Non-radiation risk factors for leukemia: A case-control study among chornobyl cleanup workers in Ukraine.

BACKGROUND: Occupational and environmental exposure to chemicals such as benzene has been linked to increased risk of leukemia. Cigarette smoking and alcohol consumption have also been found to affect leukemia risk. Previous analyses in a large cohort of Chornobyl clean-up workers in Ukraine found significant radiation-related increased risk for all leukemia types. We investigated the potential for additional effects of occupational and lifestyle factors on leukemia risk in this radiation-exposed cohort. METHODS: In a case-control study of chronic lymphocytic and other leukemias among Chornobyl cleanup workers, we collected data on a range of non-radiation exposures. We evaluated these and other potential risk factors in analyses adjusting for estimated bone marrow radiation dose. We calculated Odds Ratios and 95% Confidence Intervals in relation to lifestyle factors and occupational hazards. RESULTS: After adjusting for radiation, we found no clear association of leukemia risk with smoking or alcohol but identified a two-fold elevated risk for non-CLL leukemia with occupational exposure to petroleum (OR=2.28; 95% Confidence Interval 1.13, 6.79). Risks were particularly high for myeloid leukemias. No associations with risk factors other than radiation were found for chronic lymphocytic leukemia. CONCLUSIONS: These data - the first from a working population in Ukraine - add to evidence from several previous reports of excess leukemia morbidity in groups exposed environmentally or occupationally to petroleum or its products.

Altered glycosylation leads to Tr polyagglutination.

BACKGROUND: Polyagglutination refers to red blood cells (RBCs) that are agglutinated by a high proportion of ABO-matched adult sera but not by cord sera. Polyagglutinable RBCs have been associated with microbial infection, myeloproliferative disorders, and myelodysplasia. Lectins aid in the identification of polyagglutination. CASE STUDY: A Hispanic male infant with mild hemolytic anemia, a "Bernard-Soulier-like" syndrome, intermittent neutropenia, mitral valve regurgitation, ligament hyperlaxity, and mild mental retardation was studied. The patient's Group O RBCs were polyagglutinable; they were agglutinated by normal human sera, several lectins [including Arachis hypogea, Salvia sclarea, Salvia horminum, Glycine max, Ulex europaeus, Griffonia simplicifolia I, and Gr. simplicifolia II], and some monoclonal antibodies. His RBCs were not agglutinated by cord sera, Dolichos biflorus, or Phaseolus lunatus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the RBC membranes followed by staining with periodic acid-Schiff stain showed markedly reduced staining of glycophorins A and B. Staining with Coomassie brilliant blue revealed that Band 3 has a faster mobility than normal. CONCLUSIONS: Collectively, the results suggest that the patient's RBCs have a reduction in N-acetylneuraminic acid on both N- and O-glycans, exposing, respectively, beta1,4-galactosidase and beta1,3-galactosidase. The patient likely has an altered glycosyltransferase that results in defective glycosylation in RBCs and other cell lineages. This type of polyagglutination was named Tr.

Cell typing the sensitized transfusion-dependent patient.

Extended red cell typing is required for the management of transfusion-dependent patients to confirm the identity of suspected alloantibodies or determine the specificity of potential additional antibodies that may be formed in the future. Typing may be complicated by the presence of circulating allogeneic cells or a positive direct antiglobulin test. Phenotyping such individuals by hemagglutination is dependent on the separation of a reticulocyte-enriched fraction by differential centrifugation. Flow cytometric typing of reticulocytes is also possible. The effectiveness of these techniques is limited in those who are heavily transfused or have low reticulocyte counts. Heavily transfused patients with sickle cell anemia may be typed, however, following hypotonic lysis of allogeneic cells. In patients with a positive direct antiglobulin test, sensitized cells are usually typed with either direct agglutinating antisera and/or IgG antisera following elution of the autoantibody. Inactivation of some antigens during the elution process or the lack of some antisera specificities limit such typing. By designing appropriate oligonucleotide primers, polymerase chain reaction (PCR) amplification of target gene sequences for most blood group systems and the identification of a large number of their allelic specificities is now possible. Peripheral blood leukocytes can be used as the DNA source. Restriction fragment length polymorphism determination is widely adopted for the identification of allelic specificity of the amplified target sequence. Alternate strategies, including allele-specific PCR, are often employed if the genetic basis of the polymorphism is more complex than a single nucleotide substitution, or if it does not create or ablate a restriction endonuclease cleavage site. These techniques may permit genotyping of sensitized transfusion-dependent patients, and can improve transfusion safety and efficacy.

Hemostatic defects in massive transfusion: rapid diagnosis and management.

Severe hemostatic defects that occur during massive transfusion are related to the volume of blood transfused, preexisting hemostatic abnormalities, concomitant pathologic changes, and therapeutic maneuvers. The relative role of each factor in the bleeding can be rapidly determined by using routine clinical laboratory tests. This determination requires an understanding of the properties of selected clotting factors, what coagulation screening tests measure, how these tests behave as the levels of factors change, and test profiles characteristic of different defects. Screening tests include platelet count, prothrombin time, partial thromboplastin time, thrombin time, and fibrinogen level. These tests are generally available on an emergent basis and can be completed within 25 minutes. The pattern associated with hemodilution is universal in massive transfusion. Patterns characteristic of the other pathologic processes that may be encountered are simply superimposed on the characteristics of hemodilution. Successful management of the contributing causes of bleeding depends on the administration of the appropriate blood components in the dose necessary to ensure that the levels of platelets and clotting factors are returned to and maintained at hemostatic levels.

CD34+ cell dose requirements for rapid engraftment in a sequential high-dose chemotherapy regimen of paclitaxel, melphalan, and cyclophosphamide, thiotepa, and carboplatin (CTCb) with PBPC support in metastatic breast cancer.

Sequential high-dose chemotherapy may increase the threshold dose of CD34+ cells necessary for rapid and successful hematologic recovery. There are limited data regarding the pharmacodynamics and threshold CD34+ cell dose required for engraftment following high-dose paclitaxel. To determine the dose of CD34+ PBPC sufficient for rapid engraftment, 65 women with metastatic breast cancer undergoing a sequential high-dose paclitaxel, melphalan, and cyclophosphamide, thiotepa, and carboplatin (CTCb) chemotherapy regimen were evaluated. The intertreatment interval was a median of 27 days. Paclitaxel was escalated from 400 to 825 mg/m2, infused continuously (CI) over 24 h on day -4 with PBPC reinfusion on day 0. Following marrow recovery, 90 mg/m2/day of melphalan was given over 30 min on days -2 and -1, with PBPC reinfusion on day 0. On recovery, patients received CTCb on days -7 to -3, with PBPC reinfusion on day 0. G-CSF was administered after each cycle until WBCC recovery. For paclitaxel, an ANC >0.5 x 10(9)/L occurred at a median of 6 days (range 0-7 days) after PBPC reinfusion. The median nadir platelet count was 63 x 10(9)/L (range 6 x 10(9)/L-176 x 10(9)/L). Eight patients (12%) had platelet nadir <20 x 10(9)/L, and all recovered their counts to >20 x 10(9)/L on day 7. There was no clinical difference in days to engraftment between women receiving <2 or > or =2 x 10(6) CD34+ PBPC/kg following paclitaxel. All patients recovered neutrophil and platelet counts within 7 days after reinfusion of > or =1 x 10(6) CD34+ cells/kg and G-CSF. The data suggest that a paclitaxel dose of 825 mg/m2 is not myeloablative. For melphalan, median days to ANC >0.5 x 10(9)/L was 10 days (range 9-15), and platelet recovery to >20 x 10(9)/L was 13 days (range 0-28) after PBPC reinfusion. Median time to engraftment was more rapid in patients receiving > or =2 x 10(6) CD34+/kg versus <2 x 10(6)CD34+/kg, for both neutrophils (11 days versus 10 days, p = 0.05) and platelets (14 days versus 12 days, p < 0.01). Ninety-eight percent of patients infused with > or =2 x 10(6) CD34+/kg engrafted within 21 days. Following CTCb in this sequential regimen, a dose of > or =2 x 10(6) CD34+ cells/kg provided for significantly more rapid neutrophil engraftment than <2 x 10(6) CD34+ cells/kg (9 days versus 10 days,p = 0.01), but a dose > or =3 X 10(6) CD34+ cells/kg is necessary for reliable, rapid, and sustained neutrophil and platelet engraftment by day 21.

Effects of long-term storage at -90 degrees C of bone marrow and PBPC on cell recovery, viability, and clonogenic potential.

Autologous BM and PB HPC are usually stored from weeks to months until reinfusion after myeloablative chemotherapy. HPC have been stored for up to 16 months at -90 degrees C, using a mixture of 5% DMSO, 6% hydroxyethyl starch (HES), and 4% HSA as a cryoprotectant. Long-term storage (LTS) has usually entailed rate-controlled freezing using 10% DMSO and preservation in liquid nitrogen. The effects of LTS at -90 degrees C on the in vitro cell recovery, viability, and colony-forming unit-granulocyte macrophage (CFU-GM) clonogenic potential of autologous HPC that were not transplanted was studied. Sixteen BM and sixteen PB HPC had been cryopreserved for a median of 53 months (range 27-71) and 35 months (range 26-78), respectively. Samples of frozen HPC were thawed after 48 h, and the nucleated cell count, viability by trypan blue exclusion, and culture for CFU-GM were obtained. Following LTS, the cells were thawed and examined using the same assays. No difference in the median percentage recovery of nucleated cells was found in either the BM or PB HPC between the samples stored for 48 h and after LTS (5.73 x 10(9) versus 5.61 x 10(9) and 6.20 x 10(9) versus 5.78 x 10(9), respectively). In addition, no difference in median percentage viability was found in either the BM or PB HPC sampled at 48 h and at the end of LTS (75% versus 74% and 75% versus 76%, respectively). Finally, the median number of CFU-GM cultured from BM HPC at 48 h was 2.41 x 10(5) (range 0.33-11.01 x 10(5)) and at the end of LTS was 1.93 x 10(5) (range 0.32-10.55), representing a median recovery of 93% (range 19%-308%). Similarly, the median number of CFU-GM cultured from PB HPC was 1.66 x 10(5) (range 0-50.57) and at the end of LTS was 0.93 x 10(5) (range 0-44.9), representing a median recovery of 80% (range 36%-165%). This difference in percentage recovery was not significant (p = 0.514). There was poor correlation between the number of nucleated cells harvested and the percentage recovery of nucleated cells, cell viability, or CFU-GM for either the BM or PB HPC. Similarly, there was poor correlation between the number of CFU-GM in the harvest and their percentage recovery following LTS for both BM and PB HPC. Finally, there was poor correlation between the storage time of the BM or PB HPC and the percentage recovery of nucleated cells, cell viability, and CFU-GM. These data suggest that LTS of HPC at -90 degrees C is not associated with decreased recovery of nucleated cells or in vitro viability and is associated with only a modest decrease in clonogenic potential. This indicates that storage of HPC at -90 degrees C for periods in excess of 3 years is possible.

Harvest quality and factors affecting collection and engraftment of CD34+ cells in patients with breast cancer scheduled for high-dose chemotherapy and peripheral blood progenitor cell support.

The use of CFU-GM and CD34+ cell enumeration for assessing harvest quality and factors affecting peripheral blood progenitor cell (PBPC) harvest and engraftment were investigated in 45 women with high-risk and metastatic breast cancer scheduled for dose-intensive cyclophosphamide, thiotepa, and carboplatin (CTCb). PBPC were mobilized with standard breast cancer regimens or cyclophosphamide (1.5 g/m2) and 5 micrograms/kg/day G-CSF and used together with G-CSF for hematopoietic support post-CTCb. There was a significant correlation between peripheral blood CD34+ cells/microliter and harvest CD34+/kg (r = 0.73, p < 0.0001) and between harvest CFU-GM and CD34+ cells/kg (r = 0.5, p < 0.0001). CFU-GM clonogenic assays were of no clinical use beyond that of CD34+ cell enumeration, with the latter allowing for real-time decisions regarding harvesting. Multiple stepwise regression identified the number of prior chemotherapy cycles as the only significant clinical predictor of CD34+ cell yield. For 34 patients proceeding to CTCb with PBPC support, multiple stepwise regression identified as the best predictors for engraftment CFU-GM and CD34+ cells/kg for neutrophils and CFU-GM, CD34+ cells/kg, and the number of prior cycles of chemotherapy for platelets, respectively. A threshold dose of 1 x 10(6) CD34+ cells/kg, obtained in 87% of these heavily pretreated breast cancer patients, was adequate to ensure engraftment within 15 days. There was no significant difference in length of hospital stay or blood product use between patients receiving 1-2.5 x 10(6) CD34+ cells/kg and greater than 2.5 x 10(6) CD34+ cells/kg, although median time to engraftment of neutrophils (9 days versus 8 days, p = 0.007) and platelets (12 days versus 9 days, p = 0.006) was significantly longer. The established threshold of > or = 1 x 10(6) CD34+ cells/kg will allow for more confident consideration of using aliquots of this threshold dose for hematopoietic support in sequential high-dose regimens inclusive of CTCb.

A semiautomated technique for volume reduction of stem cell suspensions for autotransplantation.

Infusion of thawed cryopreserved autologous stem cells (SC) is associated with a variety of complications due to the presence of dimethyl sulfoxide (DMSO) and free hemoglobin and to volume overload. Commonly, the DMSO is not removed before infusion for fear that prolonged in vitro exposure of the cells to DMSO leads to loss of clonogenic activity. We describe a simple technique for the substantial reduction in volume and DMSO content of bone marrow (BM) and peripheral blood stem cell (PBSC) suspensions. Sixty-five patients were transplanted with thawed, volume-reduced SC cryopreserved according to Stiff et al. Semiautomated volume reduction was performed on a COBE 2991 cell processor. The median volumes of cryopreserved SC were 1152 ml and 933 ml for the pools of PBSC products and the mixed pools of BM and PBSC, respectively, whereas the volume of SC infused was 153 ml (78% reduction). There were no differences in cell recoveries between PBSC and BM (98%). Only 2 patients demonstrated minimal side effects during infusion. A cohort of 16 metastatic breast cancer patients underwent PBSC harvests following chemotherapy/G-CSF priming and subsequent autotransplantation. Median time to an absolute neutrophil count > 500/microliters was 8 days (range 6-14 days), and median time to a platelet count > 20,000/microliters was 11 days (range 6-18 days). Volume reduction of SC products without the risk of graft failure was performed simply and resulted in few complications during infusion.

Autologous fibrin glue from intraoperatively collected platelet-rich plasma.

A simple and inexpensive means of creating autologous fibrin glue is described that avoids the potential disadvantages of conventionally obtained material. This improvement may allow more widespread use of fibrin glue for operative bleeding.

Effects of X-linked hemoglobin on in-vitro platelet function.

The in-vitro function of platelets in 1:1 mixtures of fresh whole blood and 10% DBBF alpha-alpha cross-linked hemoglobin in Ringer's acetate buffer was assessed by quantitative aggregation after challenge with common agonists and compared to the function of platelets in similar dilutions of whole blood in saline. Whole blood aggregometry was performed after addition of ADP, collagen, and ristocetin. Aggregation was quantified by measuring the change in impedance as drawn on the chart recording. No difference in mean impedance change was noted between the groups of blood samples which were incubated and tested in the hemoglobin solution and those incubated and tested in saline. In addition, incubation and stirring of the above mixtures over a period of six minutes without the addition of agonists did not result in spontaneous platelet aggregation. We conclude that alpha-alpha cross-linked hemoglobin, in the concentration studied, does not affect in-vitro platelet function, as measured by whole blood aggregometry.

Blood grouping with the Olympus PK7100 testing system.

The Olympus PK7100, an automated microtitre blood typing machine, identifies, aliquots, and dilutes samples, adds reagents, photometrically detects agglutination, and records reaction results, with a throughput of 240 samples/h. A total of 20,147 donors was tested in parallel with the Groupamatic 360. Of these, 207 could not be ABO typed after two runs. Three samples typed as B by the Olympus were found to be A2B. Seventy-seven could not be typed for D after two runs. Of these, 55 were Rh positive and 22 negative. The Olympus identified 37 of 48 Du positive samples as Rh positive, while it typed three as Rh negative, and eight as 'uncertain'. None of these samples was identified as Rh positive by the Groupmatic 360. The Olympus PK7100 is accurate, reliable, easy to operate, and capable of high throughput with minimal operator intervention.

Partial plasma exchange in patients with AIDS and Kaposi's sarcoma. Plasmapheresis in AIDS.

Intensive plasma exchange was performed in seven male homosexual patients with AIDS and Kaposi's sarcoma. Serial 1.2 plasma volume exchange procedures were performed three times a week for six weeks. In five of the patients, plasma replacement included gamma globulin in the form of plasma (two patients), or an IV IgG preparation (three patients). No changes in the mean number of helper-inducer or suppressor-cytotoxic cells were noted during the treatment period or the weeks following completion of the last procedure. The mean mitogenic response of the patients' lymphocytes to PHA increased by 32.4% during the course of the plasmapheresis procedures (p less than .05), but returned to baseline over the eight weeks following treatment. Mitogenic responsiveness to PWM did not significantly increase during the course of treatment. No regression of Kaposi's sarcoma lesions was found in any of the patients treated.

Coagulation factor activity in platelet concentrates stored up to 7 days: an in vitro and in vivo study.

The in vitro and in vivo recovery of coagulation factor activity in platelet concentrates stored up to 172 h was studied. In vitro studies revealed that fibrinogen and antithrombin III levels do not change with storage. Factors II, V, VII, VIII, IX, X and XI all showed statistically significant falls from baseline over the 172 h storage period. However, most factor activities remained above 70%, with the major exception of factors V and VIII. These factors fell to less than 30% activity over the storage period, consistent with their known lability during whole blood storage. In vivo studies after platelet concentrate infusion in patients with concurrent thrombocytopenia and coagulation deficiencies revealed that the measured in vitro activity was recoverable in vivo. We conclude that platelet concentrates stored for 172 h are an adequate source of clotting factors. However, like stored whole blood, they may not provide therapeutic doses of factors V and VIII.

Granulocyte enriched buffy coat transfusions in neutropenic patients.

Twenty four courses of granulocyte enriched buffy coat transfusions were administered to 22 different infected neutropenic patients. Those patients who received an average of greater than or equal to 13 units per transfusion, which represented a mean of 1.02 X 10(10) granulocytes, had a survival rate of only 30% which was not significantly different from the 28.5% found among patients who received an average of less than or equal to 12 units per transfusion, which represented a mean of .63 X 10(10) granulocytes. In addition, no significant difference in survival rate was found between patients who received a course of greater than or equal to four transfusions and those who received a course of less than or equal to three transfusions. Finally, no significant difference in survival rate was found between patients with acute leukemia and those with other disorders or between patients with positive cultures and those whose cultures were negative. Given the poor clinical results associated with buffy coat transfusions, it is concluded that every effort should be made to recruit single leukapheresis donors for the support of infected neutropenic patients, rather than use granulocyte enriched buffy coats as they are presently produced.

Transfusion of granulocyte rich buffy coats to neutropenic patients.

Granulocyte rich buffy coats were transfused to infected neutropenic patients when leukapheresis donors were not available. Efficacy of transfusions was evaluated from data supplied by hospitals administering them. Buffy coats separated from ACD blood contained a mean of 4.9 X 10(8) granulocytes. Fifty-seven patients received a course consisting of a mean of 3.8 transfusions. Of these, 27 received a mean of 17.5 units per transfusion and had a survival rate of 44.4%, which was not significantly different from the 50.0% found in 30 who received a mean of 11.1 units per transfusion. No significant difference in survival rate was found between 31 patients with acute leukemia and 26 with other disorders or 38 patients with positive and 19 with negative cultures. Finally, no significant difference in survival rate was noted between patients who received a course of greater than or equal to four transfusions or less than or equal to three transfusions in any of the above groups. Survival rates were less than those generally reported following similar courses of leukapheresis units. Buffy coat transfusions consisting of a mean of approximately 17.5 units as produced during this study have therefore been shown to be not generally beneficial. The increased survival seen in some studies utilizing leukapheresis products may relate in part to the larger number of granulocytes they contained. Greater benefit from buffy coat transfusions might result if the number of granulocytes infused were increased. Evaluation of possible efficacy associated with transfusions of increased numbers of buffy coat units further enriched with granulocytes may be justified when leukapheresis donors are not available.