Biomarkers and Applications (ISSN: 2576-9588)

Article / Research Article

"CD14+CD16-Low Monocyte Subset Predicts Non-Progressive HIV Disease: Evidence of A New Prognostic and Diagnostic Biomarker"

Kabo Matlho1, Xin Maggie Wang2, Viviane Conceicao1, Suneth S. Perera1, Bin Wang1, Maly Soedjono1, Ma Jin Min3, Nitin K. Saksena1*

1Retroviral Genetics Division, Westmead Millennium Institute and Westmead Hospital, Darcy Road, Westmead NSW Sydney, Australia

2Flow Cytometry Core Facility, Westmead Institute for Medical Research, Westmead NSW 2145, Sydney, Australia

3China National Gene Bank, Beijing Institute of Genomics, Dapeng, China

*Corresponding author: Current affiliation: Nitin K. Saksena, China National Gene Bank, Beijing Institute of Genomics, Dapeng, China and IGO, Darlinghurst NSW 2010. Sydney. Australia Tel: +42-3960158; Email: nitin.saksena@bigpond.com and nitin@iggygetout.com

Received Date: 14 September, 2018; Accepted Date: 04 October, 2018; Published Date: 12 October, 2018

Monocytes are phenotypically pliable, which allows them to play several significant immunological roles in combating HIV infection. Monocytes can be subcategorized into subsets based on the expression of CD14 and CD16 antigens. Although the CD4+ T cell counts have been shown to predict HIV viremia, the actual predictive value of these monocyte subsets at different stages of plasma viremia is not known. We derived ex-vivo monocytes from HIV+ patients with detectable and below detectable plasma viremia, HIV+ Long-Term Non-Progressors (LTNP) and HIV negative individuals. We subdivided monocytes into CD14+/ CD16-low, medium and high populations and visualized the phenotypic changes in expression of both CD14 and CD16 antigens in HIV+ patients at different stages of   HIV disease.

The expression of surface markers on monocytes (CD14+/CD16) was measured from the EDTA blood of 50 HIV+ individuals [14 viremic and 29 Below Detectable Level (BDL) whilst on HAART, 7 therapy naïve, aviremic LTNP’s] and 6 HIV-negative donors using the FACSCanto (6-color) flow cytometer. Percentage of CD16/CD14+ sub-populations were measured on FACSCantoA with FACSDiva (v 6.1.2) software and analysed by FlowJo software (v10.0.7), respectively.

By categorizing monocyte population into CD14+, CD16 high, medium and low, we could clearly discriminate between viremic and aviremic HIV patients. There was considerable elevation of CD16-low population (80%) in HIV-negative individuals and LTNPS (57%), as opposed to 9% in HAART-treated group. Noteworthy was the CD16-low population failed to recover despite complete viral control during HAART therapy suggesting their definitive role as indicators of viremic control as seen with their marked prominence in LTNPs. In contrast, the HAART-treated group showed elevated CD16-high populations (34%), as opposed to relatively low percentages in the viremic group (3%).  The robust maintenance and elevation of CD16-low populations and substantial low levels of CD16-high populations distinctively in HIV-negative and non-progressing HIV+ individuals correlated with the natural control of HIV in LTNPs. This feature of CD16-low monocytic population can be exploited as a biomarker in predicting plasma viremia and the strength of the immune system.

Keywords: CD16+ Monocytes; Monocyte; Non-Progressive HIV Disease; HAART; HIV

1.    Introduction

The phenotypic pliability and the differentiation ability of monocytes empower this cell type to play a crucial role in HIV pathogenesis through cellular differentiation, phagocytosis, and antigen presentation. Compared to T cells and macrophages, monocytes are much less permissive to HIV infection [1,2], although all these cells express HIV receptor CD4 and co-receptors CCR5 and/or CXCR4. In spite of less than 1% of circulating monocytes directly infected in vivo, infectious virus can be isolated from circulating monocytes in untreated patients and HAART responders [2,3], which could become a dominant source of plasma virus in HAART responders in whom HIV replication in activated T cells is blocked [4]. In addition, monocytes represent an important cellular reservoir by harboring and trafficking HIV into various tissue compartments through differentiating into tissue macrophages or dendritic cells, which enable productive HIV replication [4]. Furthermore, undifferentiated monocytic precursor cells, such as CD34+ progenitor cells, may be infected with HIV and pass on the virus to progeny monocytes and keep on renewing the viral pool in peripheral blood monocytes [5,6]. Monocyte subpopulations exist with differing levels of maturation and functions. Monocytes that express CD14, the LPS receptor, and CD16, the Fcylll receptor, are a mature population of cells that are highly susceptible to HIV.

Based on these sub-populations, monocytes can be subcategorized into subsets based on the surface expression of CD14 and CD16 antigens. CD14+CD16+ monocytes are present in significantly greater numbers in HIV-infected people, despite viral suppression, in contrast to individuals without HIV, but the modulation of such populations, as we have visualized by sub-categorizing, has never been looked into at various stages of HIV disease. Although the CD4+ T cell counts have been shown to predict HIV viremia, the actual predictive value of surface antigen changes on monocytes, particularly CD16 at different stages of plasma viremia has also not been evaluated. In this study, we have derived ex vivo monocytes from HIV positive patients with detectable (84-231,000 copies of HIV RNA/ml plasma) and Below Detectable Levels (BDL) of plasma viremia (<40 copies of HIV RNA/ml plasma), HIV negative individuals and therapy naïve HIV+ Long-Term Non-Progressors (LTNP) who have been infected with HIV for >25 years, remained therapy naïve and have maintained below detectable levels of plasma viremia throughout the course of infection. Since CD14+CD16+ monocytes represent an important heterogeneous cell population that is often targeted, particularly for HIV-1 entry, we evaluated the effects of HIV infection and distinct subsets of ex-vivo-derived CD16+ monocytes. We subdivided monocytes into CD14+CD16-high, CD14+CD16-medium and CD14+CD16-low populations and visualized the phenotypic changes in expression of CD16 antigen in CD14+ monocytes in HIV+ patients during different stages of plasma viremia.

2.  Methods and Materials

2.1.  Derivation and Processing of Ex-Vivo Monocytes from HIV Patients

The human Peripheral Blood Mononuclear Cells (PBMCs) were obtained from the EDTA blood of 50 HIV positive individuals [14 viremic patients; 29 BDL on Highly Active Antiretroviral Therapy (HAART) and 7 therapies naïve, aviremic LTNPs] and 4 HIV negative donors (See Table 1 and supplementary File 1 for Patient clinical details and raw Excel data). The work was cleared by the human Ethics Committee of the Sydney West Western Area Health Research Committee, Westmead Hospital, Sydney, NSW. Australia. All blood samples were collected strictly after individual informed written consent.

2.2.  Immuno-Staining and Flow Cytometry Analysis on Human Peripheral Blood Mononuclear Cells Samples

A two-color antibody panel was used to identify the CD14 and CD16 antigen expression on monocytes. Cells were ficolled and stained with CD14-PE (BD Biosciences, Australia) and CD16-Alex Fluor 647 (BD Biosciences, Australia) for 20 min at 40C. Following washing with PBS, cells were fixed with (2% Paraformaldehyde) for 10 Min at room temperature, washed and then re suspended in PBS before flow cytometry. Flow cytometry was performed on a Canto A cytometer (BD Biosciences, Australia) using DIVA 6.1.2 software (BD Biosciences). Monocyte population was first identified by FSC and SSC dot plot. Following gating on the CD14 positive population, based on the fluorescent intensity, CD16 expression on CD14 positive monocytes was divided into three groups, CD16-low, CD16-med and CD16-high. The same gating strategy was used across all samples. Percentages of three CD16 populations were analyzed by FlowJo software (v10.0.7; Treestar, USA).

2.3.  Statistical Analysis

Results are expressed as mean ± standard error. Differences among groups were measured using Student’s t test with one-tailed distribution and two sample equal variance test, P<0.05 was considered significant. The relationship of CD16 with CD4 count and viral load was determined by the correlation coefficient with the formula shown below.




3. Results

By categorizing monocyte population into CD14+CD16-high, CD14+CD16-medium and CD14+CD16-low, we could discriminate between viremic and aviremic HIV patients (Figure 1). There was considerable elevation of CD14+CD16-low population (80%) in HIV-negative individuals and 57% in LTNPS (p<0.0114), as opposed to 8% in HAART-treated viremic and aviremic (BDL) groups (BDL vs neg P<0.0000374 and Viremic vs neg P<0.0000056) (Figure 2, Table 3). Notable was that the CD14+CD16-low population never recovered despite complete viral control during HAART in the BDL group, suggesting their low levels as definite indicators of quality of monocyte in BDL and viremic groups when compared against their elevated levels in the LTNPs, who control the plasma virus naturally (LTNP vs BDL p<0.0075102 and LTNP vs Viremic p<0.0015809). Moreover, the HAART-treated groups (viremic and BDL) were also characterized by the elevated levels of CD14+CD16-high population (33 and 45%, respectively) (Figure 2, Table 2,3), when compared against LTNPs (BDL vs LTNP p<0.0000180 and Viremic vs LTNP p<0.0009354) and negative controls, which showed low levels of CD14+CD16-high populations. Although LTNPs were comparable in the expression of CD14+CD16-high populations with HIV negative donors (p<0.6796), they could be segregated from LTNPs based on CD14+CD16-medium and CD14+CD16-low populations (p<0.0048514 and p<0.0114045, respectively) (Table 3).

From these data two significant aspects of phenotypic regulation of monocytes are clear that the high levels of CD14+CD16- low and low levels CD14+CD16-high of populations characterize LTNPs and HIV-negative individuals implying that although the LTNPS are closer to HIV negative individuals when compared against the viremic and BDL groups, they could be segregated from each other by both CD14+CD16-medium and -low populations. Secondly, that the CD14+CD16-low and - high populations of monocytes were strong and reliable indicators of plasma viremia, immune deterioration and the quality of monocytes during viremia. It also raises the possibility that this phenotypic modulation in monocyte may also be linked to the deterioration in quality of monocytes, which even fails to recover despite HAART as apparent in BDL HIV patients on HAART.

We also evaluated, if this modulation in monocytic subset was linked to CD4+T cell counts, but we obtained inverse relationship between monocytic deterioration and CD4+T cells counts. The correlation between CD16-med vs CD4 count was 0.31176704 for the BDL group, while in Viremic group the CD16-high vs CD4 count showed p value of 0.42996958, with R2 values for both groups at 0.0972; 0.18487, respectively, suggesting that monocytic deterioration has less bearing on overall CD4+ T cell counts (supplementary file 1).

4. Discussion

Quality of monocyte subsets and their modulation in vivo plays a vital role in guiding immune responses during HIV infection [7]. The data shown in our study not only highlights the significance of CD16+ monocytes in HIV infection but also demonstrates the vital dynamics of these monocyte subsets exhibited in viral suppression and disease progression. This novel way of visualizing the trichotomy between low, medium and high subsets of CD14+CD16+ monocytes from HIV- and HIV+ groups has allowed us for the first time in elucidating not only the immunologic relationship they hold with different stages of HIV disease, but also their association with the natural control of HIV disease in therapy naïve Elite Controllers (EC)- a phenomenon not shown previously. We believe that this could offer new insights into the roles of innate immunity in HIV pathogenesis, underpinning their role as new biomarkers in HIV disease diagnosis and prognosis.

There is a significant rise in the proportions of non-classical monocytes in HIV-1 disease [8,9]. This heterogeneous subset which represents a minor sub-population of monocytes in healthy individuals, increases in peripheral blood and may represent up to 40% of total circulating monocytes during HIV infection and in patients with AIDS [9]. An important goal in clinical manifestation and diagnosis of HIV infection is to find laboratory parameters to monitor the disease progression. By further diversifying the established classification of non-classical and intermediate monocytes population into three subsets based of on CD16+ antigen, we sub categorized them into low, medium and high clusters. Through this way of visualizing, we could clearly discriminate between viremic and aviremic HIV patients (Figure 1 and Table 3). There was considerable and statistically significant elevation of CD14+CD16-low population (80%) in HIV-negative individuals in comparison to 57% in the LTNPs (p<0.0114). In contrast, it was only 8% in HAART-treated viremic and aviremic groups (BDL vs neg P<0.0000374 and Viremic vs Negatives P<0.0000056) (Figure 2 and Table 2,3).

Notably, the CD14+CD16-low population never recovered despite complete viral control during HAART in the BDL group, suggesting their low levels as definite indicators of quality of monocyte in the BDL and viremic groups when compared against their high levels in the LTNPs, who control the plasma virus naturally (LTNP vs BDL p<0.0075102 and LTNP vs Viremic p<0.0015809). And secondly, this also highlights the fact that even in the face of complete control of viremia during HAART, the CD14+CD16-low population failed to recover, suggesting HAART has no bearing on the recovery of this population, which may be one of the underlying reasons for partial immune restoration during HAART.

The robust maintenance and elevation of CD16-low populations and substantially low levels of CD16-high populations distinctively in HIV-negative and non-progressing HIV+ individuals correlated with the natural control of HIV in the LTNPs, thereby demonstrating the ability of this subset in predicting the strength of the immune system at different stages of HIV disease and their possible role in innate immunity. Further comparing CD16-low population at complete control of plasma viremia under HAART as opposed to its natural control in LTNPs, it appears that the quality of immune cells and the overall strength of the immune system is vital for this cell subset, and even a little virus compromises their quality. This fact emerges from the comparison of LTNPs with HIV-negative individuals, where the difference becomes apparent despite the two groups sharing closeness. This further suggests that LTNPs may maintain therapy naïve and virus-free status, the overall numbers of CD16-low population in the blood can serve not only as excellent indicators of even very low and below detectable levels viremic states as seen in case of LTNPs, but also in stratifying individuals based on the strength of their immune system.

Further to this, the HAART-treated groups (viremic and BDL) were also characterized by elevated levels of CD14+CD16-high populations (45% and 33% respectively) (Figure 2 and Table 2), when compared against LTNPs (BDL vs LTNP p<0.0000180 and Viremic vs LTNP p<0.0009354) and negative controls, who displayed low levels of CD14+CD16-high populations. Although LTNPs were comparable in the expression of CD14+CD16-high populations with HIV negative donors (p<0.6796), the HIV- donors could only be segregated from the LTNPs based on CD14+CD16-medium and CD14+CD16-low populations (p<0.0048514 and p<0.0114045, respectively) (Figure 2 and Table 3), suggesting a clear demarcation between HIV- and HIV+ individuals, raising a possibility of subliminal infection in LTNPs which is under a tight natural control.

Furthermore, HIV infectivity correlated with elevated CD16-mediium monocytes population, underscoring the distinction between HIV positive patients and negative individuals, which was highlighted to a measurable extent by the expression of CD16-medium population relatively pronounced in the HIV+ group 47% (BDL) and 58% (Viremic), as opposed to 39% (LNTP) and 15% (healthy donors)- the HIV negative group, implying the functional relevance of CD16-low and CD16-med monocytic populations in discriminating LTNPs from the negative donors. Thus, for the maintenance of the LTNP status, it was the high levels of CD16-low and low-levels of CD14_CD16-high appeared essential, which essentially coincides with the levels HIV- healthy individuals.

Theiblemont et al., (1995) [9] suggested that IN HIV Infection the expansion of CD14 low CD16 high monocyte subset, which produce high amount of TNF-alpha and IL-1 alpha may participate in the immune dysfunction observed during HIV infection. Thus their elevated levels in patients with viremia, is consistent with our data. Also consistent is their low levels in healthy individuals, in addition to LTNPs-which Thieblemont et al., did not show. The CD14lowCD16high circulating monocytes co-express MAX.1, p150, 95 and HLA- DR, which are typical of tissue macrophage markers. These cells also express higher levels of Intracellular Interleukin (IL)-1 alpha and Tumor Necrosis Factor (TNF)-alpha than the CD14highCD16low monocyte population from the same patients, which could form the biological basis of natural viremia control in LTNPs as seen our study.

5. Conclusions

The robust maintenance and elevation of CD14+CD16-low populations and low levels of CD14+CD16-high populations uniquely in HIV-negative and non-progressing HIV+ individuals correlate with natural control of HIV in LTNPs and is able to predict viremia, strength of the immune system and quality of both monocytes and T cells. LTNPs and HIV-negative individuals could be segregated based on CD14+CD16-medium populations, and despite the elevation of this population in the BDL and viremic groups, they significantly differed from LTNPs (p<0.0000066), suggesting possible differences in the quality of these monocytes in the LTNP group. These data may allow the development of new diagnostic and prognostic tools for the prediction of HIV disease staging in HIV patients.

6. Financial and Competing Interest Disclosure

NKS is thankful to the NHMRC Development grant for funding the project. Viviane Conceicao is thankful to the University of Sydney for A University of Sydney Postgraduate Award (IUPA) scholarship and Westmead Medical Research Foundation for a top-up grant. SP is thankful to the Arin Apcarin WMI Scholarship for his work. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.



SUPPLEMENTARY DATA

CD16high CD16med CD16low Viral load (Log10)

1: ANCR_CD14-PE+CD16AF647.fcs

24.2

62.2

13.6

2.0:107

194

1642Viremic

3: BN-_CD14-PE+CD16AF647.fcs

1.99

66

31.6

BDL

476

571BDL

5: BERA-_CD14-PE+CD16-AF647.fcs

34.5

63.9

1.6

4.4:22900

320

1109Viremic

7: BW-CD14-PE+CD16AF647.fcs

4.35

19.4

76.2

Normal control

Negative

9: BrAl-CD14-PE+CD16-AF647.fcs

66.6

30.8

2.53

BDL<40

682

946BDL

11: BRJU-CD14-PE+CD16-AF647.fcs

73

24.2

2.84

BDL

706

706BDL

13: CRBR-Cd14-PE+CD16-AF647.fcs

7.06

66.7

26.2

BDL

363

1597BDL

15: DEBR-CD14-PE+CD16AF647.fcs

75.3

19.8

4.81

BDL<40

45

566BDL

17: DIJA-CD14-PE+CD16AF647.fcs

8.67

55.9

35.5

84

222

1112Viremic

19: DRJ-CD14-PE+CD16AF647.fcs

2.32

73.9

23.7

BDL

515

1576BDL

21: EL-CD14-PE+CD16_AF647.fcs

40.4

53

6.62

1.9:91 n/a

n/a

Viremic

23: GaWa_CD14-PE+CD16-AF647.fcs

49.6

48.3

2.04

BDL

435

435BDL

25: GTPCD14-PE+CD16AF647.fcs

20.3

65.1

14.6

231000

7

318Viremic

27: Gar_CD14-PE+CD16-AF647.fcs

8.72

61.9

29.4

BDL BDL<40 3.2:1690

437

494BDL BDL BDL

29: GS_CD14-PE+CD16-AF647.fcs

61.6

36.2

2.19

338

520

31: Gh G_CD14-PE+CD16-AF647.fcs

30.7

57.6

11.7

368

1472

33: GWJ_CD14-PE+CD16AF647.fcs

31.8

66.2

2.02

4.1:13800 n/a

n/a

Viremic

35: H_CD14-PE+CD16-AF647.fcs

59.6

36.1

4.28

BDL

615

315BDL

37: HAR_CD14-PE+CD16AF647.fcs

5.23

77.1

17.5

BDL

437

760BDL

39: HK_CD14-PE+CD16-AF647.fcs

43.3

53.8

2.83

BDL

443

586BDL

41: IAK_CD14-PE+CD16-AF647.fcs

69.3

29.3

1.34

BDL n/a

n/a

BDL

43: J_CD14-PE+CD16-AF647.fcs

4.24

7.59

87.9

Negative

45: JU_Cd14-PE+CD16-AQF647.fcs

26.2

64

9.88

2.0:104

850

975Viremic

47: KA_CD14-PE+CD16AF647.fcs

84.8

14.4

0.82

BDL BDL BDL

377

406BDL BDL BDL

48. KJ_CD14-PE+CD16AF647.fcs 51: la wu 700108_CD14-PE+CD16AF647.fcs

9.96

76.8

13.2

922

1920

LW 700108_CD14-PE+CD16AF647.fcs

79.1

19.8

1.04

592

444

53: LEJO_CD14-PE+CD16AF647.fcs

23.4

62.2

14.4

4.5:33600

156

1672Viremic

55: MS_CD14-PE+CD16-AF647.fcs

54.6

44.5

0.92

2.1:121

597

760Viremic

57: MR_CD14-PE+CD16-AF647.fcs

50.9

47.4

1.64

2.1:120

597

688Viremic

59: MP_CD14-PE+CD16-AF647.fcs

63.8

35.5

0.693

BDL n/a

n/a

BDL

61: mOJA_CD14-PE+CD16-AF647.fcs

55.9

40.9

3.13

BDL

608

496BDL

63: MOM_CD14-PE+CD16AF647.fcs

16.7

76.6

6.64

BDL<40

1078

868BDL

65: NTTH_CD14-PE+CD16AF647.fcs

0

40.2

59.2

4.2:16300

271

1747LNTP

67: OK_CD14-PE+CD16AF647.fcs

42.9

52.8

4.29

1.6:40

520

1378Viremic

69: PH_CD14-PE+CD16AF647.fcs

6.34

39.2

54.4

BDL

660

1200LNTP

71: PT_CD14-PE+CD16-AF647.fcs

83.6

15.8

0.547

BDL n/a

n/a

BDL

73: PG_CD14-PE+CD16AF647.fcs

7.45

21.2

71.2

normal control

Negative

75: SR_CD14-PE+CD16AF647.fcs

83.3

15.8

0.904

BDL n/a

n/a

BDL

77: SD_CD14-PE+Cd16-AF647.fcs

24.9

70.7

4.36

BDL

1131

485BDL

79: SD_CD14-PE+CD16AF647.fcs

0.226

39

60.3

BDL

669

491BDL

81: SP_CD14-PE+CD16AF647.fcs

75.2

22.6

2.18

BDL

907

734BDL

83: Th_CD14-PE+CD16-AF647.fcs

52.7

46.3

1.01

BDL<40

450

868BDL

85: TO_CD14-PE+CD16AF647.fcs

49.4

47.4

3.28

BDL<40 n/a

n/a

BDL

87: TY_CD14-PE+CD16-AF647.fcs

14.4

75.2

10.4

2.3:192 n/a

n/a

Viremic

89: ULE_CD14-PE+CD16AF647.fcs

39.8

56.4

3.79 no record

91: VJ_CD14-PE+CD16-AF647.fcs

18.8

70.5

10.6

BDL

934

759BDL

93: VC_CD14-PE+CD16AF647.fcs

3.82

11.7

84.3

Normal control

Negative

95: YE_CD14-PE+CD16AF647.fcs

56.2

42

1.82

2.1:121

235

706 Viremic

97: YK_CD14-PE+CD16AF647.fcs

3.7

84.3

11.9

BDL n/a

n/a

BDL

99: YY_CD14-PE+CD16-AF647.fcs

43.4

50.6

6

3.5:2820

487

502 Viremic

101: 15893_CD14-PE+CD16-AF647.fcs

40.9

48.8

10.3

BDL

834

1607 BDL

1: C122_Double +.fcs

4.9

17.1

78

40

664

920 LTNP

3: C13_Double +.fcs

1.14

12.5

86.3

40

760

1110 LTNP

5: C53_Double +.fcs

5.48

33

61.5

40

854

900 LTNP

7: HIPE_Double +.fcs

4.59

36

59.4

40

710

1900 LTNP

9: S24_Double +.fcs

7.8

42.7

49.5

40

590

1720 LTNP

We have divided the groups in to 4 groups as color coded 1. BDL, 2. Viremic ,3. LNTP (Long Term Non Progressors) and 4. Negative.


        The spread sheet includes the viral load number as a Logrithmic scale (log10) for each patient. The CD4/CD8 count was also included for each patient. N:B... those highlighted in blue are those that did not have the CD4/CD8 stats available hence (n/a) highlighted in red is that of subjects with high CD16 low percentage. normal range for CD4count (380-1390) normal range for CD8+ T cell count (200-690).


 

 

 

CD16high

 

 

CD16med

 

 

CD16low

 

Viral load (Log10)

 

 

CD4 count

 

 

CD8 count

 

 

Group

Viral Load (new)

3: Bn_CD14-PE+CD16AF647.fcs

1.99

66

31.6

BDL

476

571

BDL

40

9: BrAl_CD14-PE+CD16-AF647.fcs

66.6

30.8

2.53

BDL<40

682

946

BDL

40

11: BRJU_CD14-PE+CD16-AF647.fcs

73

24.2

2.84

BDL

706

706

BDL

40

13: CRBR_Cd14-PE+CD16-AF647.fcs

7.06

66.7

26.2

BDL

363

1597

BDL

40

15: DEBR_CD14-PE+CD16AF647.fcs

75.3

19.8

4.81

BDL<40

45

566

BDL

40

19: DMR_CD14-PE+CD16AF647.fcs

2.32

73.9

23.7

BDL

515

1576

BDL

40

23: Ga Wa_CD14-PE+CD16-AF647.fcs

49.6

48.3

2.04

BDL

435

435

BDL

40

27: GA_CD14-PE+CD16-AF647.fcs

8.72

61.9

29.4

BDL

437

494

BDL

40

29: GS_CD14-PE+CD16-AF647.fcs

61.6

36.2

2.19

BDL<40

338

520

BDL

40

31: G G_CD14-PE+CD16-AF647.fcs

30.7

57.6

11.7

3.2:1690

368

1472

BDL

40

35: HA_CD14-PE+CD16-AF647.fcs

59.6

36.1

4.28

BDL

615

315

BDL

40

37: Harris_CD14-PE+CD16AF647.fcs

5.23

77.1

17.5

BDL

437

760

BDL

40

39: HK_CD14-PE+CD16-AF647.fcs

43.3

53.8

2.83

BDL

443

586

BDL

40

41: iAK_CD14-PE+CD16-AF647.fcs

69.3

29.3

1.34

BDL

n/a

n/a

BDL

40

47: KA_CD14-PE+CD16AF647.fcs

84.8

14.4

0.82

BDL

377

406

BDL

40

49: KJ_CD14-PE+CD16AF647.fcs

9.96

76.8

13.2

BDL

922

1920

BDL

40

51: lu 700108_CD14-PE+CD16AF647.fcs

79.1

19.8

1.04

BDL

592

444

BDL

40

59: m P_CD14-PE+CD16-AF647.fcs

63.8

35.5

0.693

BDL

n/a

n/a

BDL

40

61: mJ_CD14-PE+CD16-AF647.fcs

55.9

40.9

3.13

BDL

608

496

BDL

40

63: MI_CD14-PE+CD16AF647.fcs

16.7

76.6

6.64

BDL<40

1078

868

BDL

40

71: pO T_CD14-PE+CD16-AF647.fcs

83.6

15.8

0.547

BDL

n/a

n/a

BDL

40

75: SAR_CD14-PE+CD16AF647.fcs

83.3

15.8

0.904

BDL

n/a

n/a

BDL

40

77: S D_CD14-PE+Cd16-AF647.fcs

24.9

70.7

4.36

BDL

1131

485

BDL

40

81: sp jo_CD14-PE+CD16AF647.fcs

75.2

22.6

2.18

BDL

907

734

BDL

40

83: Th_CD14-PE+CD16-AF647.fcs

52.7

46.3

1.01

BDL<40

450

868

BDL

40

85: TO_CD14-PE+CD16AF647.fcs

49.4

47.4

3.28

BDL<40

n/a

n/a

BDL

40

91: VAR_CD14-PE+CD16-AF647.fcs

18.8

70.5

10.6

BDL

934

759

BDL

40

40

40

 

97: YU_CD14-PE+CD16AF647.fcs

3.7

84.3

11.9

BDL

n/a

n/a

BDL

 

101: 15893 pl lo_CD14-PE+CD16-AF647.fcs

40.9

48.8

10.3

BDL

834

1607

BDL

 

1: ANCR_CD14-PE+CD16AF647.fcs

24.2

62.2

13.6

2.0:107

194

1642

Viremic

107

 

5: Be Ra_CD14-PE+CD16-AF647.fcs

34.5

63.9

1.6

4.4:22900

320

1109

Viremic

22900

 

17: DIJA_CD14-PE+CD16AF647.fcs

8.67

55.9

35.5

84

222

1112

Viremic

84

 

21: EP_CD14-PE+CD16_AF647.fcs

40.4

53

6.62

1.9:91

n/a

n/a

Viremic

91

 

25: GTP_CD14-PE+CD16AF647.fcs

20.3

65.1

14.6

231000

7

318

Viremic

231000

 

33: GWJ_CD14-PE+CD16AF647.fcs

31.8

66.2

2.02

4.1:13800

n/a

n/a

Viremic

13800

 

45: JU_Cd14-PE+CD16-AQF647.fcs

26.2

64

9.88

2.0:104

850

975

Viremic

104

 

53: LE(2)_CD14-PE+CD16AF647.fcs

23.4

62.2

14.4

4.5:33600

156

1672

Viremic

33600

 

55: MS_CD14-PE+CD16-AF647.fcs

54.6

44.5

0.92

2.1:121

597

760

Viremic

121

 

57: mR_CD14-PE+CD16-AF647.fcs

50.9

47.4

1.64

2.1:120

597

688

Viremic

120

 

67: ok_CD14-PE+CD16AF647.fcs

42.9

52.8

4.29

1.6:40

520

1378

Viremic

40

 

87: TK_CD14-PE+CD16-AF647.fcs

14.4

75.2

10.4

2.3:192

n/a

n/a

Viremic

192

 

95: YF_CD14-PE+CD16AF647.fcs

56.2

42

1.82

2.1:121

235

706

Viremic

121

 

99: YY_CD14-PE+CD16-AF647.fcs

43.4

50.6

6

3.5:2820

487

502

Viremic

2820

 

69: PH_CD14-PE+CD16AF647.fcs

6.34

39.2

54.4

BDL

660

1200

LNTP

40

 

79: SD_CD14-PE+CD16AF647.fcs

0.226

39

60.3

BDL

669

491

LNTP

40

 

1: C122_Double +.fcs

4.9

17.1

78

 

664

920

LNTP

40

 

3: C13_Double +.fcs

1.14

12.5

86.3

760

1110

LNTP

40

 

5: C53_Double +.fcs

5.48

33

61.5

854

900

LNTP

40

 

7: HIPE_Double +.fcs

4.59

36

59.4

710

1900

LNTP

40

 

9: S24_Double +.fcs

7.8

42.7

49.5

590

1720

LNTP

40

 

7: BW_CD14-PE+CD16AF647.fcs

4.35

19.4

76.2

Normal control

 

Negative

 

 

43: JO_CD14-PE+CD16-AF647.fcs

4.24

7.59

87.9

 

 

 

Negative

 

73: PG 2 retake_CD14-PE+CD16AF647.fcs

7.45

21.2

71.2

normal control

 

Negative

 

93: VC_CD14-PE+CD16AF647.fcs

3.82

 

 

 

11.7

84.3

Normal control

 

Negative