Leukemia-associated aberrant immunophenotype in patients with acute myeloid leukemia: changes at refractory disease or first relapse and clinicopathological findings.

INTRODUCTION: Multiparameter flow cytometry (MFC) is commonly used to detect minimal residual disease (MRD) during the course of chemotherapy or relapse. Only one study addressed the immunophenotypic changes in refractory disease. We studied changes in leukemia-associated aberrant immunophenotype (LAIP) in patients with refractory and relapsed acute myeloid leukemia (AML). METHOD: We analyzed 47 patients (refractory = 22; relapsed = 25) by MFC, morphology, and cytogenetic studies. RESULTS: Thirty-five patients (74%) showed variably changed LAIPs. The frequently altered LAIPs were lack of lineage-specific antigen and lineage infidelity. The most frequently changed marker was CD13, followed by CD33, CD56, CD7, CD4, and CD11b. Cytogenetic clonal evolution at persistence/relapse was observed in 15 patients (32%). Morphologically, three patients (6%) showed significant changes at relapse. Patients with refractory AML had a higher association with poor cytogenetic risk and classification of AML with myelodysplasia-related changes. Positive MRD at postinduction was of prognostic significance. Allogeneic stem cell transplant improved overall survival. CONCLUSIONS: LAIP alterations in refractory/relapsed AMLs are common findings. Presence of persistent disease indicates a poor prognosis, regardless of cytogenetic risk or expression of CD7 or CD56. Discordance between cytogenetic and LAIP changes suggests that gross cytogenetic clonal evolution during disease progression only partly contributes to immunophenotypic instability.

Evidence of a phospholipid binding species within human fibrinogen preparations.

Fibrinogen has been reported to interact with phospholipid; however, the properties of this binding interaction have not been characterized. Purified preparations of human fibrinogen bound to small unilamellar vesicles containing phosphatidylserine (PS) as measured by light scattering and radioisotope filtration. Binding to 100% PS was saturable (apparent Kd=5 microM, Bmax=1.9 g protein/g lipid), reversible, and involved a minor subfraction of the fibrinogen preparation (3-6% of total protein). Fibrinogen interacted minimally with phosphatidylinositol, and not at all with pure phosphatidylcholine (PC) or PC vesicles containing 5% glycosphingolipid (lactosylceramide, ganglioside GM3, ganglioside GD3). Binding efficiency decreased as the PS content of vesicles was diluted with PC. Calcium chloride (2 mM) enhanced protein binding to PS, which was reversed by EDTA. Fibrin clot formation almost quantitatively precipitated the PS binding activity. PS, but not PC, increased the final turbidity of fibrin clots. Computerized sequence analysis of fibrinogen revealed three candidate acidic phospholipid binding motifs located at position 143-210 in the alpha chain, and positions 59-77 and 101-139 in the beta chain. Further study of the PS binding activity of fibrinogen may lead to new insights about fibrinogen function.

A method for proficiency testing of small peer groups in the College of American Pathologists Coagulation Surveys.

OBJECTIVE: To develop a grading scheme for the proficiency testing of small peer groups of fewer than 10 members for the prothrombin time (PT) and activated partial thromboplastin time (APTT). METHODS: A modified target value for small peer groups was derived based on the assumption that measurement variability in the PT and APTT is more greatly influenced by variations in reagents than in instruments. Criteria for grading were established by statistical simulation to achieve misclassification errors of less than 5% for both incorrectly passing and failing participants. College of American Pathologists Coagulation Survey data were analyzed to determine the number of additional laboratories graded using the proposed scheme, as well as the failure rates among participants in the small peer groups. RESULTS: The modified target value for small peer groups is a weighted average between the mean of the peer group and the mean of all participants using the same reagent (reagent group). Peer groups with as few as 4 members can be graded provided that specific criteria are satisfied: there must be at least 5 peer groups for the same reagent, at least 3 of these 5 peer groups must have more than 3 members, and the coefficient of variation for the reagent group must be less than 10%. This proposed grading scheme decreased the number of ungraded laboratories by 44% to 46% for the PT and 42% to 55% for the APTT. The percentage of failing grades among participants in the small peer groups ranged from 1.3% to 4.1% for the PT and 1.4% to 7.2% for the APTT. These failure rates were 2.8- to 13.0-fold higher than the failure rates in large peer groups (P < or = .05). CONCLUSIONS: The proposed small peer group grading scheme can improve the effectiveness of College of American Pathologists proficiency testing for the PT and APTT and may also be generally applicable to other test methods and analytes.

Laboratory heparin resistance in burn injury complicated by venous thrombosis.

Anticoagulation with heparin is required in the management of the burn patient if their clinical course is complicated by venous thrombosis. Heparin therapy is commonly monitored by the activated partial thromboplastin time (APTT) but this assay can be unreliable in patients with acute inflammation because of an increase in plasma factor VIII levels that result in an underestimation of the heparin concentration. We report an example of heparin resistance that occurred in a patient who developed venous thrombosis following extensive second-degree burns. Heparin doses in excess of 60,000 units per day were required to produce a significant elevation in the APTT. The plasma factor VIII level was found to be markedly elevated to 455% and the plasma heparin concentration as determined by the anti-factor Xa assay was disproportionately elevated in relation to the APTT. Physicians treating patients with burn injury complicated by venous thrombosis should be aware of the potential development of factor VIII-related heparin resistance when large amounts of heparin are required to obtain a satisfactory elevation in the APTT. Measurement of the plasma heparin concentration will avoid excessive heparin administration and the serious bleeding which can result.

College of American Pathologists Conference XXXI on laboratory monitoring of anticoagulant therapy: laboratory monitoring of unfractionated heparin therapy.

OBJECTIVE: To review the state of the art as reflected in the medical literature and the consensus opinion of recognized experts in the field regarding the laboratory monitoring of unfractionated heparin therapy. DATA SOURCES, EXTRACTION AND SYNTHESIS: The authors made an extensive review of the literature. The draft manuscript was circulated to every participant in the consensus conference prior to the convening of the conference. Extensive discussion concerning all of the issues addressed in the manuscript as well as the resulting recommendations occurred. This information was then used to revise the manuscript into its final form. CONCLUSIONS: The resulting manuscript has 23 specific recommendations regarding preanalytic, analytic, and postanalytic phases of monitoring and testing for complications related to unfractionated heparin therapy. This report contains detailed discussion of these recommendations and includes literature citations that support them. A number of issues for which consensus could not be reached are also discussed. A method is provided to assist laboratories, particularly small laboratories, in providing clinicians with an appropriate therapeutic range for the activated partial thromboplastin time, the most commonly used test in monitoring heparin therapy.

Low interinstrument variability of the international normalized ratio with the coagamate X2/Simplastin excel system.

Minimizing the interlaboratory variability of the International Normalized Ratio (INR) for patients receiving coumarin therapy will require local laboratory calibration of individual coagulation instrument/reagent systems. However, some laboratories possess multiple coagulation instruments of the same model, and it is unclear if each instrument would require separate calibration. To address this question, a controlled study was performed that examined the interinstrument variability of the INR on three separate Coagamate X2 coagulometers (Organon Teknika, Durham, NC) using the Simplastin Excel reagent (Organon Teknika) (International Sensitivity Index = 2.14). The interinstrument coefficient of variation (CV) of the INR among the three instruments was 3.4% and 3.5% for patient control plasmas (n = 20) and coumarin plasmas (n = 40, INR range 1.5-4.6), respectively. The number of discordant INRs between paired instruments (one INR within and one INR out of the therapeutic range, plus a difference of at lest 0.4 INR units) was very low (0%). The interinstrument INR CVs for three commercial quality control plasmas were 2.6% (INR = 0.92), 4.1% (INR = 2.1), and 6.3% (INR = 4.5), and correlated well with the low CVs for patient samples. This study showed that the Coagamate X2/Simplastin Excel system is capable of very low interinstrument INR variability, and surpasses the interlaboratory CVs reported in the literature. Formal calibration of a single instrument may be adequate for laboratories possessing multiple instruments of the same model.

The reliability of manufacturer-determined, instrument-specific international sensitivity index values for calculating the international normalized ratio.

Some thromboplastin manufacturers are currently supplying the instrument-specific international sensitivity index (ISI) values of their reagents, allowing clinical laboratories to calculate instrument-specific international normalized ratio (INR) values on plasma samples from patients receiving coumarin therapy. However, the assumption that systematic interinstrument variability in the INR would be eliminated if manufacturer-determined ISI values were used remains unsubstantiated. This assumption was evaluated by comparing INR values obtained on one instrument that measures a mechanical endpoint (fibrometer) with one that measures a photo-optical endpoint (MLA-700). Three thromboplastin reagents with instrument-specific ISI values supplied by the manufacturer (ISI range, 1.23-2.79) were used. For two of three reagents, the fibrometer INR values were significantly higher than the MLA-700 INR values (P < .01). Analysis of log prothrombin time ratio plots showed that this systematic variability was caused by inaccurate manufacturer ISI values. Of clinical significance is that the inaccurate ISI values produced a high number of discordant INR values between these two instruments (> 47% of plasma samples had one INR value within and one out of the recommended therapeutic range). The implication of these findings for laboratory monitoring of oral anticoagulation is discussed.

Glycosphingolipid inhibition of the adhesion of thrombin-activated platelets to surfaces is potentiated by albumin.

Previous studies have shown that exogenous glycosphingolipids (GSLs) inhibit the adhesion of thrombin-activated platelets (TAP) to polystyrene plates coated with various RGD-ligands (where RGD is the peptide sequence Arg-Gly-Asp), suggesting that GSLs can modulate the platelet integrin receptor glycoprotein IIb-IIIa. However, albumin was always used as a plastic surface-blocking agent in these studies. In order to evaluate the role of albumin in these experiments, we studied the effect of various GSLs and albumin on the interaction between TAP and hydrophobic surfaces in a solid-phase assay using indium-111-labelled platelets and polystyrene plates. TAP (10(8) platelets/ml) adhered to polystyrene (half-saturation time 40 +/- 3 min) with a maximal adhesion density of 56 +/- 1 x 10(3) platelets/mm2. Platelet adhesion was only slightly affected (< 11% inhibition) by immobilized bovine serum albumin, immobilized mixed bovine brain gangliosides (MBG) or fluid-phase MBG. In contrast, fluid-phase MBG was an effective inhibitor of platelet adhesion to polystyrene (> 46% inhibition), but only after albumin was first immobilized to the plate. Covering albumin-coated polystyrene with MBG, followed by washing, was as effective as fluid-phase MBG at inhibiting platelet adhesion, thus indicating that a ganglioside-albumin interaction at the polystyrene surface was responsible for effective inhibition. When purified GSLs were substituted for MBG, it was found that all those tested (GT1b, GD1a, GM1, asialo GM1 and globoside) had similar inhibitory activity.

The role of membrane lipid in the platelet storage lesion.

Because of their hemostatic and structural importance and their chemical and physical lability, membrane lipids are likely to be involved in the development of the platelet storage lesion. Chemical analysis using the new method of high-performance liquid chromatography with laser light scattering detection (HPLC-LLS) reveals platelet lipid to be composed of more than 22 individual components, the most abundant of which are phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol (C), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylinositol (PI). Surprisingly, an asymmetric distribution of these lipids is maintained in the resting platelet with PS concentrated in the inner leaflet of the plasma membrane. The exposure of PS may be important in platelet activation because of its powerful procoagulant effect. Studies of the effect of blood bank storage on platelet lipid composition have repeatedly shown a steady loss of all components, which may be temperature dependent. Studies of platelet factor 3 activity and flow cytometry of stored platelets have revealed the lipid is lost through the process of microvesiculation. Coupled to this storage induced depletion of platelet lipid is a loss of more than half of the potential capacity of lipid-dependent platelet functions by day 5. The most likely underlying mechanism for this loss of lipid mass and functional capacity is lipid peroxidation, a process that could be blocked with antioxidants. Lipid peroxidation may also interfere with other membrane constituents such as glycoprotein IIb/IIIa and the aminophospholipid-specific translocase. Thus, lipid peroxidation should be a major focus in studies aimed at preventing or reversing the platelet storage lesion.

Comparison of the kinetic behavior of human and bovine proteins in the heparin-catalyzed antithrombin III/thrombin reaction.

Significant differences between saturation kinetic properties of heparin-stimulated reactions between thrombin and antithrombin III from human and bovine species were observed. In both systems, the apparent Km for antithrombin III was higher than the KD for antithrombin III-heparin interaction, monitored by intrinsic protein fluorescence change. The Km for thrombin and kcat were much higher for proteins of the human species than the bovine species. The apparent Km for one human protein was dependent on the concentration of the other human protein, indicating interaction of the binding events. The reaction product formed from the bovine proteins was a potent inhibitor of the reaction but the product from the human proteins was a poor inhibitor. The major differences between the two species appeared to be related to interaction of thrombin or thrombin derivatives with heparin or heparin-antithrombin III complexes.

Plasma hemoglobin determination by recording derivative spectrophotometry.

Recording derivative spectrophotometry (DS) is a technic for analyzing absorbance curves by measuring the derivative (slope) of those curves. The derivative is a mathematical function that enhances rapid changes of direction of a curve and suppresses slow changes. We are presenting a simple, rapid, and reproducible method for determining plasma hemoglobin concentration, even in the presence of bilirubin, myoglobin, or marked turbidity. No toxic reagents are used and, since the analysis is purely physicomathematic, the sample is not altered and can be used for additional tests.

Molecular weight analysis of antithrombin III-heparin and antithrombin III-thrombin-heparin complexes.

The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added to antithrombin III, the molecular weight increased to a maximum and then decreased to that of a 1:1 (antithrombin III X heparin) complex. The initial molecular weights at low heparin to antithrombin III ratios were consistent with the formation of a 2:1 (antithrombin III X heparin) complex in which only one antithrombin III molecule had undergone the conformational change measured by protein fluorescence enhancement. The peak molecular weight never reached that of a complete 2:1 complex. This behavior was observed for bovine and human antithrombin III in the presence of both unfractionated heparin and high molecular weight-high affinity heparin. Pentosane polysulfate also caused some multiple associations. Bovine antithrombin III and thrombin formed a 1:1 complex that underwent further aggregation within minutes, while the human proteins did not aggregate on this time scale after forming the 1:1 complex. In the presence of stoichiometric amounts of heparin, the bovine proteins formed an initial complex of Mr = 230,000 (corresponding to a dimer of heparin-antithrombin III-thrombin) which underwent further aggregation. The human proteins, however, formed a 1:1 (antithrombin III X thrombin) initial complex in the presence of heparin, followed by aggregation. These interactions of thrombin and antithrombin with heparin suggest complex interactions that could relate to heparin function.