BIOPRESERVATION AND BIOBANKING Volume 9, Number 4, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2011.0035

Standard PREanalytical Codes: A New Paradigm for Environmental Biobanking Sectors Explored in Algal Culture Collections Erica E. Benson,1,2 Fotini Betsou,2,3 Raquel Amaral,4 Lı´lia M.A. Santos,4 and Keith Harding1,2

The Standard PREanalytical Code (SPREC) was developed by the medical/clinical biobanking sector motivated by the need to harmonize biospecimen traceability in preanalytical processes and enable interconnectivity and interoperability between different biobanks, research consortia, and infrastructures. The clinical SPREC (01) consists of standard preanalytical variable options (7-code elements), which comprise published and (ideally) validated methodologies. Although the SPREC has been designed to facilitate clinical research, the concept could have utility in biorepositories and culture collections that service environmental and biodiversity communities. The SPREC paradigm can be applied to different storage regimes across all types of biorepository. The objective of this article is to investigate adapting the code in nonclinical biobanks using algal culture collections and their cryostorage as a case study. The SPREC (01) is recalibrated as a putative code that might be adopted for biobanks holding different types of biodiversity; it is extended to include optional coding from the point of sample collection to postcryostorage manipulations, with the caveat that the processes are undertaken by biorepository personnel.

Introduction

P

reanalytical processes1 take place between the point of biospecimen collection and their use in experimental analyses and research; they are attributed to the specimen process chain and by definition must be within the control of biobank or culture collection operations (ie, undertaken by biorepository personnel). Variations resulting from these procedures are not attributed to the intrinsic properties of samples and organisms, rather they occur as a consequence of the manipulations to which they are exposed during handling. The International Society for Biological and Environmental Repositories (ISBER) Working Group on Biospecimen Science developed the Standard PREanalytical Code (SPREC) for clinical specimens.1 The motivation being that the more precision afforded to recording a sample’s history, the more accurate and explicit will be the information that can be gained from biospecimen research, especially when it involves different collaborating institutions.1,2 Employing a SPREC facilitates traceability and enhances the current understanding of how preanalytical variables might affect sample utility, stability, and quality. Thus, cognizance of their attributes is important, because they (a) can potentially affect sample quality, (b) contribute to experimental

variation that may be difficult to attribute and identify, (c) facilitate collaborative multiple-partner projects, and (d) can help to meet the increasingly stringent requirements of clients using sensitive molecular and biochemical analyses. Preanalytical coding has particular benefits for large-scale consortia projects and research infrastructures, which require uniformity of sample process history as would be required for protocol validation, undertaking bilateral experiments across consortia,3–5 and establishing duplicate collections for security purposes. The objective of this article is to examine the wider potential of the code by adapting the clinical SPREC (01) for environmental/biodiversity sectors. Algal cryobanks have been chosen as the pilot study, because the range of manipulations involved in the process chain can be extended to preacquisition sampling (eg, from the environment and during transit) and poststorage recovery. Of particular relevance is recording the process chain history of algae and other types of biological resource/specimen retrieved from remote and inaccessible field sites and for which accurate recording of handling procedures and transit times are pertinent to downstream performance and research.6–8 The SPREC is also relevant to the supply of both nonaxenic and axenic strains for taxonomic, molecular, -omics, diagnostic,

1

Damar Research Scientists, Cuparmuir, Fife, Scotland, United Kingdom. ISBER Biospecimen Science Working (sub)-Group. Integrated Biobank of Luxembourg, Luxembourg. 4 Coimbra Collection of Algae (ACOI), Department of Life Sciences, University of Coimbra, Portugal. 2 3

399

400 biotechnological, and environmental research purposes. The significance of the axenicity of organisms cultured from environmental samples such as microalgal strains has been largely underrated, although the condition has been recently considered regarding its criticality for culture development9 and viability assessments following cryopreservation10; for these reasons, axenic state was incorporated as a SPREC element.

Materials and Methods Methods comprise (1) sample collection, transit, and culture initiation; (2) cryopreservation; and (3) SPREC codes for process chain steps. Options are incorporated to test code versatility, and to keep the SPREC practicable, it is only applied to primary cultures and not complex biotechnological procedures, altered cells, derivative samples, or biomolecular extracts. Limiting the scope of the code to the most common steps encourages its usability across different types of biorepository; however, to test its potential for developing improved practices for recalcitrant strains, greater detail is included for some elements. Variables and codes are constructed for 2 putative, 7-element-long ‘‘Algal’’ SPRECs, calibrated as SPREC (A-01) for procedures that include sample collection, processing, and culture initiation and as SPREC (A-02) for cryostorage and recovery. Practical elements are confined to the technical procedures undertaken by and within the control of culture collection personnel. Environmental parameters (eg, water, pH, temperature, conductivity, global positioning system (GPS) coordinates, site geography, habitat-niche type, prevailing conditions at time of collection) do not come within the operability of the SPREC convention. It is expected that the code will be intercalated with this crucial information as well as essential ‘‘biospecimen passport data’’ (date of collection, name of collector, provenance, taxonomic/species names) in the collection’s overarching data management system.

Sample type, collection, processing, and culture SPREC (A-01) is constructed based on procedures developed by the Algoteca de Coimbra (ACOI), University of Coimbra, Portugal (http://acoi.ci.uc.pt/), and collection methods used for remote access samples.7,8 Element 1 describes the sample type, using a term that describes a defined growth habit associated with a specific ecological zone or condition (eg, benthic, symbiont) or by environment or habitat category (eg, urban, intertidal). Element 1 directly influences handling and processing choices. Element 2 concerns the collection method and Element 3 concerns the collecting container. Element 4 describes the conditions of transit from the field site and the operational procedures used to stabilize samples during the period they are transferred from the field to the primary ‘‘holding’’ laboratory or facility for remote collections or to the biobank in the case of local sampling. Element 5 reports sample transit times, ranging from near-site collections (1–2 days) to remote expeditions7,8 (eg, to the polar regions). These can often necessitate lengthy, intermediate holding periods in field stations and transportation over several months before the samples are finally received by the biorepository. This element concurs with the date of sampling, recorded in the collection’s main data management system. Element 6 concerns isolation and culture initiation and Element 7 describes cultivation procedures for which axenic and nonaxenic practices are

BENSON ET AL. optional.10 In summary, the following final code is revealed: sample-collecting method-container-transit-transit time-isolationcultivation.

Cryopreservation Although this article specifically concerns cryostorage, the code can be modified for any type of storage regime. SPREC (A-02) is based on previously tested and validated cryopreservation methods11–16 and protocols adapted for storage-recalcitrant algae17–20; a code is applied for nonaxenic algae.10 As strain purity can critically influence culture utility and choice of cryopreservation protocol, Element 1 of SPREC (A-02) confirms culture type as axenic or nonaxenic.10 Element 2 describes pretreatments applied before cryoprotection; these facilitate handling and improve performance following cryostorage. Options include preculture in standard liquid or solid media or nutritionally supplemented media; culture under standard or low light; breaking and dispersing filamentous and thalloid organisms by cutting into small pieces; homogenization in a blender; applying ultrasound; or using centrifugation to collect cells in liquid cultures before cryoprotection. Because these procedures can evoke physiological responses caused by changes in culture conditions and the stresses incurred during mechanical injury and centrifugation, this element contains specific details of acclimatization and recovery periods.11 Element 3 is assigned to cryoprotection for which the most common variables include treatment with DMSO, methanol, or glycerol11 and vitrification strategies based on sodium alginate encapsulation, which is combined with osmotic dehydration and evaporative desiccation12,16; a further option is colligative cryoprotection combined with alginate encapsulation.12 Temperature and duration of exposure to cryoprotectants are included as variables. Element 4 comprises the cooling strategy designated as either ultrarapid cooling [direct plunge into liquid nitrogen (LN)] or controlled rate cooling using either a programmable freezer or a passive solvent ‘‘Mr. Frosty’’ cooling device. Options for starting temperature, rates of cooling, seeding (ice nucleation), terminal transfer temperatures, holds, and transfers to the cryotank are included. Element 5 describes the conditions of cryostorage, including the type of cryovial or container (volume and make of cryovial), phase of LN storage (vapor or liquid), and top-up method (automatic, manual). Element 6 concerns the rewarming regime for which variables include passive rewarming at ambient temperatures, controlled rate rewarming in a water bath, and 2-step rewarming. Element 7 describes recovery regimes in different media, dissolution of alginate beads and transfer to special intermediary conditions (eg, low light) to reduce stresses incurred by cryoinjury. Each element of the ‘‘storage code’’ SPREC (A-02) can potentially include equivalence options for different makes and models of equipment (eg, programmable freezers) and consumables (eg, type of cryovial). In summary, the following final code is revealed: axenicity-pretreatments-cryoprotectioncooling-cryostorage-rewarming-recovery.

Standard PREanalytical Code The scope of the SPREC is delineated into 2 representative types of algae: (a) filamentous, thalloid, or matt-forming strains and (b) single cell or microclump/colonial liquid

ALGAL SPREC PARADIGM suspension strains or cultures suspended from agar slopes. Two process chains have been coded as distinct components providing the potential for conjugating a 2-part SPREC, each comprising a 7-code element based on the assumption that the first part is within the control of the culture collection. The first code describes the variables involved in sample collection, transit, and culture initiation and the second pertains to cryopreservation. Each algal sample is assigned a 7-element-long code corresponding to 7 preanalytical variables within each code; this forms a ‘‘string’’ of hyphenated letters. The first elements correspond to sample type and subsequent elements to processes, treatments, and protocols, each of which is allocated an element code. If a preanalytical option is unknown or inconsistent, an ‘‘X’’ is incorporated to denote ‘‘unknown’’; if the option is known but does not correspond to any of the standard options the letter ‘‘Z’’ is used to denote ‘‘other’’ in which case a note may be annotated to ‘‘fix’’ the detailed information to the strains as an explanatory adjunct to the SPREC code.

Results and Discussion In support of ISBER’s environmental remit, this study has explored the wider potential for adapting and adopting SPREC (01) in nonhealthcare settings. Algal culture collections and cryobanks were chosen as the case study, because they represent diverse biorepository models with holdings of organisms that service different types of end users.4,20–23 Similar calibrations of SPREC may be adapted and deployed to all types (eg, environmental bacteriology, mycology, marine, forestry) of environmental and biodiversity repositories23; the clinical SPREC (01) is pertinent to animal biobanks (eg, wildlife conservation and veterinary biological resource centers). The importance of preanalytical procedures for DNA studies has been recently highlighted.24 Stringent recording and tracking of preanalytical variables1 and the robust reporting of the biospecimen process chain to improve quality25 are becoming increasingly important for medical biobanks and it is probable that this will become the case for other types of biorepository, especially those involving largescale consortia. However, there are differences between clinical and nonclinical practices, particularly the points at which the preanalytical process chain starts and finishes. In clinical scenarios, sample collection and poststorage recovery can often involve specimens being sampled by medical practitioners, after which they are sent to biorepositories for storage and dispatched to end users in the frozen state. In contrast, the extent to which preanalytical processes reside within the sphere of influence of algal and other types of biodiversity and environmental biorepositories varies, extending the possible range of the SPREC (Table 1). To provide an example of how to create a nonclinical SPREC, 2 (01, 02) putative, 7-element-long SPRECs have been devised for algae, the first describing elements assigned to sample collection, initiation, and processing before storage, SPREC (A-01), and the second comprising elements for cryostorage and recovery, SPREC (A-02). Options are described for filamentous, thalloid, or matt-forming organisms and strains of simple forms such as motile free-living suspensions, microcolonies, and coccoids. Ideally, the code should be linked to all subsamples, derivatives (eg, DNA, RNA, protein extracts), and aliquots of the corresponding algal strain throughout its processing history; this potentially

401 allows immediate recognition, tracking, and assessment of each preanalytical step in the culture collection. In practice, the SPREC can be implemented as a simple ‘‘low-tech’’ handwritten record or a digitally formatted ‘‘supermarket inventory barcode’’ intercalated with preexisting sample data and supporting Quality Management Systems. It is envisaged the SPREC will ‘‘dovetail’’ with existing tracking/ traceability systems within a culture collection.

Creating and interpreting 7-element SPRECs Using SPREC (A-01) as the first example, highlighted in bold (Table 1) are element codes (1–7) corresponding to planktonic algae (B) collected using a plankton net (A) placed into a polyethylene bottle (C) after which the samples are transported in a chilled condition (B) within 1–2 days (A) of samples being taken. Microalgae strains are isolated in drops of liquid medium (DAL) and cultured nonaxenically in cotton-capped Erlenmeyer flasks in ACOI liquid medium (NXB). This code corresponds to 7-element SPREC (A-01) BA-C-B-A-DAL-NXB. Using SPREC (A-02) as the second example, highlighted in bold (Table 1) are element codes (1–7) corresponding to the cryopreservation protocol for a nonaxenic alga (NOX), which has been precultured under standard conditions in liquid culture for 2 weeks (A), cryoprotected in 5% (w/v) methanol on ice at 0C for 30 min (MC), and cryopreserved using passive cooling at - 1C/min in a Nalgene Mr. Frosty to a temperature of - 80C, after which the samples were transferred to LN (MFA) and stored in 2.0 mL NUNC CryoTubes in liquid phase LN, which is replenished by an automatic topup system (CSC). Samples are rewarmed rapidly in a water bath at 40C until all the ice has visibly melted (RWB) and the algae recovered in standard medium under standard light conditions (REA). This code corresponds to 7-element SPREC (A-02) NOX-A-MC-MFA-CSC-RWB-REA. The 7-element codes can be applied individually for algae collected and maintained in the active growth state (SPREC A01) or for algae held in cryostores, alternatively both SPRECs (A-01) and (A-02) may be applied as 2 contiguous codes. This adds value by making explicit those factors that could potentially alter the physiological state of samples from the point of collection and during transit. The need to stabilize viable specimens at these critical stages is highlighted in SPREC (A-01), because they impinge upon sample viability and health; further, suboptimal handling can influence culture initiation and storage outcomes. This is particularly the case for algae collected from remote and inaccessible regions, which require out-of-the-ordinary sampling logistics and lengthy transit times.7,8 In the case of specimens procured on a 1-timeonly basis,26 the robust and stringent recording of process history is critical, particularly if samples are precious and rare and from endangered species or comprise unique environmental specimens (eg, collected at a specific time or following an environmental incident). In these cases, implementing codes for variables will require diligence as to which elements are included and the level of their specificity.

Potential utility in algal culture collections and wider versatility The clinical SPREC (01) is a simple, 7-element-long code, formulated using existing laboratory management tools and

402 Table 1.

BENSON ET AL. Preanalytical Variables and Codes Comprising 2 Putative, 7-Element-Long SPRECs Calibrated as SPREC-A-01 for Sample Collection, Processing, and Standard Culture and as SPREC-A-02 for Cryopreservation

SPREC element (1–7)

Element descriptor (Notes)

SPREC (A-01) 1

Sample type

2

Collecting method

3

Collecting container

4

In transit conditions and stabilization

Options can include intermediate holding in base camps and field site facilities.

Transit time Options can include intermediate holding in base camps and field site facilities.

6

Code

Sample type, collection, processing, and culture

Describes the growth habit associated with a specific ecological zone or condition or environment from which the organism is sampled.

5

Element variable options

Isolation and culture initiation

Aerophytic Plankton Periphyton Benthic Epilithic Endolithic Epiphytic Phycobiont Assemblage Snow Hot springs Intertidal Urban building Unknown Other Plankton net Squeezing Scraping Grasping (collecting by hand) Unknown Other Glass bottle Polyethylene bag Polyethylene bottle Unknown Other Ambient Chilled (on ice, refrigerated 0C to 4C) Frozen ( - 18C to - 20C) Hydrated, ambient Hydrated, chilled (on ice, refrigerated 0C to 4C) Hydrated, frozen ( - 18C to - 20C) Ambient + antimicrobial treatment Chilled + antimicrobial treatment Frozen + antimicrobial treatment Hydrated, ambient + antimicrobial treatment Hydrated, chilled + antimicrobial treatment Hydrated, frozen + antimicrobial treatment Unknown Other 1–2 days 3–5 days 1 week 2–3 weeks 1 month 1–2 months 2–3 months 3–4 months 4–5 months ‡ 6 months Unknown Other (a) Drops: inoculating alga into a drop of liquid medium, by capillary pipette drop forms isolated clonal colony

A B C D E F G H I J K L M X Z A B C D X Z A B C X Z A B C D E F G H I J K L X Z A B C D E F G H I J X Z

(continued)

ALGAL SPREC PARADIGM

403 Table 1. (Continued)

SPREC element (1–7)

Element descriptor (Notes)

SPREC (A-01)

Code

Sample type, collection, processing, and culture This is the option for most algae, particularly free-swimming forms.

Visual selection aided by a microscope and capillary pipettes.

7

Element variable options

Cultivation procedure

Dependent on different culture collection practices can also be coded as equivalent to different options.

SPREC element culture detail will be determined by criticality of factors involved in culture/cryo-recalcitrance and stringency of end user requirements for strain purity.

Transfer to new agar slopes with wire loop (option for soil, substrate algae) Vortex liquid medium, transfer to fresh liquid culture (b) Combs: streak drop on an agar plate using sterile loop; cell forms a clonal spot at tip of ‘‘comb’’ Transfer colonies to new agar slopes with wire loop (option for soil, substrate algae) Vortex liquid medium, transfer to fresh liquid culture (option for freeswimming algae) Combination of both, comb, followed by drops Unknown Other Axenic: antibiotic treatment, microalgae grown in cotton-capped glass tubes (appropriate medium) at 18C–20C, 12:12-h light:dark photoperiod (30–40 mmol m - 2 s - 1) Axenic: Dakin solution disinfection treatment, microalgae grown in cottoncapped glass tubes, (appropriate ACOI medium) at 18C–20C, 12:12-h light:dark photoperiod (30–40 mmol m - 2 s - 1) Axenic: antibiotic treatment, microalgae grown in cotton-capped Erlenmeyer flasks tubes, (appropriate culture medium) at 18C–20C, 12:12-h light:dark photoperiod (30–40 mmol m - 2 s - 1) Nonaxenic: microalgae grown in cottoncapped glass tubes, (appropriate culture medium) at 18C–20C, 12:12-h light:dark photoperiod (30–40 mmol m - 2 s - 1) Nonaxenic: microalgae grown in cottoncapped Erlenmeyer flasks (ACOI LC M7 liquid medium) at 18C–20C, 12:12-h light:dark photoperiod (30–40 lmol m22 s21) Unknown Other

DAS DAL CAS CAL DCS DCL X Z AXA

AXB

AXC

NXA

NXB

X Z

SPREC Cryopreservation (A-02) 1

Culture type

2

Pregrowth and pretreatment Standard conditions refer to usual serial subculture maintenance regime.

Axenic Nonaxenic (a) Simple form strains (coccoids, unicells, microcolonies) Preculture under standard conditions in liquid medium for 2 weeks Preculture under standard conditions in liquid medium for 2 weeks (18C, light 60 mmol m - 2 s - 1, 12:12-h light:dark photoperiod)

AXE NOX A B

(continued)

404

BENSON ET AL. Table 1. (Continued)

SPREC element (1–7)

Element descriptor (Notes)

SPREC (A-01)

Code

Sample type, collection, processing, and culture Treatments applied prior to cryopreservation to facilitate cryoprotection and improve recovery.

Culture phase usually late log-stationary.

Final cell densities *106 to 107 per mL. For centrifugation-sensitive strains, CPAs are added directly to cryovials.

For some recalcitrant stains, recovery and/or pretreatment at a low osmotic level before cryoprotection is required.

3

Element variable options

Cryoprotection Options can include equivalent cryoprotection regimes.

Cryoprotectant preparation clarified on volumetric (v/v) or gravimetric basis (w/v) where specific gravity adjustments are used for liquid additives.

Preculture under standard conditions in liquid medium for 2 weeks centrifugation (1000 rpm, 1–2 min) to harvest cells Preculture under standard conditions (20C, light 50 mmol m - 2 s - 1, 14:10-h light:dark photoperiod) in liquid medium for 5 days; centrifugation (500 g, 1–2 min) to harvest cells Preculture under standard conditions (20C, light, 20–25 mmol m - 2 s - 1, 12:12h light:dark photoperiod) in liquid medium + 1% (w/v) proteose peptone for 5 days; centrifugation (500 g, 1– 2 min) to harvest cells Preculture under standard conditions (15C–20C, light, 50 mmol m - 2 s - 1, 12:12-h light:dark photoperiod) in liquid medium for 5 days; centrifugation (1000 g, *5 min) to harvest cells Preculture for 2–3 weeks under standard conditions on agar slopes dispensed in cryovials Preculture under optimized acclimating conditions. Cultured in medium supplemented with vitamin B12 (5 · 10 - 6 g/L) for 2 weeks (18C, light 30 mmol m - 2 s - 1, 12:12-h light:dark photoperiod) followed by 5 days acclimation (18C, light 60 mmol m - 2 s - 1, 12:12-h light:dark photoperiod) without orbital shaking, homogenization with ultrasound every 3 days to disrupt cell aggregation (b) Thalloid, matt, filamentous strains cut into pieces + 48 h recovery under standard culture conditions Preculture under standard conditions in the presence of a low concentration of sucrose (osmotic pretreatment) Unknown Other Methanol 5% (w/v), 10 min ice (0C) Methanol 5% (w/v), 15 min ice (0C) Methanol 5% (w/v), 30 min ice (0C) Methanol 10% (w/v), 10 min ice (0C) Methanol 10% (w/v), 15 min ice (0C) Methanol 10% (w/v), 30 min ice (0C) Methanol 5% (w/v), 10 min ambient (22C–25C) Methanol 5% (w/v), 15 min ambient (22C–25C) Methanol 5% (w/v), 30 min ambient (22C–25C) Methanol 10% (w/v), 10 min ambient (22C–25C) Methanol 10% (w/v), 15 min ambient (22C–25C) Methanol 10% (w/v), 30 min ambient (22C–25C) Other

C

D

E

F

G H

I J X Z MA MB MC MD ME MF MG MH MI MJ MK ML MZ

(continued)

ALGAL SPREC PARADIGM

405 Table 1. (Continued)

SPREC element (1–7) SPREC (A-01)

Element descriptor (Notes)

Element variable options

Code

Sample type, collection, processing, and culture

Double-strength CPA solutions may be added as a 1:1 dilution to the equivalent culture volume to achieve desired final concentration.

Alginate SIGMA A-2158 2% viscosity Na-salt with or without 0.5 M sucrose.

Osmotic dehydration for 24–48 h (2-step osmotic treatment).

DMSO 5% (w/v), 10 min ice (0C) DMSO 5% (w/v), 15 min ice (0C) DMSO 5% (w/v), 30 min ice (0C) DMSO 10% (w/v), 10 min ice (0C) DMSO 10% (w/v), 15 min ice (0C) DMSO 10% (w/v), 30 min ice (0C) DMSO 5% (w/v), 10 min ambient (22C–25C) DMSO 5% (w/v), 15 min ambient (22C–25C) DMSO 5% (w/v), 30 min ambient (22C–25C) DMSO 10% (w/v), 10 min ambient (22C–25C) DMSO 10% (w/v), 15 min ambient (22C–25C) DMSO 10% (w/v), 30 min ambient (22C–25C) Other Glycerol 5% (w/v), 10 min ice (0C) Glycerol 5% (w/v), 15 min ice (0C) Glycerol 5% (w/v), 30 min ice (0C) Glycerol 10% (w/v), 10 min ice (0C) Glycerol 10% (w/v), 15 min ice (0C) Glycerol 10% (w/v), 30 min ice (0C) Glycerol 5% (w/v), 10 min ambient (22C–25C) Glycerol 5% (w/v), 15 min ambient (22C–25C) Glycerol 5% (w/v), 30 min ambient (22C–25C) Glycerol 10% (w/v), 10 min ambient (22C–25C) Glycerol 10% (w/v), 15 min ambient (22C–25C) Glycerol 10% (w/v), 30 min ambient (22C–25C) Other Alginate 3% (w/v) encapsulated polymerization 30 min + 0.5 M sucrose 24 h osmotic dehydration + 6–8 h silica gel desiccation (over 15 g activated silica gel; standard culture conditions) Alginate 3% (w/v) encapsulated polymerization 30 min + 0.5 M sorbitol 24 h osmotic dehydration + 6–8 h silica gel desiccation (over 15 g activated silica gel; standard culture conditions) Alginate 5% (w/v) encapsulated with + 0.5 M sucrose polymerization 30– 60 min + 0.5 M sucrose 24 h + 0.75 M sucrose 24 h osmotic dehydration + 3 h air desiccation (standard culture conditions) Alginate 5% (w/v) encapsulated with + 0.5 M sucrose polymerization 30– 60 min + 0.5 M sucrose 24 h + 0.75 M sucrose 24 h osmotic dehydration + 4 h air desiccation (standard culture conditions)

DA DB DC DD DE DF DG DH DI DJ DK DL DZ GA GB GC GD GE GF GG GH GI GJ GK GL GZ ENA

ENB

ENC

END

(continued)

406

BENSON ET AL. Table 1. (Continued)

SPREC element (1–7)

Element descriptor (Notes)

SPREC (A-01)

Code

Sample type, collection, processing, and culture Evaporative air-silica gel desiccation to 25%– 30% water (fresh weight) *0.4 g H2O/g fwt.

4

Element variable options

Cooling

Options can include equivalent cooling regimes.

Temperature at which cooling starts usually determined by CPA element temperature (ambient 20C–25C or 0C ice).

Alginate 5% (w/v) encapsulated with + 0.5 M sucrose polymerization 30– 60 min + 0.5 M sucrose 24 h + 0.75 M sucrose 24 h osmotic dehydration + 5 h air desiccation (standard culture conditions) Alginate 5% (w/v) encapsulated with + 0.5 M sucrose polymerization 30– 60 min + 0.5 M sucrose 24 h + 0.75 M sucrose 24 h osmotic dehydration + 5-8 h silica gel desiccation (over 10–15 g activated silica gel) (standard culture conditions) Alginate 5% (w/v) encapsulated with + 0.5 M sucrose polymerization 30– 60 min + 0.5 M sucrose 24 h + 0.75 M sucrose 24 h osmotic dehydration + 4 h air desiccation (standard culture conditions) + colligative cryoprotection with 10% (v/v) methanol, 15 min at 20C Unknown Other Ultra rapid freezing (URF-direct plunge in LN) Controlled rate cooling (CR) Planer Kryo 10 Programmable Freezer - 0.5C/min to - 40C, hold 8 min, transfer to LN - 0.5C/min to - 60C, hold 10 min, transfer to LN - 1.0C/min to - 35C, hold 10 min, transfer to LN - 1.0C/min to - 40C, hold 10 min transfer to LN - 1.0C/min to - 45C, hold 10 min transfer to LN - 1.0C/min to - 60C, hold 10 min transfer to LN - 1.0C/min to - 35C, hold 10 min transfer to LN - 1.0C/min to - 40C, hold 10 min transfer to LN - 1.0C/min to - 45C, hold 10 min transfer to LN - 1.0C/min to - 60C, hold 10 min transfer to LN - 0.5C/min to - 60C, hold 30 min, transfer to LN - 1.0C/min to - 35C, hold 30 min, transfer to LN - 1.0C/min to - 40C, hold 30 min transfer to LN - 1.0C/min to - 45C, hold 30 min transfer to LN - 1.0C/min to - 60C, hold 30 min transfer to LN - 1.0C/min to - 35C, hold 30 min transfer to LN - 1.0C/min to - 40C, hold 30 min transfer to LN

ENE

ENF

ENG

ENX ENZ URF

CRA CRB CRC CRD CRE CRF CRG CRH CRI CRJ CRK CRL CRM CRN CRO CRP CRQ

(continued)

ALGAL SPREC PARADIGM

407 Table 1. (Continued)

SPREC element (1–7)

Element descriptor (Notes)

SPREC (A-01)

Auto-seeding dependent upon cooling device and CPA.

Or equivalent device. Passive cooling device solvent (250 mL isopropanol).

6

Code

Sample type, collection, processing, and culture

Or equivalent instrument.

5

Element variable options

Cryostorage (CS) *1.0 mL CPA + algae dispensed per vial or 10–20 alginate beads

Rewarming Ambient *22C–25C

- 1.0C/min to - 45C, hold 30 min transfer to LN - 1.0C/min to - 60C, hold 30 min transfer to LN Other Planer Kryo 10 Programmable Freezer 2-rate programs - 1.0C/min to 0C, - 0.5C to - 40C, hold 15 min transfer to LN - 1.0C/min to 0C, - 0.5C to - 60C, hold 15 min transfer to LN Planer Kryo 10 Programmable Freezer + induced ice nucleation ‘‘autoseeding’’ - 1.0C/min to - 9C, auto-programmed seeding at - 12C, hold 15 min, resume - 1.0C/min to - 40C, hold 30 min transfer to LN Other Nalgene Mr. Frosty (MF) - 1C/min to - 80C, hold 30 min, transfer to LN - 1C/min to - 70C, hold 30 min, transfer to LN - 1C/min to - 80C, hold 60 min, transfer to LN - 1C/min to - 70C, hold 60 min, transfer to LN - 1C/min to - 80C, hold 90 min, transfer to LN - 1C/min to - 70C, hold 90 min, transfer to LN Unknown Other NUNC 2.0 mL CryoTube-Vapour Phasemanual top-up NUNC 2.0 mL CryoTube-Vapour Phaseman top-up NUNC 2.0 mL CryoTube-Liquid Phaseauto top-up NUNC 2.0 mL CryoTube-Liquid Phasemanual top-up Unknown Other Passive ambient until ice visibly melted Rapid, 40C water bath until ice visibly melted Rapid, 45C water bath until ice visibly melted Two-step, 1 min ambient, 3 min 45C water bath Alginate-encapsulated 2-step, 1 min ambient, 2 min 40C water bath Alginate-encapsulated 2–3 min 40C water bath + 1 h rehydration in liquid culture medium Alginate-encapsulated 30 min passive ambient + 1 h rehydration in liquid culture medium Unknown Other

CRR CRS Z CRT CRU

CRV

Z MF MFA MFB MFC MFD MFE MFF X Z CSA CSB CSC CSD X Z RWA RWB RWC RWD RWE REF RWG RX RZ

(continued)

408

BENSON ET AL. Table 1. (Continued)

SPREC element (1–7)

Element descriptor (Notes)

SPREC (A-01) 7

Element variable options

Code

Sample type, collection, processing, and culture Recovery Dependent on culture collection facility practices.

SPREC element detail determined by criticality of factors involved in cryorecalcitrance.

Standard medium, culture in standard light Standard medium, culture in standard light + 10 mg/L desferrioxamine for 24 to 48 h Standard medium, culture in reduced light ( < 30–40 mmol m - 2 s - 1) + 10 mg/L desferrioxamine for 24 to 48 h Standard medium, culture in reduced light 1 day before transfer to light Standard medium, culture in reduced light 2 days before transfer to light Standard medium, culture in reduced light 3 days before transfer to light Standard medium, culture in reduced light 4 days before transfer to light Standard medium, culture in dark 1 day before transfer to light Standard medium, culture in dark 2 days before transfer to light Standard medium, culture in dark 3 days before transfer to light Standard medium, culture in dark 4 days before transfer to light Alginate-encapsulated algae released by dissolution with 3% (w/v) sodium hexametaphosphate + 1 day culture shaded by overlaid filter Unknown Other

REA REB REC RED REE REF REG REH REI REJ REK REL

RWX RWZ

Elements and their derivatives have been constructed using protocols adapted and collated from the works of Day and Harding,11 Harding et al.,12,15,16 Lukesova et al.,14 Elster et al.,13 Amaral et al.,17 Fleck et al.,18,19 and Oso´rio et al.22 SPREC Codes 7-element SPREC (A-01) B-A-CB-A-DAL-NXB and 7-element SPREC (A-02) NOX-A-MC-MFA-CSC-RWB-REA are highlighted in bold. Where elements have the same starting code letter (eg, CRA cooling or CSA cryostorage) it is important to take care in deciphering the code when an alphanumeric format is used. This version is optionally designed for pretreatment before cryopreservation of (a) simple form strains (coccoids, unicells, microcolonies) and (b) thalloid, matt-forming, and filamentous algae. An inconsistent or unknown is coded X; a known option that does not correspond to a standard procedure is coded Z. Elements in bold highlight those that have been selected to demonstrate how a code is created. SPREC, Standard PREanalytical Code; DMSO, dimethyl sulfoxide; CPA, CryoProtectant additives.

technical protocols (sample preparation, centrifugation, cryoprotection, freezing, and storage regime). A similar approach has been used to test the feasibility of adapting the SPREC for biorepositories servicing environmental and biodiversity sectors, the range of which is qualified by the criteria that all elements are under biorepository control and that the code is intuitively easy to use. It is crucial that biorepository field personnel employ consistent sample collection and recording procedures and their awareness of the variables comprising SPREC (A-01) will assist adherence to standard operating procedures.26 Critical elements of protocols tracked by SPRECs will thus limit the impacts of variables that could potentially affect viability, culture, and storage outcomes. The SPREC can be implemented as a simple ‘‘low-tech’’ handwritten record; for collections that do not have a fully configured database or inventory system, code data may be collated using a simple questionnaire or electronic tick box. For larger-scale facilities, the SPREC can be formatted as a ‘‘supermarket inventory barcode.’’ To facilitate dissemina-

tion and utility, the SPREC can be included in biorepositoryspecific software such that each nonconforming modification can be flagged for follow-up.1 The code aim is that the code may be cited in scientific publications and reporting recommendations as part of the author’s guidelines and quality assurance processes. The code can enable research networking, as a robust processing history adds value to the outputs of collaborating partners and their end users. The clinical SPREC was designed to enable multipartner validation, quality control, and large-scale consortia research projects (eg, barcode of life studies, genomics, and proteomics); these applications may be relevant to all types of nonclinical biobanks, particularly those involved in environmental research.6,23 At the operational level, the SPREC could become a useful addition to culture collection quality control systems as it can help track samples, facilitate identification and authentication, and offset the risks of incorrect handling and labeling. This is particularly relevant for samples that have long and complex process chains as can often be the case for biospecimens used by environmental science and

ALGAL SPREC PARADIGM biodiversity conservation constituencies. Robust codes are especially useful for tracing samples held in very long-term cryostores or in duplicated, archived, historical, and legacy collections. Preanalytical coding can potentially support research collaboration and connectivity between different types of culture collections and their communities of practice,4,5,13–15 especially when they are required to use common strains, processes, and cryostorage systems. Comparisons of success and failure at each critical element of the SPREC can also be used in risk management and mitigation.20 The SPREC concept is in line with the standards employed in environmental specimen biobanking programs.23

Conclusions Putative preanalytical codes have been devised for algal biorepositories focusing on sample collection and transit, culture initiation, and cryostorage elements. The value of the SPREC has been noted for recalcitrant organisms on the basis that tracking variable parameters combined with knowledge of an element’s criticality could be used to identify parts of the process chain that enable the stabilization of sensitive strains. Although the benefits of implementing the SPREC with respect to quality management and sample traceability have been considered, they will need to be balanced against the risks of producing overcomplicated processing tools, which limit take up. The algal preanalytical codes have the potential to be recalibrated for the sample-processing chains and storage regimes of other types of environmental and biodiversity repositories.

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Acknowledgments The authors thank Dr. Josef Elster for kindly providing information concerning polar algae; they gratefully acknowledge colleagues in ISBER’s Biospecimen Science Work Group for which the 2010–2011 members are Fay Betsou (Chair), Garry Ashton, Michael Barnes, Erica E. Benson, Rodrigo Chuaqui, Judith Clements, Domenico Coppola, Yvonne DeSouza, James Eliason, Barbara Glazer, Fiorella Guadagni, Elaine Gunter, Keith Harding, Jae-Pil Jeon, Olga Kofanova, Sylvain Lehmann, Conny Mathay, Rolf Muller, Francesca Poloni, Kathi Shea, Amy Skubitz, Stella Somiari, and Gunnel Tybring.

Author Disclosure Statement

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No competing financial interests exist.

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BENSON ET AL. Address correspondence to: Dr. Erica Benson Damar Research Scientists Damar Drum Road Cuparmuir Cupar Fife KY15 5RJ Scotland United Kingdom E-mail: [email protected] Received 13 July, 2011/Accepted 6 August, 2011