Possible alternative strategies to implement basophil activation testing in multicentric studies.

The Basophil Activation Test (BAT) enables flow cytometry characterization of basophil reactivity against specific allergenic molecules. The focus now revolves around democratizing this tool, but, as blood sample stability could be challenging, after having developed a simplified approach, herein, we aimed to characterize two strategies for implementing BAT in multicentric studies: store and ship blood before or after sample processing. Fresh heparin- and EDTA-anticoagulated whole blood samples followed both BAT workflows: "collect, store, process & analyze" or "collect, process, store & analyze". Storage temperatures of 18-25 °C or 2-8 °C and preservation times from 0 to 7 days were considered. Interleukin-3 was also evaluated. With the "collect, store, process & analyze" workflow, heparin-anticoagulated blood and 18-25 °C storage were better than other conditions. While remaining possible, basophil activation exhibited a possible reactivity decay after 24 h. Under the conditions tested, interleukin-3 had no role in enhancing basophil reactivity after storage. Conversely, the "collect, process, store & analyze" workflow demonstrated that either heparin- or EDTA-anticoagulated blood can be processed and kept up to 7 days at 18-25 °C or 2-8 °C before being analyzed. Various strategies can be implemented to integrate BAT in multicentric studies. The "collect, store, process & analyze" workflow remains a simplified logistical approach, but depending on time required to ship from the clinical centers to the reference laboratories, it might not be applicable, or should be used with caution. The "collect, process, store & analyze" workflow may constitute a workflow improvement to provide significant flexibility without impact on basophil reactivity.

A whole blood-based functional assay to characterize immunoglobulin A effector functions.

Most investigations on the immune cell-activating potency of IgA used purified total IgA and/or specific isolated cell populations. As IgA2 has been reported to be more pro-inflammatory than IgA1, we aimed to employ a fast and convenient whole blood-based assay to individually probe the capacity of the two IgA subclasses to activate immune cells in close physiological conditions. To this end, whole blood from healthy donors (n = 10) was stimulated with immobilized IgA1, IgA2m1 or IgA2m2 (the two main allotypic variants of IgA2). Activation of major leukocyte subsets was measured using a 10-color flow cytometry panel providing access to the expression of 5 activation markers on 6 different immune cell subsets. While capturing some heterogeneity of responses among donors, IgA2m1 and IgA2m2 systematically showed a stronger activation profile compared to IgA1 in a variety of dimensions. For example, both IgA2 allotypes led to stronger modulations of CD54, CD11b, CD62L, CD66b or CD69, on both or either monocytes or neutrophils, indicating a more pronounced pro-inflammatory effect for this subclass than IgA1. By taking into account donor-specific soluble and cellular components this whole blood-based functional approach provides new perspectives to further investigate IgA effector functions in mechanistic studies and/or translational research.

Studying antigen-specific T cells through a streamlined, whole blood-based extracellular approach.

Techniques currently used for the study of antigen-specific T-cell responses are either poorly informative or require a heavy workload. Consequently, many perspectives associated with the broader study of such approaches remain mostly unexplored in translational research. However, these could benefit many fields including but not limited to infectious diseases, oncology, and vaccination. Herein, the main objective of this work was to develop a standardized flow cytometry-based approach that would combine ease of use together with a relevant study of antigen-specific T-cell responses so that they could be more often included in clinical research. To this extent, a streamlined approach relying on 1/ the use of whole blood instead of peripheral blood mononuclear cells and 2/ solely based on the expression of extracellular activation-induced markers (AIMs), called whole blood AIM (WAIM), was developed and further compared to more conventional techniques such as enzyme-linked immunospot (ELISpot) and flow cytometry-based intracellular cytokine staining (ICS). Based on a cohort of 20 individuals receiving the COVID-19 mRNA vaccine and focusing on SARS-CoV-2 and cytomegalovirus (CMV)-derived antigen T-cell-specific responses, a significant level of correlation between the three techniques was found. Based on the use of whole blood and on the expression of extracellular activation-induced markers (CD154, CD137, and CD107a), the WAIM technique appears to be very simple to implement and yet allows interesting patient stratification capabilities as the chosen combination of extracellular markers exhibited higher orthogonality than cytokines that are commonly considered in ICS (IFN-γ, TNF-α, and IL-2).

Promises and Remaining Challenges for Further Integration of Basophil Activation Test in Allergy-Related Research and Clinical Practice.

More than 20 years after having been initially proposed, the relevance and usefulness of basophil activation test (BAT) for the field of allergy research and testing were demonstrated on many occasions. Leveraging the fully open format of a flexible, whole blood-based functional assay, BAT has been shown to be equally important for fundamental research, clinical research, and diagnosis. Regardless of whether the focus of a study is on the characterization of the allergenic moiety, on the patient side, or on the study of the fundamental processes involved in the allergic disease or its treatment, BAT enables the gathering of very important insights. In spite of this, its full capabilities have yet to be leveraged. Various bottlenecks, including but not limited to assay logistics, robustness, flow cytometry access, and/or expertise, have indeed been limiting its development beyond experts and long-term users. Now, various initiatives, aiming at resolving these bottlenecks, have been launched. If successful, a broader use of BAT could then be contemplated. In such a situation, its more thorough integration in clinical practice has the potential to significantly change the allergic patient's journey.

SARS-CoV-2 spike protein induces a differential monocyte activation that may contribute to age bias in COVID-19 severity.

A strong bias related to age is observed in COVID-19 patients with pediatric subjects developing a milder disease than adults. We hypothesized that a specific SARS-CoV-2 effect conjugated with preexisting differences in the immune systems may explain this. Using flow cytometry, we investigated basal immune differences in a cohort consisting of 16 non-infected young and 16 aged individuals and further leveraged an in vitro whole blood model of SARS-CoV-2 infection so that functional differences could be mined as well. In short, blood diluted in culture media was incubated 5 or 24 h with the trimeric spike protein or controls. Following unsupervised analysis, we first confirmed that the immune lymphoid and myeloid systems in adults are less efficient and prone to develop higher inflammation than those in children. We notably identified in adults a higher CD43 lymphocyte expression, known for its potentially inhibitory role. The spike protein induced different responses between adults and children, notably a higher increase of inflammatory markers together with lower monocyte and B cell activation in adults. Interestingly, CD169, a CD43 ligand overexpressed in COVID-19 patients, was confirmed to be strongly modulated by the spike protein. In conclusion, the spike protein exacerbated the preexisting lower immune responsiveness and higher inflammatory potential in adults. Altogether, some of the markers identified may explain the marked age bias and be predictive of severity.

The Association of Low CD4 Expression on Monocytes and Low CD8+ T-Cell Count at Hospital Admission Predicts the Need for Mechanical Ventilation in Patients With COVID-19 Pneumonia: A Prospective Monocentric Cohort Study.

To identify COVID-19-associated immunophenotyping patterns at hospital admission and to determine if some patterns could predict the need for mechanical ventilation (MV). DESIGN: Prospective observational monocentric cohort study. SETTING: A university-affiliated hospital in Marseille, France. PATIENTS: Thirty patients presenting with laboratory-confirmed COVID-19 pneumonia were enrolled within the first 48 hours of hospital admission and compared with 18 healthy controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Whole-blood leukocytes were immunophenotyped with a rapid and simplified one-step flow cytometry method. Thirty-eight immune and five laboratory parameters were compared first between COVID-19 patients and controls and then between the COVID-19 patients who received or not MV during their stays. The variables that significantly discriminated MV from non-MV patients in univariate analysis were entered into a multiple stepwise logistic regression analysis. The COVID-19 patients were predominantly male (87%), aged 61 years (50-71 yr), and 93% received early corticosteroid therapy. Sixteen patients (53%) were managed with noninvasive respiratory support, and 14 (47%) required MV. Compared with controls, COVID-19 patients were characterized by an immune signature featuring: 1) decreased HLA-DR expression on monocytes; 2) reduced basophils, eosinophils, T-cells, NK cells, and nonclassical monocyte count; and 3) up regulation of CD169 on monocytes, CD64 on neutrophils, the adhesion/migration markers (CD62L and CD11b), and the checkpoint inhibitor CD274 on myeloid cells. Among the COVID-19 patients, those who received MV had lower level of CD4 and HLA-DR on monocytes, lower CD8+ T-cell count, and higher lactate dehydrogenase at hospital admission. In multivariate analysis, only CD4 on monocytes (p = 0.032) and CD8+ T-cell count (p = 0.026) were associated with MV requirement. The model combining these two variables provided an area under curve of 0.97 (95% CI, 0.83-0.99). CONCLUSIONS: The association of low CD4 on monocytes and low CD8+ T-cell count at hospital admission was highly predictive of the need for MV in hospitalized patients with COVID-19 pneumonia.

A rapid, easy, and scalable whole blood monocyte CD169 assay for outpatient screening during SARS-CoV-2 outbreak, and potentially other emerging disease outbreaks.

Objective: The COVID-19 corona virus disease outbreak is globally challenging health systems and societies. Its diagnosis relies on molecular methods, with drawbacks revealed by mass screening. Upregulation of neutrophil CD64 or monocyte CD169 has been abundantly reported as markers of bacterial or acute viral infection, respectively. We evaluated the sensitivity of an easy, one-step whole blood flow cytometry assay to measure these markers within 10 min, as a potential screening test for COVID-19 patients. Methods: Patients (n = 177) with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were tested on 10 µL blood and results were compared with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Results: We observed 98% and 100% sensitivity in early-stage (n = 52) and asymptomatic patients (n = 9), respectively. Late-stage patients, who presented for a second control RT-qPCR, were negative for both assays in most cases. Conversely, neutrophil CD64 expression was unchanged in 75% of cases, without significant differences between groups. Conclusion: Monocyte CD169 evaluation was highly sensitive for detecting SARS-CoV-2 infection in first-presentation patients; and it returns to basal level upon infection clearance. The potential ease of fingerprick collection, minimal time-to-result, and low cost rank this biomarker measurement as a potential viral disease screening tool, including COVID-19. When the virus prevalence in the tested population is usually low (1%-10%), such an approach could increase the testing capacity 10 to 100-fold, with the same limited molecular testing resources, which could focus on confirmation purposes only.

Leveraging whole blood based functional flow cytometry assays to open new perspectives for rheumatoid arthritis translational research.

Despite introduction of biological disease modifying anti-rheumatic drugs (DMARDs) for Rheumatoid arthritis (RA) treatment, therapeutic strategies do not always lead to disease control and remission. Hence, a more efficient patient stratification and monitoring biomarkers and tools are needed to enable a more personalized medicine. We used a whole blood based functional flow cytometry assay to characterize immune cells from RA patients (treated or not), healthy donors and psoriatic arthritis (PsA) patients according to their responses to LPS and/or anti-TNFα (infliximab, IFX). Activation marker expression was measured using a 10-color flow cytometry panel following a no-wash protocol. Naïve-to-treatment RA patients had a stronger inflammatory profile in comparison to healthy donors at basal level. Higher expression of activation markers (CD69 and/or CD11b) on NK, B cells and granulocytes and lower expression of the adhesion molecule CD62L were measured on monocytes, granulocytes and B cells. After LPS, naïve RA patients' cells were less capable of regulating CD69, CD11b, CD16 or CD62L showing impaired activation capabilities. Upon LPS and IFX co-incubation, hierarchical clustering analysis showed different profiles between cohorts. We believe that this whole blood-based approach should further be assessed for RA patient characterization as it provides new perspectives for stratification and/or monitoring.

One-step White Blood Cell Extracellular Staining Method for Flow Cytometry.

Flow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one-step method in which white blood cell staining and red blood cell lysis are carried out in a single step, using a gentle lysis solution mixed with fluorescent antibody conjugates or probes in a dry or liquid format. The blood sample may be obtained from a routine venipuncture or directly from a fingerprick, allowing for near-patient analysis. This procedure enables the analysis of common white blood cell markers as well as markers related to infections or sepsis. This simpler and faster protocol may help to democratize the use of flow cytometry in the research and medical fields. Graphic abstract: One-step White Blood Cell Extracellular Staining Method for Flow Cytometry.

Cell Analysis from Dried Blood Spots: New Opportunities in Immunology, Hematology, and Infectious Diseases.

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.

Development of a Flow Cytometry-Based Functional Assay to Study Anti-TNF Mechanisms of Action and Capture Donor Heterogeneity.

TNF is a key cytokine in autoimmune diseases like rheumatoid arthritis, and TNF antagonists are commonly prescribed therapeutics. Although anti-TNF drugs have enabled a very significant progress in this field, disease heterogeneity remains and causes diversity in patient response. These challenges increase the need for anti-TNF characterization tools that may open perspectives toward the development of personalized medicine. In this study, we present a novel whole blood-based flow cytometry functional assay that allows, within a given whole blood sample, the characterization of an anti-TNF molecule mechanisms of action. Whole blood from healthy human donors was employed to mimic the physiological state but also to streamline experimental workflows. Samples were incubated with LPS alone or in combination with various anti-TNF molecules such as adalimumab (ADA), etanercept (ETA), and infliximab. A 10-color flow cytometry panel including CD69, transmembrane TNF, CD16, CD62L, CD66b, CD11b, and CD54 as activation markers was used following a centrifugation-free protocol. CD69 expression decreased on NK, NKT, and T cells upon treatment with ADA, ETA, and IFX as a direct indication of forward signaling neutralization. Percentages of transmembrane TNF+ monocytes increased after incubation when using ADA or IFX but not ETA, revealing the potential of the two first molecules to trigger reverse signaling. Ab-dependent cell cytotoxicity was informed by CD16 and CD69 expressions in some donors that showed increasing levels of CD16- CD69+ NK cells when incubated with anti-TNFs. This study proposes a novel approach to assess anti-TNF mechanisms of action and provides a path toward capturing donor heterogeneity.

A Granulocytic Signature Identifies COVID-19 and Its Severity.

BACKGROUND: An unbiased approach to SARS-CoV-2-induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease. METHODS: An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis. RESULTS: Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance. CONCLUSIONS: Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker.

Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation.

BACKGROUND: Numerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption. OBJECTIVES: This work aimed at developing and characterizing a simplified and standardized whole-blood based BAT prototype procedure and at further assessing the feasibility of automating and miniaturizing the developed assay into a 96 well plate format. METHODS: A dry and room temperature stable reagent technology was used to simplify and standardize BAT. Under optimized conditions, EDTA anticoagulated whole blood samples of non-allergic and allergic donors (<24 h old) together with calcium containing buffer were added to ready-to-use dry reagent tubes or 96 well plates (negative controls, positive controls and allergen tests) containing a 5 color compensation-free antibody panel (CD45-KrO/CD3-PC7/CRTH2-A647/CD203c-PE/CD63-PB). Upon mixing and incubation at 37 °C for 15 min, erythrocytes were lysed and samples were analyzed by flow cytometry without further washing steps. While it is important to precisely control the incubation time to minimize the assay variability, herein, a 15 min incubation time was chosen as it provides a suitable compromise for both the magnitude of basophil activation and the quality of the staining. A Biomek NXP robotic platform (Beckman Coulter) was used for automation and both CD203c and CD63 levels were monitored to characterize basophil reactivity. RESULTS: This streamlined BAT protocol is no-wash, compensation free and only requires 4 pipetting steps to be completed. The assessment of assay performance characteristics showed wide applicability, satisfactory repeatability and a high degree of standardization as demonstrated by very low intra-assay and inter-operator variabilities (CVs < 10%). Leveraging these technical foundations, it was then proven that this new BAT procedure can easily be transposed into the 96 well plate format, thereby benefiting from a miniaturized format and full automation capabilities. When considering 8 dilution points to characterize the ex vivo basophil reactivity of a given whole blood sample, we found that as little as 5 µL of blood per point could be used. CONCLUSIONS: A whole blood based and simplified procedure for BAT is proposed. It relies on a dry antibody formulation technology and requires only a few manual steps to be completed. This procedure can also be transposed in a 96 well plate format, fully automated and miniaturized, when sample volume reduction, throughput increase or unattended sample preparation is required.

Peptidylarginine Deiminase Autoimmunity and the Development of Anti-Citrullinated Protein Antibody in Rheumatoid Arthritis: The Hapten-Carrier Model.

OBJECTIVE: The presence of autoantibodies to citrullinated proteins (ACPAs) often precedes the development of rheumatoid arthritis (RA). Citrullines are arginine residues that have been modified by peptidylarginine deiminases (PADs). PAD4 is the target of autoantibodies in RA. ACPAs could arise because PAD4 is recognized by T cells, which facilitate the production of autoantibodies to proteins bound by PAD4. We previously found evidence for this hapten-carrier model in mice. This study was undertaken to investigate whether there is evidence for this model in humans. METHODS: We analyzed antibody response to PAD4 and T cell proliferation in response to PAD4 in 41 RA patients and 36 controls. We tested binding of 65 PAD4 peptides to 5 HLA-DR alleles (DRB1*04:01, *04:02, *04:04, *01:01, and *07:01) and selected 11 PAD4 peptides for proliferation studies using samples from 22 RA patients and 27 controls. Peripheral blood lymphocytes from an additional 10 RA patients and 7 healthy controls were analyzed by flow cytometry for CD3, CD4, CD154, and tumor necrosis factor expression after PAD4 stimulation. RESULTS: Only patients with RA had both antibodies and T cell responses to PAD4. T cell response to peptide 8, a PAD4 peptide, was associated with RA (P = 0.02), anti-PAD4 antibodies (P = 0.057), and the shared epitope (P = 0.05). CONCLUSION: ACPA immunity is associated with antibodies to PAD4 and T cell responses to PAD4 and PAD4 peptides. These findings are consistent with a hapten-carrier model in which PAD4 is the carrier and citrullinated proteins are the haptens.

Pollen/Fruit Syndrome: Clinical Relevance of the Cypress Pollen Allergenic Gibberellin-Regulated Protein.

A pollen/food-associated syndrome (PFAS) has been described between peach and cypress pollen. Cross-reactive allergens were characterized which belong to the Gibberellin-regulated protein (GRP) family, BP14 in cypress pollen and Pru p 7 in peach. GRP are small cationic protein with anti-microbial properties. A patient suffering from a peach/cypress syndrome was explored clinically and biologically using 2 types of immunoglobulin E (IgE) multiarray microchip, immunoblots and a basophil activation test to assess the clinical relevance of various extracts and purified allergens from fruits or cypress pollen. In addition to PR10 sensitization, the patient showed specific IgE to Pru p 7, BP14 and allergen from pomegranate. These last 3 allergens and allergenic sources are able to induce ex vivo basophil activation characterized by the monitoring of the expression of CD63 and CD203c, both cell surface markers correlated with a basophil mediator release. Up to 100% of cells expressed CD203c at 50 ng/mL of BP14 protein. In contrast, snakin-1, a GRP from potato sharing 82% sequence identity with Pru p 7 did not activate patient's basophils. These results strongly suggest that, like Pru p 7, BP14 is a clinically relevant allergenic GRP from pollen. Allergen members of this newly described protein family are good candidates for PFAS where no cross-reactive allergens have been characterized.

Full antibody primary structure and microvariant characterization in a single injection using transient isotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry.

Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS.

Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes.

Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the ß ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. ß ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MDa.