A novel diagnostic algorithm for heparin-induced thrombocytopenia.

INTRODUCTION: While diagnostic algorithm using PF4-heparin enzyme-linked immunosorbent assay (ELISA) optical density (OD), and heparin neutralization assay (HNA), or 4T score have been proposed to replace serotonin-release assay (SRA) for heparin-induced thrombocytopenia (HIT), their performance against SRA is unclear. In this study, we proposed and validated the performance of a new algorithm combining PF4-heparin ELISA optical density (OD), HNA and 4T score against SRA for HIT diagnosis. METHODS: Heparin neutralization assays were performed on specimens submitted for HIT testing with positive PF4-heparin ELISA from December 2015 to September 2017, which were separated into a "training" and a "validation" data set. 4T scores were calculated for ELISA positive cases. RESULTS: A total of 123 consecutive unique patient samples had positive PF4-heparin ELISA with also HNA data, SRA data, and 4T scores available. Compared to SRA, a "laboratory criteria" (ELISA OD ≥ 1.4 and HNA ≥ 70%) had a sensitivity of 88% (14/16) and specificity of 91% (42/46), and a "combined criteria" (4T score = 8, or 4T score = 6 or 7 and ELISA OD ≥ 1.0, or 4T score = 4 or 5 and ELISA OD ≥ 2.0) had a sensitivity of 75% (12/16) and specificity of 98% (45/46) in the training data set. Laboratory and combined criteria had 90% (56/62) concordance rate. Importantly, for these concordant cases, the diagnostic specificity is 100% (46/46). Based on the data, a novel diagnostic algorithm combining these 2 criteria was proposed and validated prospectively. CONCLUSION: A novel algorithm has high diagnostic accuracy and potentially could eliminate the need for SRA testing in at least 90% patients with suspected HIT.

Hematocrit alters VerifyNow P2Y12 assay results independently of intrinsic platelet reactivity and clopidogrel responsiveness.

BACKGROUND: The VerifyNow P2Y12 assay assesses the adequacy of clopidogrel therapy by measuring ADP-induced platelet activation in whole blood. Low hematocrit is associated with high clopidogrel on-treatment platelet reactivity (HTPR) defined by this assay. OBJECTIVES: To characterize the effect of hematocrit on VerifyNow values and determine if it is due to hematocrit-dependent changes in intrinsic platelet reactivity or an in vitro assay phenomenon. PATIENTS/METHODS: Adenosine diphosphate-induced platelet activation was measured using the VerifyNow P2Y12 assay, whole blood impedance and light transmission platelet aggregometry (LTA) before and after clopidogrel loading in 113 patients undergoing elective cardiac catheterization. Iso-TRAP-induced platelet activation was additionally measured using the VerifyNow device. Multivariate modeling employing clinical and laboratory variables was used to investigate the association between hematocrit and VerifyNow values. RESULTS: VerifyNow P2Y12 reaction units (PRU) and iso-TRAP Base units before and after clopidogrel loading, but not their relative change, exhibited strong negative correlation with hematocrit (P ≤ 0.0005 for both). While hematocrit remained a strong predictor of post-clopidogrel PRU (P = 0.001) in multivariate modeling, it was independent of post-clopidogrel ADP-induced platelet reactivity as measured by LTA (P = 0.001). Correcting for the effects of hematocrit resulted in a 15-39% reduction in the prevalence of HTPR defined by thresholds of 208-236 PRU. CONCLUSIONS: The effect of hematocrit on VerifyNow PRU values is an in vitro phenomenon that is independent of intrinsic change in ADP-induced platelet reactivity and clopidogrel responsiveness. Correcting for hematocrit when using this assay may more accurately identify patients with HTPR that may benefit from alternative antiplatelet therapy.

Effect of assay specificity on the association of urine 11-dehydro thromboxane B2 determination with cardiovascular risk.

BACKGROUND: Elevated urine 11-dehydro TXB(2), an indicator of persistent thromboxane generation in aspirin-treated patients, correlates with adverse cardiovascular outcome and has recently been identified as an independent risk factor for vein graft thrombosis after cardiac bypass surgery in the Reduction in Graft Occlusion Rates (RIGOR) study. The polyclonal antibody-based ELISA used to measure 11-dehydro TXB(2) in these previous studies is no longer clinically available and has been supplanted by a Food and Drug Administration (FDA)-cleared second-generation monoclonal antibody-based ELISA. OBJECTIVES: To compare the laboratory and clinical performance of the first- and second-generation assays in a well-defined study population. METHODS: 11-dehydro TXB(2) was quantified in 451 urine samples from 229 Reduction in Graft Occlusion Rates (RIGOR) subjects using both ELISA. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and spiking studies were used to investigate discordant assay results. The association of 11-dehydro TXB(2) to clinical outcome was assessed for each assay using multivariate modeling. RESULTS: Median 11-dehydro TXB(2) levels were higher by monoclonal antibody- compared with polyclonal antibody-based ELISA (856 vs. 399 pg mg(-1) creatinine, P < 0.000001), with the latter providing values similar to UPLC-MS/MS. This discrepancy was predominantly as a result of cross-reactivity of the monoclonal antibody with 11-dehydro-2,3-dinor TXB(2), a thromboxane metabolite present in a similar concentration but with a poor direct correlation with 11-dehydro TXB(2). In contrast to the first-generation ELISA, 11-dehydro TXB(2) measured by the monoclonal antibody-based ELISA failed to associate with the risk of vein graft occlusion. CONCLUSION: Quantification of urine 11-dehydro TXB(2) by monoclonal antibody-based ELISA was confounded by interference from 11-dehydro-2,3-dinor TXB(2) which reduced the accuracy and clinical utility of this second-generation assay.

Effect of anti-platelet factor-4/heparin antibody induction on early saphenous vein graft occlusion after coronary artery bypass surgery.

BACKGROUND: Antibodies to complexes of heparin and platelet factor 4 (PF4) are capable of causing heparin-induced thrombocytopenia (HIT). Recent evidence suggests that anti-PF4/heparin antibodies may be prothrombogenic even in the absence of thrombocytopenia and clinically-recognized HIT. OBJECTIVES: To determine if induction of anti-PF4/heparin antibodies is an independent risk factor for early saphenous vein graft (SVG) occlusion or adverse clinical outcome after coronary artery bypass graft (CABG) surgery. PATIENTS/METHODS: Anti-PF4/heparin antibody titers were measured in 368 patients prior to and then 4 days, 6 weeks and 6 months after CABG surgery. Serotonin release assay (SRA) and antibody isotype analysis were also performed on 6-week samples. SVG patency was determined in 297 patients 6 months after surgery by multidetector computed tomography coronary angiography. RESULTS: Six weeks after surgery, 52% of patients were anti-PF4/heparin seropositive and 9% were SRA positive. Six months after surgery, neither the percentage of occluded SVG (19% vs. 20%, P = NS), the percentage of patients with an occluded SVG (33% vs. 33%, P = NS) nor the incidence of adverse clinical events (21% vs. 24%, P = NS) differed between seropositive and seronegative groups. Neither IgG isotype nor SRA positivity was additionally predictive of SVG occlusion or adverse clinical outcome. CONCLUSION: Induction of anti-PF4/heparin antibodies, even those capable of heparin-dependent platelet activation, is not independently associated with early SVG occlusion or adverse clinical outcomes after CABG surgery.

Ret-Y a measure of reticulocyte size: a sensitive indicator of iron deficiency anemia.

In this study the size of reticulocytes was measured, reticulocyte-Y (Ret-Y), to distinguish iron deficiency anemia from the anemia of chronic disease using a Sysmex XE2100 cell counter. We evaluated this parameter prospectively in 100 patients seen for the evaluation of anemia. A clinical diagnosis of iron deficiency anemia or anemia of chronic disease was made on the basis of a complete blood count, examination of the peripheral smear, and serum ferritin along with a history and physical examination. We analyzed the sensitivity and specificity of the Ret-Y in relationship to the clinical diagnosis. We also measured serum transferrin receptor levels to use as the gold standard laboratory test for iron deficiency against which we compared the Ret-Y. In 40 normal individuals with normal serum ferritin and transferrin receptor levels the mean Ret-Y was 1874 +/- 178 (1 SD). The mean Ret-Y in the anemia of chronic disease group (n=62) was 1722 +/- 162, not significantly different from normal. The mean Ret-Y value among iron-deficient patients (n=38), was 1407 +/- 136 (P <0.01 vs. the anemia of chronic disease group's Ret-Y value). Receiver operator curves showed that Ret-Y correlated closely to the serum transferrin receptor and was superior to the mean corpuscular volume, and ferritin level, in differentiating the type of anemia. The Ret-Y parameter has the highest overall sensitivity and specificity of the panel of tests routinely used in differentiating iron deficiency anemia from anemia of chronic disease.

Neonatal alloimmune thrombocytopenia due to anti-HPA-5b (Bra).

Neonatal alloimmune thrombocytopenia (NAIT) results from maternal immunization against fetal platelet antigens and can occur during the first pregnancy. The most common complications of NAIT are neonatal thrombocytopenia, intracerebral hemorrhage, and fetal death. Most cases of NAIT in Caucasians are caused by anti-HPA-1a (PlA1). Anti-HPA-5b (Bra) accounts for only 4.3 percent of all NAIT cases. NAIT due to anti-HPA-5b is thought to be milder and have fewer complications than NAIT caused by anti-HPA-1a because of the lower number of HPA-5b antigenic sites per platelet. This report describes a severe case of NAIT due to anti-HPA-5b that was treated by intrauterine platelet transfusion.

Cardiac troponin T and C-reactive protein as markers of acute cardiac allograft rejection.

Due to myocyte damage and an associated inflammatory response, it is possible that cardiac troponin T and C-reactive protein (CRP) concentrations may correlate with the histologic grade of rejection in endomyocardial biopsy samples obtained from patients who have received a heart transplant. In this study, 704 blood samples were obtained from 145 different heart transplant recipients just prior to endomyocardial biopsy. Plasma specimens were assayed for troponin T and CRP concentration and the results compared with the assigned International Society of Heart and Lung Transplantation (ISHLT) histologic grade. Rejection was defined as an ISHLT grade of 3A or higher. The negative predictive values were near 80% in all cases, and a statistically significant increase in median troponin T concentration was observed across ISHLT grades. After the first month posttransplantation, the specificity of the troponin T test (cutoff 0.1 ng/ml) was 95% and increased to 98% when false positives seen in renal disease patients were excluded. Both tests demonstrated poor sensitivity and positive predictive value for rejection. Neither CRP nor troponin T had sufficient sensitivity to serve as an alternative to endomyocardial biopsy in the diagnosis of acute cardiac allograft rejection. However, the troponin T test had a high specificity, especially when patients with renal insufficiency were excluded, and could serve as an adjunct test in this setting. When combined with a normal serum creatinine, a troponin T > or =0.1 ng/ml prior to endomyocardial biopsy correlated with graft rejection in almost all cases, making biopsy unnecessary.

Coagulation markers predicting cardiac transplant rejection.

BACKGROUND: Acute cellular rejection in cardiac allografts is a major cause of graft loss, and is associated with activation of the coagulation system. We investigated whether plasma markers of coagulation predict the presence of allograft rejection. METHODS: A total of 132 blood specimens and endomyocardial biopsies were collected from 35 patients, between February of 1997 and May of 1998. We measured plasma prothrombin fragment 1.2 (PF1.2) and p-selectin, fibrinogen, thrombomodulin, and d-dimer. Biopsies were graded according to the International Society of Heart and Lung Transplantation system, with a range of 0 to 4. Grades 0 and 1A were grouped as "no rejection," and the higher grades as "rejection." Linear and logistic regression, accounting for longitudinal data, were the principal analytic tools. RESULTS: p-Selectin level increased progressively with increasing rejection grade (P<0.001). With multivariate analysis, both p-selectin and prothrombin fragment levels significantly predicted rejection. p-Selectin levels were predictive of prothrombin fragment levels (P<0.0001) but not of d-dimer, fibrinogen, or thrombomodulin levels. This model allowed correct prediction of rejection, based on p-selectin and prothrombin fragment values, up to 85% of the time. Dichotomizing patients by a p-selectin level of 65 ng/ml resulted in an odds of rejection of 21.4 [95% C.I. 7.1-64.7] for the patients in the high- compared with the lower risk group. CONCLUSIONS: In heart transplant recipients, p-selectin levels and PF 1.2 levels are highly predictive of organ rejection. The elevation of PF 1.2 suggests that there is systemic generation of thrombin generation. These markers may be useful for noninvasively monitoring patients for organ rejection or for after response to treatment.

Effects of mixed hematopoietic chimerism in a mouse model of bone marrow transplantation for sickle cell anemia.

Sickle cell anemia (SCA) is an inherited disorder of beta-globin, resulting in red blood cell rigidity, anemia, painful crises, organ infarctions, and reduced life expectancy. Allogeneic blood or marrow transplantation (BMT) can cure SCA but is associated with an 8% to 10% mortality rate, primarily from complications of marrow-ablative conditioning. Transplantation of allogeneic marrow after less intensive conditioning reduces toxicity but may result in stable mixed hematopoietic chimerism. The few SCA patients who inadvertently developed mixed chimerism after BMT remain symptom free, suggesting that mixed chimerism can reduce disease-related morbidity. However, because the effects of various levels of mixed chimerism on organ pathology have not been characterized, this study examined the histologic effects of an increasing percentage of normal donor hematopoiesis in a mouse model of BMT for SCA. In lethally irradiated normal mice that were reconstituted with varying ratios of T-cell-depleted marrow from normal and transgenic "sickle cell" mice, normal myeloid chimerism in excess of 25% was associated with more than 90% normal hemoglobin (Hb). However, 70% normal myeloid chimerism was required to reverse the anemia. Organ pathology, including liver infarction, was present in mice with sickle Hb (HbS) levels as low as 16.8% (19.6% normal myeloid chimerism). Histologic abnormalities increased in severity up to 80% HbS, but were less severe in mice with more than 80% HbS than in those with 40% to 80% HbS. Therefore, stable mixed chimerism resulting from nonmyeloablative BMT may reduce the morbidity from SCA, but prevention of all disease complications may require minimizing the fraction of circulating sickle red cells. (Blood. 2001;97:3960-3965)

Acute bleeding and thrombocytopenia after bone marrow transplantation.

The relationship between hemorrhage and low platelet count was first established in patients with acute leukemia, and has been widely applied to thrombocytopenic patients, including BMT patients. Yet, the role of thrombocytopenia in bleeding post BMT has not been systematically studied. We evaluated the risk of bleeding and outcome associated with thrombocytopenia in BMT patients who had prophylactic platelet transfusions at a trigger of 20 x 10(9)/l. Thrombocytopenia was investigated in 321 patients with moderate or severe bleeding (BLD), and in a matched comparison group of 287 patients who did not bleed (NBLD). Profound thrombocytopenia (< or = 10 x 10(9)/l) was found in 8.6% of the BLD patients during the week before the bleeding onset, significantly more frequent than in NBLD patients (2.1% to 4%, P < 0.02), during weeks 2 to 6 post BMT (the period when 75% of the bleeding initiated). On the first day of bleeding, platelet counts < or = 10 x 10(9)/l were found in 13.5%, 11-20 x 10(9)/l in 20.4%, and > 20 x 10(9)/l in 66.1% of all episodes. Overall survival in BLD patients was not associated with the severity of thrombocytopenia before bleeding onset. Severity of thrombocytopenia was significantly associated with reduced survival in NBLD patients. We concluded that bleeding post BMT was significantly associated with thrombocytopenia, but the attributable risk of bleeding from profound thrombocytopenia was not large. Thrombocytopenia may be an important clinical sign in NBLD patients, and should be further explored in relation to acute toxicities other than bleeding.

Measurement of tissue factor activity in whole blood.

High circulating levels of the procoagulant molecule tissue factor (TF) are associated with thrombosis in a variety of diseases including unstable angina, cancer, and sepsis. Currently, there are no clinical assays to measure the level of TF activity in whole blood. We present an assay called Tissue Factor Clotting Time ("TiFaCT") that detects fibrin formation in human blood. The mean baseline clotting time in a healthy population was 472 +/- 94 s (mean +/- SD, n = 150). Bacterial lipopolysaccharide (LPS or endotoxin) shortened the clotting time in a time-dependent manner. Inhibitory anti-TF antibodies prolonged the clotting time of LPS-stimulated blood, indicating that the shortened clotting time was due to induction of TF expression. Patients with unstable angina had shortened mean baseline clotting time (284 +/- 86, n = 13) compared with healthy volunteers (474 +/- 98, n = 30), suggesting that these patients had elevated levels of circulating TF. The TiFaCT assay should prove clinically useful in quantifying the levels of circulating TF in patients at risk of thrombosis.

Clinical outcomes of point-of-care testing in the interventional radiology and invasive cardiology setting.

BACKGROUND: Point-of-care testing (POCT) can provide rapid test results, but its impact on patient care is not well documented. We investigated the ability of POCT to decrease inpatient and outpatient waiting times for cardiovascular procedures. METHODS: We prospectively studied, over a 7-month period, 216 patients requiring diagnostic laboratory testing for coagulation (prothrombin time/activated partial thromboplastin time) and/or renal function (urea nitrogen, creatinine, sodium, and potassium) before elective invasive cardiac and radiologic procedures. Overall patient management and workflow were examined in the initial phase. In phase 2, we implemented POCT but utilized central laboratory results for patient management. In phase 3, therapeutic decisions were based on POCT results. The final phase, phase 4, sought to optimize workflow around the availability of POCT. Patient wait and timing of phlebotomy, availability of laboratory results, and therapeutic action were monitored. Split sampling allowed comparability of POCT and central laboratory results throughout the study. RESULTS: In phase 1, 44% of central laboratory results were not available before the scheduled time for procedure (n = 135). Mean waiting times (arrival to procedure) were 188 +/- 54 min for patients who needed renal testing (phase 2; n = 14) and 171 +/- 76 min for those needing coagulation testing (n = 24). For patients needing renal testing, POCT decreased patient wait times (phases 3 and 4 combined, 141 +/- 52 min; n = 18; P = 0.02). For patients needing coagulation testing, wait times improved only when systematic changes were made in workflow (phase 4, 109 +/- 41 min; n = 12; P = 0.01). CONCLUSIONS: Although POCT has the potential to provide beneficial patient outcomes, merely moving testing from a central laboratory to the medical unit does not guarantee improved outcomes. Systematic changes in patient management may be required.

Clinical analyzers. Advances in automated cell counting.

Recently, several hematology analyzers have been developed to improve accuracy and to reduce manual work. The new analyzers use traditional impedance technology or optical detectors alone or in combination. The introduction of argon laser technology to clinical instruments is one new development that may permit integrated flow cytometry parameters leading to a whole new horizon in routine hematologic analyses.

Construction of a human platelet alloantigen-1a epitope(s) within murine glycoprotein IIIa: identification of residues critical to the conformation of the antibody binding site(s).

The human platelet alloantigen 1 system (HPA-1) is determined by a polymorphism at position 33 in the N-terminus of human glycoprotein IIIa (GPIIIa). This naturally occurring substitution creates a conformation in the HPA-1a allelic form that can be antigenic when presented to an individual expressing the HPA-1b form. Anti-HPA-1a antibodies generated by this immune response can lead to the destruction of platelets, as seen in the clinical disorders, neonatal alloimmune thrombocytopenia (NAIT) and posttransfusion purpura (PTP). To understand better the structural requirements for recognition by these pathogenic antibodies, we investigated the N-terminal 66 amino acids from the HPA-1a form of human GPIIIa and the analogous amino acids from the nonimmunogenic murine homolog. Our objectives were to define further the boundaries of the HPA-1a epitope(s) in the N-terminus of human GPIIIa, to isolate the murine 5' nucleotide sequence and compare the deduced murine N-terminal sequence to that of human, and to mutate the murine sequence systematically to include an HPA-1a epitope(s). Murine amino acids that differed from human were changed by site-directed mutagenesis to the analogous residues in the HPA-1a form of human GPIIIa, starting and radiating from murine position 33 (site of human polymorphism). This systematic approach allowed us to pinpoint amino acids critical to a conformation recognized by anti-HPA-1a antibodies. Our results show that an HPA-1a epitope can be created within the N-terminus of murine GPIIIa and raise the possibility that murine models of HPA-1a sensitization can be developed.

Neonatal alloimmune thrombocytopenia associated with maternal anti-HLA antibody: a case report.

PURPOSE: Although neonatal alloimmune thrombocytopenia (NAIT) due to maternal sensitization to human platelet antigens is well described, the role of maternal anti-human lymphocyte antigen (HLA) antibodies in NAIT is not yet firmly established. PATIENT: A 31-week-old girl born prematurely to a G2POA1 mother was noted to have thrombocytopenia which lasted 18 days without any evidence of infection. MATERIALS AND METHODS: Platelet-associated IgG, anti-platelet antibody, and platelet PL(A1) antigen typing were determined using a commercial solid-phase red cell adherent test. Antibodies to platelet glycoproteins human platelet antigen (HPA) 1 to 5 were determined using a commercial ELISA. Anti-HLA antibodies were assayed using a standard lymphocytotoxicity test. Activities and IgG subclass of anti-HLA antibodies in plasma of the mother and other postpartum mothers were measured using purified HLA antigens in an enzyme linked immunoassay. RESULTS: Both mother and infant were positive for HPA-1 (PL(A1)) antigens. The mother's HLA phenotype was A3, A31, B7, B27. The level of platelet-associated IgG was not increased on maternal platelets; however, increased platelet-associated IgG was detected on the infant's platelets. Antibodies to platelet glycoproteins HPA1 to 5 were not detectable in the maternal plasma. Maternal serum was positive for anti-HLA antibodies, which reacted to 23 of 27 panel cells. The presence of HLA antibodies was confirmed by enzyme-linked immunoassay. Of note, the maternal antibodies reacted positively to the infant's platelets and anti-IgG anti-HLA antibodies were detected in the serum sample from the infant collected at birth. When the activity and IgG subclass of the maternal anti-HLA antibodies were compared with those of other mothers known to have high anti-HLA antibody activity, no differences were noted. CONCLUSION: This report documents a patient with neonatal thrombocytopenia induced by maternal IgG anti-HLA antibody. Neither activity nor IgG subclass could explain the occurrence of NAIT. The factors that contribute to NAIT induced by maternal anti-HLA antibodies remain to be identified.

Incomplete global cerebral ischemia alters platelet biology in neonatal and adult sheep.

Platelets are implicated as etiologic agents in cerebral ischemia and as modulators of neural injury following an ischemic insult. We examined the effects of severe, transient global ischemia on platelet aggregation during 45-min ischemia and 30-, 60-, and 120-min reperfusion in adult and neonatal lambs. We also examined postischemic platelet deposition in brain and other tissues (120-min reperfusion) using indium-111-labeled platelets. Ischemic cerebral blood flow fell to 5 +/- 1 and 5 +/- 2 ml.min-1.100 g-1 in lambs and sheep, respectively. During ischemia, platelet counts fell to 47.5 +/- 5.1% of control (P < 0.05) in lambs and 59 +/- 4.9% of control in sheep (P < 0.05). Ischemia depressed platelet aggregation response (P < 0.01) to 4 micrograms collagen in lambs and sheep (20.4 +/- 29.2 and 26 +/- 44.7% of control, respectively). Marked platelet deposition occurred in brain and spleen in sheep, whereas significant platelet entrapment occurred only in brain in lambs. Our findings suggest that ischemia causes platelet activation and deposition in brain and noncerebral tissues.

Identification of a human heavy chain antibody fragment directed against human platelet alloantigen 1a by phage display library.

The human platelet alloantigen HPA-1a (PlA1) is responsible for most cases of post-transfusion purpura and neonatal alloimmune thrombocytopenia in the Caucasian population. HPA-1a and HPA-1b are two allelic forms of the platelet membrane glycoprotein IIIa (GPIIIa) gene that differ by a single amino acid. In this report, we describe the development of a recombinant heavy chain antibody fragment capable of distinguishing between the homozygous forms of HPA-1a and HPA-1b. This antibody fragment was isolated from the lymphocytes of an immunized individual through the use of a phage display library system. The recombinant antibody fragment reacted with human platelet lysates from HPA-1a homozygous donors, the HPA-1a form of recombinant N-terminal GPIIIa and intact HPA-1a platelets, but did not react with platelet lysate from HPA-1b homozygous donors, reduced HPA-1a form of platelet GPIIIa or other platelet glycoproteins. This HPA-1a specific human antibody fragment works well in common laboratory assays such as ELISA and flow cytometry, which can assist in identifying HPA-1b homozygous individuals who are known to have a higher risk for developing neonatal alloimmmune thrombocytopenia and post-transfusion purpura. Thus, selection of recombinant antibody fragment using phage display offers a promising alternative to hybridoma technology for the production of human antibodies against human alloantigens and holds potential as a technique in therapeutic applications.

Thrombocytopenia in pregnancy.

Thrombocytopenia is a common finding in normal pregnancy. The introduction of routine automated complete blood counting has clearly documented this. In the past, these patients would go undetected and be spared unnecessary testing, procedures, or medications. This article reviews the common causes of thrombocytopenia and offers criteria on which the diagnosis of clinically significant thrombocytopenia can be made. In addition, we will discuss therapeutic approaches to manage patients with pathologic thrombocytopenia.

Measurement of in vitro P-selectin expression by flow cytometry.

Measurement of in vivo platelet activation is difficult after phlebotomy and during blood processing for analysis. We used flow cytometry to measure platelet surface expression of P-selectin in the presence and absence of trimethylsphingosine (a platelet activation inhibitor) and compared the results with those from the standard methods of preventing in vitro P-selectin expression. Percent activation was calculated as a ratio of mean sample fluorescence to 100% mean fluorescence after phorbol myristate acetate treatment. Twenty-five micromoles per liter of trimethylsphingosine kept in vitro platelet activation below 5% up to 6 hours after collection and below 10% at 24 hours after collection. Trimethylsphingosine failed to prevent platelet activation caused by centrifugation, storage at 4 degrees C, or stimulation with common agonists. Addition of trimethylsphingosine to whole blood was valuable in preventing in vitro platelet activation. This compound promises to be a useful preservative for diagnostic testing of platelet activation.