Detection of antinuclear antibodies (ANA) by indirect immunofluorescence assay using HEp-2 cells (HEp-2 IFA) is used to screen for various autoimmune diseases. HEp-2 IFA suffers from variability, which hampers harmonization.
A questionnaire was developed to collect information on HEp-2 IFA methodology, computer-assisted diagnosis (CAD) systems, training, inter-observer variability, quality assessment, reagent lot change control, and method verification. The questionnaire was distributed to laboratories by Sciensano (Belgium), national EASI groups (Italy, Croatia, Portugal, Estonia, Greece) and ICAP (worldwide). Answers were obtained by 414 laboratories. The results were analysed in the framework of the recent EFLM/EASI/ICAP ANA recommendations (companion paper).
Laboratories used either HEp-2, HEp-2000, or HEp-20-10 cells and most laboratories (80%) applied the same screening dilution for children and adults. The conjugate used varied between laboratories [IgG-specific (in 57% of laboratories) vs. polyvalent]. Sixty-nine percent of CAD users reviewed the automatic nuclear pattern and 53% of CAD users did not fully exploit the fluorescence intensity for quality assurance. Internal quality control was performed by 96% of the laboratories, in 52% of the laboratories only with strongly positive samples. Interobserver variation was controlled by 79% of the laboratories. Limited lot-to-lot evaluation was performed by 68% of the laboratories. Method verification was done by 80% of the respondents.
Even though many laboratories embrace high-quality HEp-2 IFA, substantial differences in how HEp-2 IFA is performed and controlled remain. Acting according to the EFLM/EASI/ICAP ANA recommendations can improve the global performance and quality of HEp-2 IFA and nurture harmonization.
Antinuclear antibody (ANA) test by indirect immunofluorescence analysis (IFA) using HEp-2 cells (HEp-2 IFA) suffers from variability due to differences in substrate, screening dilution, conjugate and microscopic analysis (e.g. visual or automated reading). Laboratories attempt to control HEp-2 IFA variability by training of collaborators, external/internal quality assessments, reagent lot evaluation and method verification. A Belgian group of experts was interested in the way laboratories are coping with these aspects and launched a national inquiry, which was subsequently extended worldwide. The survey covers the everyday modus operandi of laboratories performing HEp-2 IFA, which we discuss in light of the European Federation of Laboratory Medicine (EFLM), European Autoimmunity Standardization Initiative (EASI) and International Consensus on Antinuclear Antibody Patterns (ICAP) recommendations on the detection of ANA (which were internationally supported through a Delphi exercise) .
Questions regarding HEp-2 IFA methodology, automatic microscopic analysis, training and controlling inter-observer variability, external quality assessment, internal quality control, reagent lot acceptance, monitoring of lot-to-lot variability, and method verification, were designed by the Expert Committee Non-infectious Serology of Sciensano and EASI Belgium. A survey was distributed to the Belgian laboratories in 2018 by Sciensano. International distribution was organised by national EASI groups (Italy, Croatia, Portugal, Estonia, Greece) and ICAP in 2019. The same set of questions was included in both surveys (Belgian and international), except for 5 questions for which only 1 answer could be selected in the Belgian inquiry and a combination of answers in the ICAP enquiry. For these questions only ICAP answers were taken into account. Data analysis was performed by the European Federation of Laboratory Medicine (EFLM) Working Group “Autoimmunity Testing”.
Results and discussion
Four hundred fifteen laboratories performing HEp-2 IFA responded to the survey, 348 (84%) via ICAP and 67 (16%) via Sciensano. Participants were mainly European (61%), American (21%) and Asian (15%) (Table 1).
|Continent||Number of responses, %|
HEp-2 IFA slide preparation was automated in 60% (n=241/401) of the laboratories. Computer assisted diagnosis (CAD) systems for HEp-2 IFA analysis were used in 27% (n=111/415) of the participating laboratories [29% (n=73/252) in Europe, 26% (n=9/34) in North-America and 17% (n=9/53) in South-America] (Table 2).
|Topic||Number of responses||Answers||%a|
|Equipment and test frequency|
|Usage of CAD||415||CAD users||27|
|2–3 times a week||37|
|Every 2 weeks||2|
|Conjugate||254||IgG heavy chain||57|
|IgG heavy and light chain||37|
|Substrate/conjugate combination||284||Kit combination||75|
|Screening dilution of children and adults||399||Not different||80|
aExpressed on the total of responses for the particular question, unless stated otherwise; bexpressed on the total of laboratories using indirect immunofluorescence microscopy in the particular geographical region; CAD, computer-assisted diagnosis.
Substrate, conjugate and screening dilution
Substrate heterogeneity can induce variability between results obtained in different laboratories. Comparative studies identified differences between substrates with respect to pattern recognition , ,  and sensitivity for particular antibodies (e.g. anti-SSA/Ro60) , , , , but results of these studies are ambiguous and probably also biased by differences in conjugates.
In our survey, 69% of the respondents used HEp-2 cells, 16% HEp-2000 cells and 15% HEp-20-10 cells (Table 2). However, geographical differences were observed. For example, 76% (n=155/204) of ICAP respondents used HEp-2 vs. 46% (n=27/59) of Belgian respondents. The use of both the classic HEp-2 substrate and the manipulated cellular substrates (HEp-2000 and HEp-20-10) was supported by the recent EFLM/EASI/ICAP Delphi exercise [92% high scores (≥7 on a scale of 9)] .
The isotype specificity of the used conjugate (polyvalent/IgG-specific) may contribute to assay variability (reviewed in ). IgG-specific conjugate is considered sufficient to detect most clinically relevant ANA in the context of ANA-associated rheumatic diseases (AARD) , , , . Polyvalent conjugate may produce background fluorescence due to the detection of clinically irrelevant antibodies [9, 10]. Our survey showed that about half of the laboratories (57%) worked with an IgG-specific conjugate, while 37% used a conjugate that detects both IgG heavy and light chains (polyvalent) (Table 2).
Seventy-five percent of the laboratories that participated in the survey utilized combinations of substrate/conjugate as proposed by the manufacturer (commercial kit-combinations). The other laboratories made their own combinations of substrate and conjugate (Table 2).
There is no consensus in literature and amongst experts on the optimal starting dilution to screen (screening dilution) for ANA by HEp-2 IFA . The majority of the respondents of the current international survey (61%) applied a 1:80 screening dilution (Table 2), which is comparable to what was observed in the 2014 EASI survey (60.5%) , but lower than the 80% reported by ICAP in 2021 . Dilution 1:160 was used for screening by 15% of the respondents.
Eighty percent of the respondents used the same screening dilution for children and adults. As summarized in the EFLM/EASI/ICAP recommendation paper , there is no evidence for screening children for AARD with a lower dilution for ANA by HEp-2 IFA than the dilution used for adults.
Automated microscopy: expert revision, use of FI score for titer estimation and added value in quality assurance
The implementation of CAD represents an important step forward in HEp-2 IFA analysis. The recent EFLM/EASI/ICAP Delphi round documented high support (i) on the added-value of CAD systems for positive/negative interpretation (in combination with expert review) [93% high scores (≥7/9)] and (ii) on the appreciation that expert review remains mandatory for pattern assignment [99% high scores (≥7/9)]. The data of the survey showed that the majority (69%) of laboratories equipped with CAD always reviewed the automatically proposed patterns. In 17% of the laboratories pattern revision was only performed in specific cases. A minority (14%) never reviewed the patterns (Table 3).
|Topic||Number of responses||Answers||%a|
|CAD pattern assignment||101||Review in all cases||69|
|Review in specific cases||17|
|CAD titer estimation||102||Not used for reporting
Used for reporting all nuclear patterns
|Used for reporting some nuclear patterns||25.5|
|QC based on FI measure||66||Not performed||53|
aExpressed on the total of responses for the particular question; CAD, computer assisted diagnosis; QC, quality control; FI, fluorescence intensity.
Many CADs provide a quantification of the fluorescence intensity (referred to as ‘FI measure’), which significantly correlates with the titer and provides information on the likelihood for AARD , , , , , , , . Some systems also provide an estimated endpoint titer based on analysis at screening dilution which is substrate-specific and pattern-dependent . The survey indicated that 49% of respondents using CAD did not use the titer estimation as provided by the system for reporting. About 25% of the laboratories relied on automatic titer estimation for all nuclear patterns and 25.5% for some nuclear patterns (Table 3). Even though >20% of the participants in the EFLM/EASI/ICAP Delphi exercise indicated inexperience on the topic, 87% of the remaining participants supported [high scores (≥7/9)] the idea that the FI measure can be used to estimate the titer and to provide information on the likelihood for AARD .
The availability of a reproducible FI measure as provided by the CAD systems brings the HEp-2 IFA to the level of a (semi)quantitative assay and therefore allows for improved evaluation of the quality of the analysis (e.g. quantitative monitoring of within- and between-run reproducibility on internal quality controls (IQC) samples and monitoring of the median FI of a run) (reviewed in ) Moreover, the international EFLM/EASI/ICAP recommendation endorsed this approach of quality assessment [92% high scores (≥7/9)] . However, our survey shows that, in daily practice, 53% of laboratories having the FI measure available did not take advantage of these new opportunities (Table 3).
Obviously, a certain threshold of ANA requests is required for a laboratory to gain and maintain sufficient expertise in reading the HEp-2 IFA slides. EASI recommends a turn-around time for HEp-2 IFA of 48–72 h in a hospital setting . This corresponds to a test frequency of at least twice a week, which is reached by 83% (n=336/407) of the laboratories that participated in the survey. A lower frequency of HEp-2 IFA was reported by the other labs: 15% of the labs performed HEp-2 IFA on a weekly basis and 2% of the labs every 2 weeks (Table 2).
Training, controlling inter-observer variability and external quality control programs
The results of our survey showed that inter-observer variation was controlled in 79% (n=218/277) of labs (Table 4). The approaches applied to control inter-observer variability were double reading (the reported result is a consensus of 2 observers) in 56% of the labs and periodic blinded reading of representative cases more than once a year (formal tests) in 16% of the labs. Some labs combined double reading and periodic blinded reading (10%) or used another approach to control inter-observer variability (18%). The formal tests were mostly organized with a combination of external quality control (EQC) samples and patient samples (68%). The laboratories that did not use both types of samples either used EQC or patient samples (Table 4).
|Topic||Number of responses||Answer||%a|
|Control inter-observer variation||277||Yes||79|
|Method for inter-observer variation controlb||160||Double reading only||56|
|Formal tests >1/year only||16|
|Combination of double reading and formal tests||10|
|Samples used for formal tests||53||Combination of both EQC and patient samples||68|
|Patients samples only||15|
|EQC samples only||17|
aExpressed on the total of responses for the particular question; bICAP only, exclusion Belgian data as for Belgian laboratories combined answers were not possible and another option (formal tests once a year) was available; cother approach than formal testing or double reading; EQC, external quality control.
Participating in EQC programs contributes to monitoring the HEp-2 IFA performance (reviewed in ). Accreditation programs or national legislation can make participation obligatory. In some countries, as in Belgium, a mandatory national EQC program is organized by national health agencies. Ninety-one percent of the laboratories (n=249/273) participated in EQC schemes, 61% subscribed to minimally 3 EQC distributions a year, and 73% evaluated yearly at least 4 EQC samples (Table 5).
|Topic||Number of responses||Answers||%a|
|Participation in EQC schemes||273||Yes||91|
|Number of distributions||239||1 Distribution/year||10|
|Number of samples||241||1 Sample/year||6|
aExpressed on the total of responses for the particular question; EQC, external quality control.
Internal quality control
IQC by monitoring the results obtained with IQC samples in each run is one of the most important cornerstones in the quality control (QC) program of clinical laboratories. Our survey shows that IQC for HEp-2 IFA was performed by 96% of the laboratories (Table 6).
|Topic||Number of responses||Answers||%a|
|Performance of run IQC||323||Yes||96|
|Details on the IQC procedure|
|Number IQC samples/run||300||1 Positive and 1 negative IQC||62|
|>1 Positive and 1 negative IQC||13|
|1 positive and no negative IQC||21|
|>1 Positive and no negative IQC||1.7|
|Position of positive IQC samples (if ≥2/run)||49||All at start or end of the run||45|
|Level of positivity of IQC samples||288||High level||52|
|Alternating patterns/specificities over different runs||276||No, always the same patterns/specificities||62|
|Yes, alternating patterns/specificities||38|
|Frequency of alternating patterns/specificities over different runs||103||Daily||20|
|>1 Week but <1 month||21|
|At the end of sample supply||17|
|Dilution of the IQC samples||275||Undiluted (different from patient samples)||63|
|Diluted (same dilution as patient samples)||30|
|Diluted (different dilution as patient samples)||7|
|Origin of the IQC samples||296||Commercial origin only||58|
|Patient origin only||13|
|Commercial and patient origin||29|
|Pooled or single patient IQC samples||128||Pooled patient sample||11|
|Single patient samples||64|
|Pooled and single patient samples||24|
|Patient sample preservation temperature if >1 weekb||127||Fridge with/without preservatives||29c|
|Target of IQC samples||256||Commercial||75|
aExpressed on the total of responses for the particular question, unless stated otherwise; bICAP only, exclusion Belgian data as for Belgian laboratories combined answers were not possible; cexpressed on the number of laboratories that use patient material for internal quality control (IQC).
Many guidelines support the need for inclusion of at least 2 IQC samples (one negative and one low positive) in each run and advice semi-quantitatively judgement of the results (either by end-point titration or automated intensity scoring) [1, 9, 24, 25]. The results of our survey indicate that 78% of laboratories indeed evaluated at least 1 positive and 1 negative control sample in every run. About 23% of the laboratories only analyzed at least 1 positive control but no negative control. Less than a fifth of the laboratories (18%) used more than 1 positive sample as run IQC. About half of the laboratories positioned all their IQC samples together at the start or end of the run; the others spread the IQC samples with some at the start and some at the end (Table 6).
The EFLM/EASI/ICAP recommendation states that the positive control should be a low positive sample matching an LR of 2–5 for ANA-associated rheumatic diseases . The survey revealed that positive samples had mostly high (52%) or medium (19%) titers. Cut-off controls were only used in 13% of the laboratories (Table 6).
Different approaches can be used for choosing the pattern(s) of the positive IQC sample(s). The 2009 German EASI recommendation suggests testing IQC samples with different patterns, in alternation over the runs . Another approach focuses on the sensitivity for fragile antigens, such as SSA/Ro-60, and proposes to include antibodies to such antigens as IQC . Still another approach is to select the patterns based on high inter-assay reproducibility of the FI measure on CAD systems, allowing optimized IQC approaches (, , , , , reviewed in ). This approach focussing on selecting a pattern with high reproducibility for IQC purposes obtained high international support during the EFLM/EASI/ICAP Delphi exercise [98% high scores ≥7/9 with median score 8] .
The survey revealed that of the labs that perform IQC, most (62%) used always the same pattern over the different runs. In those labs that alternate the patterns, the frequency of alternating the patterns differed between labs (Table 6). When labs were running more than 1 positive IQC per run, 83% (n=39/47) tested different patterns. Fifty-two percent of these labs tested at least 3 different antigen specificities/patterns per run.
Controls included in commercial kits are often pre-diluted and mostly have high titres. This precludes detection of pipetting errors. Therefore, combining IQC samples from patient origin with commercial controls is advised ([24, 25, 27, 28], reviewed in ). In our survey, 70% of laboratories used undiluted IQC samples or IQC samples that were differently diluted than the patient samples. Many labs (58%, n=172/296) exclusively used commercial IQC material. Thirteen percent of the respondents relied only on control material from patient origin, and almost one-third (29%) of the labs combined both. When using patient samples as IQC material, most laboratories (64%) used single patient samples rather than pools, while 24% used both single patient samples and pools. Laboratories using exclusively pools were a minority (11%). Seventy-five percent (n=191/256) of laboratories using commercial QC material adopted the company target, and 19% determined a laboratory target (see Table 6).
CLSI suggests long-term storage of samples at −70 °C in combination with a preservative . It is however common practice to freeze samples at −20 °C, as reported by 71% (n=90/127) of the labs participating in the survey. Ten percent of the laboratories preserved patient QC material at −80 °C (Table 6). Twenty-nine percent of the laboratories used the fridge for longer than 1-week storage, contrary to the CLSI guidelines that advises limiting preservation in the fridge to 3 days [9, 32]. Only 7% safeguarded the samples with preservatives.
Quality control approaches based on patient results
Monitoring the percentage of (low, medium, high) positive patient results in Levey-Jennings plots allows us to evaluate assay stability over time [9, 28, 29]. Such an approach is considered to be of added-value, as reflected by the high score (≥7/9) given by 87% of the experts participating in the EFLM/EASI/ICAP Delphi exercise . In the survey, however, only 17% (n=51/292) of the respondents monitored the percentage of positive patients.
Reagent lot acceptance and monitoring of lot-to-lot variability
Variations in HEp-2 IFA results have been shown between different slide brands , ,  and suggested to be present also between reagents lots [2, 29, 31, 33]. According to international guidelines and EN/ISO 15189:2012 accreditation requirements, lot-to-lot variability of different batches (of one brand/kit) should be evaluated before implementing a new lot [24,34]. According to the EFLM/EASI/ICAP recommendation this can be done by analysis of patient-derived IQC samples supplemented with samples selected for this purpose ideally covering different cell compartments (nucleus and cytoplasm) and different titer levels . Our survey revealed that lot-to-lot evaluation of substrate was performed by 68% (n=173/254) of the laboratories. This was mainly performed with commercial samples, either exclusively (42%) (n=72/173) or combined with patient material (40%) (n=69/173) (Table 7). Eighteen percent (n=31/173) of laboratories used solely patient samples. Approximately one third (37%, n=33/89) of the laboratories evaluating patient samples tested only 1 pattern. Two or 3 patterns were tested by 24% of the laboratories and more than 3 patterns by 30% of the laboratories. The EFLM/EASI/ICAP recommendation suggests testing with samples reacting with different cell compartments (nucleus and cytoplasm) of titer levels including negatives . To limit the variation linked to lot variability, 47% of the laboratories ordered reagents of 1 lot for a longer period (Table 7).
|Topic||Number of responses||Answers||%a|
|Procedure for substrate change is based on comparison of new lot with the former lot||254||Yes||68|
|Origin of samples to perform the lot change procedure||173||Using exclusively patient samples||18b|
|Using exclusively commercial samples||42b|
|Combination of patient and commercial samples||40b|
|For patient samples users: number of patterns||89||1 Pattern||37|
|2 or 3 Patterns||24|
|Limitation of lot changes||259||Yes||47|
aExpressed on the total of responses for the particular question, unless stated otherwise; bexpressed on the total of laboratories performing a procedure for lot change.
As we repeatedly mentioned, HEp-2 IFA is prone to assay variability between manufacturers , , . Therefore, each new method and/or formulation change should be verified locally (, reviewed in ). In particular one should verify that the HEp-2 IFA method detects the major clinically relevant patterns and antibodies, both in the nuclear and cytoplasmic compartments [1, 2, 9, 24, 26, 33, 35].
Our surveys showed that in daily practice, 80% (n=149/187) of the laboratories verified a new method before implementing it in routine. The other laboratories not performing verification argued to rely on the kit insert (14%) or published literature (4%) or a combination of both (2%) (Table 8).
|Topic||Number of responses||Answers||%a|
|Performing verification of commercial method b||187||Yes||80|
|No verification, rely on kit insert||14|
|No verification, rely on publications||4|
|No verification, rely on kit insert and publications||2|
|Details verification procedure:|
|Origin of samplesb||149||EQC samples exclusively||9|
|Patient and EQC samples||45|
|Patient samples exclusively||46|
|Characterization of patient samplesb||136||Clinically characterized||30c|
|Laboratory characterized (method comparison and/or follow-up tests)||44c|
|Both clinically and laboratory characterized||26c|
|Number of laboratory-characterised samples||105||<10||9d|
|≥10 and <20||20d|
|≥20 and <40||38d|
|Level of sample positivity||133||Only strongly positive samples||10|
|Only weakly positive samples||4|
|Combination strongly and weakly positive samples||86|
|Number of patterns||148||At least the 5 major patterns (homogeneous, speckled, nucleolar, centromere, cytoplasmic)||84|
|<5 Patterns, but >1 Pattern||9|
|2/Verification of precision||225||Yes||72|
|≥5 < 10 Replicates||21f|
aExpressed on the total of responses for the particular question, unless stated otherwise; bICAP only, exclusion Belgian data as for Belgian laboratories combined answers were not possible; cexpressed on laboratories using patient samples for verification; dexpressed on the number of laboratories using lab-characterized samples for verification; eexpressed on laboratories verifying precision; fexpressed on laboratories performing within/between run precision verification experiments, respectively.
It is recommended to verify the new method by method comparison with clinically relevant patterns and antibodies (i.e. those that are comprised in classification/diagnostic criteria) and negative samples . Optimally, these samples for method verification are well characterized by solid phase assays, clinical information or have a target result available of EQA . Our questionnaire revealed that laboratories used mostly patient samples (>90%) for method verification, often in combination with EQC samples (Table 8). When patient samples were used for verification purposes, clinical information was used as a reference by 30% of the laboratories, while other laboratory test results (including follow-up tests such as anti-ENA) were used as references by 44% of the laboratories. A combination of both clinical data and laboratory tests was used by 26% of the laboratories (Table 8).
The majority of the laboratories that participated in the survey performed verification with at least 20 samples (71%). Minimally 40 samples were used by 33% of labs and less than 20 samples by 29% of the laboratories (Table 8). The EASI guidelines propose to test 10–50 known positive and 10–100 known negative sera with a minimum of 30 comparisons . The majority of the labs (86%) combined strongly and weakly positive samples for method verification.
The 2009 German EASI guideline recommends evaluating minimally 3 different patterns resulting from defined antibody specificity (e.g. centromere, dsDNA, SS-A/Ro60) . CLSI LA02-A2 recommends evaluation of clinically significant autoantibodies (i.e. antibodies to dsDNA, U1RNP, Sm, SS-A/Ro60, SS-B, Scl-70, CENP, Jo-1) . The current EFLM/EASI/ICAP recommendation advices to verify for the following clinically important patterns/antibodies: nuclear homogenous (AC-1), nuclear speckled (AC-4,5), nucleolar (AC-8,9,10), centromere (AC-3), multiple nuclear dots (AC-6) and nuclear envelope pattern (AC-11,12), cytoplasmic speckled (AC-19,20), reticular/anti-mitochondrial pattern (AC-21), anti-dsDNA, anti-SSA/Ro60, anti-Sm/RNP, anti-CENPB, anti-Scl70, anti-RNA-polymerase III, anti-Jo-1, anti-Sp100, anti-gp210 and AMA-M2 . In the survey, 84% (n=125/148) of the laboratories minimally verified the nuclear homogeneous, speckled, nucleolar and centromere patterns, as well as one cytoplasmic pattern. Sixteen percent (n=23/148) of the laboratories tested less than 5 patterns, and 7% (n=10/148) tested only one pattern (Table 8).
Method verification includes verification of precision (reviewed in ). Based on our survey, precision was verified during method verification by 72% (n=161/225) of the laboratories; 56% verified both between- and within-run, 12% verified only between-run and 4% verified only within-run precision. Between-run precision was evaluated for different patterns by 77% (n=115/150) of the laboratories. In accordance with the EASI guideline , at least 10 replicates were measured in the between- or within-run precision experiments by, respectively, 31% and 38% of the respondents. About 40% of laboratories tested less than 5 replicates for either between-run or within-run precision.
We present the results of an international survey on the daily practice of HEp-2 IFA in light of the new EFLM/EASI/ICAP recommendations (companion paper). The survey focussed mainly on methodology, the usage of CAD, the procedures for controlling daily variability (e.g. QA procedures including EQC, IQC, lot change control) and approaches for method verification.
With regard to methodological aspects, most laboratories used HEp-2, HEp-2000 or HEp-20-10 cells and applied the same starting dilution for children and adults, which is according to the EFLM/EASI/ICAP recommendations. There was variability between labs regarding the use of IgG-specific vs. polyvalent conjugate.
With regard to CAD systems, the survey not only illustrated differences in how CAD systems were applied (both for reporting results and QA purposes), but also revealed that the possibilities of CAD systems for quality assessment were not fully employed yet. The EFML/EASI/ICAP recommendations stress the need for expert review for pattern recognition and recommend applying fluorescence intensity for monitoring IQC samples and/or patient medians. Laboratories should more broadly exploit the possibilities of CAD systems and the EFLM/EASI/ICAP recommendations might be a guide.
Overall, IQC and EQC were well adopted by the laboratories and inter-observer variability was controlled by a majority of the labs (79%). Many laboratories used a high-level (commercial) IQC sample, whereas the EFLM/EASI/ICAP recommendations propose to use a sample with an antibody level corresponding to the cut-off or a likelihood ratio of 2–5. Only few laboratories followed the monthly percentage of positive results, as recommended by EFLM/EASI/ICAP.
Lot-to-lot variability was evaluated by 68% of the participants. Most labs did this with a limited set of samples, whereas the EFLM/EASI/ICAP recommendations propose to verify lot variability with a more extended panel of samples covering both the nuclear and cytoplasmic compartment. Finally, the majority of the labs verified a new method before introducing it.
Overall, a majority of labs embrace basic measures to control and maintain high-quality HEp-2 IFA. However, the results of the survey also illustrated that, despite the high support documented for the current EFLM/EASI/ICAP recommendations , there is still a long road ahead of us before the majority of the labs act in accordance with all EFLM/EASI/ICAP recommendations. Complying with the recommendations will foster harmonization of HEp-2 IFA. Some EFLM/EASI/ICAP recommendations are demanding, for example those for lot validation. Therefore, we propose a close interaction and collaboration between laboratories and in vitro diagnostic companies to control lot variability, such that efforts needed for lot validation at the local laboratory level are minimized. Consistency between lots is crucial for high-quality HEp-2 IFA, especially when CAD systems are used.
Research funding: None declared.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: XB reports consultancy and/or speakers fees from Werfen/Inova, Thermo Fisher Scientific and Menarini.
Informed consent: Not applicable.
Ethical approval: Not applicable.
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