Standard PREanalytical Codes: A New Paradigm for Environmental Biobanking Sectors Explored in Algal Culture Collections

Abstract

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

Preanalytical 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, 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 expeditions^7,8 (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 optional.¹⁰ In summary, the following final code is revealed: sample-collecting method-container-transit-transit time-isolation-cultivation.

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 methods^11–16 and protocols adapted for storage-recalcitrant algae^17–20; 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 desiccation^12,16; 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-cryoprotection-cooling-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 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 large-scale 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).

Table 1.

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)	Element variable options	Code
SPREC (A-01)	Sample type, collection, processing, and culture
1	Sample type	Aerophytic	A
		Plankton	B
	Describes the growth habit associated with a specific ecological zone or condition or environment from which the organism is sampled.	Periphyton	C
		Benthic	D
		Epilithic	E
		Endolithic	F
		Epiphytic	G
		Phycobiont	H
		Assemblage	I
		Snow	J
		Hot springs	K
		Intertidal	L
		Urban building	M
		Unknown	X
		Other	Z
2	Collecting method	Plankton net	A
		Squeezing	B
		Scraping	C
		Grasping (collecting by hand)	D
		Unknown	X
		Other	Z
3	Collecting container	Glass bottle	A
		Polyethylene bag	B
		Polyethylene bottle	C
		Unknown	X
		Other	Z
4	In transit conditions and stabilization	Ambient	A
		Chilled (on ice, refrigerated 0°C to 4°C)	B
		Frozen (−18°C to −20°C)	C
		Hydrated, ambient	D
		Hydrated, chilled (on ice, refrigerated 0°C to 4°C)	E
	Options can include intermediate holding in base camps and field site facilities.	Hydrated, frozen (−18°C to −20°C)	F
		Ambient+antimicrobial treatment	G
		Chilled+antimicrobial treatment	H
		Frozen+antimicrobial treatment	I
		Hydrated, ambient+antimicrobial treatment	J
		Hydrated, chilled+antimicrobial treatment	K
		Hydrated, frozen+antimicrobial treatment	L
		Unknown	X
		Other	Z
5	Transit time	1–2 days	A
		3–5 days	B
		1 week	C
	Options can include intermediate holding in base camps and field site facilities.	2–3 weeks	D
		1 month	E
		1–2 months	F
		2–3 months	G
		3–4 months	H
		4–5 months	I
		≥6 months	J
		Unknown	X
		Other	Z
6	Isolation and culture initiation	(a) Drops: inoculating alga into a drop of liquid medium, by capillary pipette drop forms isolated clonal colony
	This is the option for most algae, particularly free-swimming forms.	Transfer to new agar slopes with wire loop (option for soil, substrate algae)	DAS
		Vortex liquid medium, transfer to fresh liquid culture	DAL
		(b) Combs: streak drop on an agar plate using sterile loop; cell forms a clonal spot at tip of “comb”	CAS
	Visual selection aided by a microscope and capillary pipettes.	Transfer colonies to new agar slopes with wire loop (option for soil, substrate algae)	CAL
		Vortex liquid medium, transfer to fresh liquid culture (option for free-swimming algae)	DCS
		Combination of both, comb, followed by drops	DCL
		Unknown	X
		Other	Z
7	Cultivation procedure	Axenic: antibiotic treatment, microalgae grown in cotton-capped glass tubes (appropriate medium) at 18°C–20°C, 12:12-h light:dark photoperiod (30–40 μmol m⁻² s⁻¹)	AXA
	Dependent on different culture collection practices can also be coded as equivalent to different options.	Axenic: Dakin solution disinfection treatment, microalgae grown in cotton-capped glass tubes, (appropriate ACOI medium) at 18°C–20°C, 12:12-h light:dark photoperiod (30–40 μmol m⁻² s⁻¹)	AXB
	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.	Axenic: antibiotic treatment, microalgae grown in cotton-capped Erlenmeyer flasks tubes, (appropriate culture medium) at 18°C–20°C, 12:12-h light:dark photoperiod (30–40 μmol m⁻² s⁻¹)	AXC
		Nonaxenic: microalgae grown in cotton-capped glass tubes, (appropriate culture medium) at 18°C–20°C, 12:12-h light:dark photoperiod (30–40 μmol m⁻² s⁻¹)	NXA
		Nonaxenic: microalgae grown in cotton-capped Erlenmeyer flasks (ACOI LC M7 liquid medium) at 18°C–20°C, 12:12-h light:dark photoperiod (30–40 μmol m⁻² s⁻¹)	NXB
		Unknown	X
		Other	Z
SPREC Cryopreservation (A-02)
1	Culture type	Axenic	AXE
		Nonaxenic	NOX
2	Pregrowth and pretreatment	(a) Simple form strains (coccoids, unicells, microcolonies)
	Standard conditions refer to usual serial subculture maintenance regime.	Preculture under standard conditions in liquid medium for 2 weeks	A
		Preculture under standard conditions in liquid medium for 2 weeks (18°C, light 60 μmol m⁻² s⁻¹, 12:12-h light:dark photoperiod)	B
	Treatments applied prior to cryopreservation to facilitate cryoprotection and improve recovery.	Preculture under standard conditions in liquid medium for 2 weeks centrifugation (1000 rpm, 1–2 min) to harvest cells	C
		Preculture under standard conditions (20°C, light 50 μmol 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	D
	Culture phase usually late log-stationary.	Preculture under standard conditions (20°C, light, 20–25 μmol m⁻² s⁻¹, 12:12-h light:dark photoperiod) in liquid medium+1% (w/v) proteose peptone for 5 days; centrifugation (500 g, 1–2 min) to harvest cells	E
	Final cell densities ∼10⁶ to 10⁷ per mL. For centrifugation-sensitive strains, CPAs are added directly to cryovials.	Preculture under standard conditions (15°C–20°C, light, 50 μmol m⁻² s⁻¹, 12:12-h light:dark photoperiod) in liquid medium for 5 days; centrifugation (1000 g, ∼5 min) to harvest cells	F
		Preculture for 2–3 weeks under standard conditions on agar slopes dispensed in cryovials	G
	For some recalcitrant stains, recovery and/or pretreatment at a low osmotic level before cryoprotection is required.	Preculture under optimized acclimating conditions. Cultured in medium supplemented with vitamin B12 (5×10⁻⁶ g/L) for 2 weeks (18°C, light 30 μmol m⁻² s⁻¹, 12:12-h light:dark photoperiod) followed by 5 days acclimation (18°C, light 60 μmol m⁻² s⁻¹, 12:12-h light:dark photoperiod) without orbital shaking, homogenization with ultrasound every 3 days to disrupt cell aggregation	H
		(b) Thalloid, matt, filamentous strains cut into pieces+48 h recovery under standard culture conditions	I
		Preculture under standard conditions in the presence of a low concentration of sucrose (osmotic pretreatment)	J
		Unknown	X
		Other	Z
3	Cryoprotection	Methanol 5% (w/v), 10 min ice (0°C)	MA
		Methanol 5% (w/v), 15 min ice (0°C)	MB
	Options can include equivalent cryoprotection regimes.	Methanol 5% (w/v), 30 min ice (0°C)	MC
		Methanol 10% (w/v), 10 min ice (0°C)	MD
		Methanol 10% (w/v), 15 min ice (0°C)	ME
		Methanol 10% (w/v), 30 min ice (0°C)	MF
		Methanol 5% (w/v), 10 min ambient (22°C–25°C)	MG
		Methanol 5% (w/v), 15 min ambient (22°C–25°C)	MH
		Methanol 5% (w/v), 30 min ambient (22°C–25°C)	MI
	Cryoprotectant preparation clarified on volumetric (v/v) or gravimetric basis (w/v) where specific gravity adjustments are used for liquid additives.	Methanol 10% (w/v), 10 min ambient (22°C–25°C)	MJ
		Methanol 10% (w/v), 15 min ambient (22°C–25°C)	MK
		Methanol 10% (w/v), 30 min ambient (22°C–25°C)	ML
		Other	MZ
		DMSO 5% (w/v), 10 min ice (0°C)	DA
		DMSO 5% (w/v), 15 min ice (0°C)	DB
		DMSO 5% (w/v), 30 min ice (0°C)	DC
		DMSO 10% (w/v), 10 min ice (0°C)	DD
		DMSO 10% (w/v), 15 min ice (0°C)	DE
		DMSO 10% (w/v), 30 min ice (0°C)	DF
		DMSO 5% (w/v), 10 min ambient (22°C–25°C)	DG
		DMSO 5% (w/v), 15 min ambient (22°C–25°C)	DH
		DMSO 5% (w/v), 30 min ambient (22°C–25°C)	DI
		DMSO 10% (w/v), 10 min ambient (22°C–25°C)	DJ
		DMSO 10% (w/v), 15 min ambient (22°C–25°C)	DK
		DMSO 10% (w/v), 30 min ambient (22°C–25°C)	DL
	Double-strength CPA solutions may be added as a 1:1 dilution to the equivalent culture volume to achieve desired final concentration.	Other	DZ
		Glycerol 5% (w/v), 10 min ice (0°C)	GA
		Glycerol 5% (w/v), 15 min ice (0°C)	GB
		Glycerol 5% (w/v), 30 min ice (0°C)	GC
		Glycerol 10% (w/v), 10 min ice (0°C)	GD
		Glycerol 10% (w/v), 15 min ice (0°C)	GE
		Glycerol 10% (w/v), 30 min ice (0°C)	GF
		Glycerol 5% (w/v), 10 min ambient (22°C–25°C)	GG
		Glycerol 5% (w/v), 15 min ambient (22°C–25°C)	GH
		Glycerol 5% (w/v), 30 min ambient (22°C–25°C)	GI
		Glycerol 10% (w/v), 10 min ambient (22°C–25°C)	GJ
		Glycerol 10% (w/v), 15 min ambient (22°C–25°C)	GK
		Glycerol 10% (w/v), 30 min ambient (22°C–25°C)	GL
		Other	GZ
	Alginate SIGMA^® A-2158 2% viscosity Na-salt with or without 0.5 M sucrose.	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)	ENA
		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)	ENB
	Osmotic dehydration for 24–48 h (2-step osmotic treatment).	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)	ENC
		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)	END
	Evaporative air-silica gel desiccation to 25%–30% water (fresh weight) ∼0.4 g H₂O/g fwt.	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)	ENE
		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)	ENF
		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 20°C	ENG
		Unknown	ENX
		Other	ENZ
4	Cooling	Ultra rapid freezing (URF-direct plunge in LN)	URF
		Controlled rate cooling (CR)
		Planer Kryo 10^® Programmable Freezer
	Options can include equivalent cooling regimes.	−0.5°C/min to −40°C, hold 8 min, transfer to LN	CRA
		−0.5°C/min to −60°C, hold 10 min, transfer to LN	CRB
		−1.0°C/min to −35°C, hold 10 min, transfer to LN	CRC
		−1.0°C/min to −40°C, hold 10 min transfer to LN	CRD
		−1.0°C/min to −45°C, hold 10 min transfer to LN	CRE
		−1.0°C/min to −60°C, hold 10 min transfer to LN	CRF
		−1.0°C/min to −35°C, hold 10 min transfer to LN	CRG
		−1.0°C/min to −40°C, hold 10 min transfer to LN	CRH
	Temperature at which cooling starts usually determined by CPA element temperature (ambient 20°C–25°C or 0°C ice).	−1.0°C/min to −45°C, hold 10 min transfer to LN	CRI
		−1.0°C/min to −60°C, hold 10 min transfer to LN	CRJ
		−0.5°C/min to −60°C, hold 30 min, transfer to LN	CRK
		−1.0°C/min to −35°C, hold 30 min, transfer to LN	CRL
		−1.0°C/min to −40°C, hold 30 min transfer to LN	CRM
		−1.0°C/min to −45°C, hold 30 min transfer to LN	CRN
		−1.0°C/min to −60°C, hold 30 min transfer to LN	CRO
		−1.0°C/min to −35°C, hold 30 min transfer to LN	CRP
		−1.0°C/min to −40°C, hold 30 min transfer to LN	CRQ
		−1.0°C/min to −45°C, hold 30 min transfer to LN	CRR
		−1.0°C/min to −60°C, hold 30 min transfer to LN	CRS
		Other	Z
	Or equivalent instrument.	Planer Kryo 10 Programmable Freezer
		2-rate programs
		−1.0°C/min to 0°C, −0.5°C to −40°C, hold 15 min transfer to LN	CRT
		−1.0°C/min to 0°C, −0.5°C to −60°C, hold 15 min transfer to LN	CRU
	Auto-seeding dependent upon cooling device and CPA.	Planer Kryo 10 Programmable Freezer+induced ice nucleation “auto-seeding”
		−1.0°C/min to −9°C, auto-programmed seeding at −12°C, hold 15 min, resume −1.0°C/min to −40°C, hold 30 min transfer to LN	CRV
		Other	Z
	Or equivalent device.	Nalgene Mr. Frosty^® (MF)	MF
		−1°C/min to −80°C, hold 30 min, transfer to LN	MFA
	Passive cooling device solvent (250 mL isopropanol).	−1°C/min to −70°C, hold 30 min, transfer to LN	MFB
		−1°C/min to −80°C, hold 60 min, transfer to LN	MFC
		−1°C/min to −70°C, hold 60 min, transfer to LN	MFD
		−1°C/min to −80°C, hold 90 min, transfer to LN	MFE
		−1°C/min to −70°C, hold 90 min, transfer to LN	MFF
		Unknown	X
		Other	Z
5	Cryostorage (CS) ∼1.0 mL CPA+algae dispensed per vial or 10–20 alginate beads	NUNC 2.0 mL CryoTube^®-Vapour Phase-manual top-up	CSA
		NUNC 2.0 mL CryoTube^-Vapour Phase-man top-up	CSB
		NUNC 2.0 mL CryoTube-Liquid Phase-auto top-up	CSC
		NUNC 2.0 mL CryoTube-Liquid Phase-manual top-up	CSD
		Unknown	X
		Other	Z
6	Rewarming	Passive ambient until ice visibly melted	RWA
		Rapid, 40°C water bath until ice visibly melted	RWB
	Ambient ∼22°C–25°C	Rapid, 45°C water bath until ice visibly melted	RWC
		Two-step, 1 min ambient, 3 min 45°C water bath	RWD
		Alginate-encapsulated 2-step, 1 min ambient, 2 min 40°C water bath	RWE
		Alginate-encapsulated 2–3 min 40°C water bath +1 h rehydration in liquid culture medium	REF
		Alginate-encapsulated 30 min passive ambient +1 h rehydration in liquid culture medium	RWG
		Unknown	RX
		Other	RZ
7	Recovery	Standard medium, culture in standard light	REA
	Dependent on culture collection facility practices.	Standard medium, culture in standard light+10 mg/L desferrioxamine for 24 to 48 h	REB
		Standard medium, culture in reduced light (<30–40 μmol m⁻² s⁻¹)+10 mg/L desferrioxamine for 24 to 48 h	REC
	SPREC element detail determined by criticality of factors involved in cryo-recalcitrance.	Standard medium, culture in reduced light 1 day before transfer to light	RED
		Standard medium, culture in reduced light 2 days before transfer to light	REE
		Standard medium, culture in reduced light 3 days before transfer to light	REF
		Standard medium, culture in reduced light 4 days before transfer to light	REG
		Standard medium, culture in dark 1 day before transfer to light	REH
		Standard medium, culture in dark 2 days before transfer to light	REI
		Standard medium, culture in dark 3 days before transfer to light	REJ
		Standard medium, culture in dark 4 days before transfer to light	REK
		Alginate-encapsulated algae released by dissolution with 3% (w/v) sodium hexametaphosphate+1 day culture shaded by overlaid filter	REL
		Unknown	RWX
		Other	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 Osório et al.²² SPREC Codes 7-element SPREC (A-01) B-A-C-B-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.

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 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) B-A-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 0°C for 30 min (MC), and cryopreserved using passive cooling at −1°C/min in a Nalgene Mr. Frosty™ to a temperature of −80°C, 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 top-up system (CSC). Samples are rewarmed rapidly in a water bath at 40°C 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 A-01) 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-time-only 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 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 dissemination and utility, the SPREC can be included in biorepository-specific 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 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.

Footnotes

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

No competing financial interests exist.

References

Betsou

, Lehmann

, Ashton

et al.

International Society for Biological and Environmental Repositories (ISBER) Working Group on Biospecimen Science: standard preanalytical coding for biospecimens: defining the sample preanalytical code (SPREC)

Cancer Epidemiol Biomarkers Prev, 2010; 19:1004–1011.

Benson

, Betsou

, Harding

. Exploring standard preanalytical coding for environmental biospecimens using algal cryobanks as a case study. Cryobiology, 2010; 61:401.

Day

, Lukavský

, Friedl

et al. Pringsheim's living legacy: CCALA, CCAP, SAG and UTEX culture collections of algae. Nova Hedwigia, 2004; 79:27–38.

Day

, Benson

, Harding

et al.

Cryopreservation and conservation of microalgae: the development of a pan-European scientific and biotechnological resource (the COBRA project)

Cryo Lett, 2005; 26:231–238.

Day

, Lorenz

, Wilding

et al. The use of physical and virtual infrastructures for the validation of algal cryopreservation methods in international culture collections. Cryo Lett, 2007; 28:359–376.

Elster

, Benson

. Life in the Polar Terrestrial Environment: a focus on algae and cyanobacteria. Fuller

, Lane

, Benson

. Life in the Frozen State. Florida: CRC Press, 2004; 111–150.

Elster

, Lukešová

, Svoboda

et al. Diversity and abundance of soil algae in the polar desert, Sverdrup Pass, central Ellesmere Island. Polar Rec, 1999; 35:231–254.

Johnstone

, Block

, Benson

et al. Assessing methods for collecting and transferring viable algae from Signy Island, maritime Antarctic, to the UK. Polar Biol, 2002; 25:553–556.

Rivas

, Vargas

, Riquelme

. Interactions of Botryococcusbraunii cultures with bacterial biofilms. Microb Ecol, 2010; 60:628–635.

10.

Amaral

, Pais

, Santos

et al. Cryopreservation of microalgae: the problem of contaminant organisms in culture. Cryobiology, 2010; 61:367–368.

11.

Day

, Harding

. Cryopreservation protocols for algae in working laboratories. Reed

. Plant Cryopreservation: A Practical Guide. Netherlands: Springer, 2008; 95–116.

12.

Harding

, Müller

, Timmermann

et al. Encapsulation dehydration colligative cryoprotective strategies and amplified fragment length polymorphism (AFLP) markers to verify the identity and genetic stability of euglenoids following cryopreservation. Cryo Lett, 2010; 31:460–472.

13.

Elster

, Lukavský

, Harding

et al. Deployment of the encapsulation/dehydration protocol to cryopreserve polar microalgae held at the Czech Republic Academy of Sciences Institute of Botany. Cryo Lett, 2008; 29:27–28.

14.

Lukešová

, Hrouzek

, Harding

et al. Deployment of the encapsulation and dehydration cryo-conservation protocol to cryopreserve diverse microalgae held at the Institute of Soil Biology, Academy of Sciences of the Czech Republic. Cryo Lett, 2008; 29:21–26.

15.

Harding

, Friedl

, Timmermann

et al. Deployment of the encapsulation/dehydration protocol to cryopreserve microalgae held at Sammlung Von Algenkulturen, Universität Göttingen, Germany. Cryo Lett, 2008; 29:15–20.

16.

Harding

, Day

, Lorenz

et al. Introducing the concept and application of vitrification for the cryo-conservation of algae-a mini review. Nova Hedwigia, 2004; 79:207–226.

17.

Amaral

, Santos

LMA

. Overcoming recalcitrance in Porphyridium aerugineum Geitler employing encapsulation-dehydration cryopreservation methods. Cryo Lett, 2009; 30:462–472.

18.

Fleck

, Benson

, Bremner

et al. Studies of free radical-mediated cryoinjury in the unicellular alga Euglena gracilis using a non-destructive hydroxyl radical assay: a novel approach for developing protistan cryopreservation strategies. Free Radic Res, 2000; 32:157–170.

19.

Fleck

, Benson

, Bremner

et al. A comparative study of antioxidant protection in the cryopreserved unicellular algae Euglena gracilis and Haematococcus pluvialis. Cryo Lett, 2003; 24:213–228.

20.

Benson

. Cryopreservation of phytodiversity: a critical appraisal of theory & practice. Crit Rev Plant Sci, 2008; 27:141–219.

21.

Benson

, Harding

, Day

. Algae at extreme low temperatures: the cryobank. Seckbach

Algae and cyanobacteria in extreme environments11 Cellular Origin, Life in Extreme Habitats & Astrobiology Environmental Series. NY: Springer, 2007; 365–383.

22.

Osório

, Laranjeiro

, Santos

LMA

et al. First attempts to cryopreserve strains from the Coimbra Collection of Algae (ACOI) and the use of image analysis to assess viability. Nova Hedwigia, 2004; 79:227–235.

23.

Pugh

, Becker

, Porter

et al. Design and applications of the National Institute of Standards and Technology (NIST's) environmental specimen biobanking programmes. Cell Preserv Technol, 2008; 6:59–72.

24.

Palmirotta

, Ludovici

, De Marchis

et al.

Preanalytical procedures for DNA studies: the experiment of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Biopreserv Biobank, 2011; 9:35–55.

25.

Moore

, Kelly

, Jewell

et al. Biospecimen reporting for improved quality. Biopreserv Biobank, 2011; 9:57–65.

26.

Holland

, Pfleger

, Berger

et al. Molecular epidemiology biomarkers—sample collection and processing considerations. Toxicol Appl Pharmacol, 2005; 206:261–268.