Cytometry Part B (Clinical Cytometry) 00B:00–00 (2016)
Original Article
Thymic Output: Assessment of CD41 Recent Thymic Emigrants and T-Cell Receptor Excision Circles in Infants Eugene Ravkov,1* Patricia Slev,1,2 and Nahla Heikal1,2 1
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah
2
Background: CD41 recent thymic emigrants (CD41 RTEs) constitute a subset of T cells recently generated in the thymus and exported into peripheral blood. CD41 RTEs have increased copy numbers of T-cell receptor excision circles (TREC). They are characterized by the expression of CD31 on na€ıve CD4 T-cells. We aimed to validate a flow-cytometry assay to enumerate CD41 RTEs and assess its performance in relation to TREC measurement. Methods: CD41 RTEs cell count in peripheral blood was measured to determine sample stability, precision, linearity, and to establish reference ranges. TRECs were measured using qPCR assay performed with DNA isolated from peripheral blood. CD41 RTEs, TRECs, and flow cytometry results for major T-cell markers were assessed in 50 infants less than 2 years of age. Results: Inter-and intra-assay precisions (% CV) were 1.5–12.2 and 1.5–7.0, respectively. Linearity studies showed that the results are linear over a range of 0.7 to 403.0 CD41 RTEs/lL of blood. There was 84% agreement (42 of 50) between CD41 RTEs and TRECs qualitative results for the infant samples. CD41 RTEs reference ranges in 17 healthy children was in agreement with published data, while that of the healthy adults were 51–609 cells/lL of blood. Conclusion: The validation results provide acceptable measures of the CD41 RTEs test performance within CAP/CLIA frameworks. CD41 RTEs and TRECs assays show high agreement in the infant population. The CD41 RTEs test can be used as a confirmation for the TREC results along with or as an alternaC 2016 International Clinical tive to T-cell phenotyping in infants with repeatedly low TRECs concentrations. V Cytometry Society
Key terms: recent thymic emigrants; T-cell receptor excision circles; thymic output; SCID; immune reconstitution
How to cite this article: Ravkov, E., Slev, P. and Heikal, N. Thymic Output: Assessment of CD41 Recent Thymic Emigrants and T-Cell Receptor Excision Circles in Infants. Cytometry Part B 2016; 00B: 000–000.
Recent thymic emigrants (RTEs), including CD41 and CD81, constitute a T-cell population that has been recently generated in the thymus and exported into peripheral blood (1–3). These cells differ from the rest of T-cells with respect to their phenotype and function. RTEs have decreased proliferation, cytokine production, and transcription factor expression (3). They are essential for maintaining peripheral T cells in sufficient diversity (4–7). The appearance of T-cell receptor excision circles (TRECs) is commonly used to identify RTEs (3). TRECs are generated during T-cell receptor (TCR) rearrangement (8,9); they are not capable of replication and eventually become diluted during cell division, which makes TRECs concentrations in the periphery correlate
C 2016 International Clinical Cytometry Society V
with thymic output (9,10). High TRECs concentrations were detected during childhood, and decreased with increasing age (11). In HIV-1 patients, TRECs were
Additional Supporting Information may be found in the online version of this article. Correspondence to: Eugene Ravkov, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah. E-mail: eugene.v.ravkov@ aruplab.com Grant sponsor: ARUP Institute for Clinical and Experimental Pathology. Received 9 June 2015; Revised 30 October 2015; Accepted 6 November 2015 Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cyto.b.21341
2
RAVKOV ET AL.
significantly lower than normal age matched controls and their level of TRECs were restored by antiretroviral treatment (11). TRECs concentration increased rapidly during T cell reconstitution in patients who received stem cell transplantation (11). Also, patients with congenital absence of the thymus, like complete Digeorge Syndrom have no detectable TRECs (11). The publication of these data suggested that TRECs concentration in the periphery is a direct marker for thymic output. CD41 RTEs are a population of na€ıve T cells that have high TRECs copy numbers indicating that they recently derived from the thymus (12–15). A number of studies have shown a progressive decrease of RTEs with age, which directly correlates with progressive physiological involution of the thymus (12,16). In humans, CD41 RTEs maturation into na€ıve T-cells is associated with decease of PTK7, CD31 (PECAM-1), and TRECs (2,5,12). It is also associated with shortening of telomeres and decreased telomerase activity (12,13). Despite thymic involution, the total T-cell count remains relatively stable due to ongoing maintenance by a variety of peripheral thymus-independent homeostatic mechanisms (2,13). Recently, it has been demonstrated that CD31 can be used as a cell surface marker to distinguish CD41 RTEs (CD41 CD45RA1 CD311) with high TRECs content from proliferated, peripheral, na€ıve T cells (CD41 CD45RA1 CD312) with diminished TRECs content that implies proliferation (12–15). CD81 RTEs express CD103, but have been difficult to identify (3). It has also been shown that CD4 : CD8 ratio is about four folds higher in RTEs than that in the total population of T cells (3). CD31 is a 130-kDa transmembrane glycoprotein that is expressed by endothelial cells, monocytes, neutrophils, platelets and certain T-cells (13,17). CD31 expression on CD41 na€ıve T-cells directly correlates with high copy numbers of TRECs and high telomerase activity, which makes it a suitable cell subset to evaluate thymic output (12,13). The same study and others found that both TRECs and telomerase activity progressively decrease and correlate with CD41 RTEs decreased numbers in aged subjects (18,19). Monitoring of thymic output provides an important parameter in establishing appropriate diagnosis and treatment strategies for patients with cellular or combined primary immunedeficiencies, and monitoring immune-reconstitution in patients undergoing anti-HIV treatment, chemotherapy and post-hematopoietic cell or thymus transplantation (20–23). In this study, we present the validation data of a flow cytometry-based test that enables enumeration of CD41 RTEs in adult and infant subjects. We also examined the agreement between CD41 RTEs and TRECs results in a cohort of infants to test the possibility of CD41 RTEs test to be used in conjunction or alternative for TRECs in this population. Several states currently include the TRECs assay in newborn screening programs. All state newborn screening programs recommend that low TRECs results are confirmed with flow cytometry for
the major T cell subset populations that include CD31, CD41, CD81, CD201, and NK cells (24). We thought that the CD41 RTEs assay could also be clinically useful as a component of flow cytometry confirmation panels for following up abnormal TRECs newborn screening results and assessing immunocompetence. MATERIALS AND METHODS Specimens and Subjects Peripheral blood samples were drawn into either heparin- or EDTA-anticoagulant tubes and stored at ambient temperature within 72 h. The deidentified samples were collected from three groups of subjects: (1) newborns and infants (27 females, 23 males; age 0–2 years old); (2) infants and children (6 females, 11 males; age 2months to 10-years-old); (3) adults (26 females, 24 males; age 16–69 years old with average age 41.9 years old). The individuals in Group 1 had test orders for the major subsets of lymphocytes, including T-cells. The reference ranges of the major T-cell populations (of CD31, CD31 CD41 and CD31 CD81 cells) were derived from the ARUP Laboratories validated tests. The specimens of the first group of subjects were used for the comparison of a flow cytometry CD41 RTEs and q-PCR TRECs assays; the specimens of the second group were used for the verification of reference ranges in infants and children with the published data (25); the third group with adults only was used to estimate CD41 RTEs reference ranges in adults. The demographics of subjects used in precision, accuracy, and linearity experiments are indicated in the result section. The patient samples included in this study were handled according to Institutional Review Board (IRB) protocol 7275, approved by the University of Utah. Enumeration of CD41 RTEs by Flow Cytometry Detection of CD41 RTEs was carried out in the whole blood, using a panel of fluorescently labeled antibodies: CD45*PerCP (BD Biosciences, Cat. # 340665), CD4*FITC (BD Biosciences, Cat. # 340133), CD31*PE (BD Pharmingen, Cat. # 560983), and CD45RA*APC (BD Pharmingen, Cat. # 550855). Following staining, the whole blood samples were treated with BD FACSTM Lysing solution (BD biosciences, Cat. # 340183) to remove red blood cells; all the steps were carried out in BD TrucountTM Tubes (BD Biosciences, Cat. # 340334). Stained cells were analyzed by flow cytometry, using a BD FACSCanto II instrument (BD Biosciences, San Jose, CA) equipped with Blue (488 nm) and Red (633 nm) lasers. The acquisition and data analysis was performed using FACSDivaTM software (BD Biosciences, San Jose, CA). The data was acquired in four dot plots, with gates set to register events of three distinct cell populations and counting beads. The side scatter (SSC) versus CD45RA*APC dot plot was used for setting up a gate on the counting beads, which were recognized by their increased side scatter signal and APC fluorescence intensity. The SSC versus CD45*PerCP dot plot was included to collect
Cytometry Part B: Clinical Cytometry
3
THYMIC OUTPUT
events of the total lymphocyte population. At least 5,000 of CD451 cells were collected. The lymphocytes were then examined in the next dot plot that shows SSC and CD4*FITC separation; a single gate in the plot was included to separate CD41 lymphocytes. The fourth dot plot (CD31*PE versus CD45RA*APC) was derived from the CD41-gated lymphocytes of the previous dot plot. It shows CD41 RTEs defined as CD311/CD45RA1 CD4 T-cells. The population hierarchy and statistics view of the acquired data is presented in a table below the dot plots (Fig. 1). The table shows events of the cell populations and counting beads. The events corresponding to CD311/CD45RA1 cells and beads were used to calculate CD41 RTEs absolute cell count by the following equation: CD41 RTEs absolute cell count 5
of CD451 =CD41 =CD311 =CD45RA1 events of Beads events of Beads per test 3 Test volume
The number of beads per test is provided by the manufacturer (BD Pharmingen, Cat. # 550855). Immunophenotyping of the major T-cell subsets was carried out using whole blood samples stained with a cocktail of CD3*FITC/CD8*PE/CD45*PerCP/CD4*APC (BD Biosciences, Cat. # 340491) antibodies, which was followed by lysing of erythrocytes with a buffer containing a mild fixative. The absolute cell count was performed using TM BD Trucount Tubes as described above. TRECs Concentration Measurement RT-qPCR for the TREC and B-actin targets was performed with DNA isolated from a whole blood sample that is spotted onto a filter paper and dried overnight. Betta (b)-actin is a reference gene and is used to ensure that the DNA sample is adequate. Briefly, a 3.2 mm disk containing 3 lL whole blood is punched from the dried blood spot (DBS) and placed in a 96-well plate format. DNA extraction is then performed using Generation DNA Purification and Elution Solutions (Qiagen, Valencia, CA). Eight (8) lL of extracted DNA were used in a 20 lL reaction for the real-time qPCR TREC assay and 1 lL in 20 lL for the b-actin reaction. The primer sequences, TaqMan probe sequence and PCR conditions have been previously described and are based on the method developed by Baker et al. (26) QuantiTect Multiplex PCR No ROX Kit (1000) mastermix (Qiagen, Valencia, CA) was used and RT-qPCR was performed on LightCycler 480 System (Roche, Indianapolis, IN). The number of copies of TREC and b-actin were determined using a standard curve based on serially diluted plasmids containing known copies of TREC and b-actin, kindly provided by Dr. Baker (26). A crossing threshold (Ct) of 35 is used as a cutoff to differentiate between normal and abnormal TREC concentrations.
Cytometry Part B: Clinical Cytometry
This study was carried out within the frameworks of CAP/CLIA. Acceptable criteria were decided according to practice guidelines from the International Council for Standardization in Hematology (ICSH) and the International Clinical Cytometry Society (ICCS) (27). The flow cytometry experiments were carried out according to the MIFlowCyt recommendations (the Minimum Information about a Flow Cytometry Experiment) (28). Statistical Analyses The precision data analysis was determined using an Excel spreadsheet. The data analysis was presented as the %coefficient of variation (%CV), which is the ratio of the standard deviation (SD) to the mean (or average) of the five replicates (intra assay) or the three independent test runs (inter assay). The analytical accuracy, linearity and the reference ranges in adults were analyzed using EP Evaluator software (David G. Rhoads Associates, Inc.). The performance standards parameters for the analytical accuracy and linearity were: Allowable Systematic Error (SEa)—10 cells/lL, Allowable Total Error (TEa)—20 cells/lL. Normal distribution of reference range data was achieved using Box-Cox transformation (29). The reference range was calculated using the transformed parametric analysis with the central interval of 95% (30). For methods comparison between CD41 RTEs and TRECs assays, we used percent agreement. RESULTS Sample Stability The stability of CD41 RTEs measurements was examined in the peripheral blood samples from three healthy individuals. The samples were drawn on the same day into EDTA and heparin tubes and stored for 72 h at both room and 48C temperatures. The CD41 RTEs, identified as CD451/CD41/CD45RA1/CD311 cells by multicolor antibody staining, were subsequently tested at 0, 24, 48, and 72 hours after being collected. The analysis of CD41 RTEs is shown in Figure 2. The results indicate that samples stored in all the conditions examined, i.e., the type of tubes and temperatures, are stable for at least 72 h. Precision of CD41 RTEs Enumeration In order to determine the precision of CD41 RTEs enumeration assay we have chosen peripheral blood samples from five healthy subjects that represented different age demographics (from 2 to 78 years old) and exhibited a wide range of CD41 RTEs counts (Table 1 and 2). Precision of the assay between runs (inter assay) was carried out in three separate stains and instrument acquisitions of the same samples (Table 1). The average of CD41 RTEs absolute cell count per lL of blood was in range from 71 (78-year-old subject # 5) to 1532 (2year-old subject # 2). The %CV between runs was found as low as 1.5% (subject # 1) and as high as 12.2% (subject # 5). Precision of the assay within a run (intra assay) was performed with the same samples and
4
RAVKOV ET AL.
FIG. 1. The gating strategy, population hierarchy and statistics of acquired data. The top two dot plots display events of the counting beads and lymphocytes. Dot plots below show the frequencies of CD4 T-cells and CD41 RTEs. The table below shows the population hierarchy and statistics that includes CD41 RTEs frequency and the number of events. Example of CD41 RTEs absolute cell count: CD41 RTEs absolute cell count5
3175
of CD451 =CD41 =CD311 =CD45RA1 events of Beads per test 3 of Beads events Test volume
978 54900 ðprovided by manufacturerÞ 3 3; 384 50 ðuL of bloodÞ
acquired in five replicates (Table 2). The subject # 3 showed the least variability with %CV of 1.5%, whereas the subject # 1 exhibited the maximum variability, displaying % CV of 7%. Precision results were acceptable
according to the guidelines from ICSH and ICCS, where a desirable target for assay %CV is
Original Article
Thymic Output: Assessment of CD41 Recent Thymic Emigrants and T-Cell Receptor Excision Circles in Infants Eugene Ravkov,1* Patricia Slev,1,2 and Nahla Heikal1,2 1
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah
2
Background: CD41 recent thymic emigrants (CD41 RTEs) constitute a subset of T cells recently generated in the thymus and exported into peripheral blood. CD41 RTEs have increased copy numbers of T-cell receptor excision circles (TREC). They are characterized by the expression of CD31 on na€ıve CD4 T-cells. We aimed to validate a flow-cytometry assay to enumerate CD41 RTEs and assess its performance in relation to TREC measurement. Methods: CD41 RTEs cell count in peripheral blood was measured to determine sample stability, precision, linearity, and to establish reference ranges. TRECs were measured using qPCR assay performed with DNA isolated from peripheral blood. CD41 RTEs, TRECs, and flow cytometry results for major T-cell markers were assessed in 50 infants less than 2 years of age. Results: Inter-and intra-assay precisions (% CV) were 1.5–12.2 and 1.5–7.0, respectively. Linearity studies showed that the results are linear over a range of 0.7 to 403.0 CD41 RTEs/lL of blood. There was 84% agreement (42 of 50) between CD41 RTEs and TRECs qualitative results for the infant samples. CD41 RTEs reference ranges in 17 healthy children was in agreement with published data, while that of the healthy adults were 51–609 cells/lL of blood. Conclusion: The validation results provide acceptable measures of the CD41 RTEs test performance within CAP/CLIA frameworks. CD41 RTEs and TRECs assays show high agreement in the infant population. The CD41 RTEs test can be used as a confirmation for the TREC results along with or as an alternaC 2016 International Clinical tive to T-cell phenotyping in infants with repeatedly low TRECs concentrations. V Cytometry Society
Key terms: recent thymic emigrants; T-cell receptor excision circles; thymic output; SCID; immune reconstitution
How to cite this article: Ravkov, E., Slev, P. and Heikal, N. Thymic Output: Assessment of CD41 Recent Thymic Emigrants and T-Cell Receptor Excision Circles in Infants. Cytometry Part B 2016; 00B: 000–000.
Recent thymic emigrants (RTEs), including CD41 and CD81, constitute a T-cell population that has been recently generated in the thymus and exported into peripheral blood (1–3). These cells differ from the rest of T-cells with respect to their phenotype and function. RTEs have decreased proliferation, cytokine production, and transcription factor expression (3). They are essential for maintaining peripheral T cells in sufficient diversity (4–7). The appearance of T-cell receptor excision circles (TRECs) is commonly used to identify RTEs (3). TRECs are generated during T-cell receptor (TCR) rearrangement (8,9); they are not capable of replication and eventually become diluted during cell division, which makes TRECs concentrations in the periphery correlate
C 2016 International Clinical Cytometry Society V
with thymic output (9,10). High TRECs concentrations were detected during childhood, and decreased with increasing age (11). In HIV-1 patients, TRECs were
Additional Supporting Information may be found in the online version of this article. Correspondence to: Eugene Ravkov, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah. E-mail: eugene.v.ravkov@ aruplab.com Grant sponsor: ARUP Institute for Clinical and Experimental Pathology. Received 9 June 2015; Revised 30 October 2015; Accepted 6 November 2015 Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cyto.b.21341
2
RAVKOV ET AL.
significantly lower than normal age matched controls and their level of TRECs were restored by antiretroviral treatment (11). TRECs concentration increased rapidly during T cell reconstitution in patients who received stem cell transplantation (11). Also, patients with congenital absence of the thymus, like complete Digeorge Syndrom have no detectable TRECs (11). The publication of these data suggested that TRECs concentration in the periphery is a direct marker for thymic output. CD41 RTEs are a population of na€ıve T cells that have high TRECs copy numbers indicating that they recently derived from the thymus (12–15). A number of studies have shown a progressive decrease of RTEs with age, which directly correlates with progressive physiological involution of the thymus (12,16). In humans, CD41 RTEs maturation into na€ıve T-cells is associated with decease of PTK7, CD31 (PECAM-1), and TRECs (2,5,12). It is also associated with shortening of telomeres and decreased telomerase activity (12,13). Despite thymic involution, the total T-cell count remains relatively stable due to ongoing maintenance by a variety of peripheral thymus-independent homeostatic mechanisms (2,13). Recently, it has been demonstrated that CD31 can be used as a cell surface marker to distinguish CD41 RTEs (CD41 CD45RA1 CD311) with high TRECs content from proliferated, peripheral, na€ıve T cells (CD41 CD45RA1 CD312) with diminished TRECs content that implies proliferation (12–15). CD81 RTEs express CD103, but have been difficult to identify (3). It has also been shown that CD4 : CD8 ratio is about four folds higher in RTEs than that in the total population of T cells (3). CD31 is a 130-kDa transmembrane glycoprotein that is expressed by endothelial cells, monocytes, neutrophils, platelets and certain T-cells (13,17). CD31 expression on CD41 na€ıve T-cells directly correlates with high copy numbers of TRECs and high telomerase activity, which makes it a suitable cell subset to evaluate thymic output (12,13). The same study and others found that both TRECs and telomerase activity progressively decrease and correlate with CD41 RTEs decreased numbers in aged subjects (18,19). Monitoring of thymic output provides an important parameter in establishing appropriate diagnosis and treatment strategies for patients with cellular or combined primary immunedeficiencies, and monitoring immune-reconstitution in patients undergoing anti-HIV treatment, chemotherapy and post-hematopoietic cell or thymus transplantation (20–23). In this study, we present the validation data of a flow cytometry-based test that enables enumeration of CD41 RTEs in adult and infant subjects. We also examined the agreement between CD41 RTEs and TRECs results in a cohort of infants to test the possibility of CD41 RTEs test to be used in conjunction or alternative for TRECs in this population. Several states currently include the TRECs assay in newborn screening programs. All state newborn screening programs recommend that low TRECs results are confirmed with flow cytometry for
the major T cell subset populations that include CD31, CD41, CD81, CD201, and NK cells (24). We thought that the CD41 RTEs assay could also be clinically useful as a component of flow cytometry confirmation panels for following up abnormal TRECs newborn screening results and assessing immunocompetence. MATERIALS AND METHODS Specimens and Subjects Peripheral blood samples were drawn into either heparin- or EDTA-anticoagulant tubes and stored at ambient temperature within 72 h. The deidentified samples were collected from three groups of subjects: (1) newborns and infants (27 females, 23 males; age 0–2 years old); (2) infants and children (6 females, 11 males; age 2months to 10-years-old); (3) adults (26 females, 24 males; age 16–69 years old with average age 41.9 years old). The individuals in Group 1 had test orders for the major subsets of lymphocytes, including T-cells. The reference ranges of the major T-cell populations (of CD31, CD31 CD41 and CD31 CD81 cells) were derived from the ARUP Laboratories validated tests. The specimens of the first group of subjects were used for the comparison of a flow cytometry CD41 RTEs and q-PCR TRECs assays; the specimens of the second group were used for the verification of reference ranges in infants and children with the published data (25); the third group with adults only was used to estimate CD41 RTEs reference ranges in adults. The demographics of subjects used in precision, accuracy, and linearity experiments are indicated in the result section. The patient samples included in this study were handled according to Institutional Review Board (IRB) protocol 7275, approved by the University of Utah. Enumeration of CD41 RTEs by Flow Cytometry Detection of CD41 RTEs was carried out in the whole blood, using a panel of fluorescently labeled antibodies: CD45*PerCP (BD Biosciences, Cat. # 340665), CD4*FITC (BD Biosciences, Cat. # 340133), CD31*PE (BD Pharmingen, Cat. # 560983), and CD45RA*APC (BD Pharmingen, Cat. # 550855). Following staining, the whole blood samples were treated with BD FACSTM Lysing solution (BD biosciences, Cat. # 340183) to remove red blood cells; all the steps were carried out in BD TrucountTM Tubes (BD Biosciences, Cat. # 340334). Stained cells were analyzed by flow cytometry, using a BD FACSCanto II instrument (BD Biosciences, San Jose, CA) equipped with Blue (488 nm) and Red (633 nm) lasers. The acquisition and data analysis was performed using FACSDivaTM software (BD Biosciences, San Jose, CA). The data was acquired in four dot plots, with gates set to register events of three distinct cell populations and counting beads. The side scatter (SSC) versus CD45RA*APC dot plot was used for setting up a gate on the counting beads, which were recognized by their increased side scatter signal and APC fluorescence intensity. The SSC versus CD45*PerCP dot plot was included to collect
Cytometry Part B: Clinical Cytometry
3
THYMIC OUTPUT
events of the total lymphocyte population. At least 5,000 of CD451 cells were collected. The lymphocytes were then examined in the next dot plot that shows SSC and CD4*FITC separation; a single gate in the plot was included to separate CD41 lymphocytes. The fourth dot plot (CD31*PE versus CD45RA*APC) was derived from the CD41-gated lymphocytes of the previous dot plot. It shows CD41 RTEs defined as CD311/CD45RA1 CD4 T-cells. The population hierarchy and statistics view of the acquired data is presented in a table below the dot plots (Fig. 1). The table shows events of the cell populations and counting beads. The events corresponding to CD311/CD45RA1 cells and beads were used to calculate CD41 RTEs absolute cell count by the following equation: CD41 RTEs absolute cell count 5
of CD451 =CD41 =CD311 =CD45RA1 events of Beads events of Beads per test 3 Test volume
The number of beads per test is provided by the manufacturer (BD Pharmingen, Cat. # 550855). Immunophenotyping of the major T-cell subsets was carried out using whole blood samples stained with a cocktail of CD3*FITC/CD8*PE/CD45*PerCP/CD4*APC (BD Biosciences, Cat. # 340491) antibodies, which was followed by lysing of erythrocytes with a buffer containing a mild fixative. The absolute cell count was performed using TM BD Trucount Tubes as described above. TRECs Concentration Measurement RT-qPCR for the TREC and B-actin targets was performed with DNA isolated from a whole blood sample that is spotted onto a filter paper and dried overnight. Betta (b)-actin is a reference gene and is used to ensure that the DNA sample is adequate. Briefly, a 3.2 mm disk containing 3 lL whole blood is punched from the dried blood spot (DBS) and placed in a 96-well plate format. DNA extraction is then performed using Generation DNA Purification and Elution Solutions (Qiagen, Valencia, CA). Eight (8) lL of extracted DNA were used in a 20 lL reaction for the real-time qPCR TREC assay and 1 lL in 20 lL for the b-actin reaction. The primer sequences, TaqMan probe sequence and PCR conditions have been previously described and are based on the method developed by Baker et al. (26) QuantiTect Multiplex PCR No ROX Kit (1000) mastermix (Qiagen, Valencia, CA) was used and RT-qPCR was performed on LightCycler 480 System (Roche, Indianapolis, IN). The number of copies of TREC and b-actin were determined using a standard curve based on serially diluted plasmids containing known copies of TREC and b-actin, kindly provided by Dr. Baker (26). A crossing threshold (Ct) of 35 is used as a cutoff to differentiate between normal and abnormal TREC concentrations.
Cytometry Part B: Clinical Cytometry
This study was carried out within the frameworks of CAP/CLIA. Acceptable criteria were decided according to practice guidelines from the International Council for Standardization in Hematology (ICSH) and the International Clinical Cytometry Society (ICCS) (27). The flow cytometry experiments were carried out according to the MIFlowCyt recommendations (the Minimum Information about a Flow Cytometry Experiment) (28). Statistical Analyses The precision data analysis was determined using an Excel spreadsheet. The data analysis was presented as the %coefficient of variation (%CV), which is the ratio of the standard deviation (SD) to the mean (or average) of the five replicates (intra assay) or the three independent test runs (inter assay). The analytical accuracy, linearity and the reference ranges in adults were analyzed using EP Evaluator software (David G. Rhoads Associates, Inc.). The performance standards parameters for the analytical accuracy and linearity were: Allowable Systematic Error (SEa)—10 cells/lL, Allowable Total Error (TEa)—20 cells/lL. Normal distribution of reference range data was achieved using Box-Cox transformation (29). The reference range was calculated using the transformed parametric analysis with the central interval of 95% (30). For methods comparison between CD41 RTEs and TRECs assays, we used percent agreement. RESULTS Sample Stability The stability of CD41 RTEs measurements was examined in the peripheral blood samples from three healthy individuals. The samples were drawn on the same day into EDTA and heparin tubes and stored for 72 h at both room and 48C temperatures. The CD41 RTEs, identified as CD451/CD41/CD45RA1/CD311 cells by multicolor antibody staining, were subsequently tested at 0, 24, 48, and 72 hours after being collected. The analysis of CD41 RTEs is shown in Figure 2. The results indicate that samples stored in all the conditions examined, i.e., the type of tubes and temperatures, are stable for at least 72 h. Precision of CD41 RTEs Enumeration In order to determine the precision of CD41 RTEs enumeration assay we have chosen peripheral blood samples from five healthy subjects that represented different age demographics (from 2 to 78 years old) and exhibited a wide range of CD41 RTEs counts (Table 1 and 2). Precision of the assay between runs (inter assay) was carried out in three separate stains and instrument acquisitions of the same samples (Table 1). The average of CD41 RTEs absolute cell count per lL of blood was in range from 71 (78-year-old subject # 5) to 1532 (2year-old subject # 2). The %CV between runs was found as low as 1.5% (subject # 1) and as high as 12.2% (subject # 5). Precision of the assay within a run (intra assay) was performed with the same samples and
4
RAVKOV ET AL.
FIG. 1. The gating strategy, population hierarchy and statistics of acquired data. The top two dot plots display events of the counting beads and lymphocytes. Dot plots below show the frequencies of CD4 T-cells and CD41 RTEs. The table below shows the population hierarchy and statistics that includes CD41 RTEs frequency and the number of events. Example of CD41 RTEs absolute cell count: CD41 RTEs absolute cell count5
3175
of CD451 =CD41 =CD311 =CD45RA1 events of Beads per test 3 of Beads events Test volume
978 54900 ðprovided by manufacturerÞ 3 3; 384 50 ðuL of bloodÞ
acquired in five replicates (Table 2). The subject # 3 showed the least variability with %CV of 1.5%, whereas the subject # 1 exhibited the maximum variability, displaying % CV of 7%. Precision results were acceptable
according to the guidelines from ICSH and ICCS, where a desirable target for assay %CV is
