Recurrent transfusion-related acute lung injury after a two-year interval.

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. The epidemiology and pathogenesis of TRALI are not well established. A Medline literature search shows only rare reports of recurrent TRALI, all occurring soon after the first episodes. We report a case of recurrent TRALI after a 2-year interval. A patient developed TRALI after transfusion of 4 units of fresh frozen plasma for gastrointestinal bleeding due to oesophageal varices in September 2002. The patient required mechanical ventilation but recovered completely. Two years later, in October 2004, the patient experienced a second episode of TRALI during liver transplantation for hepatitis C virus /alcoholic cirrhosis. Again, the patient recovered after ventilator support. Laboratory investigation of the first TRALI episode (2002) showed antibodies against class II human leukocyte antigens (HLA) in three female donors. Laboratory investigation of the second episode (2004) showed anti-DR52 (HLA class II) antibodies in one female donor matching the DR-52 HLA class II antigen in the recipient. TRALI can rarely recur. Consideration of future blood needs for patients experiencing recurrent TRALI should include preventive measures against further TRALI reactions, such as blood from male donors or blood less than 14 days old.

Plasma fibrinogen gamma' chain content in the thrombotic microangiopathy syndrome.

BACKGROUND: Human fibrinogen gamma chain variants, termed gamma' chains, contain a unique 20-residue sequence after gamma chain residue 407 that ends at gamma'427, and is designated gamma'(427L). Full-length (FL) gamma'(427L) chains are constituents of a fibrin-dependent thrombin inhibitory system known as antithrombin I, whereas a gamma' chain processed in vivo, termed gamma'(423P), lacks the C-terminal tetrapeptide EDDL, and does not bind thrombin. Together, the gamma'(423P) and gamma'(427L) chains comprise the total plasma fibrinogen gamma' chain content. OBJECTIVES: Lowered plasma gamma' chain content (i.e. gamma' chain-containing fibrinogen/total fibrinogen ratio) has been shown to correlate with susceptibility to venous thrombosis, thus prompting this study on the total and FL gamma' chain content in 45 subjects with thrombotic microangiopathy (TMA), a disorder characterized by microvascular thrombosis. METHODS: We measured by enzyme-linked immunosorbent assay the total gamma' chain-containing fibrinogen/total fibrinogen (Total gamma'-fgn/Total fgn) ratio and the FL gamma' chain-containing fibrinogen/total fibrinogen (FL gamma'-fgn/Total fgn) ratio in these plasmas and in healthy subjects (n = 87). RESULTS: In healthy subjects, the mean Total gamma'-fgn/Total fgn ratio was 0.127, whereas the FL gamma'-fgn/Total fgn ratio was somewhat lower at 0.099 (P < 0.0001), a difference reflecting the presence of gamma'(423P) chains. In TMA plasmas, both the Total gamma'-fgn and FL gamma'-fgn/Total fgn ratios (0.099 and 0.084, respectively) were lower than those of their healthy subject counterparts (P < 0.0001). CONCLUSIONS: These findings in TMA suggest that reductions in the gamma' chain content indicate reduced antithrombin I activity that may contribute to microvascular thrombosis in TMA.

Severe citrate toxicity complicating volunteer apheresis platelet donation.

We report a case of severe citrate toxicity during volunteer donor apheresis platelet collection. The donor was a 40-year-old female, first-time apheresis platelet donor. Past medical history was remarkable for hypertension, hyperlipidemia, and depression. Reported medications included bumetanide, pravastatin, and paroxetine. Thirty minutes from the start of the procedure, the donor noted tingling around the mouth, hands, and feet. She then very rapidly developed acute onset of severe facial and extremity tetany. Empirical treatment with intravenous calcium gluconate was initiated, and muscle contractions slowly subsided over approximately 10 to 15 minutes. The events are consistent with a severe reaction to calcium chelation by sodium citrate anticoagulant resulting in symptomatic systemic hypocalcemia. Upon additional retrospective analysis, it was noted that bumetanide is a loop diuretic that may cause significant hypocalcemia. We conclude that careful screening for medications and underlying conditions predisposing to hypocalcemia is recommended to help prevent severe reactions due to citrate toxicity. Laboratory measurement of pre-procedure serum calcium levels in selected donors may identify cases requiring heightened vigilance. The case also illustrates the importance of maintaining preparedness for managing rare but serious reactions in volunteer apheresis blood donors.

Age-dependent prevalence of vascular disease-associated polymorphisms among 2689 volunteer blood donors.

BACKGROUND: The development of vascular disease involves the interaction of genetic and environmental factors. Because vascular disease is a major contributor to mortality in Western societies, we hypothesized that deleterious polymorphisms associated with hemostasis decrease in frequency among a healthy population as a function of age. METHODS: The frequencies of factor V G1691A Leiden (FVL), factor II (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, glycoprotein Ia (GPIa) C807T, glycoprotein IIIa (Pl(A1)/Pl(A2)) T1565C, and angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) alleles were determined among 2689 healthy Caucasian whole-blood donors. For analysis, participants were divided into three age groups: 17-39 years (n = 979; 505 males and 474 females), 40-59 years (n = 900; 526 males and 374 females), and 60-85 years (n = 810; 530 males and 280 females). RESULTS: The Pl(A2) allele frequency decreased from 17.5% to 15.7% and 14.1% in the 17-39 years, 40-59 years, and 60-85 years age groups, respectively (n = 5094 alleles; P = 0.025). Among ACE DD males, the Pl(A2) allele frequency decreased from 20.8% to 16.1% and 9.1% in the same groups, respectively (n = 810 alleles; P = 0.001). No statistically significant decrease in genotype or allele frequency was observed among carriers of FVL, FII 20210A, MTHFR 677T, GPIa 807T, or ACE D. CONCLUSIONS: These data suggest that Pl(A2) carriers, especially those who are ACE DD, are statistically less prevalent among older healthy blood donors compared with their younger counterparts. These observations suggest an important, deleterious, time-dependent impact of the Pl(A2) allele, as well as the ACE DD/Pl(A2) allelic combination, on overall health and longevity.

Inhibition of human platelet function by sulfatides.

Sulfatides are glycolipid constituents of human platelet cell membranes and have been shown to interact with platelet-binding proteins involved in hemostasis. Because little is known about the physiological role of sulfatides in platelet function, the effect of sulfatide on platelet adhesion, aggregation, release, and ristocetin-induced platelet agglutination (RIPA) was studied. These processes are inhibited when exogenous sulfatide is present in vitro. Inhibition of aggregation induced by collagen, thrombin, and ristocetin by sulfatide was dose dependent. Adenosine diphosphate-mediated adhesion and aggregation were not significantly affected by sulfatide, nor was serotonin- and epinephrine-mediated aggregation. Collagen mediate release of serotonin was reduced sulfatide. RIPA demonstrated dose-dependent inhibition in response to sulfatide. These results suggest that sulfatide may play a role in modulating platelet activation.

von Willebrand factor and thrombotic thrombocytopenic purpura.

Recent advances in the understanding of platelet-dependent hemostasis and von Willebrand factor (vWF) functional regulation offer new insights into the pathogenesis of thrombotic microangiopathic disorders. The discovery of vWF-cleaving protease activity in normal plasma, and its deficiency in thrombotic thrombocytopenic purpura (TTP) patients, provides additional support for a pathologic role of ultra-large vWF in TTP. Although vWF-cleaving protease deficiency is highly prevalent among TTP patients, the defect has also been detected in individuals without active TTP. Therefore, vWF-cleaving protease deficiency appears to be an important risk factor for thrombotic microangiopathy rather than a specific diagnostic marker of TTP. Recent data indicate that vWF-cleaving protease activity correlates with clinical parameters in thrombotic microangiopathy patients. Therefore, determination of vWF-cleaving protease activity might prove useful in the future care of thrombotic microangiopathy patients and might be a rational basis for future classification of thrombotic microangiopathic disorders.

Expression of thrombomodulin and consequences of thrombomodulin deficiency during healing of cutaneous wounds.

Thrombomodulin is a cell surface anticoagulant that is expressed by endothelial cells and epidermal keratinocytes. Using immunohistochemistry, we examined thrombomodulin expression during healing of partial-thickness wounds in human skin and full-thickness wounds in mouse skin. We also examined thrombomodulin expression and wound healing in heterozygous thrombomodulin-deficient mice, compound heterozygous mice that have <1% of normal thrombomodulin anticoagulant activity, and chimeric mice derived from homozygous thrombomodulin-deficient embryonic stem cells. In both human and murine wounds, thrombomodulin was absent in keratinocytes at the leading edge of the neoepidermis, but it was expressed strongly by stratifying keratinocytes within the neoepidermis. No differences in rate or extent of reepithelialization were observed between wild-type and thrombomodulin-deficient mice. In chimeric mice, both thrombomodulin-positive and thrombomodulin-negative keratinocytes were detected within the neoepidermis. Compared with wild-type mice, heterozygous and compound heterozygous thrombomodulin-deficient mice exhibited foci of increased collagen deposition in the wound matrix. These findings demonstrate that expression of thrombomodulin in keratinocytes is regulated during cutaneous wound healing. Severe deficiency of thrombomodulin anticoagulant activity does not appear to alter reepithelialization but may influence collagen production by fibroblasts in the wound matrix.

Minimal evidence of platelet and endothelial cell reactive antibodies in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterized by microvascular thrombosis with thrombocytopenia and end-organ injury. Evidence suggests that platelet or endothelial cell injury may be initial pathological events in TTP. A number of factors in patient plasma, including immunoglobulins, have been proposed to mediate cellular injury in TTP. However, systematic analyses of TTP patient plasma for the presence of platelet or endothelial cell antibodies are lacking. We, therefore, analyzed 48 TTP patient plasma samples for the presence of platelet and endothelial cell antibodies by using enzyme-linked immunosorbent assay, flow cytometry, and microlymphocytotoxicity. Twelve of 48 TTP patient samples (25%) reacted against purified platelet glycoproteins. Nine (19%) also contained antibodies that bound to allogeneic target platelets in flow-cytometric assays. Nine of 48 samples (19%) contained antibodies to human umbilical vein endothelial cells in flow-cytometric assays, and seven of 48 patient samples (15%) bound to human dermal microvascular endothelial cells. Six of 48 (13%) patient plasma samples contained antibodies that bound to human umbilical vein endothelial cells activated with gamma-interferon and tumor necrosis factor-alpha. Of twenty samples that were reactive in one or more platelet or endothelial cell assay, eight contained human leukocyte antigen antibodies reactive in microlymphocytotoxicity. These studies demonstrate that antibodies reactive against platelet or endothelial cell antigens are not prevalent in TTP, and that more than a third of antibodies detected are human leukocyte antigen alloantibodies. Our findings suggest that autoantibodies against platelets or endothelial cells are not important in the pathogenesis of this syndrome.

Keratinocyte-specific expression of human thrombomodulin in transgenic mice: effects on epidermal differentiation and cutaneous wound healing.

BACKGROUND: Thrombomodulin is a cell-surface glycoprotein that regulates coagulation and fibrinolysis. Expression of thrombomodulin by epidermal keratinocytes is tightly regulated during squamous differentiation and cutaneous wound healing. METHODS: To determine the consequences of overexpression of thrombomodulin on squamous differentiation and wound healing in vivo, we expressed full-length human thrombomodulin in transgenic mice using the human keratin 14 promoter. Human thrombomodulin was detected in keratinocytes of transgenic mice by immunohistochemistry and protein C activation assays. Full-thickness cutaneous wounds were created on the dorsum of transgenic mice and nontransgenic littermates, and allowed to heal for up to 35 days. RESULTS: Transgenic mice had normal viability and appeared healthy up to one year of age. In the skin, human thrombomodulin was expressed in basal and suprabasal keratinocytes, with variable expression in the outer root sheath of hair follicles. Thrombomodulin activity in neonatal epidermis was 2.5- to 3-fold higher in transgenic mice than in nontransgenic littermates (p < 0.01). In cutaneous wounds, human thrombomodulin was expressed in migrating neoepidermal keratinocytes. No differences in keratinocyte migration or re-epithelialization were observed between transgenic and nontransgenic mice, but transgenic mice exhibited delayed collagen bundle deposition within the wound matrix. CONCLUSIONS: These findings demonstrate that keratinocyte thrombomodulin supports activation of protein C, and that thrombomodulin activity in epidermis can be increased by keratinocyte-specific expression of human thrombomodulin in transgenic mice. Expression of human thrombomodulin in keratinocytes does not impair normal squamous differentiation or re-epithelialization of cutaneous wounds, but may modulate collagen reconstitution of the wound matrix.

Adverse effects of transfusions caused by leukocytes.

The tremendous complexity of blood products gives rise to a range of adverse reactions. Transfusion reactions caused by leukocytes range from subtle and discomforting to dramatic and fatal. Although leukocyte-mediated events usually are harmful, some effects are beneficial. Leukoreduction can prevent most, but not all, adverse reactions. Awareness of the array of leukocyte-mediated transfusion complications and the appropriate preventive and therapeutic measures to be taken is a critical component of sound clinical transfusion practice.

Fingerstick blood samples in platelet donor screening: reliability and impact on predict yield programs.

Although widely used, the reliability of fingerstick platelet counts for determining donor eligibility and for use with plateletpheresis predict yield programs has not been established. We compared platelet counts obtained from fingerstick vs. venous samples in several aspects of apheresis platelet collection. Analysis of 25 paired fingerstick and venous predonation samples demonstrated a poor correlation between platelet counts (r2 = .43), with fingerstick counts having a 20% lower mean value (P < .05). The effect of using fingerstick vs. venous predonation platelet counts with apheresis instrument predict yield calculations to obtain target yields was determined. Mean yields collected using fingerstick/predict yield were 12% (Fenwal CS3000 PLUS) and 15% (Haemonetics MCS+) higher than venous/predict yield units (P < .05). The coefficients of variation (CV) of fingerstick/predict yield and venous/predict yield collections were comparable (15% vs. 14% [CS3000] and 23% vs. 21% [MCS+], respectively), indicating that possible differences in accuracy between fingerstick and venous platelet counts had little effect on the variability of predict yield collections. A retrospective analysis of the CV of 100 fingerstick/predict yield units vs. 100 units collected by processing standard volumes showed no difference: 22% vs. 20% (F = 0.99, CS3000), and 22% vs. 24% (F = 0.89, MCS+), respectively. We conclude that fingerstick platelet counts are systematically lower and correlate poorly with venous counts, though their use seldom results in false disqualification of donors. We also conclude that fingerstick count/predict yield collections do not produce more consistent yields of platelets than standard volume collections.

Regulation of thrombomodulin expression by all-trans retinoic acid and tumor necrosis factor-alpha: differential responses in keratinocytes and endothelial cells.

Thrombomodulin is a cell-surface anticoagulant glycoprotein expressed by vascular endothelial cells and epidermal keratinocytes. Thrombomodulin expression in endothelial cells is regulated by retinoic acid and tumor necrosis factor-alpha (TNF), agents that also modulate epidermal differentiation. We examined thrombomodulin function and regulation of thrombomodulin expression by all-trans retinoic acid (ATRA) and TNF in human keratinocytes and endothelial cells. Untreated keratinocytes and endothelial cells expressed thrombomodulin of comparable activity and apparent thrombin affinity. Incubation of keratinocytes with 10 mumol/L ATRA for 24 hours increased thrombomodulin activity 5.4 +/- 0.9-fold (mean +/- SE), with equivalent increases observed in thrombomodulin protein (5.5 +/- 2.1-fold) and mRNA (4.2 +/- 1.2-fold). Incubation of keratinocytes with 1.0 nmol/L TNF markedly increased expression of keratinocyte transglutaminase, but had no effect on thrombomodulin activity, protein, or mRNA. In endothelial cells, ATRA produced a small increase in thrombomodulin activity (1.9 +/- 0.1-fold), and incubation with TNF for 24 hours decreased thrombomodulin activity 83% +/- 7%. The activity profile of keratinocyte thrombomodulin exhibited a distinct maximum near 1.0 mmol/L Ca2+. These results demonstrate that keratinocyte thrombomodulin is regulated by retinoids and Ca2+, but not by TNF, and that regulation of thrombomodulin expression differs in keratinocytes and endothelial cells.

Cellular localization of thrombomodulin in human epithelium and squamous malignancies.

Thrombomodulin is a cell surface glycoprotein that functions as an anticoagulant. Although initially identified on endothelial cells, thrombomodulin is also expressed by other vascular cells, by mesothelial cells, and by epidermal keratinocytes. To determine whether thrombomodulin is expressed by epithelial cells in locations other than skin, we conducted a survey of thrombomodulin protein and mRNA in human epithelium. Thrombomodulin protein was detected by immunohistochemistry in all samples containing stratified squamous epithelium, including oral mucosa, larynx, esophagus, uterine ectocervix, and vagina. In these tissues, thrombomodulin staining localized to the suprabasal layer, with minimal staining observed in the basal or superficial layers of epithelium. Thrombomodulin was not detected in cuboidal, simple columnar, or pseudostratified columnar epithelium and was detected variably in transitional epithelium. Thrombomodulin staining was also observed in 21 of 26 cases of invasive squamous cell carcinoma and in several examples of squamous carcinoma-in-situ and squamous metaplasia. Expression of thrombomodulin mRNA was confirmed by in situ hybridization in both normal and malignant squamous epithelium. Full-length, functionally active thrombomodulin was demonstrated in cultured squamous epithelial cells. These data demonstrate that thrombomodulin expression correlates with the squamous phenotype and suggest that hemostasis is regulated by compartmentalization of procoagulant and anti-coagulant epithelial proteins.

Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation.

Thrombomodulin is an endothelial cell surface glycoprotein that inhibits the procoagulant activities of thrombin and accelerates activation of the anticoagulant protein C. Because protein C deficiency is associated with cutaneous thrombosis, we investigated the expression of thrombomodulin in human skin. Thrombomodulin was detected by immunohistochemical staining both in dermal endothelial cells and in epidermal keratinocytes. Within the epidermis, thrombomodulin staining was limited to keratinocytes of the spinous layer, suggesting that thrombomodulin is induced when basal keratinocytes begin to terminally differentiate. Thrombomodulin expression also correlated with squamous differentiation in epidermal malignancies; little or no thrombomodulin staining was seen in five basal cell carcinomas, whereas strong thrombomodulin staining was observed in each of five squamous cell carcinomas. Human foreskin keratinocytes cultured in medium containing 0.07 mM calcium chloride synthesized functional thrombomodulin with cofactor activity comparable to thrombomodulin in human umbilical vein endothelial cells. Stimulation of keratinocyte differentiation with 1.4 mM calcium chloride for 48 h produced 3.5-, 3.2-, and 5.6-fold increases in thrombomodulin cofactor activity, antigen, and mRNA, respectively. These observations suggest that thrombin is regulated by keratinocyte thrombomodulin at sites of cutaneous injury, and indicate a potential role for thrombomodulin in epidermal differentiation.

Expression of CD24 (BA-1) predicts monocytic lineage in acute myeloid leukemia.

Fifty-seven cases of acute myeloid leukemia (AML), which had been subtyped by French-American-British morphologic and cytochemical criteria as myeloid (M1, M2) or monocytic (M4, M5), were retrieved from the files of the Division of Hematopathology, University of Iowa. Corresponding immunophenotyping data were reviewed with attention to the expression of CD14 (MY4) and CD24 (BA-1). Of 20 cases expressing CD24, 19 were M4 or M5, whereas all 14 cases expressing CD14 were of monocyte lineage. Therefore, CD14 was a highly specific (100%) but only moderately sensitive (58%) marker for distinguishing classes M1 or M2 from M4 or M5. By contrast and unexpectedly, CD24 was nearly as specific (97%), but more sensitive (79%) in marking M4 or M5 cells. This appears to be true even though CD24 is apparently not expressed on normal monocytes. When positive staining for either or both antibodies (CD24 or CD14) was considered indicative of a monocytic leukemia, the sensitivity of immunophenotyping in distinguishing M4/M5 from M1/M2 AML rose to 92%, while maintaining 97% specificity. The authors discuss a recent observation that may help explain the unexpected expression of CD24 in monocytic AML. They conclude that the usefulness of CD24 in identifying monocytic AML may exceed that of CD14, and that the use of CD24 and CD14 in combination improves the ability of flow cytometry to distinguish myeloid from monocytic acute leukemias.