FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN

A correct identification of seropositive individuals for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T cell line that stably expresses the full-length native spike S protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a ratio of the mean fluorescence intensities obtained by double- staining with the sera and a monoclonal antibody specific for EGFR. We show that the method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. It can also be employed to detect neutralizing activity in the sera of individuals. Finally, the method can be used in a multiplexed format to simultaneously measure all anti-S human immunoglobulin isotypes in blood and mucosal fluids including total saliva.

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A simple, sensitive and quantitative FACS-based test for SARS-CoV-2 serology in humans and animals

10.1101/2021.09.06.21262027 ◽

2021 ◽

Author(s):

Agnes Maurel Ribes ◽

Pierre Bessiere ◽

Jean-Charles Guery ◽

Eloise Joly Featherstone ◽

Timothee Bruel ◽

...

Keyword(s):

Flow Cytometry ◽

Tissue Culture ◽

Fluorescent Protein ◽

Dynamic Range ◽

Elisa Test ◽

Negative Control ◽

Serological Tests ◽

S Protein ◽

Flow Test ◽

Better Than

Serological tests are important for understanding the physiopathology and following the evolution of the Covid-19 pandemic. Assays based on flow cytometry (FACS) of tissue culture cells expressing the spike (S) protein of SARS-CoV-2 have repeatedly proven to perform slightly better than the plate-based assays ELISA and CLIA (chemiluminescent immuno-assay), and markedly better than lateral flow immuno-assays (LFIA). Here, we describe an optimized and very simple FACS assay based on staining a mix of two Jurkat cell lines, expressing either high levels of the S protein (Jurkat-S) or the mCherry fluorescent protein (Jurkat-R, which serve as an internal negative control). We show that this Jurkat-S&R-flow test has a much broader dynamic range than a commercial ELISA test and performs at least as well in terms of sensitivity and specificity. Also, it is more sensitive and quantitative than the hemagglutination-based test HAT, which we described recently. The Jurkat-R&S-flow test requires only a few microliters of blood; thus, it can be used to quantify various Ig isotypes in capillary blood collected from a finger prick. It can be used also to evaluate serological responses in mice, cats and dogs. FACS tests offer a very attractive solution for laboratories with access to tissue culture and flow cytometry who want to monitor serological responses in humans or in animals, and how these relate to susceptibility to infection, or re-infection, by the virus, and to protection against Covid-19.

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SARS-CoV-2 serological assay and viral testing: a report of professional football setting

Postgraduate Medical Journal ◽

10.1136/postgradmedj-2021-140176 ◽

2021 ◽

pp. postgradmedj-2021-140176

Author(s):

Bahar Hassanmirzaei ◽

Zohreh Haratian ◽

Ali Ahmadzadeh Amiri ◽

Amir Ahmadzadeh Amiri ◽

Navid Moghadam

Keyword(s):

Standard Test ◽

Football Players ◽

Professional Football ◽

Serological Testing ◽

Igg Antibodies ◽

Current Standard ◽

Serological Tests ◽

The Mean ◽

Viral Testing ◽

Pcr Test

Purpose of the studyPCR is the current standard test for the diagnosis of SARS-CoV-2 infection. However, due to its limitations, serological testing is considered an alternative method for detecting SARS-CoV-2 exposure. In this study, we measured the level of SARS-CoV-2 IgM and IgG antibodies of male professional football players and compared the results with the standard PCR test to investigate the association between the two tests.Study designParticipants were male professional football players and team officials. Nasopharyngeal swabs and peripheral blood samples were collected for the PCR and serological tests, respectively. Also, previous records of COVID-19 testing and symptoms were gathered. Those with previous positive PCR tests who tested negative for the second time were considered to be recovered patients.ResultsOf the 1243 subjects, 222 (17.9%) were seropositive, while 29 (2.3%) tested positive for the SARS-CoV-2 PCR test. Sixty percent of symptomatic cases with a negative PCR were found to be seropositive. The mean level of IgM was significantly higher in PCR-positive and symptomatic subjects, whereas the recovered cases showed significantly higher levels of IgG.ConclusionOur study revealed an inconsistency of results between the two tests; therefore, although application of serological assays alone seems insufficient in diagnosing COVID-19 disease, the findings are beneficial in the comprehension and the management of the disease.

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Efficacy of SSRIs on cognition of Alzheimer's disease patients treated with cholinesterase inhibitors

International Psychogeriatrics ◽

10.1017/s1041610209990184 ◽

2009 ◽

Vol 22 (1) ◽

pp. 114-119 ◽

Cited By ~ 27

Author(s):

Luca Rozzini ◽

Barbara Vicini Chilovi ◽

Marta Conti ◽

Erik Bertoletti ◽

Marina Zanetti ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cholinesterase Inhibitors ◽

Mmse Score ◽

Joint Effect ◽

Negative Effects ◽

Multidimensional Assessment ◽

Degree Of Protection ◽

Depressed Patients ◽

The Mean

ABSTRACTBackground: This study examines the joint effect on cognition of selective serotonin re-uptake inhibitors (SSRIs) and cholinesterase inhibitors (AChEIs) in depressed patients affected by Alzheimer's disease (AD) living at home.Methods: The study was conducted in two different outpatient neurological clinics. 338 patients with probable AD were treated with ChEis (donepezil, rivastigmine and galantamine) as per the clinician's judgment and were observed for nine months. At study entry, participants underwent a multidimensional assessment evaluating cognitive, functional and psychobehavioral domains. All patients were evaluated at baseline, after one (T1), three (T2) and nine months (T3). Patients were grouped in three different categories (patients not depressed and not treated with SSRIs, patients depressed and treated with SSRIs, and patients depressed but not treated with SSRIs).Results: At baseline 182 were diagnosed as not depressed and not treated with SSRIs, 66 as depressed and treated with SSRIs, and 90 as depressed but not treated with SSRIs. The mean change in MMSE score from baseline to nine months showed that depressed patients not treated worsened in comparison with those not depressed and not treated with SSRIs (mean change −0.8 ± 2.3 vs 0.04 ± 2.9; p = 0.02) and patients depressed and treated with SSRI (mean change −0.8 ± 2.3 vs 0.1 ± 2.5; p = 0.03).Conclusions: In AD patients treated with AChEIs, SSRIs may exert some degree of protection against the negative effects of depression on cognition.

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Detection of the Human Erythrocyte Surface Antigen Gerbich by Flow Cytometry using Human Antibodies and Phycoerythrin for Extreme Immunofluorescence Sensitivity

Vox Sanguinis ◽

10.1111/j.1423-0410.1987.tb05072.x ◽

1987 ◽

Vol 53 (4) ◽

pp. 231-234 ◽

Cited By ~ 13

Author(s):

Thomas M. McHugh ◽

Marion E. Reid ◽

Daniel P. Stites ◽

Eric S. Chase ◽

Conrad H. Casavant

Keyword(s):

Flow Cytometry ◽

Surface Antigen ◽

Human Erythrocyte ◽

Human Antibodies ◽

Erythrocyte Surface

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New evidence of spontaneous cure in human Chagas' disease

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822003000100014 ◽

2003 ◽

Vol 36 (1) ◽

pp. 103-107 ◽

Cited By ~ 22

Author(s):

Sonia S. Francolino ◽

Antonio Fernandez Antunes ◽

Rodolfo Talice ◽

Rachel Rosa ◽

Joel Selanikio ◽

...

Keyword(s):

Flow Cytometry ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Acute Phase ◽

Clinical Data ◽

Specific Treatment ◽

Serological Tests ◽

Spontaneous Cure ◽

New Evidence ◽

Cruzi Infection

A new case of spontaneous cure of human Chagas' disease is described in Uruguay. An 87-year-old man who had a typical acute phase of Trypanosoma cruzi infection in 1947 and never received specific treatment against the disease, when examined in 1998 revealed several completely negative parasitological and serological tests, including traditional serology, PCR and flow cytometry. As a whole, such findings fulfill the current criteria to define the cure of Chagas' disease. Clinical data suggest the possibility of a benign evolution of Chagas' disease in this case, but the basic findings (slight cardiac and esophageal impairment) could also be due to the advanced age of the patient.

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Effect of Erythropoietin on Platelet Function and Glycoprotein Expression in Patients with MDS.

Blood ◽

10.1182/blood.v106.11.3993.3993 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3993-3993

Author(s):

Fotis F.I. Girtovitis ◽

Dimitrios D. Bougiouklis ◽

Michel M.P. Makris ◽

Elefteria E. Pithara ◽

Pantelis P.E. Makris

Keyword(s):

Flow Cytometry ◽

Platelet Function ◽

Normal Subjects ◽

Mean Value ◽

Special Treatment ◽

Recombinant Erythropoietin ◽

Mean Values ◽

Human Recombinant Erythropoietin ◽

The Mean ◽

The Difference

Abstract Aim: We decide to study the effect of erythropoietin on the platelet function and glycoproteins expression in patients (pts) with myelodysplastic syndrome (MDS). All patients suffered from primary MDS but none of them received any special treatment. From our study pts that suffered from diseases or they were receiving medication that affect the function of platelets were excluded. Furthermore pts with platelet count<50,000/μl were excluded also because we were unable to check the aggregation of platelets. Material: 41 subjects were studied, 15 normal subject (10 men and 5 women mean age 66,6±14 years old)and 26 pts (17 men and 9 women with mean age70,9±7,4 years old) suffered from all types of MDS according to FAB criteria (9 with RA, 3 with RARS, 7 RAEB, 4 with CMML and 3 with RAEB-t). We divided them in 2 groups: 1st - 7 Pts receiving human recombinant erythropoietin (rEPO) with mean dose 30.000 iu subcutaneous weekly and 2nd - 19 without rEPO. Methods. 1- The platelet function was studied in Platelet Ionized Calcium Aggregometer (PICA) using Ristocetin, ADP, Collagen and Adrenalin as stimulators. 2- The expression of platelet glycoproteins (GPIb, IX, IIb, IIIa and P-selectin) was studied using the flow cytometry and special monoclonal antibodies. This way the percentage of glycoprotein expressed in platelet membrane and MFI were estimated. We performed the statistical analysis of our results using the t-test with common standard deviation.. Results: our results concerning the aggregation test and flow cytometry are presented in tables 1,2 and 3. From the study of our results we can see that while the decrease of aggregation between the patients under EPO and normal subjects is statistically non significant (p<0.1)the decrease of the corresponding values between the pts that did not received EPO and normal subjects was statistically very significant for all stimulators (p <0,001). pts under EPO show an important increase of platelet expressing GPIIb percentage grater than the expressed percentage of patients without EPO (60% vs. 46,1% correspondingly, p<0.001). The difference of expressed MFI was not statistically significant. Conclusion: The findings of our study show us that erythropoietin improves the function of platelets in patients with MDS, probably through the increase of platelet percentage which express glycoproteins. Table 1 Comparison of the mean value of aggregation between the groups Table 2 Comparison of the mean values of the platelets expressing corresponding glycoproteins percentage between the groups Table 3 Comparison of the mean value of MFI between the groups

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FLT-3 Activity and Its Response to Drugs Can Be Determined in AML Blast Cells by FLT-3 Phosphorylation Status Using Flow Cytometry.

Blood ◽

10.1182/blood.v108.11.2308.2308 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2308-2308

Author(s):

Tiziana Grafone ◽

Michela Palmisano ◽

Emanuela Ottaviani ◽

Alberto Maria Martelli ◽

Alessandra Cappellini ◽

...

Keyword(s):

Flow Cytometry ◽

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Receptor Activation ◽

Molecular Defects ◽

Activating Mutation ◽

Blast Cells ◽

The Mean

Abstract One of the most common molecular defects identified in acute myeloid leukemia (AML) patients is an activating mutation of FLT3 tyrosine kinase. The identification of activated FLT3 as a contributor to the cause and progression of much leukemia has led to its consideration as a potential target for therapy. Since small molecule FLT3 kinase inhibitors are actually in clinical trials; a robust and standardized method for screening of FLT3 receptor activation is necessary. We evaluated the expression level of FLT3 receptor (CD135) by Facs analysis. We developed a flow cytometry method to measure FLT3 phosphorylation (P-FLT3) in samples with <10 (e)5 cells. The amount of P-FLT3 in the samples was determined as the mean fluorescence intensity (MFI). The P-FLT3 status of the treated samples was expressed as a percentage of the untreated control (100%). The method was first validated in FLT3 wild-type (HL-60) and mutant (MV4-11/ITD+) as well as FLT3 negative (K562) cell lines. The method also provides to be reproducible with samples AML from patients. Analysis was performed after exposure to drugs, in vitro and in vivo. In response to increasing drugs concentration (CEP-701 and SU11657) there was a linear reduction in P-FLT3. The results validate a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry, and enable an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.

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Elevated Complement (C) and IgG Bound Microparticles (MP) in Immune Thrombocytopenic Purpura (ITP) and Hemolytic Anemia (HA).

Blood ◽

10.1182/blood.v114.22.1313.1313 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1313-1313

Author(s):

Wenche Jy ◽

Lawrence L Horstman ◽

Andrew Lin ◽

Carlos Bidot ◽

Yeon-Soong Ahn

Keyword(s):

Flow Cytometry ◽

Cell Activation ◽

Conflicts Of Interest ◽

Linear Regression Analysis ◽

Red Cell ◽

Major Mechanism ◽

Cell Destruction ◽

The Mean ◽

Underlying Mechanisms ◽

Normal Controls

Abstract Abstract 1313 Poster Board I-337 Background Cell derived microparticles (MP) are shed during cell activation and apotosis and MP profiles reflect the status of cell disturbances in various forms of pancytopenias. It was demonstrated that when antibodies fix complement (C) on the membrane, red cells evade C mediated lysis by shedding MP with bound C. We analyzed C and IgG bound to MP to gain insight on the underlying mechanisms of cell destruction. We measured C1q fragment and IgG on cell-derived (MP) in plasmas of patients with ITP, hemolytic anemias (HA) and thrombosis (TBS). Methods (1) Patient population. Consenting patients consisted of 18 TBS, 14 ITP and 6 HA (5 AIHA and 1 TTP), as well as 20 normal controls (NC). (2) Flow cytometry. MP were centrifuged from 1 mL fresh (not frozen) platelet-poor plasma (PPP), washed twice with saline, resuspended in 100 μL, then incubated with fluorescent mAb to C1q and IgG, then analyzed by flow cytometry. In addition, MP in the PPP were analyzed for MP from RBC (RMP) using marker glycophorin A, and MP from platelets (PMP) by CD42b. Values were considered elevated if >2SD above the mean of NC. Control values were (mean ±SD per μL): C1q+ MP = 536 ±151; IgG+ MP = 5,542 ±2,081; RMP = 823 ±246; PMP = 7,520 ±2,084. Results We observed significant elevation of C1q+ and IgG+ MP in patients with ITP (2-3 fold) and hemolytic anemias (6-10 fold) but not in those with thrombosis. These findings indicate that complement mediated cell destruction or disturbance in these disorders is frequent. (1). The ITP group consisted of 2 subgroups, one of which had elevated C1q+ and IgG+ MP, the other not, and these subgroups also differed in RMP. Specifically, six ITP with high C1q+ MP also had high RMP (1,899 ±682 /μL) and PMP (16,602 ±4,216 /μL) while those with normal C1q+ MP had normal RMP (504 ±186 /μL) and PMP (3,472 ±1196 /μL). This suggests that platelet destruction in ITP can proceed via C in some but not all cases, probably depending on the autoantibodies. We have previously reported high RMP in ITP. These findings suggest subclinical C mediated hemolysis in a subset of ITP. (2) In HA patients, all 6 had elevated C1q+ MP (3,934 ±1,419 /μL, p<0.001) as compared to normal controls (536 ±151 /μL). The mean in HA was nearly 6-fold greater than NC. The HA group also had higher IgG+ MP, with mean counts about 10-fold higher than NC (61,531 ±20,733 vs. 5,542 ±2,081 /μL, p<0.001). Furthermore, the HA patients also had elevated RMP (2,191 ±635 /μL, p<0.01). This suggests that C-mediated destruction of RBC is a major mechanism in HA. (3) Linear regression analysis showed that C1q+ MP is well correlated with IgG+ MP (R = 0.84, p<0.0001) and RMP (R = 0.79, p<0.001). (4) The TBS group did not show higher levels of any of the measures assayed. It is widely believed that phagocytosis is the mechanism of cell destruction in ITP and AIHA. Our findings support the concept that complement (C) -mediated platelet or red cell destruction play an important role and is common in these disorders. Assay of C and IgG on MP can provide new insight to underlying mechanisms of immune mediated platelet and red cell destruction. Disclosures No relevant conflicts of interest to declare.

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FISH Redoux — A Novel Way to Emulate CLL Leukemic Cells From Buffy Coat Samples

Blood ◽

10.1182/blood.v116.21.3606.3606 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3606-3606

Author(s):

Stephanie A. Smoley ◽

Patricia T. Greipp ◽

Neil E. Kay ◽

Tait D. Shanafelt ◽

Jeanette E. Eckel-Passow ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Cell Population ◽

Cell Sorting ◽

Mayo Clinic ◽

Buffy Coat ◽

Fish Analysis ◽

Cell Type ◽

The Mean

Abstract Abstract 3606 Background: Cytogenetic abnormalities are important prognostic indicators in CLL. At Mayo Clinic, CLL FISH analyses are derived from buffy coat samples not purified for lymphocytes. Scoring is therefore performed on consecutive qualifying nuclei regardless of cell size, shape or morphology. This scoring may not reflect the percent of abnormal nuclei of the affected cell type, but rather the entire population of cells present in the sample. We sought to determine whether the percent abnormal nuclei in only the affected cell type (e.g. lymphocytes) of patients with CLL differed from that of the general cell population. Scoring only B-lymphocytes could increase the sensitivity of the test in patients with low B-cell counts, either early in their disease or after treatment. Cell sorting techniques could be used to reach this goal but can be expensive, labor intensive and add to completion time. We propose a “Poor Man's Cell Sorting” technique based on cell morphology when stained with 4',6-diamidino-2-phenylindole (DAPI). In CLL samples, both round and so called “lobed” cells are seen by DAPI staining but we hypothesize that the CLL B-cells typically present as perfectly round in shape (Fig. 1). Thus our hypothesis: if only the round cells are scored for a genetic defect, would this more accurately represent the malignant leukemic B-cell population and allow for enhanced disease status by FISH? Methods: After IRB approval, we identified 87 CLL patients (75% male; 25% female, mean age 62.4 y, range 43–89 y) in the Mayo Clinic CLL Database, who were seen at Mayo Clinic between March, 2002 - July, 2010. We selected patients known to have low level FISH abnormalities and who therefore should have a significant population of both lymphocytes and neutrophils. Selection criteria included <20×106 B-cells per microliter (by flow cytometry) of peripheral blood and ≤40% of interphase nuclei expressing a specific FISH abnormality. Although most of the 87 patients exhibited more than one FISH abnormality, we focused on only one FISH defect per patient for this study. The original specimen (slide or fixed cell pellet) was rescored for % abnormal nuclei among 100 consecutive round and 100 consecutive lobed (or multilobular) nuclei, using DAPI to identify nuclear architecture. These scores were compared to each other and to the original clinical FISH analysis (scored for 200 consecutive nuclei and not selected for nuclear morphology). Results: Among 87 cases, FISH signals were scored for 6q-(1), 11q-(9), +12(15), 13q-(58), 17p-(2) and 2 IGH rearrangements. In 86/87 cases, the abnormal lobed nuclei did not have FISH defects. One patient exhibited 35% +12 by clinical assay, 52% +12 in the round nuclei, and 11% by lobed nuclei. For all cases, the mean percentage of abnormal nuclei was greater in the round cells (46%) vs original scoring (23.6%). In 79/87 patients (91%), the % of abnormal nuclei was greater in the round cells vs original FISH (mean increase 24.3%; range 1.5–57%). One patient's score was the same in round cells vs original, and 7 patients exhibited fewer abnormal nuclei in the round cells vs original. For these 7 cases, the mean % abnormal nuclei was 5.6% for round nuclei vs 8.4% for original score (score differences ranged from 1 – 5.5%). Overall, by univariate regression analysis, round cells (p=0.0043) have a better correlation with % B-cells, ascertained by flow cytometry, than either the current clinical approach (p=0.0462) or the lobed-cell approach (p=0.4058). Discussion: In virtually all cases (99%), the abnormal FISH patterns were confined to the round nuclei, and the lobed nuclei were virtually always normal by FISH. For patients with <20×106 lymphocytes, selecting for round nuclei uniformly resulted in identification of a higher percentage of abnormal cells. Further studies, including comparison of our round nucleus approach to CLL FISH analysis to actual sorted B-lymphocyte cell selection and the association of this leukemic restricted estimation of abnormal FISH levels to clinical outcome are necessary. Given our findings, we believe that estimating FISH defects restricted to the leukemic B-cell population will become an important adjunct to cytogenetic analysis for patients with low lymphocyte counts, including those in clinical remission and those with minimal residual disease. Disclosures: No relevant conflicts of interest to declare.

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Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Blood ◽

10.1182/blood.v122.21.2030.2030 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2030-2030

Author(s):

Nishant Tageja ◽

Neha Korde ◽

Constance Yuan ◽

Kristen Cole ◽

Jennifer Hsu ◽

...

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

High Risk ◽

Tumor Cell ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cell ◽

Myeloma Cells ◽

Cd34 Cells ◽

The Mean

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare.

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