Effect of cold agglutinins on red blood cell parameters in a trauma patient: a case report.

The presence of cold agglutinins (CAs) in samples intended for complete blood count (CBC) using automated haematology analysers might cause serious preanalytical errors. In this report we describe the case of a 90-year old female patient admitted to the Emergency department following trauma injuries. A blood testing on admission revealed surprisingly low red blood cell count (0.99 x 1012/L), low haematocrit (0.102 L/L) which did not correlate with haemoglobin concentration (100 g/L), and high erythrocytes indices (mean corpuscular haemoglobin, 101 pg; mean corpuscular haemoglobin concentration, 980 g/L). In the second sample, after repeated collection, almost equal results were observed. Blood smear examination under the microscope revealed clusters of erythrocytes. Cold agglutinins presence was suspected and, in order to get valid results, sample was warmed to 37 °C. Correction of CBC was observed. Furthermore, we performed some additional analysis to confirm the presence of CAs in this patient. The aim of this report was to present the laboratory findings in a case of CAs and propose a laboratory procedure for whole blood samples with suspected CAs.

An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbß3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbß3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

70-year old female patient with mismatch between hematocrit and hemoglobin values: the effects of cold agglutinin on complete blood count.

INTRODUCTION: There are a number of pre-analytical and analytical factors, which cause false results in the complete blood count. The present case identifies cold agglutinins as the cause for the mismatch between hematocrit and hemoglobin values. MATERIALS AND METHODS: 70-year old female patient had a history of cerebrovascular diseases and rheumatoid arthritis. During routine laboratory examination, the patient had normal leukocyte and platelet counts; however, the hemoglobin (Hb: 105 g/L) and hematocrit (HCT: 0.214 L/L) results were discordant. Hemolysis, lipemia and cold agglutinin were evaluated as possible reasons for the mismatch between hematocrit and hemoglobin values. RESULTS: First blood sample was slightly hemolysed. Redrawn sample without hemolysis or lipemia was analyzed but the mismatch became even more distinct (Hb: 104 g/L and HCT: 0.08 L/L). In this sample, the titration of the cold agglutinin was determined and found to be positive at 1:64 dilution ratios. After an incubation of the sample at 37°C for 2 hours, reversibility of agglutination was observed. CONCLUSION: We conclude that cold agglutinins may interfere with the analysis of erythrocyte and erythrocyte-related parameters (HCT, MCV, MCH and MCHC); however, Hb, leukocyte and platelet counts are not affected.

'Agglutination and flocculation' of stem cells collected by apheresis due to cryofibrinogen.

Collection of peripheral stem cells by apheresis is a well-described process. Here, investigations concerning 'agglutination and flocculation' of stem cells collected from two patients are described. In both cases, cryoproteins were observed and cryofibrinogen was identified using high-resolution two-dimensional electrophoresis. In one case, peripheral stem cells were collected after a second course of mobilization, and the cells were immediately washed at 37 degrees C before being frozen, allowing their use, despite the presence of cryofibrinogen. In the other case, 'agglutination' was reversed by warming the bag, and plasma was removed before freezing.

Effect of cryoglobulin and crystalcryoglobulin on TNF-alpha production by normal human monocytes.

OBJECTIVES: We recovered an IgG1-kappa cryocrystalglobulin in synovial fluid and membrane specimens from a patient with destructive arthropathy. In the present study, we investigated its proinflammatory properties by measuring its effects on TNF-alpha production by normal human monocytes. MATERIALS AND METHODS: Normal human monocytes isolated by plastic adhesion were cultured in microtiter plates. Adherent monocytes were cultured for 6, 8, and 24 hours with sterile cryocrystalglobulin (150 microg/mL and 2 mg/mL), type I noncrystallised cryoglobulin (same concentrations), monosodium urate (MSU) crystals (2 mg/mL), LPS (10 microg/mL), or medium alone. Supernatant TNF-a concentrations were assayed using an ELISA. RESULTS: Cryocrystalglobulin had no effect on TNF-alpha production by normal human monocytes. Noncrystallised cryoglobulin increased TNF-alpha levels in supernatants in a time-dependent and concentration-dependent fashion. This increase was significantly less marked than the increases achieved with MSU crystals or LPS. CONCLUSION: IgG1kappa cryocrystalglobulin has no effect on TNF-alpha production by normal human monocytes. Fc region changes within the cryocrystalglobulin molecule may explain this finding.

The supercooling ability of ticks (Acari, Ixodoidea).

The supercooling capacity of nine laboratory-held species of ticks originating from different geographical areas, as well as five field-collected species from Germany, was investigated. All but one tick species showed mean supercooling points between about -17 and -23 degrees C, suggesting that the capacity to supercool to temperatures of < or = -17 degrees C might be an inherent property of many tick species unrelated to their geographic origin. Photoperiod did not influence the mean supercooling point in any of the species and there was also no distinct seasonal pattern of supercooling in seasonally acclimatized Dermacentor marginatus. Thus, the supercooling ability was independent of the presence/absence of diapause. The finding of thermal hysteresis in D. marginatus hemolymph raises the question of whether or not anti-freeze proteins are involved in the supercooling capacity of that species. An interspecies comparison revealed a weak negative correlation between relative water content and supercooling point of the ticks and an even weaker correlation between body mass or body water mass and the supercooling point. Since the ticks exhibited low supercooling points both before and shortly after feeding, the blood used as food should lack potent ice nucleators.

Fc gamma receptor activation of neutrophils in cryoglobulin-induced leukocytoclastic vasculitis.

OBJECTIVE: The role of Fc gamma receptors (Fc gamma R) in type I cryoglobulinemia was investigated to characterize novel mechanisms of neutrophil activation in the pathogenesis of leukocytoclastic vasculitis. METHODS: Neutrophils from healthy donors were incubated with purified monoclonal IgG1 kappa cryoglobulin complexes in vitro. Changes in surface antigen expression and mechanisms of intracellular hydrogen peroxide production and calcium release were measured by flow cytometry. RESULTS: After incubation for 2 hours, surface expression of Fc gamma RI (CD64), CD66, and CD67 was up-regulated; Fc gamma RII (CDw32), Fc gamma RIII (CD16), and LAM-1 were down-regulated. Using solubilized and complexed cryoglobulins, it was demonstrated that complex formation is necessary to induce intracellular H2O2 production and calcium release from intracellular stores. Both H2O2 generation and calcium mobilization could be inhibited by pretreatment with F(ab')2 fragments of monoclonal antibodies (MAb) against Fc gamma RIII. In contrast, Fab fragments of anti-Fc gamma RII MAb failed to block these activations. Neither the cryoglobulin complex-induced production of H2O2 nor the increase in cytoplasmic calcium was affected by treatment with pertussis toxin, which suggests that pertussis toxin-sensitive G proteins are not involved in signal transduction. CONCLUSION: These results indicate that Fc gamma RIII plays a major role in the pathogenesis of leukocytoclastic vasculitis.

A cryoglobulin with cold agglutinin and erythroid stem cell suppressant properties.

A 93-year-old woman presented with profound anemia (hematocrit 23% [0.23]); there was clumping of her red cells in test tubes and on peripheral blood smears. There was also a marked decrease in erythroid precursors in the bone marrow and reticulocytopenia in the peripheral blood. An IgM kappa monoclonal gammopathy was found in low concentration (approximately 1%) in her serum, and the cold agglutinins had a titer of 2560. However, the cold agglutinin titer of the supernatant after cryoglobulin precipitation was 40. Redissolving the cryoglobulin in the supernatant resulted in a cold agglutinin titer of 1280. Moreover, the addition of the patient's whole serum inhibited erythroid colony formation in culture. The inhibition was removed by cryoprecipitation of the cryoglobulin. The patient was given steroid therapy, to which she responded with reticulocytosis and an elevation of hematocrit. By 3 months, the cold agglutinin titer had fallen to 10. She remained well 4 years later. Whereas reports of cryoprecipitable cold agglutinins are rare, this case is unique because there have been no previous reports that these cold active proteins also have erythroid stem cell-suppressant properties.

Adduct formation between soluble fibrin monomers and elastin.

Monomers of fibrin generated from fibrinogen by thrombin reacted with elastin to give a new addition product or adduct. The adduct formation results from a covalent bond between both proteins, formation of which is dependent on elastin, fibrinogen and Ca2+ concentrations; but, Factor XIIIa did not intervene. Addition of fibronectin, together with fibrinogen, as cold insoluble proteins from human plasma, and of soluble collagen from rat tail tendon did not inhibit the first reaction. This enabled us to elaborate a new artificial connective matrix.

Preparation and characterization of murine monoclonal antibodies that express both cold agglutinin and cryoglobulin activities.

To obtain murine cold agglutinin (CA) and cryoglobulin antibodies, BALB/c mice were hyperimmunized with heat-killed type XIV Streptococcus pneumoniae. The spleen cells of these mice were fused with either the P3 NS1/Ag4. 1 or P3 X63/Ag.653 cell line. Several stable hybridomas were obtained that produced monoclonal antibodies (Mab) that reacted with rabbit and human erythrocytes only at temperatures below 37 degrees C. Three of these Mab were also cryoglobulins, as evidenced by their insolubility at reduced temperature. All of the antibodies studied were IgM(k) and reacted with purified type XIV S. pneumoniae polysaccharide at room temperature. With one exception, all antibodies were specific for N-acetyl-lactosamine, the immunodominant sugar residue expressed on type XIV polysaccharide. Inhibition experiments demonstrated that both CA activity and cryoprecipitation were inhibited by the same sugar compounds in the same order of efficiency. The data presented strongly suggest these CA antibodies are cross-reactive members of a S. pneumoniae-specific population. Cryoprecipitation persisted in antibodies purified under conditions that would exclude the presence of trapped serum antigens. It is therefore proposed that the cryoprecipitation observed is a result of the interactions of the antibody combining sites with carbohydrate residues of adjacent antibody molecules.

Effects of fresh-frozen plasma and its cryosupernatant fraction on von Willebrand factor multimeric forms in chronic relapsing thrombotic thrombocytopenic purpura.

Remission plasma samples of some patients with chronic relapsing thrombotic thrombocytopenic purpura (TTP) contain unusually large von Willebrand factor (vWF) multimers similar to those produced by normal human endothelial cells in culture. The infusion of the cryosupernatant fraction of normal plasma is as effective as normal fresh-frozen plasma (FFP) in the treatment or prevention of TTP episodes in patients with the chronic relapsing form of TTP. Three patients with chronic relapsing TTP during remission have unusually large vWF multimers present in their plasma. Two of the patients were transfused once with FFP, one of the two received cryosupernatant on three occasions, and the third patient was studied before and immediately after plasma exchange. Unusually large vWF multimers decreased or disappeared from patient plasma samples within 1/2 to 1 1/2 hours following the transfusion of FFP (on two occasions) or cryosupernatant (on two of three occasions), and immediately after plasma exchange (on one occasion). The patient who received cryosupernatant was studied serially after the infusions. Unusually large vWF multimers returned to her plasma within ten to 24 hours and persisted thereafter. Unusually large vWF multimers did not disappear from patient remission plasma samples, or from the culture medium removed from normal human endothelial cells, when these fluids were incubated in vitro with either normal FFP or cryosupernatant. We conclude that an activity in FFP, and its cryosupernatant fraction, promoted the rapid in vivo disappearance of unusually large vWF multimers from the plasma of two patients with chronic relapsing TTP in remission, and plasma exchange reversed the abnormality in a third patient who was in partial remission. Neither FFP nor cryosupernatant directly converted unusually large multimers to smaller vWF forms in vitro in the fluid phase. These results indicate that an activity in the cryosupernatant fraction of normal plasma is involved in vivo in controlling the metabolism of unusually large vWF multimers, and that this process is defective in some chronic relapsing TTP patients.

Pancytopenia with mixed cryoglobulinemia: evidence for anti-percursor cell activity of cryoglobulin--effects of plasmapheresis.

The efficacy of plasmapheresis in a patient with mixed cryoglobulinemia and pancytopenia is shown. The cryoglobulin was shown to have percursor cell suppressing activity and its depletion by plasmapheresis resulted in improvement of blood counts, Indications, limitations and guidelines for plasmapheresis in various diseases are discussed.

Influence of factor VIII/von Willebrand factor protein (F VIII/vWF) and F VIII/vWF-poor cryoprecipitate on post-ischemic microvascular reperfusion in the central nervous system.

To test the hypothesis that plasma contains native constituents capable of impairing microcirculatory flow in zones of acute ischemic tissue damage, we performed 14C-antipyrine autoradiographic blood flow studies in splenectomized dogs subjected to 35 min of cerebrospinal fluid compression ischemia followed by 30 min of recirculation to the neuraxis. The animals were anticoagulated with heparin and were divided into 4 groups by exposure to various measures before induction of ischemia. Groups 1 and 2 served for comparison with the other groups and underwent, respectively, no glass-wool filtration and glass-wool filtration via an arteriovenous shunt. Post-ischemic brain blood flows in Group 1 were low and focal zones of greatly impaired reperfusion were present. In Group 2, post-ischemic brain blood flows were high and focal perfusion impairment did not occur. Group 3 received homologous purified factor VIII/von Willebrand factor protein (F VIII/vWF) after glass-wool filtration but before induction of ischemia; Group 4 received F VIII/vWF-poor cryoprecipitate at the same time point. The purpose of administering the plasma preparations was to check for the presence of activity that nullified the enhancement of post-ischemic reperfusion expected after exposure to glass-wool. The results indicate that activity deleterious to post-ischemic reperfusion primarily resides in the F VIII/vWF fraction of cryoprecipitate. The F VIII/vWF-poor cryoprecipitate infusate, containing 250 to 800-fold more protein than the F VIII/vWF fraction, produced an intermediate reduction of blood flow.

Attachment and spreading of baby hamster kidney cells to collagen substrata: effects of cold-insoluble globulin.

Studies have been carried out to determine the effects of cold-insoluble globulin (CIG) on the attachment and spreading of baby hamster kidney cells on various collagen substrata. Cell attachment to native collagen substrata occurred in the absence of CIG just as fast as attachment to dried collagen or gelatin substrata occurred in the presence of CIG. On the other hand, cell attachment to dried collagen or gelatin was markedly reduced in the absence of CIG. Cell spreading also occurred on native collagen in the absence of CIG; however, CIG was absolutely required for cell spreading to occur on dried collagen or gelatin. Finally, anti-CIG antiserum or lactoperoxidase treatment inhibited cell spreading on CIG-coated substrata but not on native collagen substrata. The data are discussed in terms of the interaction of fibroblasts with collagen in situ.

Relationship between the anticoagulant and antithrombotic effects of heparin in experimental venous thrombosis.

The relationship between the antithrombotic and anticoagulant effects of heparin was assessed using venous thrombi in rabbits. Accretion of 125I-fibrinogen onto jugular vein thrombi was used to assess the antithrombotic effect of heparin, and the protamine sulfate titration test (heparin activity) and the activated partial thromboplastin time (APTT) were used to measure its anticoagulant effect. The effect of heparin on jugular vein bleeding times was also measured in a separate group of animals. Fibrinogen accretion was significantly lower with continuous infusion than with intermittent injection. Heparin, given by continuous infusion, produced marked inhibition of fibrinogen accretion (to less than 10% of control accretion) at an APTT value of between 75 and 80 sec (control 34 sec) and at a level of heparin activity of 0.4-0.5 U/ml. Infusion of cryoprecipitate reduced the effect of heparin on the APTT relative to its effect on heparin activity. In these cryoprecipitate-treated animals, marked inhibition of fibrinogen accretion occurred at a similar level of heparin activity (0.4-0.6 U/ml) but at a significantly lower APTT (35-50 sec) than in normal animals. On the other hand, there was a progressive increase in jugular vein bleeding time with both increasing APTT values and heparin levels in both groups of animals.