Serial measurement of lymphocytotoxic antibody and response to nonmatched platelet transfusions in alloimmunized patients

Serial evaluations of lymphocytotoxic antibody (LCTAb) and responsiveness to random donor platelet transfusion were reviewed in 234 patients who had developed LCTAb at some time during their treatment course. Seventy (30%) of these patients had significant falls in antibody levels. In 44 patients these declines occurred after further antigenic exposure was reduced either because no transfusions were administered or only histocompatible platelets were transfused. Forty patients with declines in LCTAb levels who were previously refractory to platelet transfusion were rechallenged with random donor platelets. Thirty-four of 35 clinically evaluable patients had good responses to these unmatched transfusions for 2 weeks to 36 months, and in 21 patients antibody did not return despite repeated transfusions. Thus, serial LCTAb measurements are helpful in the management of alloimmunized patients. Many patients will have decreases or a loss of LCTAb, either permanently or transiently, and can be successfully supported with more easily available unmatched random donor platelet transfusions.     

ABO compatibility and platelet transfusions of alloimmunized thrombocytopenic patients.

With data on 91 alloimmunized thrombocytopenic patients and 389 donor-recipient pairs matched or selectively mismatched for HLA antigens, it was observed that ABO incompatibility significantly reduced the effectiveness of platelet transfusions. The mean 24-hr recovery of platelets from histocompatible donors and from donors selectively mismatched for cross-reactive HLA antigens was decreased by approximately 23% if the donor typed for blood group A and/or B not found in the recipient. Thus, the reduction in platelet recovery associated with ABO incompatibility is not of a magnitude that would contraindicate transfusion of ABO-mismatched platelets.     

The effect of lymphocytotoxic antibody reactivity on the results of single antigen mismatched platelet transfusions to alloimmunized patients

Despite selection strategies that attempt to maximize the platelet donor pool, significant numbers of alloimmunized patients have few if any available donors. Although the number of potential donors increases when one antigen mismatched platelet transfusions (OAMPT) are considered, transfusions from such donors are often cited to fail to produce satisfactory platelet count increments. The presence of lymphocytotoxic antibody (LCTAB) correlates well with responsiveness to random donor platelet transfusions and serves as a good serologic screen for the diagnosis of alloimmunization. We therefore reviewed the results of OAMPT to alloimmunized patients and assessed the relationship between LCTAB levels in the recipient and posttransfusion platelet count increments. We noted an unexpectedly high percentage of good responses in our patient population: 73% of all OAMPT to recipients with LCTAB < 60% reactive, resulted in successful increments. In recipients with LCTAB > or = 60%, 58% of all transfusions were still successful. Despite a statistically significant inverse relationship between the level of LCTAB and the response of OAMPT to alloimmunized patients, 58% to 73% of recipients will have a satisfactory platelet recovery posttransfusion. These data support extending donor searches for alloimmunized patients to include any single mismatch particularly if a recipient's LCTAB has lower reactivity.     

Arachidonic acid-stimulated platelet tests: Identification of patients less sensitive to aspirin treatment

Serum thromboxane-B2 (TxB2), together with arachidonic acid (AA)-induced platelet aggregation, are, at the moment, the most used tests to identify patients displaying high on-aspirin treatment platelet reactivity (HAPR). Both tests are specific for aspirin action on cyclooxygenase-1. While the correlation between serum TxB2 assay and clinical outcome is established, data are conflicting with regard to aspirin treatment and a possible association with AA-stimulated platelet markers and clinical outcome. To understand such discrepancy, we performed a retrospective study to compare both assays. We collected data from 132 patients receiving a daily dose of aspirin (100?mg/day) and data from 48 patients receiving aspirin on alternate days. All Patients who received a daily dose of aspirin were studied for AA-induced platelet aggregation together with serum TxB2 levels and AA-induced TxB2 formation was also studied in 71 patients out of entire population. Consistent with recommendations in the literature, we defined HAPR by setting a cut-off point at 3.1?ng/ml for serum levels of thromboxane B2 and 20% for AA-induced platelet aggregation. According to this cut-off point, we divided our overall population into two groups: (1) TxB2?<?3.1?ng/ml and (2) TxB2?>?3.1?ng/ml. We found low agreement between such tests to identify patients displaying HAPR. Our results show that AA-induced platelet aggregation >20% identify a smaller number of HAPR patients in comparison with TxB2. A good correlation between serum TxB2 and arachidonic acid-induced TxB2 production was found (r?=?0.76619).     

HLAMatchmaker-driven analysis of responses to HLA-typed platelet transfusions in alloimmunized thrombocytopenic patients

This study describes a novel application of HLAMatchmaker to determine platelet compatibility in 16 alloimmunized patients with aplastic anemia refractory to random donor platelet transfusions. HLAMatchmaker is a software algorithm that predicts HLA compatibility by identifying immunogenic epitopes represented by amino acid triplets in antibody-accessible regions of human leukocyte antigen (HLA) molecules and determines the number of triplet mismatches (TMMs) and highly immunogenic triplet mismatches (HIMMs). Corrected count increments (CCIs) and molecular HLA typing were available for 523 transfusions. Conventional compatibility assessment based on cross-reactive group (CREG) determination was not predictive of transfusion outcome. Low HIMMs and TMMs numbers were associated with a higher likelihood of satisfactory (CCIs > or = 8) compared with unsatisfactory (CCIs < 8) outcomes (median HIMMs = 4 vs 6, p2 value < .001; median TMMs = 11 vs 13, p2 value < .001). Although receiver operator characteristic curves revealed that HIMMs or TMMs number are not powerful predictors of individual transfusion outcome, a threshold of at least 3 HIMMs or at least 9 TMMs appeared to be associated with successful transfusions. Triplet-matched transfusions were successful, regardless of CREG matching. Our data validate HLAMatchmaker for platelet transfusions and demonstrate its potential to refine and expand donor selection for HLA-alloimmunized patients.     

Attempts to standardize platelet aggregation measurements: simplified screening tests

We present a standardized measurement of collagen and ADP-induced platelet aggregation with the help of a personal computer. The calculator improves the recording of measurements and the automatic calculation of parameters to define the strength of aggregation in various aggregation phases, as well as aggregation and disaggregation velocities. The dose-effect relationships between aggregants and five calculated parameters after addition of ADP and four after addition of collagen are demonstrated in the light of measurements taken in 40 healthy persons. Comparison of these measurements with those of 8 volunteers 24 h after they had taken 250 mg of acetylsalicylic acid and those of of 5 patients with typical illnesses associated with hypo- and hyperaggregability shows that analysis of the aggregation with two reagent concentrations (1, 2 and 4 micrograms/ml collagen, 1, 2 and 4 mumols/ml ADP) and the resulting values allow one to distinguish between normal, hypo- and hyperaggregable platelets.     

Relationship of HLA and platelet-reactive antibodies in alloimmunized patients refractory to platelet therapy

Platelet crossmatching assays have been used to predict the outcome of platelet transfusions in alloimmunized patients by detecting antibodies against platelets. The transfusion failure of HLA-matched platelets predicted by platelet crossmatching may be related to HLA antibodies undetected by lymphocytotoxicity but detected by platelet immunoglobulin-binding assays or platelet-specific antibodies (both antibodies defined here as platelet-reactive antibodies). To differentiate platelet-reactive antibodies from lymphocytotoxic HLA antibodies, we used HLA characterized lymphocytes in parallel with platelets from individuals to form separate frozen panels. Sera from 10 allosensitized patients were studied in the lymphocyte panel by lymphocytotoxicity and in the platelet panel by enzyme-linked immunoassay (ELISA). By comparing pattern and percent wells reacting in each panel, lymphocytotoxic HLA antibodies and antibodies reactive with platelets in ELISA were detected separately. In all 10 allosensitized patients, platelet-associated antibodies were present and 7 had additional lymphocytotoxic HLA antibodies. Using this double parallel panel technique, we found platelet-reactive antibodies important in platelet alloimmunization, unrecognized by lymphocytotoxicity. These data indicate platelet-crossmatching be solely used in the selection of platelets for allosensitized patients.     

Successful treatment of platelet transfusion refractoriness: the use of platelet transfusions matched for both human leucocyte antigens (HLA) and human platelet alloantigens (HPA) in alloimmunized patients with leukaemia

Six patients, 4 with acute myeloid leukaemia and 2 with a myelodysplasia syndrome who were refractory to random donor platelet transfusions and alloimmunized to human leucocyte antigens (HLA) and human platelet alloantigens (HPA), were treated with HLA-and HPA-matched platelet transfusions. In all the patients refractoriness and alloantibodies to HLA as well as HPA-1b or HPA-5b were detected simultaneously. Sixty-seven transfusions (445 units) of HLA-and HPA-matched platelets were given and responses to them were, in general, satisfactory in all the patients. No major spontaneous bleeding occurred. Four patients underwent bone marrow transplantation despite alloimmunization. The percentages of platelet transfusion days with a platelet nadir below 20×10⁹/l were 88% for the last 3 random donor platelet transfusions and 39% for the first 3 HLA-and HPA-matched platelet transfusions, respectively (p=0.009, Fisher's exact test). Four patients received also HLA-matched platelets, but responses to them were poor. The small number of transfusions with HLA-matched platelets precluded comparisons to either the random donor or HLA-and HPA-matched platelet transfusions. It seems that HLA-and HPA-alloimmunized patients can be successfully supported with HLA-and HPA-matched platelet concentrates.     

The use of radiolabeled and fluorescein-labeled antiglobulins in assays to predict platelet transfusion outcome

We used both radiolabeled and fluorescein-labeled antiglobulins in assays to detect antibodies against platelets in multiply transfused patients to determine the value of these tests in predicting the outcome of platelet transfusion in such patients. In 15 allosensitized patients, we studied 68 single-donor platelet transfusions, 43 (63%) of which had a poor outcome, defined as a corrected count increment (CCI), less than 10,000. The results obtained with either test were significantly correlated with the CCI following transfusion (p less than 0.001), but the assay using the radiolabeled antiglobulin had slightly better sensitivity, specificity, and predictive value. When the assays were used in combination, there was again significant correlation with the CCI of the transfusion, p less than 0.001. When both assays predicted failure of the transfusions, 31/31 (100%) such transfusions resulted in a CCI of less than 10,000, and when both assays predicted success of the transfusions, 14/15 (93%) such transfusions resulted in a CCI of greater than 10,000. Both assays are useful in predicting the outcome of the platelet transfusions; when the assay results were concordant, almost total predictive accuracy was obtained.     

Influence of HLA-A2 on the effectiveness of platelet transfusions in alloimmunized thrombocytopenic patients

Platelet transfusions from donors selectively mismatched for cross- reactive and certain non-cross-reactive HLA antigens were found to be more effective in HLA-A2 negative than in HLA-A2 positive, alloimmunized thrombocytopenic patients. The two groups of patients responded equally well to platelets matched for antigens of the HLA-A and B loci. Certain alloimmunized patients negative for HLA-A2 continued to respond satisfactorily to platelets selectively mismatched for non-cross-reactive HLA antigens as long as platelets containing HLA- A2 were avoided. The data indicate that platelet transfusion support can be provided within a broader range of donor-recipient HLA antigenic disparity to HLA-A2 negative alloimmunized patients than to those who are positive for this antigen.     

Bernard-Soulier syndrome: quantitative characterization of megakaryocytes and platelets by flow cytometric and platelet kinetic measurements

Abstract: Platelets and megakaryocytes have been characterized in a Bernard-Soulier syndrome (BSS) kindred with respect to glycoprotein (GP) membrane receptors and measurements of thrombocytopoiesis. The index patient exhibited lifelong bleeding tendency, moderate thrombocytopenia (35 × 10⁹/1), giant platelets (mean platelet volume 12.5 μm³ compared to 7.5 ± 1.5 μm³ in normals), absent ristocetin-induced platelet agglutination and absent binding of von Willebrand factor (vWF). Flow-cytometric analysis revealed absent platelet binding (0–2%) of monoclonal antibodies (mAb, LJ-P3, LJ-Ibl and LJ-Ibl0) directed against distinct epitopes on membrane GPIbα of the GPIb-IX complex, and normal binding of LJ-P4 mAb directed against GPIIb/IIIa complex (relative to increased platelet surface area). Marrow megakaryocytes also failed to express GPIb-IX complex, but demonstrated normal expression of GPIIb/IIIa. Among 6 asymptomatic family members, the patient's mother and 2 of his 4 children exhibited approximately 50% binding of anti-GPIbα mAb to their platelets by both flow cytometry and direct binding studies using ¹²⁵I-vWF, ¹²⁵I-LJ-Ibl and ¹²⁵I-LJ-Ibl0 mAb. Marrow megakaryocytes were increased in the average cell volume and cytoplasmic granularity with a corresponding increase in ploidy (46% > 16N compared to 22 ± 5% in normal individuals), a pattern typical of megakaryocytes stimulated by thrombocytopenia. Autologous ¹¹¹In-platelet life span was shortened to 4.1 days (compared with 9.5 ± 0.5 days in normal subjects), and the turnover of platelet mass in the circulation was near normal. The data directly demonstrate that the platelet membrane GPIb-IX defect in BSS originates in megakaryocytes at all levels of cell maturation, and exclude the possibility that the receptor abnormality is acquired during cell maturation or after platelets are released into the circulation. Since marrow megakaryocytes exhibited cellular changes consistent with stimulated megakaryocytopoiesis, these results also suggest that thrombocytopenia in this kindred of BSS is a consequence of both decreased platelet survival and ineffective platelet production.     

Long-term follow-up patients with leukemia receiving platelet transfusions: identification of a large group of patients who do not become alloimmunized

Alloimmunization is the major complication of platelet transfusion therapy in patients with acute leukemia. To evaluate whether alloimmunization continues to be a long-term problem in patients surviving induction therapy, 114 patients with acute nonlymphocytic leukemia (ANLL) who survived more than 6 mo and who received multiple courses of chemotherapy and abundant platelet transfusions were studied. Clinical response to random donor platelets and lymphocytotoxic antibody (LCTAb) were measured pretreatment and serially throughout the study period. Fourteen patients (12%) were alloimmunized upon admission, 34 (30%) patients became alloimmunized during remission induction therapy, and 66 (58%) patients did not become alloimmunized during that period. Sixty-one of these 66 patients (92%) never became alloimmunized and responded to random donor platelets during their subsequent course despite the fact they received multiple further platelet transfusions, whereas the alloimmunized patients tended to remain alloimmunized for their entire clinical course. There was no difference in age or sex between groups, and prognostic factors predicting alloimmunization could not be detected. In greater than 90% of patients not alloimmunized at admission, the presence or absence of LCTAb after induction predicts later alloantibody production. This information can be used to plan the type of platelet transfusions (HLA-matched or random donor) needed for subsequent maintenance and induction therapy. It may also help to identify a group of patients to whom more aggressive maintenance chemotherapy may be more safely administered.     

Clinical review 3: The clinical use of thyrotropin receptor antibody measurements

There are relatively few circumstances wherein the assay of TRAb, as TSAb or TBIAb, is merited in the routine clinical management of patients with autoimmune thyroid disease. These include: 1. the prediction of neonatal hyperthyroidism; 2. the confirmation, by assay of maternal serum, and perhaps newborn blood, that neonatal hypothyroidism, identified on routine screening, may be transient and due to transplacental passage of TBIAb; 3. the prediction of relapse of hyperthyroidism at the end of a course of antithyroid drugs; this information should lead to more careful observation to identify relapse as early as possible; 4. the confirmation that adult agoitrous hypothyroidism reflects the effect of an antibody (TBIAb) blocking the action of TSH although, as discussed, there is little, if any, practical gain from this information. Additional applications of the routine use of TRAb assays will require the development of a procedure that combines the sensitivity, specificity, availability, and ease found variably in current TSAb and TBI procedures, and at much less cost than what is provided today.     

Identification of the target platelet glycoprotein in autoimmune thrombocytopenia occurring after allogeneic bone marrow transplantation

Summary We report the case of a patient who developed autoimmune thrombocytopenia after allogeneic bone marrow transplantation from her HLA-identical sister. An IgG autoantibody was detected that bound to the platelet glycoprotein IIb/IIIa and to the HLA class I proteins. Immunoprecipitation studies with radiolabeled platelets revealed additional antibody binding sites on proteins of 214 kDa molecular weight under nonreduced conditions and 65 and 56 kDa molecular weight under reduced conditions.     

血小板输注中抗体检测和配合试验的应用

目的：提高血小板制剂对血小板减少、尤其是血小板输注无效症（PTR）患者的输注疗效,避免宝贵血源的浪费。方法：应用单抗固相微孔板（MASPAT）法检测患者血清中的血小板抗体,进行血小板供者与患者之间的配合试验。结果：2005年6月-2007年11月对109例患者进行了血小板抗体的检测,其中42例患者检出血小板抗体（阳性率38．5％）,对含有血小板抗体的患者经适合性血小板输注后,血小板计数有明显上升。结论：MASPAT法在特异性、敏感性、重复性方面良好,操作快速、简便,判断可靠;易做到规范化,程序化,标准化;据此建立的“适合性血小板输注”对含有血小板抗体的患者是有效的,可用于临床血小板抗体的检测和配合试验。     

Transient platelet and HLA antibody formation in multitransfused patients with malignancy

Fifty-nine patients receiving platelet transfusions for bone marrow failure secondary to malignancy were screened at regular intervals for the presence of antibodies to human leucocyte (HLA) and platelet specific antigens. HLA antibodies occurred in 19 patients, 10 of whom also developed platelet specific antibodies. The HLA antibodies disappeared in 10 of 15 patients followed for periods of 2-14 months. In two patients this occurred whilst still receiving platelet transfusions. Antibody reappeared in only two of six patients subsequently transfused. Antibodies to platelet specific antigens were detected in 28 patients. They were transient, often appeared in association with infection, and in 50% of cases tested demonstrated autoantibody activity. There was no association with antibiotic drug therapy, or PFA/EDTA-dependent cryptantigens. Platelet recovery at 1 h or 20 h post transfusion was not significantly reduced in the presence of platelet specific antibodies. These findings have important implications for the selection of platelet donors for alloimmunized recipients.