Ian Chin-Yee

Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines

In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we previously described a set of guidelines for detection of CD34+ cells based on a four-parameter flow cytometry method (CD45 FITC/CD34 PE staining, side and forward angle light scatter). With this procedure, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser (two-platform method). In the present study, we modified the basic ISHAGE method with the addition of a known number of Flow-Count fluorospheres. To reduce errors inherent to sample washing/centrifugation, we implemented ammonium chloride lyse, no-wash no-fix sample processing. These modifications convert the basic protocol into a single-platform method to determine the absolute CD34 count directly from a flow cytometer and form the basis of the Stem-Kit from Coulter/Immunotech. A total of 72 samples of peripheral blood, apheresis packs, and cord blood were analysed and compared using the ISHAGE protocol with or without the addition of fluorescent microspheres. Comparison of methods showed a high correlation coefficient (r=0.99), with no statistically significant difference or bias between methods (P > 0.05). Linearity of the absolute counting method generated an R2 value of 1.00 over the range of 0-250/microl. Precision of the absolute counting method measured at three concentrations of CD34+-stabilised KG1 a cells (Stem-Trol, COULTER) generated a coefficient of variation (C.V.) ranging from 4% to 9.9%. In a further modification of the single-platform method, the viability dye 7-amino actinomycin D was included and demonstrated that both viable and nonviable CD34+ cells could be identified and quantitated. Together, these modifications combine the accuracy and sensitivity of the original ISHAGE method with the ability to produce an absolute count of viable CD34+ cells. It is the accurate determination of this value that is most clinically relevant in the transplant setting. These modifications may improve the interlaboratory reproducibility of CD34 determinations due to the reduction in sample handling and calculation of results.

Effects of storage on efficacy of red cell transfusion: When is it not safe?

ObjectiveTo review the literature on red blood cell storage and its relationship to the efficacy of transfusion. ResultsWell-documented changes occur to the red blood cell product during ex vivo storage. These changes include a reduction in red blood cell deformability, altered red blood cell adhesiveness and aggregability, and a reduction in 2,3-diphosphoglycerate and ATP. Bioactive compounds with proinflammatory effects also accumulate in the storage medium. These changes reduce posttransfusion viability of red blood cells. The clinical effects beyond posttransfusion viability are uncertain, but a growing body of evidence suggests that the storage lesion may reduce tissue oxygen availability, have proinflammatory and immunomodulatory effects, and influence morbidity and mortality. There are no published randomized, control trials examining the effect of storage duration on morbidity and mortality. Leukoreduction improves the quality of stored red blood cell products and in some studies has been shown to reduce morbidity and mortality. ConclusionAlthough storage duration influences the quality of red blood cell product, there is currently insufficient evidence to advocate shorter storage periods for red blood cell products.

A comparison of biochemical and functional alterations of rat and human erythrocytes stored in CPDA-1 for 29 days: implications for animal models of transfusion

. Animal models of transfusion are employed in many research areas yet little is known about the storage‐related changes occurring in the blood used in these studies. This study assessed storage‐related changes in red blood cell (RBC) biochemistry, function and membrane deformability in rat and human packed RBCs when stored in CPDA‐1 at 4 °C over a 4‐week period. Human blood from five volunteers and five bags of rat RBC concentrates (five donor rats per bag) were collected and stored at 4 °C. RBC function was assessed by post‐transfusion viability and the ability to regenerate adenosine triphosphate (ATP) and 2,3‐diphosphoglycerate (DPG) when treated with a rejuvenation solution. Membrane deformability was determined by a micropipette aspiration technique. ATP in rat RBCs declined more rapidly than human RBCs; after 1 week rat ATP fell to the same level as human cells after 4 weeks of storage (rat, 2·2 ± 0·2 µmol g−1 Hb; human, 2·5 ± 0·3 µmol g−1 Hb). Baseline DPG concentrations were similar in rat and human RBCs (16·2 ± 2·3 µmol g−1 Hb and 13·7 ± 2·4 µmol g−1 Hb) and declined very rapidly in both species. Human RBCs fully regenerated ATP and DPG when treated with a rejuvenation solution after 4 weeks of storage. Rat RBCs regenerated ATP but not DPG. Post‐transfusion viability in rat cells was 79%, 26% and 5% after 1, 2 and 4 weeks of storage, respectively. In rats, decreased membrane deformability became significant (− 54%) after 7 days. Human RBC deformability decreased significantly by 34% after 4 weeks of storage. The rejuvenation solution restored RBC deformability to control levels in both species. Our results indicate that rat RBCs stored for 1 week in CPDA‐1 develop a storage lesion similar to that of human RBCs stored for 4 weeks and underscores significant species‐specific differences in the structure and metabolism of these cells.

Thrombotic Thrombocytopenic Purpura Associated with Ticlopidine

OBJECTIVE: To report a case of possible ticlopidine-induced thrombotic thrombocytopenic purpura (TTP). CASE SUMMARY: A 68-year-old man was started on ticlopidine therapy for transient ischemic attack because of aspirin intolerance. Three weeks after starting therapy, the patient developed TTP, manifest by severe thrombocytopenia, renal failure, microangiopathic hemolytic anemia, and neurologic symptoms. The condition reversed over three to five days coincident with the discontinuation of ticlopidine and therapy with daily plasma infusion and exchange. No evidence of relapse was found at one-year follow-up. DISCUSSION: TTP is a well-defined clinical entity with varied etiologies. Ticlopidine has been reported as causing neutropenia and isolated thrombocytopenia. Case reports have linked ticlopidine to the development of TTP. CONCLUSIONS: Including this case there are now seven patients who have been documented as developing TTP while receiving ticlopidine. In addition to monitoring for neutropenia and isolated thrombocytopenia, patients receiving ticlopidine also should be monitored for the development of TTP.

Use of recombinant activated factor VII in patients without hemophilia: a meta-analysis of randomized control trials.

Context:Benefits of recombinant activated factor VII (rFVIIa) in hemorrhage may be lost because of thromboembolic events (TAE). Method:MEDLINE, EMBASE, BIOSIS, CINAHL, Science Citation Index Expanded, clinicaltrials.gov were searched for placebo controlled trials of rFVIIa in patients without hemophilia. Reports of 22 randomized controlled trials were selected for analysis. Results were pooled using random effects models to calculate the odds ratios (OR) with 95% confidence interval (CI). Subgroup analyses were predetermined. Results:Among 3184 participants, 478 (15.0%) died and 249 (7.8%) had TAE. Additional blood transfusion was required in 517 (41.2%) of 1256 subjects. Patients receiving rFVIIa were less likely to need additional blood transfusions (OR, 0.54; 95% CI, 0.34–0.86) than patients receiving placebo. Mortality was not increased but may be reduced (OR, 0.88; 95% CI, 0.71–1.09). Reduction in mortality was more likely if rFVIIa was given therapeutically (OR, 0.87; 95% CI, 0.70–1.09) rather than prophylactically (OR, 1.00; 95% CI, 0.37–2.68). Differences in the pooled analysis of TAE were not statistically significant (OR, 1.17; 95% CI, 0.87–1.58) but the incidence of arterial TAE was likely higher in patients receiving rFVIIa (OR, 1.50; 95% CI, 0.93–2.41) although no differences were seen with respect to venous TAE (OR, 0.76; 95% CI, 0.49–1.15). Conclusions:Use of rFVIIa reduces the need for blood transfusion and it may reduce mortality, especially if the dose of rFVIIa is limited to therapeutic doses of 90 μg/kg. It does not increase the risk of venous thrombosis but it may increase the risk of arterial thrombosis.

Wbc reduction reduces storage-associated RBC adhesion to human vascular endothelial cells under conditions of continuous flow in vitro

BACKGROUND : The effects of storage duration, WBC reduction, and irradiation on RBC adherence to vascular endothelia are unknown and are investigated under conditions of continuous flow.

Diagnosing PNH with FLAER and multiparameter flow cytometry.

PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl‐inositol (GPI)‐linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI‐linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement‐mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI‐linked molecules and causes lysis of normal but not GPI‐deficient PNH cells. FLAER is an Alexa488‐labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER‐based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders.

Detection and quantification of circulating tumor cells in mouse models of human breast cancer using immunomagnetic enrichment and multiparameter flow cytometry

Circulating tumor cells (CTCs) in the peripheral blood of breast cancer patients may be an important indicator of metastatic disease and poor prognosis. However, the use of experimental models is required to fully elucidate the functional consequences of CTCs. The purpose of this study was to optimize the sensitivity of multiparameter flow cytometry for detection of human tumor cells in mouse models of breast cancer.

Abrogation of MRL/lpr Lupus Nephritis by Dietary Flaxseed

A diet supplemented with flaxseed, rich in alpha-linolenic acid and plant lignans (the latter, potent platelet-activating factor receptor antagonists), was tested in a murine model of lupus nephritis. MRL/lpr female mice (n = 25) were fed 15% flaxseed diet for 14 weeks commencing at 10 weeks of age. As controls, 30 MRL/lpr mice received a standard rodent diet without flaxseed. Isotope-glomerular filtration rate (14C-inulin clearance) was measured at 9, 16, and 24 weeks of age. Proteinuria was assessed at 2-week intervals. Spleen lymphocyte proliferation, quantitated by DNA analysis, was evaluated using flow cytometry at 9, 13, 19, and 21 weeks of age. Mortality was recorded throughout the study. Glomerular filtration rate at 16 weeks was greater in flaxseed-fed mice (0.15 +/- 0.03 mL/min) compared with controls (0.06 +/- 0.04 mL/min; P = 0.01). The onset of proteinuria (Albustix, Ames Division, Miles Laboratories, Rexdale, Ontario, Canada; > or = 2+) was delayed by 4 weeks in the flax-treated mice. The percentage of flaxseed-fed mice with proteinuria was lower than the control mice up to 21 weeks of age (39% v 58%; P = 0.01). Spleen lymphocyte proliferation (percentage of cells in S-phase) at 13 weeks of age was significantly higher in the control group (22.9 +/- 5.0, P = 0.01) but not in the flaxseed group (17.2 +/- 4.9) compared with baseline (9 weeks of age) values (13.0 +/- 3.5). Mortality was lower in the flaxseed-fed mice versus the control mice (assessed by Mantel-Haenszel (log-rank) test; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Isotype controls in the analysis of lymphocytes and CD34+ stem and progenitor cells by flow cytometry—time to let go?

The isotype control has long been considered a useful part of both microscopic and flow cytometric immunologic assays, and, consequently, is still routinely used in clinical laboratories. In flow cytometry, the isotype control has traditionally been used to distinguish between fluorescent positive and fluorescent negative cell populations. Additionally, it has been used to estimate the number of cells reacting non-specifically with the target antibody under investigation. Over the past 10 years, the widespread use of directly conjugated monoclonal antibodies (mAb) and multiparameter analysis in clinical flow cytometry has reduced the need for a separate ‘‘negative control’’ tube. This tendency has materialized in guidelines recommending that the isotype control is irrelevant and potentially misleading in commonly used flow cytometric assays (3, 14). This perspective summarizes the rationale for omitting isotype control staining for surface membrane marker analysis, focusing on lymphocyte and CD341 hematopoietic stem/progenitor cell analyses. Consequently, these points also pertain to the immunophenotyping of leukemia/lymphoma samples (14). Prior to the development of directly conjugated mAb, pre-immune sera were used in microscopic and flow cytometric studies to estimate the level of ‘‘non-specific staining’’ of the specific antibody to its target cell, i.e., the binding of that specific antibody by mechanisms other than specific antibody-to-antigen interactions. Such nonspecific binding is usually, but not exclusively, mediated by receptors that bind the Fc portion of the various immunoglobulin subclasses (19). In flow cytometry, an estimate of the number of cells reacting non-specifically is typically determined by placing a cursor at the foot of the isotype control negative population on a fluorescence (FL) histogram such that less than 2% of events are assessed as positive. This cursor position is maintained to determine the ‘‘percent positive cells’’ in the experimental stainings. Currently, many isotype controls are produced by fusion of antibody producing cells with a myeloma-derived cell line to form a hybridoma. By the very nature of mAb production, antibodies produced by hybridomas will differ structurally from each other, even within the same immunoglobulin subclass or isotype. Thus mAb that ‘‘specifically’’ bind to the same antigen on the cells under study might each additionally bind ‘‘non-specifically’’ to other leukocytes, platelets, etc. in an unpredictable manner. Other issues to consider in the use of monoclonal isotypes include differences in protein concentration and FL to protein (F/P) ratio between test antibody and isotype control. Different manufacturers use different protocols to produce, purify and chemically conjugate antibodies with a variety of fluorochromes which almost certainly impact the reliability with which experimental and isotype control mAb can be used to distinguish specific from nonspecific binding. A compounding problem is that in a panel with several surface markers each would need their own isotype control matched for the above criteria. This is rarely done in the clinical laboratory.