Apart from RBCs, there is increasing evidence about the presence of RBC microparticles in platelet concentrates. Microparticles are cell-derived membrane vesicles that range in size between 0.1 and 1 μm , and RBC microparticles can express D antigen on their surface . RBC microparticles have been detected in PCs prepared from both whole blood [28,29][28,29] and apheresis [29–31][29–31][29–31] collections. Moreover, it has also been found that platelet concentrates prepared from whole blood contain platelet-derived microparticles [28,29][28,29]; platelet concentrates collected by apheresis contain not only platelet-derived microparticles but also endothelial cell-derived microparticles . However, the clinical consequences of those observations remain to be investigated.
RECIPIENTS CAN BE IMMUNIZED BY THE RED BLOOD CELLS FOUND IN PLATELET CONCENTRATES
The propensity for D alloimmunization among D negative recipients of D positive platelet concentrates is greatly affected by the dose of D positive RBCs in the platelet concentrate unit that they receive and their immune status . When 200 ml or more of D positive RBCs are transfused to healthy D negative volunteers, anti-D can be detected in the plasma of approximately 85% of the recipients within 2–5 months . The remaining 15% of D negative subjects fail to make detectable anti-D within the following 6 months. About half of these latter volunteers fail to produce a detectable anti-D even after further injections of D positive RBCs and are termed nonresponders . Apart from the quantity of D positive RBCs that the D negative patients are receiving, the importance of their immune status is reflected by data from recent studies showing that the D alloimmunization rate amongst hospitalized D negative patients after receiving D positive RBC units is 21–22% [35–37][35–37][35–37], a figure much lower than the 85% rate cited in textbooks .
However, the range of the RBC content is 0.036–0.59 ml per pooled platelet concentrate prepared from whole blood [22,25][22,25], and 0.00017–0.009 ml per platelet concentrate collected by apheresis [21,24][21,24]. Although small doses of D positive RBCs can provoke primary immunization in healthy volunteers, the fraction of recipients receiving platelet concentrates for therapeutic purposes who become alloimmunized is smaller, and the titers of anti-D that they produce are lower than is observed after exposure to large doses of D positive RBCs . There is some evidence that the minimum quantity of RBCs necessary for primary immunization is as low as 0.03 ml .
From these data, it can be concluded that platelet concentrates prepared from whole blood collections generally contain a sufficient number of RBCs to produce an antibody response. Platelet concentrates collected by modern apheresis devices, however, contain only traces of RBCs, a quantity perhaps insufficient to provoke an immune response, particularly in immunocompromised patients (Fig. 2) [4,12][4,12]. However, recent data from Kitazawa et al. showed that platelet concentrates collected by apheresis contained a residual RBC volume and RBC-derived microparticle volume in the range of 0.0004–0.0008 ml and 0.0001–0.001 ml, respectively . Volumetrically, these potential antigenic stimuli are within an order of magnitude of each other and authors hypothesized that the smaller, more numerous RBC-derived microparticles may be more immunogenic than RBCs themselves, because they could be more easily phagocytosed by recipient antigen-presenting cells. However, authors concluded that the alloimmunization potential of such a small volume of RBC-derived microparticles was speculative, but the fact that the RBC-derived microparticles are found in platelet concentrates requires further research aimed at understanding the immunological side-effects associated with their transfusion .
DEVELOPMENT OF ANTI-D ALLOANTIBODY AFTER TRANSFUSING D-MISMATCHED PLATELET CONCENTRATES
Sixteen published studies address D alloimmunization in D negative patients after receiving D-mismatched platelet concentrates (Table 2). Several authors found no D alloimmunization in this group of patients [15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ][15,19–24,38,39 ▪▪ ,40 ▪▪ ]; other authors, however, reported a frequency of D alloimmunization ranging from 1.4 to 18.7% [14,16–18,25,41 ▪ ,42 ▪▪ ][14,16–18,25,41 ▪ ,42 ▪▪ ][14,16–18,25,41 ▪ ,42 ▪▪ ][14,16–18,25,41 ▪ ,42 ▪▪ ][14,16–18,25,41 ▪ ,42 ▪▪ ][14,16–18,25,41 ▪ ,42 ▪▪ ][14,16–18,25,41 ▪ ,42 ▪▪ ]. One limitation of the majority of these studies was the small number of included patients and the generally short serological follow-up . Cid et al. reported the largest single center retrospective series with the longest serological follow-up, and 49 of 1014 (4.8%) of the D negative recipients developed anti-D after a median follow-up of 17 weeks (range 1–718 weeks).
The immune status of the recipients affects the alloimmunization rate
Immunogenicity refers to the likelihood that an antigen induces antibody formation in an exposed person. Immunogenicity is influenced by factors surrounding the immunization event, such as the route of antigen exposure, and the recipient's underlying medical conditions, such as being iatrogenically immunosuppressed .
In the current clinical scenario, the majority of D negative patients who received D-mismatched platelet concentrates are immunosuppressed (Fig. 1). Therefore, it is not surprising that 11 of 16 of the studies listed in Table 2 included only immunosuppressed patients [14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ][14–23,38,41 ▪ ]. The remaining five studies included both immunosuppressed and immunocompetent patients, and three of them reported no D alloimmunization in any group of patients [24,39 ▪▪ ,40 ▪▪ ][24,39 ▪▪ ,40 ▪▪ ][24,39 ▪▪ ,40 ▪▪ ]. However, in two studies, the authors clearly reported the frequency of D alloimmunization in both groups of patients: Cid et al. reported a frequency of D alloimmunization of 4.8 and 1.9% in immunosuppressed and immunocompetent patients, respectively, in a single-center study (P = 0.3). Finally, Cid et al. reported a frequency of 1.5 and 1.7% in immunosuppressed and immunocompetent patients, respectively, in a multicenter study (P value not reported) [42▪▪].
Time course to D alloimmunization
The shortest time interval after which a primary anti-D can be detected in D negative immunocompetent individuals ranges from 4 to 10 weeks [45,46][45,46]. The production of anti-D within 2 weeks of the first D antigen stimulus has been observed only after injection of specially treated D positive RBCs designed to enhance alloimmunization .
In previously nonimmunized D negative healthy volunteers, anti-D could not be produced more rapidly by giving a series of injections of D positive RBCs compared with the administration of a single D positive RBC injection. For example, among 121 D negative subjects given an initial injection of 5 ml of D positive RBCs (ccDEe), followed by 2 ml every 5 weeks, 8 of 121 formed anti-D within 10 weeks, and 27 of 121 within 15 weeks .
It is important to take into account the observation interval (i.e., the length of serological follow up) after transfusing D positive RBCs to D negative recipients. The response times ranged from approximately 1–5 months in immunocompetent healthy volunteers, with the majority of responding volunteers producing anti-D within 3 months . Some studies detailed in Table 2 did not report the length of follow-up [23,24,41 ▪ ][23,24,41 ▪ ][23,24,41 ▪ ] or the authors reported no D alloimmunization after a median follow-up less than 2 months, an interval perhaps insufficient to detect antibody formation [17,20,41 ▪ ][17,20,41 ▪ ][17,20,41 ▪ ].
REANALYSIS OF REPORTED D ALLOIMMUNIZATION FREQUENCY AFTER TRANSFUSING D-MISMATCHED PLATELET CONCENTRATES
Taking into account that D negative recipients are receiving small quantities of D positive RBCs with the transfusion of D positive platelet concentrates, and the majority of D negative patients who received D positive platelet concentrates are immunosuppressed because of their underlying disease itself, or receipt of immunosuppressive therapy, 4 weeks should be considered the minimum length of time required to develop anti-D after a first stimulus in D negative individuals. In order to interpret the published data correctly, it is important to exclude those recipients whose anti-D represents a secondary immune response, which generally appears within 4 weeks after the second antigen stimulus. If we exclude patients whose antibodies reflect a secondary immune response, the frequency of D alloimmunization varies as outlined in Table 2. We can exclude one patient in the series reported by Goldfinger who was followed for only 2 weeks . Baldwin et al. reported nine patients who formed anti-D, however five of them developed anti-D within 28 days after the first D-mismatched platelet transfusion, thereby likely reflecting a secondary immune response. McLeod et al. reported three patients who formed anti-D at 13, 24 and 83 days from the first D-mismatched platelet transfusion. Of note, two women who developed anti-D within 4 weeks had an obstetrical history suggesting a possible earlier exposure to D positive RBCs. Cid et al. reported the largest retrospective series with 1014 patients, but only 315 patients had a follow-up longer than 4 weeks. Of them, 12 (3.8%) patients developed anti-D. Finally, Solves et al.[41▪] reported two patients who formed anti-D but one of them developed anti-D 11 days after receiving one D-mismatched PC.
As a result of the confusion caused by the short follow-up of D negative patients after receiving D-mismatched platelet transfusions in many of the previous studies, the Biomedical Excellence for Safer Transfusion (BEST) Collaborative designed an international, retrospective study to collect data from D negative patients who received D-mismatched platelet concentrates. Eleven centers from five different countries participated. The goal of this study was to determine the frequency of D alloimmunization (ADAPT study) in immunologically naïve recipients. To this end, an important inclusion criterion in this study was that recipients must not have previously received D positive RBCs or platelet concentrates, thereby greatly enhancing the probability that any anti-D response elicited by the D positive platelet concentrate transfusion would be a primary response. A primary D alloimmunization event was defined as one in which the anti-D was detected greater than 28 days after receiving the D-mismatched platelet concentrate. Another important strength of this study was that all D negative recipients of D positive platelet concentrates were included. Thus its results are generalizable across a wide range of in-patient and out-patient. In this study, the frequency of D alloimmunization was 7 of 485 (1.4%; 95% confidence interval 0.58–2.97) [42▪▪].
As it is not possible to rule out a secondary immune response in patients who developed anti-D within 4 weeks after receiving the first D-incompatible platelet transfusion, thus the frequency of D alloimmunization after D-mismatched platelet transfusions could be lower than the 18.7% figure currently cited . Keeping these new data in mind, we can conclude that the frequency of D alloimmunization ranges between 0 and 7.1% (Table 2).
Stern et al. first reported that there might not be an antibody response if D positive RBCs were coated in vitro by RhIG before being injected into D negative healthy volunteers. Later, Pollack et al. reported the results of a very important experiment that helped to define the dose of RhIG required to prevent D alloimmunization. A fixed amount of 267 μg of RhIG was given to D negative healthy volunteers who received 11.6–37.5 ml of D positive RBCs. Control subjects received the same dose of RBCs without RhIG. All of the patients in both groups were given a challenge dose of 0.2 ml of D positive whole blood 6 months after the RhIG injection, and they were tested for anti-D 1 week later. Among the control subjects who did not receive RhIG, 49 out of 86 (57%) formed anti-D. In the treatment group, 15 out of 92 (16%) formed anti-D. Authors concluded that 267 μg of RhIG was completely effective in preventing alloimmunization against about 13 ml of D positive RBC in whole blood and was partially effective against larger amounts. From this and other studies it was concluded that about 20 μg of RhIG/ml of D positive RBCs is sufficient to avoid D immunization [45,50,51][45,50,51][45,50,51].
RhIG administered intramuscularly is widely used for anti-D prophylaxis in pregnancy. The availability of intravenous RhIG preparation provides a convenient approach to Rh immunoprophylax is when a D negative person has been inadvertently transfused with D positive RBCs, although it is unclear how many milliliters of RBCs can be hemolyzed following the administration of RhIG before end organ damage occurs. Intravenous RhIG can be useful when the administration of larger doses is needed if the patient has low platelet counts.
The manufacturer's recommended dose for intravenous RhIG is 18 μg/ml of D positive RBC, administered as 600 μg (3000 IU) every 8 h until the total dose is reached. According to this recommendation, an injection of a 120 μg (600 IU) vial of intravenous RhIG should be adequate for preventing D alloimmunization with a wide margin of safety after transfusion of multiple doses of platelet concentrates prepared from whole blood collections or from current apheresis devices. There are two reports that support this view. Heim et al. reported the successful prevention of D alloimmunization in 36 D negative patients who received 200 μg (1000 IU) of intravenous RhIG just before the transfusion of D-mismatched platelet concentrates. In the other report, Zeiler et al. used only 20 μg (100 IU) of intravenous RhIG to prevent D alloimmunization, and none of the 20 patients developed anti-D within a median follow-up of 5.5 months (range: 3–13 months).
The content of RBCs in platelet concentrates and the immune status of the recipients are the two major factors influencing D alloimmunization in D negative recipients after receiving D-mismatched platelet transfusions. The frequency of primary D alloimmunization has been reported to be as high as 18.7% in D negative immunocompromised patients; however, the true incidence ranges between 0 and 7.1% because some of the reported observations should be considered to be secondary D alloimmunization events. To prevent this complication, we recommend administering RhIG, if D-mismatched PCs obtained from whole blood collections are transfused to women of childbearing potential.
The authors thank Jordi Bozzo for drawing Figure 1.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
1. Cid J, Harm SK, Yazer MH. Platelet transfusion
- the art and science of compromise. Transfus Med Hemother 2013; 40:160–171.
2▪. Cid J, Lozano M, Klein HG, Flegel WA. Matching for the D antigen in haematopoietic progenitor cell transplantation: definition and clinical outcomes. Blood Transfus 2014; 12:301–306.
A recent review introducing the concept of D-mismatched hematopoietic progenitor cell transplantation.
3. Dunstan RA, Simpson MB, Rosse WF. Erythrocyte antigens on human platelets. Absence of Rh, Duffy, Kell, Kidd, and Lutheran antigens. Transfusion 1984; 24:243–246.
4. Lozano M, Cid J. The clinical implications of platelet transfusions associated with ABO or Rh(D) incompatibility. Transfus Med Rev 2003; 17:57–68.
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. British Committee for Standards in Haematology, Blood Transfusion Task Force. Guidelines for the use of platelet transfusions. Br J Haematol 2003; 122:10–23.
7. Roback JD, Grossman BJ, Harris T, Hillyer CD. Technical manual. 17th edBethesda, MD: AABB Press; 2011.
8. Carson TH. Standards for blood banks and transfusion services. 28th ed.Bethesda, MD: AABB Press; 2012.
9. Schmidt PJ. Blajchman M, Cid J, Lozano M. Blood component therapy from the beginning. Blood component preparation. From benchtop to bedside 1st edBethesda, MD: AABB Press; 2011. 1–22.
10. European Committee on Blood Transfusion. Guide to the preparation, use and quality assurance of blood components. 16th ed. Strasbourg: EDQM; 2010.
11. Cid J. Blajchman M, Cid J, Lozano M. Automation in blood component preparation methods. Blood component preparation. From benchtop to bedside 1st edBethesda, MD: AABB Press; 2011. 271–286.
12. Cid J, Lozano M. Risk of Rh(D) alloimmunization
after transfusion of platelets from D+ donors to D− recipients. Transfusion 2005; 45:453–454.
13. Santana JM, Dumont LJ. A flow cytometric method for detection and enumeration of low-level, residual red blood cells in platelets and mononuclear cell products. Transfusion 2006; 46:966–972.
14. Goldfinger D, McGinniss MH. Rh-incompatible platelet transfusions - risks and consequences of sensitizing immunosuppressed patients. N Engl J Med 1971; 284:942–944.
15. Lichtiger B, Surgeon J, Rhorer S. Rh-incompatible platelet transfusion
therapy in cancer patients: a study of 30 cases. Vox Sang 1983; 45:139–143.
16. Baldwin ML, Ness PM, Scott D, et al. Alloimmunization
to D antigen and HLA in D-negative immunosuppressed oncology patients. Transfusion 1988; 28:330–333.
17. McLeod BC, Piehl MR, Sassetti RJ. Alloimmunization
to RhD by platelet transfusions in autologous bone marrow transplant recipients. Vox Sang 1990; 59:185–189.
18. Heim MU, Bock M, Kolb HJ, et al. Intravenous anti-D
gammaglobulin for the prevention of rhesus isoimmunization caused by platelet transfusions in patients with malignant diseases. Vox Sang 1992; 62:165–168.
19. Zeiler T, Wittmann G, Zingsem J, et al. A dose of 100 IU intravenous anti-D
gammaglobulin is effective for the prevention of RhD immunisation after RhD-incompatible single donor platelet transfusion
. Vox Sang 1994; 66:243.
20. Atoyebi W, Mundy N, Croxton T, et al. Is it necessary to administer anti-D
to prevent RhD immunization after the transfusion of RhD-positive platelet concentrates? Br J Haematol 2000; 111:980–983.
21. Molnar R, Johnson R, Sweat LT, Geiger TL. Absence of D alloimmunization
in D- pediatric oncology patients receiving D-incompatible single-donor platelets. Transfusion 2002; 42:177–182.
22. Cid J, Ortin X, Elies E, et al. Absence of anti-D alloimmunization
in hematologic patients after D-incompatible platelet transfusions. Transfusion 2002; 42:173–176.
23. Misso S, Feola B, Paesano L, et al. Anti-D
alloimmunisation following Rh-incompatible platelet transfusions. Blood Transfus 2006; 4:102–105.
24. Bartley AN, Carpenter JB, Berg MP. D+ platelet transfusions in D− patients: cause for concern? Immunohematology 2009; 25:5–8.
25. Cid J, Carbasse G, Pereira A, et al. Platelet transfusions from D+ donors to D− patients: a 10-year follow-up study of 1014 patients. Transfusion 2011; 51:1163–1169.
26. Greenwalt TJ. The how and why of exocytic vesicles. Transfusion 2006; 46:143–152.
27. Oreskovic RT, Dumaswala UJ, Greenwalt TJ. Expression of blood group antigens on red cell microvesicles. Transfusion 1992; 32:848–849.
28. Bode AP, Orton SM, Frye MJ, Udis BJ. Vesiculation of platelets during in vitro aging. Blood 1991; 77:887–895.
29. Krailadsiri P, Seghatchian J. Are all leucodepleted platelet concentrates equivalent? Comparison of Cobe LRS Turbo, Haemonetics MCS+ LD, and filtered pooled buffy-coat-derived platelets. Vox Sang 2000; 78:171–175.
30. Kitazawa J, Nollet K, Morioka H, et al. Non-D Rh antibodies appearing after apheresis platelet transfusion
: stimulation by red cells or microparticles? Vox Sang 2011; 100:395–400.
31. Rank A, Nieuwland R, Liebhardt S, et al. Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles. Vox Sang 2011; 100:179–186.
32. Seager A, Sandler SG. Immunosuppressive protocols for transplantation and certain hematologic malignancies can prevent the primary immune response to the D blood group antigen. Immunohematology 2013; 29:110–114.
33. Klein HG, Anstee DJ. Blood transfusion in clinical medicine. 11th ed.London: Blackwell Publishing; 2005.
34. Young NT, Street J, Darke C. Immunoglobulin allotype frequencies in responders and nonresponders to the Rh(D) antigen. Exp Clin Immunogenet 1995; 12:88–91.
35. Frohn C, Dumbgen L, Brand JM, et al. Probability of anti-D
development in D− patients receiving D+ RBCs. Transfusion 2003; 43:893–898.
36. Yazer MH, Triulzi DJ. Detection of anti-D
in D− recipients transfused with D+ red blood cells. Transfusion 2007; 47:2197–2201.
37. Gonzalez-Porras JR, Graciani IF, Perez-Simon JA, et al. Prospective evaluation of a transfusion policy of D+ red blood cells into D− patients. Transfusion 2008; 48:1318–1324.
38. Cid J. PLT transfusions from D+ blood donors to D− patients with hematologic diseases: an update. Transfusion 2003; 43:1759–1760.
39▪▪. O’Brien KL, Haspel RL, Uhl L. Anti-D alloimmunization
after D-incompatible platelet transfusions: a 14-year: single-institution retrospective review. Transfusion 2014; 54:650–654.
One of the largest retrospective studies about D-mismatched platelet transfusions, including 130 D− patients who received 565 D+ apheresis PCs, and none of them formed anti-D after a follow-up greater than or equal to 4 weeks after the last D-incompatible platelet transfusion.
40▪▪. Weinstein R, Simard A, Ferschke J, et al. Prospective surveillance of D− recipients of D+ apheresis platelets: alloimmunization
against D is not detected. Transfusion 20152015; 55:1327–1330.
The first prospective study about D-mismatched platelet transfusions.
41▪. Solves P, Carpio N, Gomez I, et al. Risk of RH (D) alloimmunisation after RH (D) positive platelet transfusions in patients undergoing haematopoietic stem cell transplantation. Transfus Med 2015; 25:49–50.
A retrospective study about D-mismatched platelet transfusions in patients undergoing hematopoietic progenitor cell transplantation.
42▪▪. Cid J, Lozano M, Ziman A, et al. Low frequency of anti-D alloimmunization
following D+ platelet transfusion
: the Anti-D Alloimmunization
after D-incompatible Platelet Transfusions (ADAPT) study. Br J Haematol 2015; 168:598–603.
The largest retrospective study with the longest follow-up about D-mismatched platelet transfusions.
43. Menitove JE. Immunoprophylaxis for D− patients receiving platelet transfusions from D+ [correction of D−] donors? Transfusion 2002; 42:136–138.
44. Flegel WA. Immunogenicity reloaded. Blood 2009; 114:3979–3980.
45. Pollack W, Ascari WQ, Crispen JF, et al. Studies on Rh prophylaxis. II. Rh immune prophylaxis after transfusion with Rh-positive blood. Transfusion 1971; 11:340–344.
46. Gunson HH, Stratton F, Cooper DG, Rawlinson VI. Primary immunization of Rh-negative volunteers. Br Med J 1970; 1:593–595.
47. Gunson HH, Stratton F, Phillips PK. The use of modified cells to induce an anti-Rh response. Br J Haematol 1971; 21:683–694.
48. Archer GT, Cooke BR, Mitchell K, Parry P. [Hyperimmunization of blood donors for the production of anti-Rh (D) gamma globulin]. Rev Fr Transfus 1969; 12:341–350.
49. Stern K, Goodman HS, Berger M. Experimental isoimmunization to hemoantigens in man. J Immunol 1961; 87:189–198.
50. Pollack W, Gorman JG, Hager HJ, et al. Antibody-mediated immune suppression to the Rh factor: animal models suggesting mechanism of action. Transfusion 1968; 8:134–145.
51. Pollack W, Ascari WQ, Kochesky RJ, et al. Studies on Rh prophylaxis. I. Relationship between doses of anti-Rh and size of antigenic stimulus. Transfusion 1971; 11:333–339.
Keywords:Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
alloimmunization; anti-D; platelet transfusion