Titration of monoclonal antibodies and stability of tandem dyes in cocktail preparation for flow cytometry processing

INTRODUCTION

Multicolor flow cytometry (MFC) helps measure the physical properties of the cells based on light scatter and the antigenic properties of the cells with the help of antibodies labeled with fluorochrome. Antibodies are the key components of MFC analysis.^[1] For reproducible analysis, high-quality antibody performance is essential. To use these antibodies judiciously in routine practice, the appropriate volume should be determined through titration experiments. The individual antibodies should be checked for their stability in cocktail preparation after titration as a part of the validation procedure.^[2-4]

Antibody titration is the process of identifying and determining the correct concentration of antibody. It helps establish the optimal antibody volume that provides the best separation of cell populations and the most accurate measurement of expression levels. By optimizing the concentration and amount of antibody needed for a specific test, non-specific antibody binding (noise) can be minimized, reducing background interference.^[2-5] However, not all antibodies can be assessed by simple signal-to-noise ratio (SNR) or signal-intensity graphs, as internal negative or unreactive cell populations are not always present.^[6]

The next step is to prepare antibody cocktails for different panels by combining the pre-titrated volumes of each fluorochrome- conjugated antibody. The performance and stability can be impaired when stored as antibody cocktails. Moreover, this problem gets exaggerated when using tandem dyes, which comprise two fluorochromes, namely one donor (phycobiliprotein) and one receiver/acceptor (such as Alexa Fluor or cyanine dye). In clinical practice, commonly used phycobiliproteins include phycoerythrin (PE), allophycocyanin (APC), and the peridinin–chlorophyll–protein complex. Frequent re-compensation may be required due to the possible spectral spillover between tandem dyes, which can reduce the robustness of clinical MFC panels. Since tandem dyes are essential for standard 8–10 color or higher-order MFC, several studies have emphasized the importance of assessing their stability in cocktail preparations.^[7] Therefore, the primary aim was to evaluate the optimal titration volume and the stability of the pre-titrated volume of tandem dyes in cocktail preparations.

MATERIALS AND METHODS

The present quantitative study was carried out after obtaining approval from the Institute’s Ethics Committee from November 2021 to August 2022. The inclusion criteria were that all monoclonal antibodies in the acute leukemia orientation tube (ALOT) were used for titration and for checking the stability of tandem dyes in the ALOT cocktail. Clotted/lysed samples, samples received in the wrong anticoagulant tube, outdated monoclonal antibodies, and samples from acute leukemia patients on chemotherapy or follow-ups were excluded from the study.

This study was conducted in a phased manner, in which a titration experiment was carried out in part 1 and stability testing for tandem dyes was performed in part 2. A convenience sampling technique was used, and peripheral blood samples (2 mL) received in ethylenediaminetetraacetic acid were screened for normal white blood cell (WBC) count and distribution. Ten such samples were used for the titration experiment (part 1), and CytoComp^TM lyophilized cells were used for stability testing (part 2). Finally, the validation of the antibody cocktail was performed using acute leukemia cases for routine practice.

RESULTS

Titration experiment (part-1)

The titration experiment was carried out for all the antibodies included in the ALOT tube. Tables 2 and 3 summarize the titration results for CD19 energy coupling dye (ECD) and sCD3-pacific blue (PB), and their graphical interpretations are shown in Figure 1. The final titrated volume of each antibody and the corresponding cost–benefit analysis have been shown in Table 4.

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Figure 1:

(A-N) Histogram, and (O) double Y-plot showing titration experiment for cluster of differentiation (CD) 19 energy coupling dye (ECD); (P) double Y-plot showing titration experiment for surface CD3 pacific blue (PB)

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Table 2: CD19-ECD titration.

Antibody	Conjugate	Clone	Isotype	Species	Vendor	Cat. no	Lot no
CD19	ECD	J3-119	IgG1	Mouse	Beckman	A07770	200131
VOLUME (μl)	MFI +	MFI -	MFP-MFN	SD NEG	S/N	SI	% gated
10	18982	312	18670	157	60.78	59.6	23.01
5	16740	301	16440	146	55.71	56.5	23.09
2.5	12466	292	12174	137	42.72	44.5	22.56
1.25	7210	293	6917	138	24.63	25.2	23.52
0.625	3979	286	3693	139	13.90	13.3	22.82
0.31	2277	288	1989	138	7.91	7.2	23.41

ECD: Energy coupling dye, CD: Cluster of differentiation, Cat no: Catalog number, Ig: Immunoglobulin, MFI: Mean fluorescent intensity, MFP: Mean fluorescent intensity positive, MFN: Mean fluorescent intensity negative, SD NEG: Standard deviation of negative signal, S/N: Sound to noise ratio, SI: Stain index, μL: microliter

Table 3: sCD3-PB titration.

Antibody	Conjugate	Clone	Isotype	Species	Vendor	Cat. no	Lot no
CD3	PB	UCHT1	IgG1	Mouse	Beckman	A93687	200033
Volume (μL)	MFI +	MFI -	MFP-MFN	SD NEG	S/N	SI	% gated
10	14924	400	14524	206	37.32	35.19	36.03
5	13827	396	13432	164	34.94	40.98	36.3
2.5	12043	396	11647	161	30.44	36.22	37.36
1.25	10144	386	9758	156	26.28	31.31	36.17
0.625	7838	390	7448	161	20.11	23.17	36.54
0.31	4788	414	4375	188	11.58	11.60	36.55

PB: Pacific blue, ‘s’ for surface processing, CD: Cluster of differentiation, Cat no: Catalog number, Ig: Immunoglobulin, MFI: Mean fluorescent intensity, MFP: Mean fluorescent intensity positive, MFN: Mean fluorescent intensity negative, SD NEG: Standard deviation of negative signal, S/N: Sound to noise ratio, SI: Stain index, μL: Microliter

Table 4: Final titrated volume and cost-benefit analysis for each antibody.

Antibody-conjugate	Cost per vial (rupees)	Number of tests/volume per test as per the manufacturer	CPT as per manufacturer (rupees)	Titrated volume (µL)	Cost per test after titration (rupees)	Percentage reduction in CPT (%)
cMPO-FITC	11,750	100/20	118	10	59	50
cCD79a-PE	43,853	100/20	439	5	110	75
CD19-ECD	33,747	100/10	338	2.5	84	75
CD34-PC5.5	58,000	100/10	580	0.6	36	84
cCD3-PC7	47,493	100/10	475	2.5	119	75
nTdT-APC (BD)	41,076	100/5	411	2.5	205	50
CD45-APC700	36,276	100/10	363	0.6	23	84
CD7-APC750	45,448	50/10	909	2.5	227	75
sCD3-PB	19,638	50/10	393	5	196	50
HLA-DR-KO	51,912	50/10	1,038	1.25	130	88
Total	3,89,193		5,064		1,189	77

μL: Microliter, FITC: Fluorescein isothiocyanate, PE: Phycoerythrin, ECD: Energy coupling dye, PC: Phycoerythrin-cyanin, APC: Allophycocyanin, PB: Pacific blue, KO: Krome orange, CD: Cluster of differentiation, MPO: Myeloperoxidase, c, s, n indicates cytoplasmic, surface and nuclear staining, TdT: Terminal deoxynucleotidyl transferase, HLA-DR: Human leucocyte antigen-DR isotype, BD: Becton, Dickinson biosciences company and all the other antibodies from Beckman Coulter company, CPT: Cost per test

Stability testing for tandem dyes (part-2)

Pre-titrated volumes of monoclonal antibodies were aliquoted to prepare antibody cocktails. The daily MFI values for granulocytes, lymphocytes, and monocytes were calculated, and their mean, SD, and CV have been shown in Table 5. The CV was consistently <5% during the initial 5 days. Subsequently, the weekly MFI values, along with mean, SD, and CV, have been shown in Table 6 and Figure 2. Similarly, the CV was consistently <5% throughout the four weeks, indicating that the tandem dyes were stable in the cocktail when tested with lyophilized cells. Finally, the validation of the cocktail was performed using acute leukemia cases during routine diagnostic practice. The compensation matrices used for analyzing the ALOT cases during days 1-5 and during the four weeks have been shown in Figures 3 and 4, respectively.

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Figure 2:

(A and B) week 1 and 4 stability check with CytoComp^TM lyophilized sample on every Wednesday.

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Figure 3:

(A-E) Compensation matrix from day 1 to day 5 for the acute leukemia orientation tube.

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Figure 4:

(A-D) Compensation matrix from week 1 to week 4 for acute leukemia orientation tube using CytoComp^TM lyophilized cells.

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Table 5: Stability of tandem dyes in cocktail using CytoComp^TM lyophilized sample for the initial 5 days.

Antibody-conjugate	Day 1 MFI value	Day 2 MFI value	Day 3 MFI value	Day 4 MFI value	Day 5 MFI value	Mean	SD	CV (%)
cMPO-FITC	3.2	3.0	6.6	3.0	2.1	3.6	1.7	0.48
cCD79a-PE	5.8	5.1	12.1	5.1	7.8	7.2	3.0	0.41
CD19-ECD	10.6	10.9	8.5	8.1	10.5	9.7	1.3	0.13
CD34 – PC5.5	NA	NA	NA	NA	NA	NA	NA	NA
cCD3-PC7	1.9	1.7	2.4	2.2	1.8	2.0	0.3	0.14
nTdT-APC	NA	NA	NA	NA	NA	NA	NA	NA
CD45-APC700	112.9	92.3	92.6	195.1	170.7	132.7	47.4	0.35
CD7-APC750	13.8	24.6	21.7	21.6	15.4	19.4	4.6	0.23
sCD3-PB	11.2	13.2	12.3	13.0	12.9	12.5	0.8	0.06
HLA-DR-KO	16.5	14.9	26.8	30.4	34.4	24.6	8.6	0.34

FITC: Fluorescein isothiocyanate, PE: Phycoerythrin, ECD: Energy coupling dye, PC: Phycoerythrin-cyanin, APC: Allophycocyanin, PB: Pacific blue, KO: Krome orange, CD: Cluster of differentiation, MPO: Myeloperoxidase, c, s, n indicates cytoplasmic, surface and nuclear staining, TdT: Terminal deoxynucleotidyl transferase, HLA-DR: Human leucocyte antigen–DR isotype, MFI: Mean fluorescent intensity, SD: Standard deviation, CV: Co-efficient of variation, NA: Not available

Table 6: Stability of tandem dyes in cocktail using CytoComp^TM lyophilized sample on every Wednesday for a continuous 4 weeks.

Antibody-conjugate	Week 1 MFI value	Week 2 MFI value	Week 3 MFI value	Week 4 MFI value	Mean	SD	CV (%)
cMPO-FITC	11.14	14.16	16.31	11.07	13.17	2.5	0.19
cCD79a-PE	3.73	4.98	4.12	4.17	4.25	0.52	0.12
CD19-ECD	4.06	5.65	4.44	4.55	4.67	0.59	0.13
CD34 – PC5.5	NA	NA	NA	NA	NA	NA	NA
cCD3-PC7	4.61	5.70	6.44	3.70	5.11	1.04	0.20
nTdT-APC	NA	NA	NA	NA	NA	NA	NA
CD45-APC700	69.18	125.25	81.32	107.99	95.93	21.9	0.23
CD7-APC750	15.01	17.95	14.48	15.28	15.68	1.3	0.08
sCD3-PB	10.41	11.66	10.39	10.23	10.67	0.57	0.05
HLA-DR-KO	18.14	17.88	16.94	18.10	17.76	0.54	0.03

FITC: Fluorescein isothiocyanate, PE: Phycoerythrin, ECD: Energy coupling dye, PC: Phycoerythrin-cyanin, APC: Allophycocyanin, PB: Pacific blue, KO: Krome orange, CD: Cluster of differentiation, MPO: Myeloperoxidase, c, s, n indicates cytoplasmic, surface, and nuclear staining, TdT: Terminal deoxynucleotidyl transferase, HLA-DR: Human leucocyte antigen-DR isotype, MFI: Mean fluorescent intensity, SD: Standard deviation, CV: Coefficient of variation, TM: Trademark, NA: Not available

DISCUSSION

The major pre-analytical variable that should be addressed in MFC is the validation of monoclonal antibodies. The initial step in this process includes titration of each antibody, as endorsed by the EuroFlow Consortium for standardization in MFC.^[3] Most manufacturers recommend a higher concentration of the antibody for MFC testing; however, following these recommendations directly may not be cost-effective.

The need to titrate the antibody is to optimize signal intensity, achieve staining saturation, and conserve reagents. This is usually done using objective methods such as SNR or SI, or using a subjective method such as visual interpretation of histograms or plots to determine the lowest effective antibody concentration.^[3-6] In this study, both of these approaches were used, as both of them were found to be complementary to each other, thereby negating the disadvantages associated with using either method alone. Titration experiments not only improve multicolor panel experiments but also optimize fluorescent staining in MFC.^[4]

As part of the initial phase of the study, the individual antibodies in the ALOT tube were titrated. It was found that there was a greater reduction in the final antibody concentrations required for MFC testing. This optimization proved to be highly cost-effective in our laboratory, resulting in approximately a 77% reduction in the cost of ALOT tube testing alone. We also extended the same titration experiment to the other panels for the sub-categorization of acute leukemias.

In the second phase of the study, the pre-titrated volumes of monoclonal antibodies were combined to prepare antibody cocktails before adding them to the test samples. The initial titration experiment also reduced the workforce requirements by decreasing the time spent on each test and minimizing non-conformities related to the omission or incorrect addition of individual antibodies, although this was not objectively evaluated.^[3-5]

In general, it is recommended to store antibody cocktails for the expected maximum number of MFC tests, typically for 1–2 weeks.^[3-5] In our laboratory, the prepared antibody cocktails are stored for a period of 7–10 days. The major concern is whether tandem dyes conjugated to monoclonal antibodies remain stable during prolonged storage. This stability varies within and between laboratories.^[7-11] This was the stimulus for us to check for the stability of tandem dyes in antibody cocktail preparations in our laboratory.

There are several studies that have discussed and debated this issue.^[7-15] We conducted this study like that of Johansson and Macey^[9], using both fresh peripheral blood samples during the initial five days to check for the stability and CytoComp^TM lyophilized cells for up to four weeks. The study results were in agreement with those of these authors. We had tested for the maximum of only four weeks, beyond which the utility of maintaining antibody cocktails was minimal; therefore, further evaluation was not performed.

The main practical difficulty encountered in this study was the unavailability of acute leukemia cases daily for five days during the stability analysis. Consequently, objective evaluation of CD34-PC5.5 and nTdT-APC was not performed in the daily testing, as CytoComp^TM lyophilized samples predominantly showed mature cells for weekly stability assessment. However, this specific difficulty was addressed by validating the same antibody cocktail in routine diagnostic practice using acute leukemia samples, which demonstrated clinically acceptable stability for 10–14 days.

Rawstron et al.^[16] tested two separate antibody cocktails, each containing six antibodies (including APC-H7- and PECy7-conjugated antibodies), over 28 days. They observed no loss of PE-Cy7 or APC-H7 signal intensity during this time; however, there was an increase in background APC-H7 intensity on APC-positive cells. In a similar study, Le Roy et al.^[10] showed that APC tandems may undergo degradation depending on the cell types present in the sample. The effect occurred relatively immediately (within 30 min) and was also observed in single antibody-stained samples. However, it was difficult to determine how the results were influenced by cell type and whether the APC tandem dyes were stored individually or as part of a cocktail.

Hulspas et al.^[11] studied the photosensitivity of PE-based tandem dyes, particularly PE-Cy7. They were stored as antibody cocktails in light-protective bottles and subjected to a single daily light exposure of 10 min (for preserved cells) or 20 min (for fresh bone marrow), reflecting the average time spent on the bench for each type of cocktail. This level of light exposure did not appear to cause tandem degradation. In this study, we did not analyze the tandem degradation after light exposure.

O’Donahue et al.^[12] studied the validation of antibody cocktails for MFC. They observed that to improve laboratory efficiency, they recommend using the antibody cocktails rather than adding individual antibodies before testing. They found that pipetting errors were the most common problem during cocktail preparation. To avoid these issues, they recommend using electronic pipettes and regular calibration, to be performed and monitored at least twice per year. They concluded that antibody cocktails, when performed with proper standardization and appropriate quality control, enhance the accuracy of the results and improve overall performance in the MFC laboratories.

CONCLUSIONS

Titration of monoclonal antibodies resulted in a substantial reduction in the final volume of antibodies required for the cocktail preparation compared to the manufacturer’s recommendation. This experiment led to an approximate 77% reduction in cost per test, representing a significant improvement in resource utilization. Although not objectively measured, there was also an observed improvement in the need for manpower, including reduced time per test and fewer non-conformities related to the omission of individual antibodies. The pre-titrated antibody cocktail remained stable for the initial five days and demonstrated tandem dye stability for four weeks when tested using CytoComp^TM lyophilized samples. This stability was further validated in acute leukemia samples during routine daily reporting.