When blood runs cold: cold agglutinins and cardiac surgery

Cold agglutinins are particular cold-reactive antibodies that react with red blood cells when the blood temperature drops below normal body temperature causing increased blood viscosity and red blood cell clumping. Most individuals with cold agglutinins are not aware of their presence, as these antibodies have little effect on daily living, often necessitating no treatment. However, when those with cold agglutinins are exposed to hypothermic situations or undergo procedures such as cardiopulmonary bypass with hypothermia during cardiac surgery, lethal complications of hemolysis, microvascular occlusion and organ failure can occur. By identifying those suspected of possessing cold agglutinins through a comprehensive nursing assessment and patient history, cold agglutinin screening can be performed prior to surgery to determine a diagnosis of cold agglutinin disease. With a confirmed diagnosis of cold agglutinin disease, the plan of care can be focused on measures to maintain the patient's blood temperature above the thermal amplitude throughout their hospitalization including the use of normothermic cardiopulmonary bypass with warm myocardial preservation techniques to prevent these fatal complications. Using a case report approach, the authors review the mechanism, clinical manifestations, detection and nursing management of a patient with cold agglutinins undergoing scheduled cardiac surgery. Cold agglutinin disease is rare. However, the risk to patients warrants an increased awareness of cold agglutinins and screening for those who are suspected of carrying these antibodies.     

Molecular characterization of cold agglutinins

An unusual bias involving the exclusive usage of the V4-34 gene segment by pathogenic antibodies with I/i antigen specificity has been documented in the literature. In addition, all unmutated and several mutated V4-34 encoded antibodies have been shown to be reactive with the anti-idiotypic monoclonal antibody 9G4. The 9G4 Id, therefore, is a marker for V4-34 gene segment expression. Based on these two correlations, it became vital to localize and characterize the nature of the 9G4 Id and to determine the relationship between the Id and I binding. Mutational analysis indicated that the 9G4 Id is located in framework region 1 (FR1) of V4-34 encoded antibodies. Two distinct sections of FR1, encompassing amino acid residues 6-12 and 23-25, form the 9G4 Id. Mutational analysis demonstrated that both FR1 and CDRH3 were required for I binding. When either one was disrupted, the mutant antibody could not bind I. This indicates that I binds through a framework region, and not exclusively through CDRH3. This renders the I interaction with the V4-34 encoded portion of immunoglobulins unconventional, with characteristics similar to superantigen binding to immunoglobulin through FR. When the FR1 DNA sequence of V4-34 was exchanged for FR1 sequences from other VH families I binding was lost, providing a structural explanation for this restricted VH usage. An understanding of the localization and structure of the 9G4 Id and the requirements of V4-34 encoded antibodies for I binding provide insights into the structure of pathogenic antibodies and their requirements for binding antigen. This information should be useful in analyzing new interactions such as the lytic activity of some V4-34 encoded antibodies for B cells.     

Diagnostic utility of red cell flow cytometric analysis.

Although most of the diagnostic applications of flow cytometry bring forth examples of leukocyte immunophenotyping for immunodeficiency diseases and leukemia-lymphoma diagnosis, the same technology has improved medical assessment of diseases affecting the red cell and erythropoiesis. Flow cytometric methods were first applied to laboratory hematology with the improvement in reticulocyte counting and the creation of the immature reticulocyte fraction for better anemia evaluation and therapeutic monitoring. A more recent improvement attributable to flow cytometry is accurate detection of fetal red cells in the evaluation of FMH hemorrhage. The same method used in the detection of fetal RBCs based on HbF content measurement using monoclonal antibodies also offers the potential for enumeration of F cells, which promises to have use in therapeutic monitoring of patients with sickle cell disease and the evaluation of other hemoglobinopathies and myelodysplasia. Other clinical uses of flow cytometric RBC analysis include nonisotopic red cell survival studies, sensitive blood group typing, sensitive detection of immune-mediated hemolytic diseases, and evaluation of parasitic diseases whose life cycle involves intracellular RBC infestation. This article summarizes red cell flow cytometry, particularly as it impacts the areas of immunohematology and laboratory hematology, and points to areas of potential future contribution of this technology to diagnostic medicine.     

Clinical syndromes associated with cold agglutinins

Auto-immune haemolysis (AIHA) occurs when an individual's own red blood cells are destroyed by an autoantibody. Anaemia ensues if bone marrow production of red cells cannot compensate for the increased loss of red blood cells.     

Characterization of various anti-Pr cold agglutinins

Forty -one samples of anti-Pr cold agglutinins (CA) were studied. The titers ranged from 4 to 32,000. As is the case with cold agglutinins of other specificities, immunoglobulin class M and k-type light chains predominated with anti-Pr CA. On the other hand, the few IgM lambda, IgG, and IgA found were associated preferentially with anti-Pr specificity. All anti-Pr CA were inhibited by human red cell membrane sialoglycoproteins. On the basis of an increased inhibition by sialoglycoproteins after periodate oxidation and carbodiimide treatment, respectively, three anti- Pr2 and five anti-Pr3 were found. Among 24 anti-Pr1 CA subclassified on the basis of agglutination with dog red cells, six were anti- Pr1h, 16 were anti- Pr1d , and two did not fit into the subclassification. Similar to the anti- Pr1h , - Pr1d subclassification, anti-Pr3 CA could be subclassified into anti- Pr3h , - Pr3d. Three anti-Pra examples were found. None of the anti-Pr CA was inhibited by N-acetylneuraminic acid; some (5/35) were inhibited by sialyllactose, NeuAc alpha 2–3 2–6 Gal beta 1–4Glc, in high doses (5.0- 20.0 mM).     

Head and neck paragangliomas: a clinicopathologic study with DNA flow cytometric analysis

A total of 11 head and neck paragangliomas were the subject of pathologic study, including histologic, immunohistochemical, and DNA flow cytometric analyses. We cannot absolutely predict aggressive clinical behavior using histologic parameters alone, but we can use such parameters to segregate patients into low-risk and high-risk groups. Several trends were observed in the current study. Tumors with higher S-phase fractions, G2/M fractions, or aneuploid cell populations tended to behave "aggressively." The presence of sustentacular cells in the primary tumors cannot be used as an absolute indicator of tumor metastatic potential, as two metastatic paragangliomas in this study contained sustentacular cells in both the primary and metastatic lesions. DNA ploidy status cannot be used as an absolute prognostic parameter as the two metastatic tumors were composed of diploid primary and metastatic lesions. The three tumors with aneuploid cell populations showed "aggressive" histologic and clinical features, but the length of the follow-up period for these cases is too limited to draw any conclusions. Although no absolute criteria can be used at present to gauge aggressiveness, close follow-up of these patients is essential, especially if pathologic findings suggest an "aggressive" course (ie, "malignant" histology, higher S-phase fractions, G2/M fractions, aneuploid cell populations, or decreased sustentacular cell density).     

Automated flow cytometric analysis of cerebrospinal fluid

BACKGROUND: Recently, the UF-100 (Sysmex Corporation) flow cytometer was developed to automate urinalysis. We evaluated the use of flow cytometry in the analysis of cerebrospinal fluid (CSF). METHODS: UF-100 data were correlated with microscopy and biochemical data for 256 CSF samples. Microbiological analysis was performed in 144 suspected cases of meningitis. RESULTS: Good agreement was obtained between UF-100 and microscopy data for erythrocytes (r = 0.919) and leukocytes (r = 0.886). In some cases, however, incorrect classification of lymphocytes by the UF-100 led to underestimation of the leukocyte count. UF-100 bacterial count positively correlated (P < 0.001) with UF-100 leukocyte count (r = 0.666), CSF total protein (r = 0.754), and CSF lactate concentrations (r = 0.641), and negatively correlated with CSF glucose concentration (r = -0.405; P < 0.001). UF-100 bacterial counts were unreliable in hemorrhagic samples and in samples collected by ventricular drainage where interference by blood platelets and cell debris was observed. Another major problem was the UF-100 "bacterial" background signal in sterile CSF samples. Cryptococcus neoformans yeast cells and cholesterol crystals in craniopharyngioma were detected by the flow cytometer. CONCLUSIONS: Flow cytometry of CSF with the UF-100 offers a rapid and reliable leukocytes and erythrocyte count. Additional settings offered by the instrument may be useful in the diagnosis of neurological disorders.     

Oxidative product formation in irradiated neutrophils. A flow cytometric analysis

The effect of irradiation on neutrophil oxidative function was evaluated using a flow cytometric assay of intracellular hydrogen peroxide (H2O2) production. This assay quantitates the H2O2-dependent conversion of the nonfluorescent compound, 2'-7'-dichlorofluorescein (DCFH), into fluorescent 2'-7'-dichlorofluorescein (DCF) on a single-cell basis. Intracellular H2O2 production in response to stimulation with phorbol myristate acetate was not affected by neutrophil irradiation at doses up to 2500 rad. In addition, irradiation of intracellular DCFH and aqueous 2'-7'-dichlorofluorescein diacetate (DCFH-DA) resulted in DCF production, which suggested that oxidative molecules produced by aqueous radiolysis were detected by this assay. This study indicates that radiation doses of 1500 to 2500 rad, which are sufficient to prevent induction of graft-versus-host disease by transfused blood components, are not deleterious to neutrophil oxidative metabolism.     

Stage C prostatic adenocarcinoma: flow cytometric nuclear DNA ploidy analysis

Flow cytometric nuclear DNA ploidy analysis was used to study pathologic stage C prostatic adenocarcinoma (pT3, N0, M0) in 146 patients who underwent radical retropubic prostatectomy and bilateral pelvic lymphadenectomy between 1967 and 1981. Of these tumors, 46% had a DNA diploid pattern, 47% had a DNA tetraploid pattern, and 7% had a DNA aneuploid pattern. Abnormal ploidy patterns were associated more frequently with histologic high-grade tumors than with low-grade tumors. Considered alone, DNA ploidy pattern showed a strong association with subsequent prognosis. The median interval to progression for tumors with DNA tetraploid and DNA aneuploid patterns was 7.8 and 3.5 years, respectively. For the DNA diploid tumors, only 23% progressed within 18 years, the longest follow-up. At 10 years, only 10% of patients with DNA diploid tumors had died of prostatic cancer, in comparison with 28% of the DNA tetraploid and 36% of the DNA aneuploid groups (P less than 0.01). By analysis of a combination of histologic tumor grade and nuclear DNA ploidy pattern, an even stronger association with prognosis was demonstrated. For the 38 patients with histologic low-grade and DNA diploid tumors, progression-free survival was 92% at 10 years, in comparison with 57% for 23 patients with low-grade DNA nondiploid tumors. Patients with high-grade tumor had a poorer prognosis whether the DNA ploidy pattern was diploid or nondiploid. Nuclear DNA ploidy pattern is an important and independent prognostic variable for patients with pathologic stage C prostatic cancer treated by radical prostatectomy.     

Infant feeding effects on flow cytometric analysis of blood

Flow cytometric analysis was performed on purified mononuclear cells isolated from whole blood samples of 11 adults, 7 breast-fed (BF) infants and 11 formula-fed (FF) infants, mean ages 34.2 +/- 4.3 years, 6.3 +/- 1.3 months, and 6.2 +/- 1.2 months, respectively. Infants were receiving at least 70% of calories from formula or breast milk. Infant mononuclear cell populations contained a higher percentage of lymphocytes and a lower percentage of monocytes compared with adults. Within the lymphocyte population, infants had a higher CD4+/CD8+ ratio (T helper-inducer/T cytotoxic-suppressor), a higher percentage of CD19+ (pan B) and CD4+ cells, and a lower percentage of CD8+ and CD16+ (natural-killer) cells compared with adults. CD3+ (pan T) and CD4+ lymphocyte percentages were higher and CD19+ lymphocyte percentages were lower in FF compared with BF infants. Although sample size is small, our data indicate that diet may influence lymphocyte subset distribution during infancy when the majority of calories is derived from infant formula or human milk.     

Progress and application of multiple-parameter flow cytometric analysis]

Multiple-parameter flow cytometric analysis for the expression of cell surface markers has been generally used in various clinical and fundamental fields. This was made possible by the development of many monoclonal antibodies and new fluorescence dyes coordinately with the progress in hardwares. Especially, the development of new fluorescence dyes enabled us simultaneously analysis of more than two markers, not only surface markers but also intracellular events, such as Ca2+ concentration and cell cycle. As a result, fine characterization of cells of interest is now possible even with a limited number of cells within a shorter time. The high resolution and sensitivity of current multiple-parameter flow cytometer make possible the isolation of diverse and unique cell populations that can be obtained by no other way.     

Microfluidics for flow cytometric analysis of cells and particles

This review describes recent developments in microfabricated flow cytometers and related microfluidic devices that can detect, analyze, and sort cells or particles. The high-speed analytical capabilities of flow cytometry depend on the cooperative use of microfluidics, optics and electronics. Along with the improvement of other components, replacement of conventional glass capillary-based fluidics with microfluidic sample handling systems operating in microfabricated structures enables volume- and power-efficient, inexpensive and flexible analysis of particulate samples. In this review, we present various efforts that take advantage of novel microscale flow phenomena and microfabrication techniques to build microfluidic cell analysis systems.     

The flow cytometric analysis of hemopoietic cell proliferation. The methodological questions]

Studies of hemopoietic cell (myelokaryocyte) cycle and adequacy of applying flow DNA-cytometry for such analysis are discussed. Heterogeneity of the bone marrow, possibility of diluting a blood sample during puncture, reproducibility and stability of parameters, reliability of computer analysis of histograms demonstrating cell distribution, and correspondence of cell distribution by the cell cycle stages to the level of proliferative activity are the factors which may cause difficulties in the course of studies and while interpreting the results. Probable effects of these factors on the final results and efficacy of the method in clinical laboratory diagnosis are discussed.     

Flow cytometric DNA analysis.

Flow cytometric DNA analysis, including principles, techniques, and applications, is reviewed. Flow cytometry, a relatively recently developed technology, is being increasingly used in the diagnosis and treatment of a wide variety of benign and malignant diseases. Current major applications include phenotypic analysis of cells, cell sorting, and DNA analysis. DNA analysis by flow cytometry is rapid, reliable, and reproducible. Flow cytometric DNA analysis offers a quick, reliable method of analyzing cellular DNA content capable of identifying cell populations with abnormalities in total DNA content.     

Platelet function testing in apheresis products: flow cytometric, resonance thrombographic (RTG) and rotational thrombelastographic (roTEG) analyses.

During storage of platelet concentrates, quality control of the units is mandatory. This includes the important testing of the hemostatic function of platelets. So far, mostly platelet aggregation analyses have been performed. In this study, new approaches were tested to evaluate the applicability of modern techniques for quality monitoring. Plateletpheresis was performed with two different cell separators (AMICUS cell separator, Fenwal, Baxter Healthcare, Deerfield, USA; COBE Spectra, COBE BCT, Lakewood, USA). In each procedure split products (n = 22) were prepared and stored for 1-2 days (n = 22) or 3 5 days (n = 22). Platelet hemostatic capacity was tested by applying flow cytometry. platelet aggregation (platelet-rich-plasma [PRP]+agonist), resonance thrombography (RTG; PRP, no agonist) and rotational thrombelastography (roTEG; PRP+agonist). Flow cytometric analyses did not reveal significant changes in structural (CD41a. CD42b) or activation-dependent antigens (CD62p, CD63, LIBS, RIBS). Also, differences in the data from the flow cytometric reactivity tests were not significant between the two groups. In platelet aggregation assays, shape change (p = 0.8), maximum aggregation (p = 0.4), and maximum gradient (p = 0.8) did not show significant differences between the two groups. In the RTG test, differences between r-time (reaction time; p = 0.4), and f-time (clot formation time [fibrin influence]; p = 0.3), and in roTEG r-time (coagulation time; p = 0.1) and k-time (clot formation time; p = 1.0) were not significant. P-time (clot formation time [platelet influence]) and M (maximum amplitude) in RTG, and k-time and MA (maximum amplitude) in roTEG showed a slight decrease in platelet function (p < or = 0.05). We conclude that platelet function is well maintained during storage. This is reflected by the results of immunological and platelet function assays. Rotational thrombelastography (in the case of PRP) and especially resonance thrombography represent promising methods for quality control of platelet concentrates and rapidly provide information about the status of platelet function and the whole clotting process.     

Changes in ABH antigen expression on red cells during in vivo aging: a flow cytometric analysis

BACKGROUND: During aging of red cells they undergo a multitude of physical and chemical changes. STUDY DESIGN AND METHODS: Age-dependent changes in the expression of ABH major blood group antigens on red cells were studied in a two-variable flow cytometric analysis. The expression of the antigens was measured after staining with fluorescence antibodies (for A and B) or lectin (for H). Cell age was related to size as measured by forward light scattering. Aging senescent cells are smaller and have low forward light scattering properties. RESULTS: The results indicated that, within a given population, the A and/or B and H antigens decreased with the decrease in forward light scattering (i.e., increase in age), but A and B antigen expression decreased to a greater extent than did H. CONCLUSION: During aging, red cells lose the antigens of the major blood groups, but the degree of this loss is related to the fact that these antigens are located on the cell surface.