-24 hours of <5,000 platelets x [m.sup.2]/ uL or <7,500 platelets x [m.sup.2]/uL at 10 minutes to 1 hour following two consecutive platelet transfusions.15, 17 In our case however, the patient's daily complete blood count (CBC) revealed an initial decrease in the platelet count followed by no increment upon subsequent platelet transfusions between hospital day 1 to 6 clearly indicative of refractoriness to platelet transfusion. It was on hospital day 7 that the patient eventually began to show response to platelet transfusion with a CCI of 5, 7000 platelets x [m.sup.2]/uL at 24 hours. The patient was then monitored with daily complete blood counts (CBC), which showed a steady rise in platelets with no need for further transfusion.

The poor recovery and survival of transfused platelets can be due to immune and non-immune causes. Studies in patients with AML or hematopoietic progenitor cell transplants have shown that fever, sepsis, splenomegaly, disseminated intravascular coagulation (DIC), bleeding, venoocclusive disease, graft versus host disease (GVHD), and medications can all be causes of refractoriness to platelet transfusion. (18,19) Altogether non-immune causes can account for approximately two-thirds of refractory episodes. (20,21)

Non-immune factors pertinent to our case include fever (Tmax 102F) and gram-positive sepsis that were first noted two days prior to the onset of inadequate responses to platelet transfusion. Although fever is likely not an independent cause of refractoriness, it is a manifestation of underlying infection which is clearly associated with shortened platelet survival and thrombocytopenia. (5,22) In our case, blood cultures revealed alpha-hemolytic streptococcal bacteremia to be the source of neutropenic fever. Empiric antibiotic coverage with vancomycin and cefepime was started soon after the patient first became febrile and she subsequently began to show steady clinical improvement within a few days.

In addition to the above, medications provide another etiology of refractoriness in our case. Drug-induced thrombocytopenia is generally produced by antibody-mediated destruction or decreased production. Immune-mediated destruction is most common and transfused platelets are subject to the same process resulting in a shortened lifespan. Vancomycin and cephalosporin beta-lactam antibiotics are amongst the more wellknown drugs to cause thrombocytopenia. (23,24,25) Both of these classes of antibiotics are thought to produce thrombocytopenia by an antibody mediated mechanism. In our case both of these agents were given as stated above prior to the onset of transfusion refractoriness. In addition as prophylaxis during myeloablative therapy the patient also received ciprofloxacin which is also associated with thrombocytopenia. (11) However, the clinical presentation of drug induced thrombocytopenia involves a median time for daily exposure of six days before the onset of thrombocytopenia, and a median recovery in five to seven days following discontinuation of the inciting drug. (26,27) In our case the patient began to respond to platelet transfusions on hospital day 7 during the course of antibiotic treatment, which argues against drug induced thrombocytopenia as a primary cause of transfusion refractoriness.

In the diagnostic approach to refractoriness to platelet transfusion a CCI at one hour of >7,500 platelets x [m.sup.2]/uL but a 24-hour CCI of <4,500 platelets x [m.sup.2]/uL is suggestive of non-immune causes such as sepsis and medications. In our case the CCI was <7,500 platelets x [m.sup.2]/uL at 10 minutes to 1 hour post transfusion on more than one occasion, which in part defines alloimmune refractoriness. Laboratory testing in this case revealed the patient to have antibody to Class I HLA antigen by Antigen Capture ELISA II but not to other platelet specific antigens. Platelet antigens causing alloimmunization can be separated into two groups, those that are shared with other blood cells (i.e. HLA system) and those that are specific to platelets, such as the human platelet antigen (HPA) system. (28) Immunization to HLA antigens is a major risk factor for refractoriness to platelet transfusions as most often refractoriness results from the production of antibodies to HLA class I antigens. In contrast, platelet-specific antibodies occur infrequently and are not usually associated with a significant reduction in the CCI. (15,29)

Knowledge of the presence of alloimmunization is essential as there are various strategies to consider when selecting platelets for these patients. These include provision of human leukocyte antigen (HLA)-matched platelets or HLA "compatible" (antigen-negative) platelets, platelets selected by cross-matched tests, and maneuvers to reduce alloimmunization. The incidence of refractoriness has been reduced by the use of leukocyte-reduced platelets. The Trial to Reduce Alloimmunization to Platelets (TRAP), a study conducted in patients with AML receiving standard induction chemotherapy, showed that 16 percent of patients receiving non-leukocyte-reduced products met the criteria for refractoriness as opposed to 7-10 percent of patients receiving some form of leukodepleted product, with no significant difference between single donors and pooled random donor platelets. (15) In addition, given the transient nature of antibody production, patients diagnosed with refractoriness need to be regularly reassessed for the presence/specificity of antibodies. It has been shown that anti-HLA antibodies in patients receiving induction chemotherapy for acute leukemia frequently disappeared during treatment. (29,30) Thus patients need to be regularly reassessed in order to avoid unnecessary use of more expensive and difficult to obtain HLA-matched or antigen-negative units.

In conclusion, screening for HLA and HPA alloantibodies is an important adjunctive laboratory test in the diagnosis of platelet refractoriness. Of the various causes for platelet refractoriness, alloimmunization is the one for which there is greatest potential for prevention and management. Although the patient's lack of response to platelet transfusions in this case is likely due to a combination of alloimmunization and her complex underlying disease state and its sequelae, knowledge of the presence of an antibody mediated component provides a point of intervention should the patient ever again exhibit platelet refractoriness.


(1.) Pappalardo PA, Secord AR, Quitevis P, et al. Platelet transfusion refractoriness associated with HPA-1a (PlA1) alloantibody without coexistent HLA antibodies successfully treated with antigen-negative platelet transfusions. Transfusion. 2001; 41:984-987.

(2.) Bernstein, S.H., Nademanee, A.P., Vose, J.M., et al. A multicenter study of platelet recovery and utilization in patients after myeloablative therapy and hematopoietic stem cell transplantation. Blood. 1998; 91:3509-3517.

(3.) Bayer, W.L., Bodensteiner, D.C., Tilzer, L.L. & Adams, M.E. Use of platelets and other transfusion products in patients with malignancy. Seminars in Thrombosis and Hemostasis. 1992; 18:380-391.

(4.) Doughty, H.A., Murphy, M.F., Metcalfe, P., et al. Relative importance of immune and non-immune causes of platelet refractoriness. Vox Sanguinis. 1994; 66:200-205.

(5.) Davis, R.B., Meeker, W.R. & Mc, Q.D. Immediate effects of intravenous endotoxin on serotonin concentrations and blood platelets. Circ Res.1960; 8:234239.

(6.) Neame, P.B., Kelton, J.G., Walker, I.R., et al. Thrombocytopenia in septicemia: the role of disseminated intravascular coagulation. Blood. 1980; 56:88-92.

(7.) Stephan, F., Cheffi, M.A., Kaplan, C., et al. Autoantibodies against platelet glycoproteins in critically ill patients with thrombocytopenia. Am J Med. 2000; 108: 554-560.

(8.) Francois, B., Trimoreau, F., Vignon, P., et al. Thrombocytopenia in the sepsis syndrome: role of hemophagocytosis and macrophage colony-stimulating factor. Am J Med.1997; 103114-120.

(9.) George MR, Herman JH, Holdbrook T, et al. Platelet refractoriness in acquired hemophagocytic syndrome. Transfusion. 2011; 51:2319-2326.

(10.) Visentin, G.P., Wolfmeyer, K., Newman, PJ. & Aster, R.H. Detection of drug-dependent, platelet-reactive antibodies by antigen-capture ELISA and flow cytometry. Transfusion. 1990; 30:694-700.

(11.) Aster, R.H. & Bougie, D.W. Drug-induced immune thrombocytopenia. N Engl J Med. 2007; 357:580-587.

(12.) Claas, F.H., Smeenk, R.J., Schmidt, R., et al. Alloimmunization against the MHC antigens after platelet transfusions is due to contaminating leukocytes in the platelet suspension. Exp Hematol. 1981; 9:84-89.

(13.) Klingemann, H.G., Self, S., Banaji, M., et al. Refractoriness to random donor platelet transfusions in patients with aplastic anaemia: a multivariate analysis of data from 264 cases. Br J of Haematol. 1987; 66:115-121.

(14.) Legler, TJ., Fischer, I., Dittmann, al. Frequency and causes of refractoriness in multiply transfused patients. Ann Hemato. 1997; 74:185-189.

(15.) Slichter, SJ. 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.

(16.) Daly, P.A., Schiffer, C.A., Aisner, J. & Wiernik, P.H. Platelet transfusion therapy. One-hour posttransfusion increments are valuable in predicting the need for HLA-matched preparations. JAMA. 1980; 243:435-438.

(17.) O'Connell BA, Lee EJ, Rothko K, et al. Selection of histocompatible apheresis donors by crossmatching random donor platelet concentrates. Blood. 1992; 79:527-531.

(18.) Ishida, A., Handa, M., Wakui, M., et al. Clinical factors influencing post-transfusion platelet increment in patients undergoing hematopoietic progenitor cell transplantation-a prospective analysis. Transfusion. 1998; 38:839847.

(19.) Slichter, SJ., Davis, K., Enright, H., et al. Factors affecting post-transfusion platelet increments, platelet refractoriness, and platelet transfusion intervals in thrombocytopenic patients. Blood. 2005; 105:4106-4114.

(20.) Hod E, Schwartz J. Platelet transfusion refractoriness. Br J Haematol. 2008; 142:348-360.

(21.) Bishop, J.F., McGrath, K., Wolf, M.M., et al. Clinical factors influencing the efficacy of pooled platelet transfusions. Blood. 1988; 71:383-387.

(22.) Bock M, Muggenthaler KH, Schmidt U, Heim MU. Influence of antibiotics on post-transfusion platelet increment. Transfusion. 1996; 36:952-954.

(23.) Von Drygalski A, Curtis B, Bougie D, et al. Vancomycin-Induced Immune thrombocytopenia. N Engl J Med 2007; 356:904-910.

(24.) Reese JA, Li X, Hauben M, et al. Identifying drugs that cause acute thrombocytopenia: an analysis using 3 distinct methods. Blood. 2010; 116:21272133.

(25.) George JN, Raskob GE, Shah SR, et al. Drug-induced thrombocytopenia: a systematic review of published case reports. Ann Intern Med. 1998; 129:886890.

(26.) Rousan TA, Aldoss IT, Cowley BD Jr, et al. Recurrent acute thrombocytopenia in the hospitalized patient: sepsis, DIC, HIT, or antibiotic-induced thrombocytopenia. Am J Haematol. 2010; 85:71-74.

(27.) Pedersen-Bjergaard U, Andersen M, Hansen PB. Drug-induced thrombocytopenia: clinical data on 309 cases and the effect of corticosteroid therapy. Eur J Clin Pharmacol. 1997; 52:183-189.

(28.) Tinmouth, A.T., Semple, E., Shehata, N. & Branch, D.R. Platelet immunopathology and therapy: a Canadian Blood Services Research and Development Symposium. TransfusMedRev. 2006; 20:294-314.

(29.) McGrath, K., Wolf, M., Bishop, J., et al. Transient platelet and HLA antibody formation in multi transfused patients with malignancy. Brit J Haematol. 1988; 68:345-350.

(30.) Murphy, M.F., Metcalfe, P., Ord, J., et al. Disappearance of HLA and platelet-specific antibodies in acute leukaemia patients alloimmunized by multiple transfusions. Brit J Haematol. 1987; 67:255-260.

Drs. Vora and Al-Delfi are residents in the Department of Pathology, LSUHSC, Shreveport, LA. Dr. Cruz is an instructor, Dr. Ong is an Associate Professor of Clinical Pathology, Dr. Veillon is a Professor of Clinical Pathology and Dr. Cotelingam is a Professor of Pathology, all associated with the Department of Pathology, LSUHSC-Shreveport, LA.

Table 1. Characteristics of single donor platelet units issued
(LR-leukocyte-reduced; IR-irradiated).

Hospital Day   # of Units   Category   ABO type

1              1            LR/IR      [O.sup.+]
2              1            LR/IR      [A.sup.-]
3              2            LR/IR      [A.sup.+]
4              1            LR/IR      [A.sup.+]
5              2            LR/IR      [A.sup.+]
6              2            LR/IR      [A.sup.+]
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Title Annotation:human leukocyte antigen
Author:Vora, Moiz; Ong, Menchu; Al-Delfi, Firas; Dela Cruz, Nestor; Veillon, Diana; Cotelingam, James
Publication:The Journal of the Louisiana State Medical Society
Article Type:Clinical report
Date:Jan 1, 2016
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