The 'cherry buffy-coat syndrome', a cause of decreased platelet yield in platelet concentrates obtained from buffy-coats

BACKGROUND AND OBJECTIVES: A large number of European blood centres, including our own, use the buffy-coat method for platelet production. In this article we describe a previously unnoticed phenomenon shown by a proportion of buffy-coats, which display an unusually bright cherry colour and low platelet counts. MATERIALS AND METHODS: We performed bacterial cultures, platelet counts, pO2, pCO2 and pH, and evaluated platelet activation by flow cytometry in cherry versus normal-colour (control) buffy-coats. In addition, we compared donor characteristics in the two groups and platelet counts in the packed red blood cells (RBC) obtained from the original donations. Finally, we monitored the frequency of cherry buffy-coats in the bags of three manufacturers, and determined the concordance rate of two trained technicians in detecting cherry buffy-coats. RESULTS: Bacterial cultures were negative. Cherry buffy-coats contained significantly fewer platelets, more O2, less CO2 and had a significantly higher pH than normal buffy coats. Platelet activation was slightly higher in cherry buffy-coats. RBC from donations yielding cherry buffy-coats contained a significantly higher number of platelets than controls. Donor characteristics were not significantly different. Cherry buffy-coats were significantly more frequent with bags from one manufacturer (24%) than from others (9% and 11.6%). The concordance study showed excellent agreement. CONCLUSIONS: Our hypothesis is that the cherry colour is caused by O2 accumulation in buffy-coats with low platelet counts. The latter may be caused by platelet activation and aggregation during blood processing. Further work is needed to determine the cause of this phenomenon, its frequency in different laboratories and means to prevent it.     

急性白血病患者血小板无效输注的原因分析

目的：探讨急性白血病（AL）血小板输注无效的原因。方法：观察106例AL患者的263例次单采血小板输注效果并检测血小板抗体,分析讨论血小板无效输注的影响因素。结果：①AL血小板无效输注率为43．35％;②血小板抗体阳性检出率为32．32％;③血小板输注有效组与无效组的抗体阳性检出率的差异有统计学意义（P〈0．01）。④AL发热组输注无效率高于未发热组（P〈0．01）;脾脏肿大与无肿大组无效输注率差异有统计学意义（P〈0．01）。结论：引起血小板输注无效的病因复杂。血小板输注前应进行血小板抗体的筛选,避免或减少造成血小板输注无效的原因,提高血小板输注的有效率。     

Comparative assessment of prophylactic transfusions of platelet concentrates obtained by the PRP or buffy-coat methods,in patients undergoing allogeneic hematopoietic stem cell transplantation

ABSTRACT

Objectives: Whole blood-derived platelet concentrates can be obtained by the platelet-rich plasma (PRP-PCs) or the buffy-coat (BC-PCs) method. Few studies have shown that BC-PCs display lower in vitro platelet activation, but scarce information exists regarding transfusion efficacy. We have performed a retrospective study assessing platelet transfusion in patients undergoing allogeneic hematopoietic cell transplantation (AHCT) in our clinic, before and after the implementation of BC-PCs.

Methods: We reviewed clinical records corresponding to 70 PRP-PCs and 86 BC-PCs prophylactic transfusions, which were performed to 55 AHCT patients. Transfusion efficacy was assessed by the 24-h post-transfusion corrected count increment (24-h CCI) and bleeding events. Clinical factors affecting transfusion outcome were also investigated.

Results: Clinical characteristics and the total number of platelet transfusions were similar among groups. Mean donor exposure was 5.8 and 5.0 in each single PRP-PCs and BC-PCs transfusion, respectively (p?<?0.01). The 24-h CCI was significantly higher in patients transfused with BC-PCs than in those receiving PRP-PCs (8.3[2.7–13.4] vs. 4.7[1.3–8.1]; p?<?0.01). Independent predictors of poor platelet transfusion response included diagnosis other than acute leukemia (HR 8.30; 95% CI 1.96–35.22; p?=?0.004), splenomegaly (HR 8.75; 95% CI 2.77–27.60; p?<?0.001), graft versus host disease prophylaxis different from cyclosporine A and methotrexate (HR 3.96; 95% CI 1.55–10.14; p?=?0.004) and PRP-PCs transfusion (HR 4.54; 95% CI 1.72–12.01; p?=?0.002). There were no differences between both groups regarding the bleeding events.

Conclusion: In the AHCT setting, we hypothesize that BC-PCs transfusion, when compared to PRP-PCs, results in higher CCI and reduced donor exposure, but provides no significant benefit regarding bleeding outcome.     

A randomized study of buffy coat platelets in platelet additive solution stored 1–5 versus 6–7 days prior to prophylactic transfusion of allogeneic haematopoietic progenitor cell transplant recipients

Background and Objective  Storage of platelets > 5 days provides improved availability, logistical management and decreased outdating. Promising results on in vitro parameters and on in vivo post-transfusion recovery and survival of autologous platelets in healthy volunteers have earlier been shown. To provide additional verification, randomized patient transfusion studies are needed.
Materials and Methods  Sixty allogeneic haematopoietic progenitor cell transplant recipients were randomized to receive buffy-coat (BC) platelets stored in platelet additive solution (PAS) for 1–5 days the first time a prophylactic transfusion was needed after transplantation, followed the second time by platelets stored for 6–7 days or vice versa. The corrected count increment (CCI) for 1 and 24 h were calculated.
Results  CCI 1 h and CCI 24 h were higher for platelets stored 1–5 days as compared to 6–7 days, 10·4 ± 5·1 vs. 7·4 ± 3·8 ( P <  0·001) and 5·4 ± 4·1 vs. 2·6 ± 2·6 ( P <  0·001), respectively. Time to next platelet transfusion was significantly longer after a transfusion of platelets stored for 1–5 days as compared to platelets stored for 6–7 days: 2·2 ± 1·1 vs. 1·6 ± 0·8 days, respectively ( P <  0·005). No differences in bleeding events and no transfusion reaction were recorded.
Conclusion  The advantage of an extension of platelet storage time beyond day 5 should be balanced against the increased need for platelet transfusions that may occur and the conceivable risk of transfusion failure.     

Multiple HLA-matched platelet transfusions for a single patient with broad anti-HLA antibodies: a case report

The efficacy of 30 platelet concentrate (PC) products transfused to a patient with myelodysplastic syndrome (MDS) was evaluated by calculating the 1-hour post-transfusion corrected count increment (1h-CCI). Of the 30 transfusions, all HLA-A/B-matched, the cross-match (CM) test was negative in 23 (CM(-)-PC) and weakly positive (CM(+)-PC) in 2, and the CM test was not conducted in 5 (non-CM-PC). The effective rate was higher with CM(-)-PC compared to non-CM-PC (82.6% vs 60%), but statistical significance was not achieved, which suggested that the CM test of PC may still be a not satisfactorily effective predictor of PC refractoriness. Studies are ongoing in Japan to confirm on the importance of CM test of PC.     

The platelet count accuracy of platelet concentrates obtained by using automated analysis is influenced by instrument bias and activated platelet components

BACKGROUND AND OBJECTIVES: The blood platelet content (in numbers) of platelet concentrates is required for production quality control and to predict clinical responses. MATERIALS AND METHODS: This study compared the performance of automated counting from impedance and optical instruments to data from immunoplatelet reference analysis. RESULTS: All methods showed good linearity with evidence of significant instrument-specific deviations from the line of agreement. Relational formulae largely corrected bias, but did not resolve platelet count variability. A second confounding factor, related to the proportion of small (activated) platelets, was also shown to contribute to intermethod discrepancies. CONCLUSIONS: Blood processing centres should establish correction factors for each instrument compared to reference methods, such as the immunoplatelet count.     

Platelet count evaluation using three automated haematology analysers compared with the immunoplatelet reference method, and estimation of possible inadequate platelet transfusion

The accuracy of three automated haematology analysers [Sysmex XE-2100 (both optical and impedance mode), Bayer Advia 120, and Beckman Coulter LH-750] was compared with the immunoplatelet reference method for platelet measurement. A total of 165 blood specimens were obtained from patients and platelet counts were determined using the four-automated haematology analyser methods and the immunoplatelet reference method. The coefficients of determination (R(2)) between the automated haematology analyser methods and the immunoplatelet reference method for the overall platelet range were >0.98. A bias study, however, showed some disagreement. The use of a coincidence correction calculation for the immunoplatelet method did not improve the correlation between the immunoplatelet method and the automated haematology analyser methods. To estimate the possibility of inadequate platelet transfusion, the number of prophylactic platelet transfusion indications determined by the automated haematology analyser platelet counts were compared with the number of transfusion indications according to the platelet counts determined by the immunoplatelet method. An additional 48 blood specimens were included in this analysis. All of the automated haematology analysers showed some disagreement in the transfusion indications when compared with the immunoplatelet method, suggesting the possibility of inadequate platelet transfusion.     

Blood collection,platelet isolation and measurement of platelet count and size in mice—a practical guide

Inherited or acquired disorders of platelet production and function can result in thrombocytopenia and bleeding. Mouse models have proven useful for investigating the mechanisms that underlie these defects in humans. Precise methods for blood withdrawal, platelet isolation and measurement of platelet parameters are key for the generation of reproducible and conclusive data.

Here, we provide three different protocols for mouse platelet isolation to encourage research knowledge transfer between experienced laboratories, while at the same time enabling less experienced researchers to implement a protocol that best suits their local expertise and equipment. We also address the issue that reported mouse platelet count and size vary considerably in the literature by investigating different factors that influence these important platelet parameters, namely: 1) genetic background and gender, 2) choice of analysis method (hematological analyzer or flow cytometry), 3) dilution of the blood sample and 4) choice of anticoagulant. The herein presented results and considerations may serve as a practical guide for both experienced and new researchers in the platelet field.     

Incidence and pattern of 12 years of reported transfusion adverse events in Zimbabwe: a retrospective analysis

Background

Haemovigilance hinges on a systematically structured reporting system, which unfortunately does not always exist in resource-limited settings. We determined the incidence and pattern of transfusion-related adverse events reported to the National Blood Service Zimbabwe.

Materials and methods

A retrospective review of the transfusion-event records of the National Blood Service Zimbabwe was conducted covering the period from 1 January 1999 to 31 December 2011. All transfusion-related event reports received during the period were analysed.

Results

A total of 308 transfusion adverse events (0.046%) were reported for 670,625 blood components distributed. The majority (61.6%) of the patients who experienced an adverse event were female. The median age was 36 years (range, 1–89 years). The majority (68.8%) of the adverse events were acute transfusion reactions consisting of febrile non-haemolytic transfusion reactions (58.5%), minor allergies (31.6%), haemolytic reactions (5.2%), severe allergic reactions (2.4%), anaphylaxis (1.4%) and hypotension (0.9%). Two-thirds (66.6%) of the adverse events occurred following administration of whole blood, although only 10.6% of the blood was distributed as whole blood. Packed cells, which accounted for 75% of blood components distributed, were associated with 20.1% of the events.

Discussion

The incidence of suspected transfusion adverse events was generally lower than the incidences reported globally in countries with well-established haemovigilance systems. The administration of whole blood was disproportionately associated with transfusion adverse events. The pattern of the transfusion adverse events reported here highlights the probable differences in practice between different settings. Under-reporting of transfusion events is rife in passive reporting systems.     

In vitro combinations of red blood cell,plasma and platelet components evaluated by thromboelastography

Background

Thromboelastography is increasingly used to evaluate coagulation in massively bleeding patients. The aim of this study was to investigate how different combinations of blood components affect in vitro whole blood clotting measured by thromboelastography.

Materials and methods

Packed red blood cells, plasma and platelets from fresh and old blood components were mixed in vitro, in proportions of 4:4:1, 5:5:2, 8:4:1 and 2:1:0, and analysed with thromboelastography. For the ratio 4:4:1 the experiment was done at both 37 °C and 32 °C.

Results

Thromboelastography curves were within normal reference values for the blood component proportions of 4:4:1 and 5:5:2. For 8:4:1, the angle and maximal amplitude were reduced below normal values, indicating low levels of fibrinogen and/or platelets. For the 2:1:0 proportion, all parameters were affected resulting in severely impaired in vitro clot formation. The reaction-time, reflecting the coagulation factor-dependent, initial clot formation, was slightly increased at a low temperature. Prolonged storage of the components did not affect the curve.

Discussion

With the introduction of guidelines on the management of massive bleeding it is important to have tools for the assessment of the new protocols. In vitro evaluation of mixtures of packed red blood cells, plasma and platelets by thromboelastography may be relevant in the prediction of in vivo clot formation and haemostasis.     

Long-term monitoring of platelet count, as a non-invasive marker of hepatic fibrosis progression and/or regression in patients with chronic hepatitis C after interferon therapy

Background: Platelet count has been shown to correlate with the hepatic fibrosis stage in chronic hepatitis C (CHC). The aim of the present study was to assess hepatic fibrosis progression or regression of CHC patients by long‐term monitoring of the platelet count. Methods: A total of 429 interferon (IFN)‐treated CHC patients were studied. Follow‐up data on the platelet count were collected every 6 months after IFN therapy. The IFN response was defined as follows: complete responders (CR, n = 121) demonstrating persistent clearance of serum hepatitis C virus (HCV) RNA; biochemical responders (BR, n = 94) demonstrating alanine aminotransferase (ALT) normalization for ≥6 months without eradication of HCV‐RNA; and non‐responder (NR, n = 214) demonstrating all other patterns. Results: In comparison with the baseline level, mean platelet count increased in the CR group from 0.5 years after IFN therapy (for each point, P < 0.01), but significantly decreased in the NR group from 1 year after IFN therapy (for each point, P < 0.01). In the BR group, an increase in mean platelet count was observed from 0.5 to 3.5 years following IFN therapy (for each point, P < 0.01), followed by a gradual decrease. Conclusion: An increase from baseline values in platelet count was observed, regardless of the presence of HCV‐RNA, in both the CR and BR groups, suggesting the importance of ALT normalization in preventing hepatic fibrosis progression in IFN‐treated CHC patients.     

Evaluation of platelet function during extended storage in additive solution, prepared in a new container that allows manual buffy-coat platelet pooling and leucoreduction in the same system

Background

A novel and practical storage container designed for manual buffy-coat pooling and leucodepletion was evaluated to assess its filtration performance and to analyse the quality of stored leucoreduced buffy-coat-derived platelet pools.

Materials and methods.

To analyse the Grifols Leucored^® Transfer PL system, blood was collected from random donors into standard triple bag systems, and fractionated using standard procedures to obtain buffy-coats. Ten leucodepleted platelet pools were prepared each from five units of buffy-coats in additive solution. Concentrates were stored for 10 days at 22 °C on an end-over-end agitator. On days 0, 5, 7, and 10 of storage, samples were tested using standard in vitro platelet parameters.

Results

The use of this novel system for volume reduction and leucodepletion of buffy-coats resuspended in additive solution led to platelet pools that met the European requirements. pH was maintained well, declining from an initial value of 7.11±0.04 to 6.88±0.08 after 10 days. Parameters of cell lysis, response to a hypotonic stimulus and aggregation induced by agonists (arachidonic acid, ristocetin, collagen or thrombin receptor activating peptide) were also well-preserved. During storage, the quality profile of the platelet pools remained very similar to that previously reported in platelet concentrates in terms of metabolism, platelet activation (CD62, CD63, sCD62), expression of glycoproteins Ib and IIb/IIIa, capacity of glycoprotein IIb/IIIa to become activated upon ADP stimulation, and release of biological response modifiers (sCD40L and RANTES).

Discussion.

This new system allows the preparation of leucodepleted buffy-coat platelet pools in additive solution with good preservation of platelet function. The logistics of the procedure are relatively simple and it results in good-quality components, which may reduce costs and ease the process of buffy-coat pooling and leucocyte reduction in transfusion services.