Hereditary thrombocytopenia with platelet sialic acid deficiency and mutations in the GNE genes.

BACKGROUND: The inherited macrothrombocytopenias are rare disorders and the underlying cause can be identified in many cases but in some, this can remain enigmatic. Platelet transfusions are often administered during hemorrhagic events. METHODS: A patient with previously unexplained inherited macrothrombocytopenia with a platelet count between 3-20 × 109 /L is described in which studies were performed using exome sequencing (ES) and platelet flow cytometry. RESULTS: Both the hemoglobin and white cell counts were normal. ES revealed two suspicious variants, one likely pathogenic and one a variant of uncertain significance, in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, and flow cytometry showed diminished expression of surface platelet sialic acid (about 5%) but normal red cell sialic acid. The Thrombopoietin (TPO) level was low, and the patient responded to TPO-mimetic treatment with an increase in the platelet count. CONCLUSION: Two variants in the GNE gene were able to be upgraded to pathogenic with apparently restricted expression to the megakaryocyte lineage. Platelet transfusion may be avoided in these patients with TPO-mimetic treatment.

In vitro comparison of CPD whole blood with conventional blood components.

INTRODUCTION: Resuscitation of severely injured trauma patients is commonly performed using red blood cells in additive solution supplemented with plasma and platelet concentrates. There is an increasing interest in the use of low anti-A titer Group O whole blood (LTOWB) in the early management of the resuscitation. It is unclear whether clinical outcome is improved using this approach. METHODS: Expired units of CPD-LTOWB were studied on Day 22 and expired units of thawed plasma on Day 6 and Day 7. LTOWB was assessed for hemoglobin content, clotting factor levels and platelet numbers and function using thromboelastography (TEG) and impedance aggregation. Assays of fibrinogen and FV, FVIII, FVII and FX were performed on the expired plasma. The LTOWB hemoglobin was compared to red cells in additive solution (AS-RBCs) and the clotting factor levels to those of expired thawed plasma. Platelet function was compared to fresh whole blood samples from healthy subjects. RESULTS: LTOWB contained slightly more hemoglobin than the AS-RBCs (Medians, 66 v 59 G), and the plasma content of fibrinogen was similar. Other clotting factors were reduced by approximately 15% except for FVIII which was 30% less. Both TEG and impedance aggregometry showed evidence of residual platelet function despite the prolonged period of refrigerator storage. CONCLUSION: LTOWB contains higher hemoglobin and adequate clotting factors, and residual platelet function is demonstrated indicating that this product would be expected to be at least equivalent to a single unit of each of the conventional components commonly used in trauma resuscitation.

Interaction Between Platelet and Fibrinogen on Clot Strength in Healthy Patients.

OBJECTIVE: The purpose of this study was to explore the relationship between platelet concentration (PLT) (× 109/L) and clot strength measured by thromboelastography maximum amplitude (TEG-MA) in healthy volunteers without a history of coagulation abnormalities. Secondarily, the relationship between fibrinogen (mg/dL) and TEG-MA was analyzed. DESIGN: A prospective study. SETTING: At a university's tertiary-care center. MEASUREMENTS AND MAIN RESULTS: Using whole blood, PLT was reduced in the first part, and hematocrit was reduced in the second part of the study by hemodilution with platelet-rich and -poor plasma. Thromboelastography (TEG 5000 Haemonetics) was performed to measure clot formation and strength. Spearman correlation coefficients regression analyses and receiver-operating characteristics (ROC) were obtained to analyze the relationships among PLT, fibrinogen, and TEG-MA. Strong correlations were found in univariate analysis between PLT and TEG-MA (r = 0.88; p < 0.0001) and between Fibrinogen and TEG-MA (r = 0.70; p = 0.003). A biphasic relationship between PLT and TEG-MA was linear below a PLT 90 × 109/L, followed by a plateau above 100 × 109/L (p = 0.001). A linear relationship between fibrinogen (190-474 mg/dL) and TEG-MA (53-76 mm) was found (p = 0.0007). The ROC analysis found that PLT = 60 × 109/L was associated with a TEG-MA of 53.0 mm. The product of PLT and fibrinogen concentrations was more strongly correlated (r = 0.91) to TEG-MA than either PLT (r = 0.86) or fibrinogen (r = 0.71) alone. A ROC analysis revealed that a TEG-MA of 55 mm was associated with a PLT × fibrinogen of 16,720. CONCLUSION: In healthy patients, a PLT of 60 × 109/L was associated with normal clot strength (TEG-MA ≥53 mm), and there was little change in clot strength with PLT >90 × 109/L. Although prior analyses described the contributions of platelets and fibrinogen toward clot strength, they are presented and discussed independently. The data above described clot strength as an interaction among them. Future analyses and clinical care should evaluate and recognize the interplay.

A Pilot Study to Assess the Clinical Onset of IV Heparin in Interventional Cardiology and Cardiac Surgery.

OBJECTIVES: To determine the onset of heparin anticoagulation, using 2 different measures of activated clotting times (ACT), thromboelastography (TEG; R-time), and anti-Xa levels, after administering low- (100 U/kg) and high- (300 U/kg) dose intravenous (IV) heparin to patients undergoing transcatheter aortic valve replacement (TAVR) and cardiac surgery, respectively. DESIGN: Prospective study. SETTING: Single academic institution. PARTICIPANTS: Patients with normal baseline coagulation presenting for TAVR or cardiac valve surgery. INTERVENTIONS: Coagulation studies were performed at baseline, 30 seconds, 90 seconds, and 180 seconds after IV heparin administration. The tests included iSTAT (iACT) and Hemochron ACT (hACT), TEG R-Time, and anti-Xa levels. At the authors' institution, anti-Xa is the preferred measure of heparin anticoagulation when time permits. ACT, a rapid point- of-care test, is used to assess intraprocedural anticoagulation. MEASUREMENTS AND MAIN RESULTS: After both low- and high-dose heparin, there are peak increases in ACT and anti-Xa at 30 seconds, followed by a decline at 90 seconds and plateau at 180 seconds. The TEG R-time remained elevated (>80 minutes) throughout. For TAVR cases, all anti-Xa was >1.5 IU/mL, and was associated with an iACT >180 seconds and an hACT >200 seconds. For cardiac valve surgery cases, all anti-Xa was >2.4 and associated with an iACT >420 seconds and and hACT >340 seconds. Compared with hACT, iACTs were significantly lower at all time points after low-dose heparin, but not after high-dose heparin. CONCLUSIONS: In this pilot study, heparin anticoagulation was detected as early as 30 seconds after IV administration, based on ACT, anti-Xa levels, and TEG R-time.

Assessment of Heparin Anticoagulation Measured Using i-STAT and Hemochron Activated Clotting Time.

OBJECTIVE: Adequate anticoagulation, measured using activated clotting time (ACT), is important during vascular and cardiac surgeries. Unfractionated heparin is the most common anticoagulant used. The purpose of this analysis was to compare the i-STAT ACT (iACT) to the Hemochron ACT (hACT), both of which were then compared to anti-factor Xa (anti-Xa) assay, a representation of heparin level and activity. DESIGN: Prospective study. SETTING: Tertiary care cardiovascular center. PARTICIPANTS: Eleven consecutive elective adult cardiac surgical patients. INTERVENTIONS: Prior to cardiopulmonary bypass, ACTs were measured using i-STAT and Hemochron technologies and compared to each other and to anti-Xa assay prior to and during a cumulative administration of heparin. Data were compared using bias analyses. MEASUREMENTS AND MAIN RESULTS: Heparin (300 U/kg) was administered in quarterly doses. Coagulation labs were collected prior to and 3 minutes after each quarterly dose of heparin. The baseline ACTs for i-STAT and Hemochron were 147 and 142 seconds, respectively. A significant association was found between iACT and hACT (p = 0.002). The iACT measurements underestimated hACT at ACT levels >180 seconds or anti-Xa levels >0.75 U/mL. No significant difference was found between ACT data at anti-Xa levels <0.5 U/mL. CONCLUSION: There was a good association between the iACT and hACT; however, the 2 tests are not equivalent. Overall, the iACT underestimated the hACT. Agreement between the ACT technologies was good at lower ACTs and anti-Xa levels, but declined with an anti-Xa >0.75 U/mL.

Effect of ADAMTS13 activity turnaround time on plasma utilization for suspected thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) due to deficiency of the von Willebrand-cleaving protease ADAMTS13 is a hematologic emergency that requires prompt initiation of therapeutic plasma exchange (TPE). Long turnaround times (TATs) have precluded the use of pre-TPE measurement of ADAMTS13 activity for the initial diagnosis in most institutions. STUDY DESIGN AND METHODS: An in-house rapid TAT (r-TAT) assay for ADAMTS13 activity was implemented after 18 months of validation. In a quasi-experimental design using interrupted time series analysis, patterns of plasma utilization in patients with suspected TTP were assessed after implementation of this assay for ADAMTS13 activity and compared to utilization patterns for patients who received plasma exchange before r-TAT assay implementation designated the standard TAT period. RESULTS: In the 18 months after implementation of the r-TAT ADAMTS13 assay, there was a significant reduction in plasma utilization per patient suspected of having TTP (mean, 144.5 units vs. 63.3 units of plasma per patients suspected of having TTP; p = 0.002). The mean number of exchanges per patient and mean number of exchanges after achieving a platelet count of at least 150 × 10(9) /L were lower in the r-TAT cohort (p < 0.001 for both). There was no significant difference in 30-day mortality. CONCLUSIONS: Implementation of a rapid turnaround assay for ADAMTS13 resulted in a significant reduction in plasma utilization for patients with suspected TTP, without an increase in mortality. This study demonstrates that these data, provided in a timely fashion, can avoid unnecessary plasma exchange in patients who do not have TTP.

Sulfamethoxazole-induced thrombocytopenia masquerading as posttransfusion purpura: a case report.

BACKGROUND: Drug-induced immune thrombocytopenia (DITP) is a rare clinical disorder characterized by accelerated platelet (PLT) clearance in the presence of drug-dependent antibodies. Distinguishing DITP from other immune-mediated disorders such as posttransfusion purpura (PTP) and autoimmune thrombocytopenia can represent a clinical challenge. CASE REPORT: A 68-year-old male with no prior transfusion history presented to the emergency department (ED) with dyspnea, epistaxis, and severe thrombocytopenia (<10 × 10(9)/L) 12 days after discharge from a hospital admission for a coronary artery bypass graft. Evaluation of the degree of thrombocytopenia and the temporal association between the peri- and postoperative receipt of multiple transfusions and the acute decrease in PLT count indicated PTP as a possible cause of the severe thrombocytopenia. Treatment with 1 g/kg intravenous immunoglobulin (IVIG) was initiated and followed by a rapid 48-hour increase in the PLT count. PLT antibodies lacking serologic specificity were subsequently identified in a sample collected upon presentation. Two weeks later he again presented to the ED with epistaxis and severe thrombocytopenia (<10 × 10(9)/L). Clinical history now revealed that the patient had been treated with trimethoprim-sulfamethoxazole by his primary care physician after his first hospitalization for a "cellulitic-appearing" leg and again before his final presentation for surgical site erythema and edema. IVIG was administered again with a rapid return of PLT count to baseline. Sulfamethoxazole-dependent PLT antibodies were subsequently identified in the original patient sample. CONCLUSION: This case report documents a case of IVIG-responsive DITP initially misdiagnosed as PTP, highlighting the clinical overlap of these immunologic-mediated phenomena.

Discordance in tests used to detect inhibition of the P2Y12 receptor in patients undergoing interventional neuroradiology procedures.

BACKGROUND: Clopidogrel is an inhibitor of the P2Y12 platelet receptor but testing to demonstrate a drug effect is controversial since there are often discordant results between different tests methods. METHODS: Samples from patients taking clopidogrel prior to intracranial flow-diversion procedures were tested using light transmission aggregometry (LTA), whole blood impedance aggregometry (WBIA) and the VerifyNow device (VND). Samples were classified as concordant if all test results were either responsive (inhibition) or resistant. Discordant results were separated using the VND into those with a responsive versus a resistant test result. RESULTS: Samples from 96 patients were studied. Concordance for all three tests was seen in 53/96 (55%) of samples, of which 41 (43%) were responsive and 12 (12%) were resistant. Discordance was observed in 43 samples (45%), 37 (28%) of which were caused by responsive VND and either a resistant WBIA or LTA and 6 (7%) of which were caused by a resistant VND but a responsive test result using either WBIA or LTA. These two discordant groups differed in both platelet count and hematocrit, but no such difference was present between the two concordant groups. CONCLUSION: Discordance in P2Y12 inhibition testing may be partly explained by sample platelet count and hematocrit.

Laboratory Methods in the Assessment of Hereditary Hemostatic Disorders.

In patients presenting with a suspect hereditary bleeding disorder a detailed bleeding history is first obtained. Testing proceeds in a tiered manner with platelet count, platelet morphology, platelet histogram, PFA-100, fibrinogen, prothrombin time, and activated partial thromboplastin time. More detailed testing includes von Willebrand factor, individual clotting factor assays, and platelet function testing. Next, testing for a dysfibrinogenemia, FXIII, or a fibrinolytic defect is considered. Hemostatic abnormality is not demonstrated in a fraction of patients. An approach to management in these patients, such as desmopressin or antifibrinolytic therapy, may be required and empiric use of blood component therapy is discouraged.

Red Cell Exchange as Adjunctive Therapy for Babesiosis: Is it Really Effective?

Human babesiosis is a parasitic disease prevalent in the Northeastern and Midwestern United States (US). Treatment with antibiotics is the standard of care but red cell exchange (RCE) has been used as an adjunctive treatment in more severe disease. Data for the efficacy of RCE in the treatment of babesiosis has been based on case reports and case series. An English language literature search was conducted for cases of babesiosis treated with RCE since 1980 and relevant laboratory and clinical outcome data were extracted. Similar data were obtained on severe cases of babesiosis referred for RCE in our hospitals in the time period 2000 to 2020. Fifty reports including forty-one individual case reports and nine case series were retrieved. There were 108 patients that underwent RCE with an overall mortality rate of 20%. Some patients had more than one RCE. The patients varied in the level of anemia and evidence of compromise of renal or pulmonary function. The pre-RCE level of parasitemia varied between 1.7% to 85% with the vast majority >10%. The post-RCE level of parasitemia varied between 1% to 10%. Since 2000, 32 patients were referred for RCE in our hospitals and RCE was performed on 23 of 32. There were more patients treated with RCE in the second decade as compared to the first decade, 19 versus 4 respectively. The overall mortality was 22% similar to the national data. Comparing the cohort treated with RCE to the 9 patients who were treated only with antibiotics, there were similar levels of parasitemia and laboratory parameters. The overall number of days needed to achieve a parasite count <1% was similar between the two cohorts and mortality for the antibiotics only cohort was 0%. More than 40 years after the first reported case of RCE in severe babesiosis it cannot be concluded that this adjunctive therapy favorably influences the clinical outcome. Since there is largely equipoise, a registry of severe patients treated with or without RCE could identify a benefit or otherwise.

Stability of coagulation factors in plasma prepared after a 24-hour room temperature hold.

BACKGROUND: The manufacture of fresh-frozen plasma (FFP) requires that plasma be frozen within 8 hours of collection and 24-hour frozen plasma requires 1 to 6°C refrigeration before freezing. Manufacture of plasma after a room temperature hold for 24 hours, while convenient, could compromise clotting factor levels. STUDY DESIGN AND METHODS: Pairs of FFP and 24-hour room temperature-frozen plasma (PLT-rich plasma [PRP]-24HRTFP) were manufactured from PRP after a room temperature hold for 8 and 24 hours, respectively. Additional whole blood (WB) donations were kept at room temperature for 24 hours before plasma manufacture (WB-24HRTFP). The frozen plasma products were stored at -18°C, thawed, and then stored at 1 to 6°C, with coagulation factor assays performed for up to 7 days. RESULTS: On the day of thaw, Factor (F)VIII was lower in PRP-24HRTFP by 13% (p = 0.002) but not in WB-24HRTFP (p = 0.3) compared to FFP. All other clotting factors were within normal range. During the postthaw period FVIII and FV declined 25 and 6%, respectively, in WB-24HRTFP and 23 to 50% in the paired products; however, the difference between both types of 24HRTFP and FFP is insignificant by Day 7 (p > 0.05). Other clotting factors either were unchanged or showed minimal reduction (< 15%). CONCLUSION: Plasma manufactured after a 24-hour room temperature hold contains coagulation factors comparable to FFP except for a possible reduction of up to 20% in FVIII. This plasma appears suitable as a transfusable product and extension of liquid storage to 7 days merits consideration.

Hemolysis in In-Date RBC Concentrates.

OBJECTIVES: Hemolysis is one of the most prominent changes that occur during the liquid storage of RBCs in additive solution (AS), but most studies have measured hemolysis only on day 42. METHODS: Prestorage leukoreduced RBCs in AS-1 and AS-3 were studied, one group on day 42 and a second group between day 0 and day 40. Each product was sampled for direct measurement of supernatant hemoglobin and hematocrit. RESULTS: Ninety day 42 and 218 day 7 to day 39 RBCs showed a mean ± SD supernatant hemoglobin of 75 ± 100 vs 25.5 ± 16 mg/dL respectively (P < .01). Supernatant hemoglobin correlated weakly with storage age (r = 0.2, P < .01) but more strongly with hematocrit (r = 0.4, P < .01). CONCLUSIONS: There are minimal differences in supernatant hemoglobin until the final days of liquid storage when some high hematocrit RBCs show excessive hemolysis.

The effect of specimen hemolysis on coagulation test results.

Hemolyzed specimens are rejected for coagulation testing based on concerns of artifactual interference. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and selected factor assay test results for consecutive pairs of hemolyzed and subsequently recollected (mean, 72 minutes later) nonhemolyzed patient specimens were compared. Specimens from healthy human subjects were subjected to mechanically induced hemolysis, and PT and aPTT results compared with concurrently drawn nonhemolyzed control samples. In 50 paired patient specimens, there were statistically significant differences in PT (15.8 +/- 8.4 vs 16.3 +/- 8.7 seconds, P < .01) and aPTT (31.6 +/- 18 vs 32.5 +/- 19 seconds, P < .01) between hemolyzed and nonhemolyzed specimens, respectively. Specimens from healthy subjects showed no difference in PT and a minor difference in aPTT. A policy of rejecting hemolyzed specimens for coagulation tests should be revisited because the observed difference, when present, is unlikely to be considered clinically meaningful.

The effect of filtration on residual levels of coagulation factors in plasma.

Leukoreduced blood components are commonly manufactured via filtration. There are specifications for the residual leukocyte content of any final cellular blood component but not for residual clotting factors. Leukoreduced and nonleukoreduced platelet-poor plasma products were manufactured from filtered vs unfiltered platelet-rich plasma, respectively, using platelet leukoreduction filters. The leukoreduced plasma showed lower levels of factor VIII (75% ± 16% vs 88% ± 18%, P ≤ .05), factor XI (86% ± 9% vs 96% ± 10%, P ≤ .01) and factor VII (87% ± 14% vs 98% ± 11%, P ≤ .01). No difference was seen with factor X, factor V, or fibrinogen. Plasma filtered through a whole blood filter showed a reduction in factor V (105% ± 12% vs 124% ± 10%, P ≤ .01) but a minimal reduction in factor VIII (80% ± 5% vs 82% ± 6%, P = .04). Filtration can alter the residual levels of clotting factors to a variable extent in manufactured plasma, most noticeably factors V, VII, VIII, and XI.

Manufacture of pooled platelets in additive solution and storage in an ELX container after an overnight warm temperature hold of platelet-rich plasma.

The processing of whole blood-derived platelet-rich plasma (PRP) to a platelet concentrate and platelet-poor plasma is currently performed within 8 hours to comply with the requirements to manufacture fresh frozen plasma. Maintaining PRP at room temperature for a longer period can have the advantage of shifting the completion of component manufacture onto day shifts. Pairs of ABO-identical prepooled platelets were manufactured by the PRP method, using the current approach with platelet storage in a CLX HP container (Pall Medical, Covina, CA) and plasma, or a novel approach with an 18- to a 24-hour room temperature hold of the PRP and the manufacture of pooled platelets in a glucose-containing additive solution (AS) and storage in a new ELX container (Pall Medical). Standard in vitro assays were performed on days 2, 5, and 7. The results showed that the AS platelets in ELX have in vitro characteristics that are equivalent or superior to those of the standard product.

The suitability of hemolyzed specimens for compatibility testing using automated technology.

BACKGROUND: Blood banks have historically rejected hemolyzed specimens for ABO type and antibody screen based on concerns of artifactual interference with test performance or the detection of incompatibility. Samples from emergency departments (EDs) are commonly discarded due to hemolysis or mislabeling. STUDY DESIGN AND METHODS: This study investigated whether hemolysis produced via experimental mechanical stress has a threshold for introducing discrepancy in ABO-Rh typing and antibody screening using an automated gel testing system (ProVue, Ortho-Clinical Diagnostics, Inc.). RESULTS: Twenty-three samples from healthy subjects were shown to have threshold supernatant hemoglobin (Hb) levels producing discrepancy in the results for both ABO reverse type and antibody screen at 75 and 125 mg per dL, respectively. Above these levels of Hb, both tests became uninterpretable and reported as "no result determined." Twenty of 31 positive antibody screens became uninterpretable after experimental hemolysis without any threshold supernatant Hb being evident, likely explained by the duration of specimen storage before experimental hemolysis. No false-positive or false-negative samples were observed in the antibody screen of the hemolyzed specimens. CONCLUSIONS: Properly collected hemolyzed specimens present an opportunity for ABO-Rh forward typing, and a significant proportion of such specimens give valid (concordant) results for reverse type and antibody screen. Our data, although device-specific, are adequate to suggest that sample discard and recollection due to hemolysis in blood bank specimens should be reconsidered with potential benefits for patient safety and efficiency.

In vitro evaluation of prestorage pools consisting of mixed A and O platelet concentrates.

BACKGROUND: Current FDA regulations allow the prestorage pooling of whole blood-derived platelet concentrates (PCs) of identical ABO type in a recently cleared platelet (PLT) pooling bag (Acrodose, Pall Medical). It is unclear how ABO-mixed PC pools would store if pooled before storage. STUDY DESIGN AND METHODS: Pools consisting of ABO-identical PLTs and mixed A and O PLTs in varying proportions were evaluated on Days 1, 5, and 7 of storage with measures of the PLT storage lesion, lymphocyte activation, and activation of the complement and coagulation system. Data were analyzed by analysis of variance. RESULTS: Pools did not differ on Day 7 in pH (p = 0.63), hypotonic shock response (p = 0.25), extent of shape change (p = 0.26), morphology score (p = 0.18), or white cell count (p = 0.79), but surface P-selectin expression was more evident in the ABO-mixed pools (p = 0.02). Small microscopic clumps of PLTs were observed in all pools, but were more prominent in the ABO-mixed pools (p < 0.01). PLT counts, however, did not differ between pools (p = 0.93), indicating that only a small proportion of PLTs were clumped. Surface A-antigen expression was proportional to the number of A PCs in each pool and did not vary between study days. Anti-A(1) titers were either unchanged or decreased by one dilution. Complement and coagulation activation markers did not differ between pools. CONCLUSION: Pooling A and O PCs is associated with evidence of increased microscopic PLT clumping and activation, but these differences are not exacerbated with 7-day storage. Other major measures of PLT quality do not differ, and there was no evidence of a mixed lymphocyte reaction.