Prolonged profound abciximab associated immune thrombocytopenia complicated by transient multispecific platelet antibodies.

Patients receiving abciximab occasionally develop transient severe thrombocytopenia within a few hours of receiving the drug. Thrombocytopenia has been reported to resolve within 10 days of abciximab administration, but in this case profound thrombocytopenia lasted 21 days before a slow spontaneous recovery. Management was complicated by the presence of HLA antibodies and the transient production of antibodies directed at major platelet glycoproteins IIb/IIIa, Ib/IX, and Ia/IIa. The patient remained refractory to platelet transfusion and two courses of intravenous gammaglobulin for the duration of her admission.

HPA-1, 3, 5 genotyping to establish a typed platelet donor panel.

Platelet antigen typing of blood donors is not routinely performed. Usually, ABO-matched platelets are transfused. In situations where the recipient has a human platelet-associated (HPA) antibody resulting in refractoriness to platelet transfusions, post-transfusion purpura or neonatal alloimmune thrombocytopenia, HPA-typed platelets may be required. Having a panel of HPA-typed donors would be helpful. This study utilised PCR-RFLP and PCR-SSP methods to determine the HPA-1, 3, 5 genotypes of 1,000 blood donors to establish a panel of HPA homozygous donors who could donate platelets for patients with anti-HPA-antibodies. The study demonstrates the HPA genotype frequencies in a Western Australian blood donor population and has identified donors who are homozygous for the "aa" and "bb" genotypes for HPA-1, 3 and 5. The Australian Red Cross Blood Service-NW Region can now use these data to develop a panel of HPA-1, 3 and 5 "aa" and "bb" homozygous platelet donors, which can be accessed for patients with HPA antibodies requiring platelet transfusion.

Monocyte activation in platelet concentrates.

BACKGROUND AND OBJECTIVES: Cytokines in platelet concentrates contribute to febrile transfusion reactions. Activated monocytes are a major source of inflammatory cytokines, however the role of monocytes in cytokine production in platelet concentrates has not been clarified. This study undertook to quantitate monocytes, determine whether monocyte activation occurs and identify an association with IL-6 and IL-1beta concentrations in platelet concentrates. MATERIALS AND METHODS: 17 platelet concentrates were analysed for total leucocyte and monocyte counts, CD14 and CD16 monocyte-associated antigen expression and IL-1beta and IL-6 measurements on days 1, 2, 3, 4 and 5. RESULTS: Monocytes in all platelet concentrates expressed increased levels of CD14 and CD16 from day 1 of storage. 10/17 platelet concentrates had elevated IL-6 levels by day 3. Platelet concentrates with IL-6 levels above 15 pg/ml on day 5 had monocyte counts between 0.14 and 15.6 x 10(6)/unit on day 1, while those with IL-6 levels below 15 pg/ml had low monocyte counts of < 0.01 to 1.2 x 10(6)/unit on day 1. CONCLUSION: Monocytes present in platelet concentrates exhibit features of activation. Monocyte activation is present following the preparation of platelet concentrates, implicating the manufacturing process in its development. Increased IL-6 and IL-1beta levels during platelet concentrate storage are commonly associated with a higher monocyte count. However, no direct association could be identified between the extent of monocyte activation and the level of cytokine release.

White cells in fresh-frozen plasma: evaluation of a new white cell-reduction filter.

BACKGROUND: Fresh-frozen plasma (FFP) has generally been regarded as an acellular component. Recently, viable lymphocytes have been detected in this component and the question of irradiation of FFP for certain patients has been raised. Whether the numbers of white cells (WBCs) in FFP are sufficient to require WBC-reduction of acellular components for patients receiving WBC-reduced cellular components has not been determined. WBC numbers in FFP were examined, and the performance of a new commercial WBC-reduction filter for FFP was assessed. STUDY DESIGN AND METHODS: WBC numbers in plasma processed for use as FFP and in thawed FFP were counted before and after WBC-reduction filtration by the use of flow cytometry Fast and slow filtration was used to simulate laboratory and bedside filtration, respectively. Three different methods for plasma harvesting (soft-spin, hard-spin, and second-spin methods) were assessed. The filter capacity was also examined. RESULTS: The numbers of WBCs in plasma covered a three-log10 range (soft-spin method, 0.04-3.6 x 10(6); hard-spin method, 0.47-45.4 x 10(6); second-spin method, 0.4-37.2 x 10(6)). For the hard-spin and second-spin methods which produced the greatest plasma yields, 92 percent and 85.7 percent of bags, respectively, had counts >1 x 10(6) and 43 percent (hard-spin method) and 45.7 percent (second-spin method) had counts >5 x 10(6). There was no significant difference between the counts obtained in plasma and thawed FFP. The filter reduced WBC numbers to <1 x 10(5) in all but 3 of 49 bags. In the remaining three, there were <2 x 10(5) WBCs. Five bags of plasma could be processed effectively through each filter. CONCLUSION: FFP may contain WBC numbers above the threshold at which the use of WBC-reduction filters for cellular components in some patients is necessary. Confirmation of these findings and similar investigations on plasma prepared by other methods may help in defining a role for the use of WBC-reduction filters for FFP

Use of unrefrigerated fresh whole blood in massive transfusion.

OBJECTIVE: To establish the efficacy of transfusion of fresh unrefrigerated whole blood in surgical patients with ongoing massive bleeding despite adequate blood-component replacement therapy and adequate surgical haemostasis. DESIGN: A retrospective review of patients who received fresh unrefrigerated whole-blood transfusions, noting blood usage and outcome. SETTING: A tertiary care teaching hospital with a major cardiac surgical and trauma service. PATIENTS: All patients (n=11) receiving fresh unrefrigerated whole-blood transfusions from March 1992 to February 1995. RESULTS: Mean blood usage in the 24 hours before the decision to transfuse fresh unrefrigerated whole blood was 16.5 units of packed cells (range, 6-27), 17.1 units of platelets (8-32), 14.5 units of fresh frozen plasma (6-26) and 13.5 units of cryoprecipitate (4-36). After the transfusion of fresh whole blood there was an immediate and substantial reduction in the rate and volume of blood loss in all patients. This was sustained in seven patients, who had a successful outcome; the other four patients died within 24 hours from recurrent uncontrollable haemorrhage. The reduction in blood usage in the 24 hours after the transfusion of fresh whole blood was statistically significant in the surviving patients but not in the patients who died. None of the surviving patients showed evidence of viral seroconversion six months after the transfusion. CONCLUSIONS: Our study provides preliminary evidence that there is a role for transfusion of fresh unrefrigerated whole blood in surgical patients with unresponsive life-threatening haemorrhage.

Application of the proteolytic enzyme papain in routine platelet serology.

The use of proteolytic enzymes is well established in red cell serology. These enzymes modify some antigen structures and remove sialic acid from the red cell membrane. Enzyme-sensitive structures have also been identified on the platelet membrane. The effect of papain, a proteolytic enzyme used widely in red cell serology, on the detection of various platelet alloantibodies was examined to determine its usefulness in platelet serology. Antisera with the specificities anti-HPA-la, -2b, -3a, -4a, -5a, -5b, and -Naka were examined. HLA antibodies were also included. All sera were tested by a solid-phase red cell adherence technique in parallel with untreated platelets (UP) and platelets treated with papain (PP) for 15 minutes at 37 degrees C. The reactivity of anti-HPA-2b was eliminated and that of anti-HPA-3a was either eliminated or almost eliminated with PP. Antisera specific for the other alloantigens tested reacted similarly or more strongly with PP compared with UP. These findings were confirmed by flow cytometry. The reactivity of HLA antibodies with PP was generally enhanced. Inactivation by papain of platelet alloantigens in the HPA-2 and HPA-3 systems, but not in other systems, may assist in resolving mixtures of platelet alloantibodies. Also, detection of weak antibodies of other specificities may be enhanced. The use of PP may be a simple and useful serologic tool for investigating platelet alloantibodies.

Cross-matched platelets in bone marrow transplantation.

Antibodies to HLA-antigens remain a problem in multiply-transfused patients. Over a 2 yr period 44 bone marrow transplant recipients were screened at weekly intervals for the presence of HLA-antibodies using a solid phase red cell adherence technique. An adaptation of this method was used to provide cross-matched random donor platelets (XMRDP) when screening proved positive. Twelve of the 44 patients were antibody positive, 6 prior to transplantation and 6 following the transplant. Those 4 patients who developed an antibody following the transplant had a significant increase in platelet increments following the change from random donor platelets (RDP) to XMRDP even though only one patient was refractory to platelets at the time the antibody was first detected. No significant bleeding occurred during the study period. The use of routine platelet antibody screening followed by platelet cross-matching allows excellent support of thrombocytopenic patients without requiring HLA-typed blood donors and avoiding clinical platelet refractoriness.

Feto-maternal alloimmune thrombocytopenia associated with anti-HPA-5a--the first Australian case.

Maternal alloantibodies to platelet specific antigens are a rare but important cause of severe thrombocytopenia in the fetus and neonate. Feto-Maternal Alloimmune Thrombocytopenia (FMAT) is most often due to anti-HPA-1a (PLA-1) or anti-HPA-5b (Bra). Anti-HPA-5a (Brb) however is extremely uncommon, with only 4 cases previously described. We report the first Australian case of feto-maternal alloimmune thrombocytopenia due to anti-HPA-5a.

A fatal case of necrotizing enterocolitis in a neonate with polyagglutination of red blood cells.

An infant of 30 weeks gestation developed necrotizing enterocolitis (NEC) 8 days after birth and died 2 days later after a fulminating course. During her illness she received two blood transfusions, both of which produced sub-optimal rises in her haemoglobin and were associated with evidence of haemolysis. Retrospective analysis demonstrated T antigen (Tk) polyagglutination of the infant's red blood cells and donor plasma. Although bacterial cultures were negative throughout the course of the illness in this case, T antigen exposure is associated with certain anaerobic infections and with severity of NEC. Infants with NEC should be regularly screened for T antigen exposure and if this test is positive, plasma (immunoglobulin) containing infusions should be avoided.

Evaluation of a solid phase red cell adherence technique for platelet antibody screening.

Solid-phase red-cell adherence (SPRCA) techniques in platelet serology are used mainly for crossmatching. A SPRCA method for general diagnostic application was evaluated in parallel with the platelet suspension immunofluorescence test (PIFT). Of 149 patient sera sent for investigation of thrombocytopaenia, 76 were negative and 59 positive when studied by both methods, eight positive by PIFT only and six positive by SPRCA only. The reactivity observed for 24 sera containing HLA antibodies tested with chloroquine-treated and untreated platelets was similar for both methods. All of 14 sera containing quinine-associated antibodies reacted strongly to both techniques in the presence of added quinine. In comparison, however, whereas all sera were nonreactive to SPRCA in the absence of added quinine, and with PIFT, seven of the sera reacted weakly. Titration studies with three examples of anti-PlA1 and five sera containing HLA antibodies generally showed a one doubling dilution lower titre with the SPRCA procedure. End-point interpretation, however, was more readily achieved with the SPRCA method. The SPRCA technique displays similar sensitivity and specificity to the PIFT and is recommended for use by routine hospital laboratories to screen platelet antibodies.

Polybrene technique for red cell antibody screening using microplates.

The manual polybrene technique was adapted as a microplate test for antibody screening to determine its sensitivity and specificity and compared with conventional tube testing using various antibodies and serologically inert sera. Equivalent results were obtained for both techniques and it is concluded that this adaptation of the polybrene technique is useful in pretransfusion testing.

Eighteen months' experience with a manual polybrene crossmatch in a large hospital transfusion laboratory.

For 18 months in this laboratory the manual polybrene technique (MP) has been used as the only crossmatching procedure preceded or accompanied by an antibody screen comprising two-stage papain and LISS antiglobulin techniques. There were 17,161 requests representing 43,006 blood units crossmatched and 20,841 units transfused. Non-specific reactivity with the MP required use of an antiglobulin crossmatch in approximately 0.2% of patient samples. Heparin in excess of 20 IU/ml reduced polybrene aggregation of red cells necessitating an antiglobulin crossmatch for 2 patients. Of 288 antibodies detected 20 reacted exclusively by MP compared with 18 by the papain procedure. The data supported the use of MP as an alternative to enzymes in antibody screening protocols. The polybrene technique was found to be a superior abbreviated crossmatch compared with the immediate spin technique and was applicable to all patients including those with known antibodies.

A reappraisal of pre-transfusion testing procedures in a hospital blood bank.

In recent years, experience with screening protocols for the detection of red cell antibodies has led to a reappraisal of pre-transfusion testing procedures. In the U.S.A. in particular, it has become accepted practice to perform only an antiglobulin crossmatch when an antibody screen has been negative. There is now debate in that country regarding the necessity of retaining even the antiglobulin crossmatch. In this laboratory, as is usual in Australia, room temperature, enzyme and antiglobulin tests are used for both antibody screening and crossmatching. A review was therefore conducted of the results of screening 58,227 samples from approximately 40,000 patients, involving 126,771 crossmatched blood units over a 53 mth period. Eight hundred and seventy-two red cell antibodies were detected in 718 patients. Forty-four of these antibodies were detected only in the crossmatch, and of these 23 were of potential clinical significance. Fourteen of the 23 were detected by the antiglobulin test, 8 by an enzyme test and only 1 by the saline method. Of 107 antibodies detected only in the room temperature phase, none were of clinical significance. The results indicate that exclusion of room temperature tests from all pre-transfusion testing, and deletion of enzyme tests from the crossmatch will not compromise patient safety.

Inhibition of serological reactions with enzyme-treated red cells by complement binding alloantibodies.

Complement-binding antibodies (2 examples of anti-Jka and 2 anti-kell) which inhibit agglutination of enzyme-treated cells have been investigated in the sera of 4 patients. The sera containing anti-Jka also contained rhesus antibodies (c + E and D + C) which could not be readily identified by an enzyme technique due to failure of the Jka+ cells of the corresponding Rh phenotype to react. The inhibition of the enzyme test reactivity was revealed using a control reagent added to each test after the initial examination for agglutination. Addition of ethylenediaminetetraacetic acid to the sera prior to testing resulted in identifiable reactivity with panel red cells. Failure to recognise this phenomenon in sera containing antibodies reacting only by an enzyme technique could result in misidentification, delay in providing compatible blood and transfusion of incompatible blood. Routine control of enzyme tests to detect false-negative results is recommended.

Evaluation of an enzyme-linked antiglobulin test for the detection of red cell antibodies.

An enzyme-linked antiglobulin test (ELAT) using low ionic strength saline for the initial red cell sensitisation phase, and alkaline phosphatase conjugated antiglobulin (AP-AHG), has been compared with a conventional low ionic strength antiglobulin test in testing 222 red cell antibodies of various specificities. A wide variation in absorbance values was observed at all levels of haemagglutination strength. Relatively higher absorbance values were obtained with anti-K compared with the agglutination gradings. Haemolysis was eliminated by modifying the substrate buffer used for diluting the AP-AHG, since fixation of red cells prior to sensitisation significantly reduced the sensitivity of the ELAT. Five commercial AP-AHG reagents compared to tests with D, Fya, K and Jka antibodies varied markedly in performance, some being unsatisfactory. The ELAT can be effectively used for antibody detection as well as quantitative determinations but requires automation to realise its full potential.