WBC Subsets Research Articles

Thrombosis and lymphocyte subsets of COVID-19 omicron BA.2 variant patients with cancer.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused an ongoing global pandemic of COVID-19. It has been found that COVID-19 has an influence on the changes of blood coagulation parameters and the high incidence of thrombosis. Changchun experienced the epidemic of the Omicron BA.2 variant SARS-CoV-2 in March 2022 in China. Once infected, BA.2 spreads rapidly and most of them are asymptomatic. The purpose of this study is to research venous thrombosis and laboratory changes (including PLT, PT, APTT, DD, FDP, CRP, WBC, IL-6 and lymphocyte subsets) among 92 cancer patients with COVID-19 and 73 COVID-19 patients with non-cancer by Mann-Whitney U and Chi-square test. It was found that the levels of D-dimer, FDP, CRP and IL-6 in cancer patients were significantly higher than those in the COVID-19 cohort. There were 9 (9.8%) cancer patients and 2 (2.7%) non-cancer patients found VTE, with no significant difference. The results showed that WBC, lymphocytes and B cells in cancer patients were significantly lower than those in the other group. Prophylactic anticoagulation was recommended for cancer patients with high risk factors, while paying attention to the occurrence of bleeding events. The detection of leukocyte classification, D-dimer, prothrombin time and fibrinogen at different time points are helpful for the diagnosis and anticoagulation of COVID-19 patients with cancer.

Rapid and Safe T Cell Immune Reconstitution By T Cell Progenitor Injection Following Haploidentical Transplantation for Severe Combined Immunodeficiency (SCID)

Severe Combined Immunodeficiencies (SCID) are defined by a complete absence of T lymphocytes in the blood and lymphoid organs, with variable defects in other WBC subsets depending on the gene defect. From a clinical perspective SCIDs are characterized by early development of life-threatening infections accounting for early death if untreated. The treatment of choice is allogeneic HSCT with very high success rates if a HLA identical sibling (MRD) or unrelated donor (MUD) is used. However, due to the scarcity of matched-related donors, SCID can benefit from haploidentical HSCT. In contrast to the continuous improvement of HLA compatible donor transplantations, no significant improvements have been obtained over the last twenty years for haploidentical HSCT. The profound immunodeficiency during the first months following haploidentical HSCT exposes patients to opportunistic viral, bacterial and fungal infections, which account for approximately 30-40% of the transplant related mortality (TRM). The rapid restoration of the T-cell compartment is the main aim of stem cell therapy in this setting. To this end we have recently set up a phase I/II clinical trial (ClinicalTrials.gov Identifier: NCT03879876) aiming to accelerate the immune reconstitution by injection of ex vivo generated Human T lymphoid progenitors (ProTcell TM) following haploidentical HSCT. T cell progenitors in this trial are generated in vitro within 7 days from mobilized peripheral blood (mPB) CD34 + hematopoietic stem and precursor cells (HSPCs) using our Notch ligand Delta-like 4 GMP culture platform so called SMART Immune's SMART101 product.This open-label, non-randomized study evaluates safety and efficacy of the SMART101 injection following CD34 + selected, haploidentical HSCT in SCID patients and is designed as a dose-escalation study comprising 6 doses of the SMART101 product obtained from the patient's haploidentical stem cell graft. The aim of this protocol is to define the highest efficacy dose without any toxicity.The conditioning regimen is based on Busulfan and Fludarabine according to IE-WP/EBMT guidelines with upfront administration of ATG to prevent graft rejection. Tight monitoring of ATG serum levels is applied in order to assure injection of SMART101 when ATG is below the lymphotoxicity threshold.Here we report the results of the first two SCID patients. P1 presented a homozygous Artemis deficiency. At diagnosis he had an ALC of 341/µl, with complete absence of T cells (CD3 + < 4/µl, CD4 + < 1/µl, CD8 + < 2/µl) and B cells (CD19 + 0/µl). NK cells were present in the normal range for age (CD16 +CD56 + 331/µl).In the absence of an HLA compatible donor, the patient's father was chosen as haploidentical stem cell donor.P1 received upfront ATG (5 mg /kg total dose), Busulfan (AUC of 16058 microM.min) and Fludarabine (160 mg/m²). He received Defibrotide prophylaxis from D0 until D+21, as well as Ursodeoxycholic acid until D+80. The CD34 + immunoselected graft contained 1.04 x 10 8 nucleated cells/kg with 24.15 x10 6 CD34 + cells/kg and 4000 CD3 + cells/kg on D0. After ATG monitoring 0.12x10 6 Smart101 cells were administered at D+14 post- HSCT.In the follow-up P1 didn't develop any acute or chronic SAEs, no acute or chronic GVHD, and no infection. He was discharged at D+121 post HSCT.The day +100 post transplantation CD4 + cell count/microliter exceeded 10 times the CD4 + count of our historical cohort of RAG1/2 or Artemis deficient patients transplanted with haploidentical HSCT alone following the same conditioning regimen.At 6 months post HSCT this difference remains important (851 versus 300 CD4 + cells/µl); Ig replacement therapy could be stopped as early as 9 months post transplantation and vaccinations have been started. At last follow up; almost 14 months post HSCT P1 is alive and well.P2 had an undefined molecular SCID diagnosis. She has been treated with the same conditioning regimen and received the second dose of 0.2x10 6 CD7 + cells, but unfortunately died from severe VOD emphasizing the need to replace chemotherapy with less toxic myeloablative agents.The preliminary results obtained after injection of Human T lymphoid progenitors in P1 are encouraging. While deserving confirmation in larger numbers of patients they could represent an important step forward in improving the outcome of haploidentical HSCT for SCID. DisclosuresCavazzana: Smart Immune: Other: co-founder.

Immunomodulatory activities of pixatimod: emerging nonclinical and clinical data, and its potential utility in combination with PD-1 inhibitors

BackgroundPixatimod (PG545) is a novel clinical-stage immunomodulatory agent capable of inhibiting the infiltration of tumor-associated macrophages (TAMs) yet also stimulate dendritic cells (DCs), leading to activation of natural killer (NK) cells. Preclinically, pixatimod inhibits heparanase (HPSE) which may be associated with its inhibitory effect on TAMs whereas its immunostimulatory activity on DCs is through the MyD88-dependent TLR9 pathway. Pixatimod recently completed a Phase Ia monotherapy trial in advanced cancer patients.MethodsTo characterize the safety of pixatimod administered by intravenous (IV) infusion, a one month toxicology study was conducted to support a Phase Ia monotherapy clinical trial. The relative exposure (AUC) of pixatimod across relevant species was determined and the influence of route of administration on the immunomodulatory activity was also evaluated. Finally, the potential utility of pixatimod in combination with PD-1 inhibition was also investigated using the syngeneic 4T1.2 breast cancer model.ResultsThe nonclinical safety profile revealed that the main toxicities associated with pixatimod are elevated cholesterol, triglycerides, APTT, decreased platelets and other changes symptomatic of modulating the immune system such as pyrexia, changes in WBC subsets, inflammatory changes in liver, spleen and kidney. Though adverse events such as fever, elevated cholesterol and triglycerides were reported in the Phase Ia trial, none were considered dose limiting toxicities and the compound was well tolerated up to 100 mg via IV infusion. Exposure (AUC) up to 100 mg was considered proportional with some accumulation upon repeated dosing, a phenomenon also noted in the toxicology study. The immunomodulatory activity of pixatimod was independent of the route of administration and it enhanced the effectiveness of PD-1 inhibition in a poorly immunogenic tumor model.ConclusionsPixatimod modulates innate immune cells but also enhances T cell infiltration in combination with anti-PD-1 therapy. The safety and PK profile of the compound supports its ongoing development in a Phase Ib study for advanced cancer/pancreatic adenocarcinoma with the checkpoint inhibitor nivolumab (Opdivo®).Trial registrationClinicalTrials.gov Identifier: NCT02042781. First posted: 23 January, 2014 - Retrospectively registered.

지적장애인의 성별에 따른 혈액의 구성성분과 hematocrit의 차이

The purpose of this study was to investigate the difference of formed elements(RBC, Hb, WBC, WBC subsets) and hematocrit of blood according to the gender of the intellectual disabilities. Fourteen mentally retarded males(n=7) and females(n=7) participated in this study. All subjects were classified as a light and moderate mental retardation(mean IQ 48.0±3.63). All subjects performed low intensity submaximal exercise(75%HRmax) by treadmill during 30minutes. Blood sample was obtained at rest, immediately after exercise and 60minute later after immediately exercise. No significant interaction effects(group×time of measurement) were observed for RBC, Hb, HCT. Between-group analysis of each group reveals that RBC, Hb, HCT value of the male were significantly higher than the female at all measurement point. No group×time interaction for WBC was determined. Between-group analysis of each group reveals that WBC was significantly increased after immediately exercise and returned baseline level after 60minutes recovery period. The group×time interaction effect for neutrophil and lymphocyte did not reach significance but there was a trend toward a difference.

CD4/CD8 ratio during natalizumab treatment in multiple sclerosis patients

Although improved, the risk of progressive multifocal leukoencephalopathy is a constant threat for patient affected by multiple sclerosis treated with natalizumab.We performed a 24months longitudinal study aimed to evaluate the total WBC and lymphocytes subsets modifications and their correlations with anti-JCV antibody index after 1 and 2years of natalizumab treatment.Natalizumab induced an increase of WBC, total and C19+ lymphocytes together with a decrease of CD3+, CD4+ T lymphocytes and CD4/CD8 ratio, which was positively related to anti-JCV antibodies index at month 0, 12 and 24.Our study confirms that lymphocytes subsets are modified under NAT therapy. Implications of lymphocyte subsets alterations in the pathogenesis of PML are under analyses.

Effects Of Octacosanol Intake During Weight-loss On Body Composition, Wbc Subsets, Cortisol In Taekwondo Competitors

Effects Of Octacosanol Intake During Weight-loss On Body Composition, Wbc Subsets, Cortisol In Taekwondo Competitors

Novel Integrated Autologous Hematopoietic Stem Cell Tracking in Nonhuman Primates Reveals Successive Pattern of Multi-Lineage Reconstitution after Total Body Irradiation

Hematopoietic stem cell transplantation (HSCT) has been used to cure patients for over four decades, yet the kinetics and clonal dynamics of repopulation by multipotent HSCs remains unresolved. Two sequence-based methods have been used to measure HSC contribution in preclinical models, integration site (IS) analysis and DNA barcoding. Both methods are accomplished by retrovirus tagging of HSCs and each confers different methodological constraints. We hypothesized that IS analysis and DNA barcoding together would provide more robust HSCT reconstitution data if applied to the clinically relevant nonhuman primate autologous HSCT model. We developed a high complexity (~1.2 million), DNA-barcoded LV library encoding the chemotherapy resistance gene MGMT(P140K) and enhanced green fluorescent protein (GFP) to measure hematopoietic reconstitution following total body irradiation (1020 cGy) and autologous HSCT in two pigtailed macaques. In both animals, hematopoietic recovery from TBI was achieved within 1-2 months after HSCT. We observed ~1 and 12% GFP+ peripheral blood white blood cells (PB WBCs) following hematopoietic recovery in these animals, respectively. We performed barcode and IS retrieval from PB WBCs collected early (1 month) after HSCT and observed <0.001% of infused clones contributing to hematopoiesis by DNA barcoding. We identified 42% fewer clones by IS analysis of the same sample, but with similar contributions of the most dominant clones identified by each method. This suggests DNA barcoding provides greater retrieval of low abundance clones. Over 1 year following HSCT, a total of 5,089 (0.4%) and 20,799 (1.7%) infused clones contributed to hematopoiesis in each animal, respectively, with polyclonal contribution patterns. At 1 year post-transplant, we analyzed PB WBC subsets and identified a total of 116 and 2,246 clones respectively contributing to each of three lineages (T, B, and granulocyte), suggesting <0.2% of infused, gene-modified CD34+ cells displayed multi-lineage potential. Some of these clones emerged in the periphery as early as 1 month after HSCT, indicating a successive pattern of multi-lineage clonal behavior. Adjusting for marking levels, we estimate that 1 in 10,000 transplanted CD34+ cells gave rise to trilineage hematopoiesis at 1 year post-HSCT. We then challenged these animals with chemotherapy (O6-benzylguanine and BCNU) to provide gene modified cells with a selective advantage in vivo. We observed moderate myelosuppression following chemotherapy with stable increases in GFP+ PB WBCs to ~2.5 and 22% in each respective animal. Analysis of the clonal repertoire after chemotherapy identified a significant number of new clones not previously identified in the first year after transplant. This suggests that >1 in 10,000 infused CD34+ cells is capable of trilineage hematopoiesis. In sum, the combination of retrovirus integration site analysis with molecular barcode analysis provides synergistic methods in understanding the kinetics and clonal dynamics of hematopoietic reconstitution following autologous HSCT and revealed a successive pattern of multi-lineage clonal behavior. DisclosuresNo relevant conflicts of interest to declare.

Severe Eosinophilia in Children

The differential diagnosis of hypereosinophilia includes both primary (clonal and idiopathic) and secondary medical conditions. Here we raise the awareness of physicians to the unusual causes of hypereosinophilic states and describe the molecular assays used in the diagnosis of hypereosinophilia. Two unusual cases of hypereosinophilia in children that were initially misdiagnosed are reported. T-cell receptor gene rearrangement, skewed X inactivation, fluorescence in situ hybridization analysis, and chromosomal karyotyping were used to reach the final correct diagnosis. Both patients displayed significant eosinophilia and were initially misdiagnosed as having parasitic infection. Nonspecific T-cell clonal expansion was diagnosed in 1 patient based on the clonality of the T-cell receptor variable γ-chain and the skewed chromosome inactivation. The second patient was diagnosed with B-lineage acute lymphoblastic leukemia with a translocation (5;14) (q13;q32) that is well known to be associated with hypereosinophilia. The level of awareness to clonal expansion of WBC subsets which can cause hypereosinophilia should be high when evaluating a patient with extreme eosinophilia. Advanced molecular assays to detect clonal expansion should be used to exclude aberrant clonal processes in such patients.

Real-Time Identification of Leukocyte Subsets and Cell Surface Receptor Microdomains in the Microvasculature of Wild-Type and Sickle Cell Mice In Vivo.

Intravital microscopy has contributed enormously to the recent mechanistic advances in leukocyte (WBC) trafficking. However, current methods have provided little knowledge about differential WBC subset behavior or the spatial redistribution of surface adhesion molecules during recruitment and activation. Here, we use digital high-speed multichannel fluorescence intravital videomicroscopy to identify WBCs and localize cell surface molecules. Adherent WBCs were ascertained using a series of near-simultaneous 4-channel images in randomly selected venular segments following the injection of low-dose (0.02–0.12 mg/kg) fluorescently labeled Mab against CD45, Gr-1, and F4/80. Preliminary experiments on FAC-sorted WBCs revealed that Gr–1+F4/80- cells were highly enriched in neutrophils (PMNs, ~99%), whereas the F4/80+ fraction was mostly monocytes (~70%) and the fraction negative for both markers was largely lymphocytes (~88%). In the cremaster muscle of C57BL/6 mice (n=6), 39 ± 4% of adherent CD45+ cells recruited in venules ~90 min following surgery were Gr–1+F4/80- (PMNs), and 45 ± 7% were F4/80+ (monocytes), while 18 ± 3% were Gr–1-F4/80- (lymphocytes). In contrast, the proportion of PMNs was significantly increased in cremasteric venules of sickle cell (SS) mice (Berkeley) (79 ± 7%, n=5; p<0.001 compared to C57BL/6). Our previous studies have revealed that the interactions between erythrocytes (RBCs) and adherent WBCs correlate with vascular occlusion and mortality in SS mice (Turhan et al, PNAS, 2002). To determine which WBC subset can capture RBCs in vivo, we monitored RBC-WBC interactions in high-resolution brightfield movies acquired immediately after the capture of fluorescence images. We found that the vast majority of interactions (~93%) occurred with CD45+Gr–1+F4/80- PMNs. The high proportion of lymphocytes in systemic venules was unexpected. We thus analyzed directly lymphocyte subclasses with antibodies specific for B and T cell markers (B220 and CD4/CD8). The proportion of B and T cells was consistent with the above results (total ~20%), similar between C57BL/6 and sickle cell mice, with B cells predominating in both groups (70–76% of all lymphocytes). Since the bone marrow (BM) is a target of SS disease, we examined differential WBC recruitment in this vascular bed. In contrast to the cremasteric microcirculation, CD45+Gr–1-F4/80- lymphocytes represented the major WBC subset in both C57BL/6 and SS venules (49 ± 2% and 63 ± 5%, respectively.) Adherent CD45+B220+ B lymphocytes were significantly more numerous than CD45+CD4/CD8+ T lymphocytes in C57BL/6 (6-fold ↑, p<0.001) and SS (9-fold ↑, p<0.005) mice. We next evaluated the spatial distribution of LFA-1 and PSGL–1 on Gr–1+ WBCs recruited in TNF-α-stimulated cremasteric venules. We observed that PSGL–1 distribution was polarized in the great majority of adherent Gr–1+ cells (93 – 97%), whereas neither LFA–1 nor Gr–1 was polarized. The majority of PSGL–1 clusters was luminal rather than adjacent to the endothelium in both stationary and slowly migrating cells. In stationary cells, PSGL–1 appeared random in relation to the direction of blood flow. By contrast, in migrating cells, PSGL–1 redistribution was strongly associated with the direction of cell migration, with PSGL–1 clusters being almost exclusively located at the trailing edge. In summary, our results reveal a profound difference in the WBC subsets recruited in the cremastric and BM venules, and a powerful method to analyze the formation of discrete microdomains in vivo during cell activation.

Effects of Lactobacillus acidophilus DSM13241 as a probiotic in healthy adult cats

To evaluate the effect of dietary supplementation with the probiotic strain Lactobacillus acidophilus DSM13241 in healthy adult cats. 15 adult cats. Cats were fed a nutritionally complete dry food for 5 weeks. Fecal character was assessed daily, and a single fecal sample and 3-mL blood sample were collected for bacterial enumeration and hematologic analysis, respectively. Cats were then fed the same diet supplemented with L acidophilus DSM13241 (2 x 10(8) CFU/d) for 4.5 weeks. Repeat fecal and hematologic measurements were taken prior to the return to control diet for a 4-week period. The probiotic species was recovered from feces, demonstrating survival through the feline gastrointestinal tract. Probiotic supplementation was associated with increased numbers of beneficial Lactobacillus and L acidophilus groups in feces and decreased numbers of Clostridium spp and Enterococcus faecalis, indicating an altered bacterial balance in the gastrointestinal tract microflora. Fecal pH was also decreased suggesting a colonic environment selective for the beneficial lactic acid bacterial population. Systemic and immunomodulatory effects were associated with administration of L acidophilus DSM13241 including altered cell numbers within WBC subsets and enhanced phagocytic capacity in the peripheral granulocyte population. In addition, plasma endotoxin concentrations were decreased during probiotic feeding, and RBCs had a decreased susceptibility to osmotic pressure. Probiotic strain L acidophilus DSM13241 fed at 2 x 10(8) CFU/d can alter the balance of gastrointestinal microflora in healthy cats. Furthermore, administration of this probiotic results in beneficial systemic and immunomodulatory effects in cats.

WBC content of platelet concentrates prepared by the buffy coat method using different processing procedures and storage solutions.

The number of WBCs in platelet concentrates (PCs) prepared by the buffy coat (BC) method with different storage solutions can result in low (5 x 10(6)/unit) WBC levels by the use of careful centrifugation techniques without filtration. At present, most blood banks use filtration steps to meet these requirements. The difference in processing methods and suspension solutions prompted the investigation of the influence of the various procedures on the WBC and platelet content of PCs. PCs from 5 BCs were harvested without or with inline filtration (AutoStop BC, Pall Corp.) in either plasma (PCs-plasma) or platelet additive solution (PCs-PAS-2). After preparation, samples were taken for counting WBCs and platelets and for analyzing WBC subsets by flow cytometry using specific MoAbs. The WBCs were concentrated before analysis of the WBC subsets. Results less than 2.5 cells per microL were considered below the limit of accuracy of the subset analysis. All filtered PCs met the AABB standard of 5 x 10(6) per unit and the European guidelines of 1 x 10(6) per unit. None of the nonfiltered PCs met the European guidelines, but all met the AABB guidelines. All filtered units gave residual WBC counts below the detection limit for subset analysis. Filtered PCs-plasma gave significantly higher platelet counts than filtered PCs-PAS-2 or nonfiltered PCs (p<0.01, ANOVA). Careful centrifugation of pooled BCs, with plasma or PAS-2, can result in PCs with low WBC contamination levels. However, filtered PCs are superior, because of better WBC removal and higher platelet counts.

Homozygous C1qa Deficiency Alters Pulmonary Clearance of Apoptotic Cells

Clearance of dying (apoptotic) cells from inflamed tissues is an integral step in the control of inflammation, and defective processing of autoantigens present in apoptotic cells may contribute to the initiation of autoimmune syndromes. The complement system is one mechanism that has been implicated in apoptotic cell removal. C1q, the first component of the classical complement cascade, binds to apoptotic cells, potentially facilitating their removal by phagocytes. In support of this hypothesis, mice homozygous for C1qa deficiency (C1qa−/−) spontaneously accumulate apoptotic cells in the kidney, and do not effectively clear apoptotic cells during thioglycolate-induced peritonitis. Interestingly, humans homozygous for C1qa−/− develop an early and severe form of systemic lupus erythematosus. We sought to determine whether C1qa−/− delayed resolution of pulmonary inflammation through defective removal of apoptotic cells. Baseline differences between C1qa−/− mice and control mice were assessed by CBCs, BAL WBC subsets, and apoptotic cells. Inflammation in C1qa−/− mice was evaluated in BAL by measurement of WBC subsets at 24, 48, and 72 h posttreatment with 20 μg of intratracheal endotoxin lipopolysaccharide [LPS]. Spontaneous clearance of apoptotic cells in BAL was assessed at these same time points by nuclear morphology, terminal deoxynucleotidyl nick-end labeling, and propidium iodide/annexin V staining. LPS-treated, C1qa−/− mice were also challenged intratracheally with apoptotic human polymorphonuclear leukocytes (PMNs) at 72 h, and clearance was ascertained. No differences were found in baseline blood cell counts, BAL WBC subsets, or BAL apoptotic cells in C1qa−/− mice vs control mice. LPS increased pulmonary PMN, but not macrophage, influx at 48 h and 72 h in C1qa−/− mice compared to control mice. BAL from LPS-challenged C1qa−/− mice and control mice contained only small percentages of spontaneous apoptotic cells (1 to 3%), and no differences were observed between groups. However, when C1qa−/− mice were challenged intratracheally with apoptotic human PMNs, clearance was decreased. These results demonstrate that C1qa−/− impairs clearance of apoptotic cells and enhances pulmonary inflammation. The impact of dysregulated apoptotic cell removal on interstitial lung disease is as yet unknown, but may provide a basis for ongoing inflammation.EST

Leukocyte rolling in rat mesentery venules: distribution of adhesion bonds and the effects of cytoactive agents.

A new method of analyzing in vivo measurements of leukocyte WBC rolling along venular endothelium (EC) has been developed to extract insightful information on the dynamics of WBC-EC bond formation and disruption. The rolling velocity of WBCs was obtained by intravital microscopy of rat mesenteric venules. For the "spontaneous" rolling observed following exteriorization of the mesentery, we estimated that the average distance between clusters of adhesion bonds which tether a rolling cell to the venular wall was about 2 microm, and that the average lifetime of a bond cluster at the trailing edge of the rolling cell, from its exposure to the tensile force to its release, was on the order of 0.05 s. Both the inter-cluster distance and the lifetime were significantly reduced by treatments with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine and the cytokine interleukin-1, while the average lifetime of the stretched bond clusters was not significantly changed by treatment with the cytoskeleton-modifying agents cytochalasin B and colchicine. Each of the four treatments significantly reduced the heterogeneity in the cell rolling velocity, presumably by the selective recruitment of WBC subsets from the circulating WBC population or by a reduction in the heterogeneity of endothelial adhesiveness. These results were analyzed in the context of in vitro data in the literature on molecular bonds of cell adhesion. The findings suggest that, in the case of "spontaneous" rolling, there are on average approximately 2-3 clusters of adhesion bonds between a rolling cell and the vessel wall, and approximately five bonds in each cluster.

Longitudinal monitoring of WBC subsets in packed RBC units after filtration: implications for transfusion transmission of infections

Specific subsets of peripheral blood WBCs are reservoirs for infectious agents, such as CMV and EBV, and can serve as vectors for transfusion transmission of these agents. While filter WBC reduction has been used to prevent transfusion transmission of infections, its effectiveness has not been documented for many infectious agents and in some instances may be difficult to demonstrate in clinical trials. Because the effectiveness of filtration depends on the number of infected WBCs remaining at transfusion, WBC subpopulations in packed RBC units were quantitated after filtration and storage. Packed RBC units (n = 14) were filtered and stored at 4(o)C for 42 days or were stored without filtration. Serial samples were subjected to flow cytometric immunophenotyping of WBC subsets: neutrophils, monocytes, CD4+ and CD8+ T cells, B cells, and NK cells. Filtration produced a mean reduction in total WBCs of 3.2 log. Monocytes, lymphocytes, and neutrophils were reduced by 4.1, 3.8, and 2.5 log, respectively. Lymphocyte subsets also demonstrated differential reduction with filtration. All WBC subsets showed ongoing loss during storage. Monocyte and lymphocyte subsets are removed most effectively by prestorage filtration. Postfiltration storage leads to further significant reductions in WBC subsets. The implications of these findings for the mitigation of transfusion transmission of infection are discussed.

Differences in Residual White Blood Cell Subset Counts in Buffy Coat‐Depleted Red Cell Concentrates Prepared with Bottom and Top or Quadruple‐Bag Systems

Abstract Background: For preparation of buffy coat‐depleted red cell concentrates (RCCs) in additive solution whole blood is currently collected in The Netherlands both in quadruple‐bag and bottom and top bag systems. By using the quadruple‐bag system both plasma and buffy coat cells are transferred into integrated satellite bags while the red cells remain in the collection bag. When bottom and top bags are used, the buffy coat remains in the collection bag while both red cells and plasma are transferred into satellite bags. The difference in processing prompted us to perform quantitative analysis of residual WBC subsets in buffy coat‐depleted RCCs. Differences in removal of specific cells with the buffy coat could improve the outcome of additional filtration procedures aiming at complete removal of specific WBC subsets. Study Design and Methods: The buffy coat was removed in semiautomated procedures (Optipress I; Compomat G4) from units of whole blood collected in both bag systems. Paired samples were taken before and after removal of the buffy coat for counting and analyzing WBC subsets by flow cytometry using subset specific monoclonal antibodies. Results: All RCCs met the criteria from the guidelines of the Council of Europe. The percentage of residual total WBCs was lower (p &lt; 0.001) in RCCs processed in bottom and top bag systems (26% Compomat and 18% Optipress) as compared to RCCs processed in quadruple‐bag systems (43% compomat). WBC subset analysis in RCCs processed in quadruple‐bag systems showed approximately 25% of residual T cells, B cells and monocytes and 60% of residual granulocytes. In contrast, WBC subset analysis in RCCs processed in bottom and top bag systems showed approximately 2% residual T cells, B cells, and monocytes and 35% residual granulocytes; in about 45% of units, lymphocytes and monocytes were even below the detection limit of flow cytometry analysis. Conclusion:Buffy coat‐depleted RCCs are currently processed in bottom and top bag or quadruple‐bag systems, the former being superior to the latter due to selective depletion of lymphocytes and monocytes by 98% (2 logs). The bottom and top procedure is an evident contribution to leukodepletion in blood transfusion, both with and without additional filtration.

Survival of Donor Leukocyte Subpopulations in Immunocompetent Transfusion Recipients: Frequent Long-Term Microchimerism in Severe Trauma Patients

We recently reported detection of a transient increase in circulating donor leukocytes (WBCs) in immunocompetent recipients 3 to 5 days posttransfusion (tx) (Blood 85:1207, 1995). We have now characterized survival kinetics of specific donor WBC subsets in additional tx populations. Eight female elective surgery patients (pts) were sampled pre-tx and on days 1, 3, 5, 7, and 14 post-tx. Ten female trauma pts transfused with a total of 4 to 18 U of relatively fresh red blood cells were sampled up to 1.5 years post-tx. WBC subsets from frozen whole blood were isolated using CD4, CD8 (T cell), CD15 (myeloid), and CD19 (B cell) antibody-coated magnetic beads. Donor WBCs were counted by quantitative polymerase chain reaction (PCR) of male-specific sex determining region (SRY) sequences. PCR HLA typing and mixed leukocyte reaction (MLR) between recipient and donor WBCs were performed on two of the trauma tx recipients who had long-term chimerism of donor cells post-tx. In 6 of 8 female surgery pts, circulating CD4(+) male donor cells peaked at day 3 or 5 (0.01 to 1 cell/microL), followed by clearance by day 14. In 7 of 10 female trauma pts, we observed multilineage persistence of male donor WBCs (CD4, CD8, CD15, CD19) for 6 months to 1.5 years post-tx at concentrations of 10 to 100 cells/microL. In 2 trauma recipients studied, MLR showed no, or very low, response to WBC of the single donor implicated as the source of microchimerism by HLA typing. Establishment of long-term multilineage chimerism in trauma recipients is probably caused by engraftment of donor stem cells and mutual tolerance between recipient and donor leukocytes. A better understanding of factors determining clearance versus chimerism of transfused leukocytes is critical to prevention of alloimmunization and transfusion-induced graft-versus-host disease, and, potentially, to induction of tolerance for transplantation.

Survival of Donor Leukocyte Subpopulations in Immunocompetent Transfusion Recipients: Frequent Long-Term Microchimerism in Severe Trauma Patients

AbstractWe recently reported detection of a transient increase in circulating donor leukocytes (WBCs) in immunocompetent recipients 3 to 5 days posttransfusion (tx) (Blood 85:1207, 1995). We have now characterized survival kinetics of specific donor WBC subsets in additional tx populations. Eight female elective surgery patients (pts) were sampled pre-tx and on days 1, 3, 5, 7, and 14 post-tx. Ten female trauma pts transfused with a total of 4 to 18 U of relatively fresh red blood cells were sampled up to 1.5 years post-tx. WBC subsets from frozen whole blood were isolated using CD4, CD8 (T cell), CD15 (myeloid), and CD19 (B cell) antibody-coated magnetic beads. Donor WBCs were counted by quantitative polymerase chain reaction (PCR) of male-specific sex determining region (SRY) sequences. PCR HLA typing and mixed leukocyte reaction (MLR) between recipient and donor WBCs were performed on two of the trauma tx recipients who had long-term chimerism of donor cells post-tx. In 6 of 8 female surgery pts, circulating CD4+ male donor cells peaked at day 3 or 5 (0.01 to 1 cell/μL), followed by clearance by day 14. In 7 of 10 female trauma pts, we observed multilineage persistence of male donor WBCs (CD4, CD8, CD15, CD19) for 6 months to 1.5 years post-tx at concentrations of 10 to 100 cells/μL. In 2 trauma recipients studied, MLR showed no, or very low, response to WBC of the single donor implicated as the source of microchimerism by HLA typing. Establishment of long-term multilineage chimerism in trauma recipients is probably caused by engraftment of donor stem cells and mutual tolerance between recipient and donor leukocytes. A better understanding of factors determining clearance versus chimerism of transfused leukocytes is critical to prevention of alloimmunization and transfusion-induced graft-versus-host disease, and, potentially, to induction of tolerance for transplantation.

White cell subsets in apheresis and filtered platelet concentrates.

White cell (WBC)-reduced platelet concentrates (PCs) are defined by their absolute WBC count, a criterion which provides no information regarding the various WBC subsets contained in the PC. These heterogeneous cells are known to mediate different physiologic and pathophysiologic functions and account for distinct adverse transfusion responses. This study describes a method which allows the detection and quantification of these subsets and characterizes their presence in a variety of platelet components. Random-donor pooled PCs (RD PCs) and single-donor apheresis PCs (SD PCs) were studied. RD PCs consisting of 6 units of 2- to 3-day old PCs were randomly assigned to be filtered with one of four WBC-reduction filters from three different manufacturers (n=34). The residual WBCs were pelleted by centrifugation and isolated on a density gradient. The various WBC subsets were quantified by flow cytometry in unfiltered and filtered PCs using fluorescence and two-angle light scatter. SD PCs obtained with two manufacturer's systems and three processing protocols (n=30) were studied in like manner. WBC counts for non-WBC-reduced PCs averaged 3 x 10(8) in RD PCs and ranged from 8.6 to 9.6 x 10(6) per SD PC. Residual WBC counts in filtered PCs ranged from 2.3 x 10(4) to 2.2 x 10(5) and those in WBC-reduced SD PCs averaged 2.2 x 10(5) per unit. The data demonstrate significant phenotypic differences among PCs produced with various procedures. All SD PCs and two of four filtered RD PCs contained five WBC populations including granulocytes and monocytes, while RD PCs filtered with the remaining manufacturer's devices contained only lymphocytes. The data confirm that distinct phenotypic differences exist among PCs prepared with different devices and/or procedures. It is suggested that as for non-generic pharmaceuticals, the clinical benefits of these various PCs should be individually proved.

Quantitation of white cell subpopulations by polymerase chain reaction using frozen whole-blood samples. Viral Activation Transfusion Study.

Previous methods for processing whole blood (WB) for nucleic acid analyses of white cells (WBCs) required fresh blood samples. A simple protocol that involves the freezing of WB for quantitative polymerase chain reaction (PCR) analyses was evaluated. Controlled studies were conducted in which paired fresh and frozen WB preparations were analyzed. The integrity of WBCs in the frozen WB samples was first assessed by flow cytometry using CD45 fluorescence, and calibration beads to quantitate recovery of WBC subsets. PCR of an HLA-DQ-A sequence was used to quantitate residual WBCs in a double-filtered red cell (RBC) component spiked with serial dilutions of WBCs, as well as in 51 filtered RBCs and 19 filtered platelet concentrates. Y-chromosome-specific PCR was used to quantitate male WBCs in five female WB samples spiked with serial dilutions of male WBCs and in serially collected frozen WB samples from four females transfused with male blood components. By flow cytometry, all major WBC subpopulations in frozen-thawed WB were quantitatively recovered and immunologically intact, although they were nonviable. HLA-DQ-A PCR quantitation of a dilution series from 8 to 16,700 per mL of WBCs spiked into double-filtered RBCs showed linear correlation of the results with both fresh and frozen preparations of the expected WBC concentrations (r2 = 0.98, p<0.0001 for both), without significant difference between observed and expected values (p>0.05). Y-chromosome-specific PCR results in female WB samples spiked with male WBCs were not significantly different in fresh and frozen preparations over a 3 log10 range of male cells. The results of WBC survival studies on frozen WB samples were consistent with previous observations in fresh blood samples. Direct freezing of WB enables subsequent recovery of WBCs for quantitative PCR analyses, with results comparable to those of fresh preparations.This protocol should facilitate wider implementation of nucleic acid-based analyses for quality control of WBC-reduced components, as well as for prospective clinical studies of microchimerism in transfusion and transplant recipients.

Target antigen(s) in endometrial autoimmunity of endometriosis.

To obtain the molecular weights (MW) of endometrial antigens eliciting immunoglobulin (Ig) G auto-antibodies in all endometriosis patients irrespective of their place of origin or race, and to verify their specificity and immunogenicity. We tested the serum and peritoneal fluid (P.F.) of 76 endometriosis patients and 24 controls from 4 cities against endometrial and implant antigens by Western blot analysis. Endometrial and implant antigens with MW of 34, 46/48, 64, 84, 94 and 120 kDa bound with IgG in serum and PF of most patients, but not the controls. Antigen(s) with MW of 64 kDa was reactive against serum or P.F. IgG of patients from all cities. Specificity: Endometrial and implant extracts did not react with monoclonal antibodies to WBC subsets and 5 sera with nuclear antibodies. Also, the presence of nuclear and endometrial antibodies did not correlate in 20 other patients with endometriosis. Immunogenicity: We immunized rabbits with the native and eluted (MW 29 to 68 kDa and > or = 68 kDa) endometrial and implant proteins. The antiserum had specific IgG binding to the same glandular epithelial antigens as those bound by the patient's serum. Endometrial antigens with MW of 34, 46/48, 64, 94 and 120 kDa, especially 64 kDa appear to be specific, immunogenic and relevant to endometrial autoimmunity in all patients with endometriosis.