**1. Introduction**

The Development of cryopreservation techniques, the increase in demand for cryopreserved cells or tissues and the use of these techniques in cells or tissues from patients with infectious diseases, has forced us to reduce the risk of contamination during the freezing process and the risk of cross-contamination during the storage of this material. Recent publications that demonstrate the survival of pathogens at low temperatures and possible contamination of the cells or tissues stored have changed the laws of each country and the customs and protocols used so far in the cryopreservation.

To understand the problem of contamination in cryopreservation we need to have an overview of the current problem in which all researchers are concerned about, seeking a cryopreservation protocol with good results but without contamination problems. Discussing the cryopreservation's different techniques such as slow freezing, vitrification, kinetic vitrification (extra-, hyper-, super-, ultra-fast vitrification) and the various components that help us understand the difficult balance between technique, device used and the risk of contamination. We need to use new products and new protocols' to have good results ensuring biological samples and patient safety.

Dr. Katkov's idea of find the *"Universal crypreservation protocol"* (see the other Chapter in the Book) by Katkov at al. that we can use worldwide with all the possible biological samples would lower considerably the price of cryopreservation process and we would have better results because everybody would work with the same protocol and the same results.

We have to comprehend the difference between open, closed or semi-closed devices and the importance of choosing one device or another both in morphological survival post thaw cell or tissue as on non-contamination of these samples. The device used, the protocol used and the cooling solution used can change the outcome of cryopreservation and therefore we have to find a protocol for cryopreservation with a cooling solution and a secure device to provide us good results free of contamination.

#### **2. Contamination and cross-contamination**

The first thing we must learn is to differentiate their respective importance are the concepts of contamination and cross contamination of samples. The first relates to the contamination

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 107

recommendations of the AEBT for cryopreservation of semen (21).

 Use of contaminated culture media. In these cases the degree of cross-contamination would reach very high levels having an impact on many patients. Although the preparation of embryo culture media and sperm extenders from specific ingredients are avoided in human clinics, it continues to be a common practice in animal ART (23). Nevertheless, many ingredients of embryo culture media and sperm extenders act as stabilizers for many micro-organisms at freezing temperatures ( milk, serum or serum albumin, sucrose, sorbitol and other sugars). Unfortunately, the most common cryoprotectors (CPs) in applied oocyte cryopreservation and embryo (glycerol, DMSO, ethylene glycol, propylene glycol, methanol etc.) are toxic for cells. Also bacteria and viruses efficiently protect from cryoinjuries, eg Concentrations of DMSO as low as 5% enveloped viruses defend against the trauma of freezing (24). The Fact That microorganisms survive in association with germplasm is not only important from the potential of disease transmission by embryo transfer to recipients, but also in approaches to the storage of samples for testing and health certification of embryos for international movement. On the other hand we must also bear in mind that all culture

media containing antibiotics to prevent or limit survival of microorganisms.

(cryotube, straw, etc.). It is very important to disinfect and clean the container before filling it with LN2 (16). In this regard to ensure an adequate level of biosafety a study is needed of infectious diseases transmissible from any patient or donor who wants to freeze any samples. According to Castilla (17) the clinic policy for a donor with infectious diseases is radically different to that of a patient with any of these diseases wanting to freeze biological material for autologous use. In the first situation, the biological material at hand will not freeze. In the second, the biological material should be frozen but with measures that we discuss later. Screenings for infectious diseases that normally must be submitted are: To analyse serological studies for syphilis, hepatitis and HIV. To analyse the clinical studies infective clinical phases: toxoplasmosis, rubella, herpes virus, cytomegalovirus (CMV), Neisseria gonorrhoeae and Chlamydia trachomatis. These tests are required for donors of semen every 6 months. As the risk of disease transmission during storage in LN2 is mainly viral. Interestingly, the American Society of Fertilisation (18), ESHRE (19), British Andrology Society (BSA) (20) and the Spanish Association of Tissue Banks (AEBT) (21) also recommend serologic screening for CMV, not just clinical. The presence of CMV in semen has been associated with active disease (anti-CMV IgM + or recent seroconversion anti-CMV IgG +). Similarly, these companies recommend performing serologic tests for HTLV-I and HTLV-II. But although it is clearly demonstrated the transmission of human papilloma virus by using LN2 cryotherapy and has been shown IUI transmission of herpes simplex virus (HSV) (22), none of the scientific associations mentioned above recommend a culture for detection or serological studies of HPV donors or patients with infection who are going to freeze biological material because the analysis to detect these deceases are not very sensitive. As rubella serologic screening of donors, its low prevalence in this population means that serological tests have a low positive predictive value, making it unadvisable. Finally, we believe a patient who needs to freeze some reproductive biological material should have at least one serology for HIV, hepatitis B and C. This proposal is consistent with the

of the sample by freezing or by direct contact with the cooling solution and the second refers to the contamination of the sample within the common container which is in contact with all cryopreserved samples, some samples may be contaminated or the liquid nitrogen (LN2) might be contaminated producing a possible cross-contamination. The potential for disease transmission and pathogen survival through contaminated LN2 has been proposed by many authors (1-3), and the evidence of contamination in human patients has been described for different pathogens (4-10). It has to be stated that none of the reported infections after insemination or ET in humans and domestic animals can be clearly attributed to the applied cryopreservation and storage procedure but the use of safe cryopreservation protocol is very important to avoid human cell contamination or crosscontamination in common LN2 tanks.

Although cryopreservation had a boom in the mid 70's and early 80's with the opening of the first sperm banks in America and Europe, it was not until the mid-80's when we saw the need for biological samples cryopreserved in quarantine and the lack of screening leads to infection of several recipients that had been inseminated with semen samples from donors HIV+ those unaware of their disease (11). In these cases it was found that samples stored in the same containers with frozen HIV+ samples were not contaminated, otherwise in 1995, six patients undergoing cytotoxic treatments hermetic problems developed an outbreak of acute hepatitis B after undergoing an autologous cryopreserved material that had been stored in the same cryogenic container as other patients infected with hepatitis B (12).

#### **2.1 Cells and tissue contamination**

In the field of assisted reproduction, although it hasn't been detected any contamination in the cryopreservation of gametes and embryos, the probability and the occurrence is low, the risk is not zero so it is recommended to follow the rules in biosecurity manuals for both the physical and chemical risk as well as the risk of contamination and cross contamination of samples.

The case in 1985 where there was infection with hepatitis B in the cryopreserved samples (12) the infection was due to an error in packaging and storage of samples. With time a deterioration of the bags containing infectious material causing the infection of the LN2 and other samples was observed.

Further studies have shown that the storage of samples is decisive. There is evidence that frozen samples in hermetically sealed straws are not contaminated even if they are in contaminated containers with contaminated LN2 and LN2 does not contaminate infective biological samples that were frozen in a sealed container (13,14)

During the cryopreservation, biological samples go through many processes before being cryopreserved. In the case of IVF cells are subjected to a phase of procurement, fertilization, development, transfer and finally cryopreservation. This represents an approximate 6-day process in which many factors can affect the contamination of the sample at the end of the process. We can find contamination or cross-contamination in the following cases (15):

 Handling contaminated biological samples (semen, follicular fluid, tissue, etc.). Without precautions to avoid contamination outside the base plate to be used for conservation

of the sample by freezing or by direct contact with the cooling solution and the second refers to the contamination of the sample within the common container which is in contact with all cryopreserved samples, some samples may be contaminated or the liquid nitrogen (LN2) might be contaminated producing a possible cross-contamination. The potential for disease transmission and pathogen survival through contaminated LN2 has been proposed by many authors (1-3), and the evidence of contamination in human patients has been described for different pathogens (4-10). It has to be stated that none of the reported infections after insemination or ET in humans and domestic animals can be clearly attributed to the applied cryopreservation and storage procedure but the use of safe cryopreservation protocol is very important to avoid human cell contamination or cross-

Although cryopreservation had a boom in the mid 70's and early 80's with the opening of the first sperm banks in America and Europe, it was not until the mid-80's when we saw the need for biological samples cryopreserved in quarantine and the lack of screening leads to infection of several recipients that had been inseminated with semen samples from donors HIV+ those unaware of their disease (11). In these cases it was found that samples stored in the same containers with frozen HIV+ samples were not contaminated, otherwise in 1995, six patients undergoing cytotoxic treatments hermetic problems developed an outbreak of acute hepatitis B after undergoing an autologous cryopreserved material that had been stored in the same cryogenic container as other patients infected with hepatitis B (12).

In the field of assisted reproduction, although it hasn't been detected any contamination in the cryopreservation of gametes and embryos, the probability and the occurrence is low, the risk is not zero so it is recommended to follow the rules in biosecurity manuals for both the physical and chemical risk as well as the risk of contamination and cross contamination of

The case in 1985 where there was infection with hepatitis B in the cryopreserved samples (12) the infection was due to an error in packaging and storage of samples. With time a deterioration of the bags containing infectious material causing the infection of the LN2 and

Further studies have shown that the storage of samples is decisive. There is evidence that frozen samples in hermetically sealed straws are not contaminated even if they are in contaminated containers with contaminated LN2 and LN2 does not contaminate infective

During the cryopreservation, biological samples go through many processes before being cryopreserved. In the case of IVF cells are subjected to a phase of procurement, fertilization, development, transfer and finally cryopreservation. This represents an approximate 6-day process in which many factors can affect the contamination of the sample at the end of the process. We can find contamination or cross-contamination in the following cases (15):

 Handling contaminated biological samples (semen, follicular fluid, tissue, etc.). Without precautions to avoid contamination outside the base plate to be used for conservation

biological samples that were frozen in a sealed container (13,14)

contamination in common LN2 tanks.

**2.1 Cells and tissue contamination**

other samples was observed.

samples.

(cryotube, straw, etc.). It is very important to disinfect and clean the container before filling it with LN2 (16). In this regard to ensure an adequate level of biosafety a study is needed of infectious diseases transmissible from any patient or donor who wants to freeze any samples. According to Castilla (17) the clinic policy for a donor with infectious diseases is radically different to that of a patient with any of these diseases wanting to freeze biological material for autologous use. In the first situation, the biological material at hand will not freeze. In the second, the biological material should be frozen but with measures that we discuss later. Screenings for infectious diseases that normally must be submitted are: To analyse serological studies for syphilis, hepatitis and HIV. To analyse the clinical studies infective clinical phases: toxoplasmosis, rubella, herpes virus, cytomegalovirus (CMV), Neisseria gonorrhoeae and Chlamydia trachomatis. These tests are required for donors of semen every 6 months. As the risk of disease transmission during storage in LN2 is mainly viral. Interestingly, the American Society of Fertilisation (18), ESHRE (19), British Andrology Society (BSA) (20) and the Spanish Association of Tissue Banks (AEBT) (21) also recommend serologic screening for CMV, not just clinical. The presence of CMV in semen has been associated with active disease (anti-CMV IgM + or recent seroconversion anti-CMV IgG +). Similarly, these companies recommend performing serologic tests for HTLV-I and HTLV-II. But although it is clearly demonstrated the transmission of human papilloma virus by using LN2 cryotherapy and has been shown IUI transmission of herpes simplex virus (HSV) (22), none of the scientific associations mentioned above recommend a culture for detection or serological studies of HPV donors or patients with infection who are going to freeze biological material because the analysis to detect these deceases are not very sensitive. As rubella serologic screening of donors, its low prevalence in this population means that serological tests have a low positive predictive value, making it unadvisable. Finally, we believe a patient who needs to freeze some reproductive biological material should have at least one serology for HIV, hepatitis B and C. This proposal is consistent with the recommendations of the AEBT for cryopreservation of semen (21).

 Use of contaminated culture media. In these cases the degree of cross-contamination would reach very high levels having an impact on many patients. Although the preparation of embryo culture media and sperm extenders from specific ingredients are avoided in human clinics, it continues to be a common practice in animal ART (23). Nevertheless, many ingredients of embryo culture media and sperm extenders act as stabilizers for many micro-organisms at freezing temperatures ( milk, serum or serum albumin, sucrose, sorbitol and other sugars). Unfortunately, the most common cryoprotectors (CPs) in applied oocyte cryopreservation and embryo (glycerol, DMSO, ethylene glycol, propylene glycol, methanol etc.) are toxic for cells. Also bacteria and viruses efficiently protect from cryoinjuries, eg Concentrations of DMSO as low as 5% enveloped viruses defend against the trauma of freezing (24). The Fact That microorganisms survive in association with germplasm is not only important from the potential of disease transmission by embryo transfer to recipients, but also in approaches to the storage of samples for testing and health certification of embryos for international movement. On the other hand we must also bear in mind that all culture media containing antibiotics to prevent or limit survival of microorganisms.

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 109

The cooling solution plays a significant role in avoiding contamination of biological samples. It means that we will freeze the sample and we will deposit it for a long storage until thawed and used. Normally the LN2 cooling solution is the most widely used in cryopreservation and survival of pathogens at high temperatures (-196 º C) has already been proven by many studies (1-3,27,28) cases also involved in seeing cross-contamination of

The need for better cooling rates to avoid formation of crystals in cryopreservation has resulted in the discovery and use of new cooling solutions (slush, slurry, etc.). So there are more components to consider when contamination is to be avoided. Using these new cooling solutions gives a lower temperature than the LN2 temperature and much faster transmission. The Slush nitrogen is obtained by a vacuum pump (Telstar TOP-3; Telstar S.A., Terrassa, Spain) that solidifies part of the LN2 in a few minutes. On return to normal atmospheric pressure, the nitrogen collapses, and the subcooled LN2 has solid particles in it commonly referred to as ''slush'' (30).The advantage of Slush nitrogen lies not only in the temperature difference with respect to LN2 (-196ºC Vs -210ºC) but also in the reduction of the Leiden frost effect, which is the formation of a layer of vapor around the sample when immersed in the cryogenic liquid from room temperature decreasing the cooling rate (31,32). It has not yet been demonstrated the survival or non survival of pathogens in this cooling solution of 15-20 ° C difference in LN2, this is obtained by vacuum pressure, which can lead to rupture of the cell wall of pathogens to balance internal and external pressure of these in the process of forming Slush. The 'Slurry' nitrogen is a mix of LN2 with different particles for example copper powder. At present investigations are being carried out as an alternative to LN2 to increase the cooling rate because with this cooling solution the thermal conduction is increased. Likewise, experiments are ongoing with various solutions to increase the

These cooling solutions "alternatives" are only used at the time of freezing the sample and once frozen, it passes to the general container that is filled with LN2, although these solutions where they freeze cool samples have to be sterile we have to ensure that the general LN2 container does not have contact with the frozen sample in order to not

Retrospective studies in which commercial LN2 cryotanks were examined after 35 continuous years of service revealed various bacterial and fungal contaminations in the LN2 detritus (23). Many of the identified bacteria isolated in these studies were ubiquitous environmental micro-organisms and were rare opportunistic pathogens of low significance in producing disease in humans or animals (Table I). It should be acknowledged that some of the isolates may have been derived from laboratory contamination during semen and embryo processing for cryopreservation rather than genuinely being present within the sample. In agreement with Bielansky and Vajta the risk of contamination by human pathogens seems to be rather low. Components of the standard LN2 production system comprise a compressor, a cryogenerator and containers. From a practical point of view, the complete sterilization and maintenance of sterility in such a robust system might be a very demanding task, if possible at all. Accordingly, some ubiquitous bacterial agents can be expected in any commercially produced LN2. Nevertheless, it is an 'in and out' system and only air-borne contaminants are supposed to enter it (LN2 compressor) via air used for LN2

contaminate the sample and the LN2 if the sample is positive for any pathogen.

**2.2 Cooling solution contamination**

human papillomavirus (14,29).

thermal conduction and the cooling rate.

#### **2.2 Cooling solution contamination**

108 Current Frontiers in Cryopreservation

 Conservation of contaminated material or straws cryotubes closed or sealed badly flawed causing the breakdown of the frozen straw, leaving the contaminated sample directly exposed to the LN2 tank risking contaminating the other samples. Closed systems can be sealed in many ways (thermal sealer, ultrasound sealer, radiofrequency sealer, polyvinyl alcohol powders, and solid caps). Given the sealing time and the temperature reached does not affect the cryopreserved sample, we have to ensure that the seal is airtight and that the device is built of resistant material to low temperatures of LN2 (Ionomeric resins, quartz glass capillary, Polyvinyl chloride, Polyethylene glycol

Using contaminated LN2 during the freezing process. In this case we have proposed

 Poor source management of LN2 from our supplier contaminating commercial LN2 that comes to our lab in the process of manufacture or transportation and filling our

 For transportation of contaminated material in containers. Storage containers should be emptied and cleaned periodically due to the risk of lost straws or small particles of contaminated material that falls to the bottom of a large container (25,26). Most of the companies of LN2 containers provide cleaning protocols. The main problem is the cleaning of transport cylinders called "dry" because the material that absorbs the LN2 in these bottles is difficult to sterilize. Bielanski (27) describes a method of disinfection of commercial dry shippers with two different types of a LN absorbent. Based on the results presented, it appears that solutions of sodium hypochlorite and ethylene oxide are equally useful for the disinfection of dry shippers constructed with a hydrophobic LN absorbent. In contrast, for dry shippers without a hydrophobic LN absorbent it is advisable to use gas only for decontamination in order sterilization to avoid their

 The air in the room. If the air that reaches the lab comes from another area that could be contaminated and there isn't a good filter. Some laboratories do not have filtration

 Operators. If they are infected then that can lead to contamination by contact or peeling during processing of samples or the handling of cryogenic tanks. Staff must meet certain health and hygiene conditions: negative serology for HIV, HCV, HBV and vaccination against hepatitis B and other viral diseases for which there is a vaccine available. We must also have a detailed description of their jobs, tasks and responsibilities. In addition the centre must provide the worker training in freezing

 Use of open devices. In recent times there is much talk of closed or open system and the possibility of contamination, so many countries have banned open systems and the trend is to ban the high risk of sample contamination. In a closed or semi-closed device the nitrogen of common container is never in contact with biological material frozen on the inside so cross-contamination cannot produced. In the open system, the biological material is in contact with the common nitrogen so contamination from the sample is very easy if the LN2 is contaminated or contamination of LN2 if the sample is contaminated. The latest study done by Criado and his group (28) showed 45% of contamination in an open device (Cryotop) Vs 0% of contamination in a semi-close

systems or positive pressure to prevent air contamination.

techniques for updating and improving procedures.

device (Ultravit) equal and using a contaminated laboratory LN2.

tetralato, etc).

containers.

some solutions that we will see later.

damage by liquid disinfectants.

The cooling solution plays a significant role in avoiding contamination of biological samples. It means that we will freeze the sample and we will deposit it for a long storage until thawed and used. Normally the LN2 cooling solution is the most widely used in cryopreservation and survival of pathogens at high temperatures (-196 º C) has already been proven by many studies (1-3,27,28) cases also involved in seeing cross-contamination of human papillomavirus (14,29).

The need for better cooling rates to avoid formation of crystals in cryopreservation has resulted in the discovery and use of new cooling solutions (slush, slurry, etc.). So there are more components to consider when contamination is to be avoided. Using these new cooling solutions gives a lower temperature than the LN2 temperature and much faster transmission. The Slush nitrogen is obtained by a vacuum pump (Telstar TOP-3; Telstar S.A., Terrassa, Spain) that solidifies part of the LN2 in a few minutes. On return to normal atmospheric pressure, the nitrogen collapses, and the subcooled LN2 has solid particles in it commonly referred to as ''slush'' (30).The advantage of Slush nitrogen lies not only in the temperature difference with respect to LN2 (-196ºC Vs -210ºC) but also in the reduction of the Leiden frost effect, which is the formation of a layer of vapor around the sample when immersed in the cryogenic liquid from room temperature decreasing the cooling rate (31,32). It has not yet been demonstrated the survival or non survival of pathogens in this cooling solution of 15-20 ° C difference in LN2, this is obtained by vacuum pressure, which can lead to rupture of the cell wall of pathogens to balance internal and external pressure of these in the process of forming Slush. The 'Slurry' nitrogen is a mix of LN2 with different particles for example copper powder. At present investigations are being carried out as an alternative to LN2 to increase the cooling rate because with this cooling solution the thermal conduction is increased. Likewise, experiments are ongoing with various solutions to increase the thermal conduction and the cooling rate.

These cooling solutions "alternatives" are only used at the time of freezing the sample and once frozen, it passes to the general container that is filled with LN2, although these solutions where they freeze cool samples have to be sterile we have to ensure that the general LN2 container does not have contact with the frozen sample in order to not contaminate the sample and the LN2 if the sample is positive for any pathogen.

Retrospective studies in which commercial LN2 cryotanks were examined after 35 continuous years of service revealed various bacterial and fungal contaminations in the LN2 detritus (23). Many of the identified bacteria isolated in these studies were ubiquitous environmental micro-organisms and were rare opportunistic pathogens of low significance in producing disease in humans or animals (Table I). It should be acknowledged that some of the isolates may have been derived from laboratory contamination during semen and embryo processing for cryopreservation rather than genuinely being present within the sample. In agreement with Bielansky and Vajta the risk of contamination by human pathogens seems to be rather low. Components of the standard LN2 production system comprise a compressor, a cryogenerator and containers. From a practical point of view, the complete sterilization and maintenance of sterility in such a robust system might be a very demanding task, if possible at all. Accordingly, some ubiquitous bacterial agents can be expected in any commercially produced LN2. Nevertheless, it is an 'in and out' system and only air-borne contaminants are supposed to enter it (LN2 compressor) via air used for LN2

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 111

 UV Sterilization: This method is based on emitting the minimum dose on UV radiation necessary to kill micro-organisms that can survive at the boiling point of nitrogen (- 196°C) and which is irradiated in a temperature-controlled regimen, within a short time interval, before the LN2 completely evaporates. The extremely radiation-resistant

Fig. 1. CERALIN

the pressure drop and avoids the vaporization of the LN2. Thus it avoids nitrogen losses. Several sizes are available, depending on the nitrogen flow. The efficiency of this equipment was investigated and proved in laboratory. The filter is located downstream of the nitrogen vessel. During operation, LN2 flows through the filter and over the ceramic membrane. The result is high-purity LN2 with a bacteria count of less than 1 CFU/L gas. Additionally, the large filtration area of the membrane and low level of contamination of LN2 means it is likely to be several decades before filter saturation.

production. As they are not air-borne, it is unlikely that viral agents of human concern such as HIV, hepatitis and herpes viruses would enter the LN2 production system.

Table 1. Microbiological contamination of embryos and semen during storage in LN2 (23)

One of the biggest discussions recently in the world of cryopreservation focuses on the importance of the sterility of LN2. As shown in Table I and in total agreement with Bielansky and Vajta and many other authors the commercial LN2 reaching our lab is not contaminated enough to cause any infection to freeze biological material. The major problem is common containers where the samples are deposited with a LN2 stored for months, years or even decades in contact with many samples, which, many clinics do not empty and do not disinfect, so it is in common containers where we can find the highest risk of contamination and cross contamination.

As a possible solution to minimize the risk of freezing biological material some systems have been proposed where we sterilize the LN2 and where we ensure that the sample is not in contact with LN2 with the use of semi-close devices or devices that are the only ones that guarantee a hermetic sealing of the device and avoid any risk of breakage of the solder thus ensuring the aseptic samples. The fact that LN2 can be quickly and safely sterilized could encourage the clinical application of human cell/tissue vitrification, both with open carriers and with closed systems. The problem is that if this device is an open device and is passed to the general container where all the other cryopreserved samples there is a huge risk of cross contamination, so it has not helped.

 LN2 Filtration: One of the solutions that have been developed is the filtration of LN2. Air Liquid has marketed CERALIN a liquid filtration system through LN2 ceramic filters. The CERALIN ON LINE consists of two elements of liquid filtration connected in series and inserted into a section of vacuum transfer line. The ceramic membrane is made from multiple layers formed into a multi-channel element. It is housed in a vacuum insulated pipe, itself installed close to the end-use point. The filter minimizes the pressure drop and avoids the vaporization of the LN2. Thus it avoids nitrogen losses. Several sizes are available, depending on the nitrogen flow. The efficiency of this equipment was investigated and proved in laboratory. The filter is located downstream of the nitrogen vessel. During operation, LN2 flows through the filter and over the ceramic membrane. The result is high-purity LN2 with a bacteria count of less than 1 CFU/L gas. Additionally, the large filtration area of the membrane and low level of contamination of LN2 means it is likely to be several decades before filter saturation.

Fig. 1. CERALIN

110 Current Frontiers in Cryopreservation

production. As they are not air-borne, it is unlikely that viral agents of human concern such

Table 1. Microbiological contamination of embryos and semen during storage in LN2 (23)

of contamination and cross contamination.

cross contamination, so it has not helped.

One of the biggest discussions recently in the world of cryopreservation focuses on the importance of the sterility of LN2. As shown in Table I and in total agreement with Bielansky and Vajta and many other authors the commercial LN2 reaching our lab is not contaminated enough to cause any infection to freeze biological material. The major problem is common containers where the samples are deposited with a LN2 stored for months, years or even decades in contact with many samples, which, many clinics do not empty and do not disinfect, so it is in common containers where we can find the highest risk

As a possible solution to minimize the risk of freezing biological material some systems have been proposed where we sterilize the LN2 and where we ensure that the sample is not in contact with LN2 with the use of semi-close devices or devices that are the only ones that guarantee a hermetic sealing of the device and avoid any risk of breakage of the solder thus ensuring the aseptic samples. The fact that LN2 can be quickly and safely sterilized could encourage the clinical application of human cell/tissue vitrification, both with open carriers and with closed systems. The problem is that if this device is an open device and is passed to the general container where all the other cryopreserved samples there is a huge risk of

 LN2 Filtration: One of the solutions that have been developed is the filtration of LN2. Air Liquid has marketed CERALIN a liquid filtration system through LN2 ceramic filters. The CERALIN ON LINE consists of two elements of liquid filtration connected in series and inserted into a section of vacuum transfer line. The ceramic membrane is made from multiple layers formed into a multi-channel element. It is housed in a vacuum insulated pipe, itself installed close to the end-use point. The filter minimizes

as HIV, hepatitis and herpes viruses would enter the LN2 production system.

 UV Sterilization: This method is based on emitting the minimum dose on UV radiation necessary to kill micro-organisms that can survive at the boiling point of nitrogen (- 196°C) and which is irradiated in a temperature-controlled regimen, within a short time interval, before the LN2 completely evaporates. The extremely radiation-resistant

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 113

At European level, all documents related to transport are based on the recommendations of the Committee of Experts of the United Nations Dangerous Goods (UN) (45). There is also a european agreement on international transport of dangerous goods by road (ADR), approved by RD 2115/9838 (46). We will describe some aspects of the mentioned regulations on the

1. Primary container, watertight, leak proof, labeled and contains the sample. This container should be wrapped in absorbent material. In terms of labeling, according to AEBT, if it is a semen sample from a donor, must contain an alphanumeric code that identifies the donor and the sample number of the donor. On the other hand, if the

sample is for autologous use may be noted also the surname of the patient (21). 2. Secondary container, sealed, leak-proof and protects the primary container. You can place multiple primary containers wrapped in a secondary container. This should be sufficient absorbent material used to protect all primary containers and avoid collisions

3. Outer shipping container: the secondary container is placed in a shipping package that protects the secondary container and its contents from outside elements, such as

The data forms, letters and other identifying information of the sample should be placed

taped outside the secondary container. The label for submitted materials consists of:

 Name, address of destination, as detailed as possible, and phone number. Name, address, telephone number and contact person at the semen bank. 6. The documents included with the storage conditions and special instructions for shipping. One of the special considerations that we must have in mind when transporting a sample of semen is not breaking the cold chain, so you must use a

7. Vehicle with kit (gloves, absorbent material, disinfectant, waste container, etc.).

You must ensure perfect coordination of transport between the sender, carrier and recipient to ensure delivery. Thus, each party involved should carry out its part perfectly and

transport of diagnostic specimens. The basic system consists of packaging:

between them.

1. Basic triple packaging.

5. Tag address:

8. Label orientation.

1. Sealed and resistant containers. 2. Threaded tubes upright (rack, tray ...). 3. Use of resistant boxes and perfect closure. 4. Secured box in the transport vehicle.

6. Have the forms with necessary details.

5. Appropriate Labeling.

physical damage and water.

2. Does not require signs from United Nations (UN).

7. Permission for import / export and declaration.

9. Date and time of departure of Semen Bank (21).

The requirements to be met for local transport are as follows:

4. "Biological material for clinical use" must be indicated.

3. No substances require pictogram or declaration from the sender.

container or LN2 as well as avoiding the possible use of dry ice.

bacterium Deinococcus radiodurans is inactivated (>- 4log) by administe-ring 400.000 µWs/cm² per each sterilization cycle. An adequate amount of UV radiation deactivates the growth of all kinds of micro-organisms, from viruses like Hepatitis (which require an 8.000 UV dose) to fungi like Aspergillus Niger (330.000 UV dose) (33). At CRYO 2011 Dr. Parmegiani spoke about a new dispositive of UV sterilization of the common containers with cells or tissues inside but the scientific community thinks that is too dangerous biological samples exposed to UV rays without any protection. Although his group is proposing special canisters "not transparent" I think they have to do many more tests to rule out damage to the samples because the common view is confirmed that UV light is harmful, even if used just overnight decreased embryo developmental rates.

#### **2.3 Contamination in transport**

To carry out a safe transportation of biological material we should clearly distinguish a number of concepts (17).

Most often transported biological reproductive materials are cryopreserved semen donor and follicular fluid when the laboratory is separated from the follicular puncture site. In both cases, we consider the recommendations to follow are those of diagnostic specimens. There are several documents related to the transport of biological material, such as the Universal Postal Union (UPU), the International Aviation Organization (ICAO) and International Air Transport Association (IATA) (42-44).

At European level, all documents related to transport are based on the recommendations of the Committee of Experts of the United Nations Dangerous Goods (UN) (45). There is also a european agreement on international transport of dangerous goods by road (ADR), approved by RD 2115/9838 (46). We will describe some aspects of the mentioned regulations on the transport of diagnostic specimens. The basic system consists of packaging:

The data forms, letters and other identifying information of the sample should be placed taped outside the secondary container. The label for submitted materials consists of:

112 Current Frontiers in Cryopreservation

 LN2 Steam: As an alternative to hermetical storage in LN2, cryostorage contamination might be avoided by storing the carrier containing the vitrified oocytes in LN2 vapour (34, 35). However, Grout and Morris (36) maintain that storage in the vapour phase of LN2 still carries a risk of sample contamination. Storage of semen in LN2 vapours was discarded early in the development of sperm cryopreservation techniques and it was found that long-term viability of sperm was reduced compared with LN2 storage (37,38). However, recent experiments with new materials have succeeded in developing the technique with acceptable results for both semen and embryos (39,40) and in our last experiment we demonstrated 0% of contamination in vapor nitrogen in a experimental contaminated laboratory LN2 (non published). The drawback of the generalization of this form of storage is the need for careful monitoring of temperature in different parts of the container, which makes the marketing of these containers type

 Before entering discussions regarding the sterility in LN2 used for vitrification, we should debate the use of communal containers, which is where cross-contamination can be found, as there is a possibility that the "contaminated cells" could come into contact with each other, and where a number of viruses and bacteria are found, which would

To carry out a safe transportation of biological material we should clearly distinguish a

1. Infectious substances: those that contain viable microorganisms (bacteria, virus, prions, parasite, fungus) or bacterial toxins that are known or believed to cause disease in

2. Diagnostic specimens: human or animal materials (body fluids, blood, tissue, tissue

Most often transported biological reproductive materials are cryopreserved semen donor and follicular fluid when the laboratory is separated from the follicular puncture site. In both cases, we consider the recommendations to follow are those of diagnostic specimens. There are several documents related to the transport of biological material, such as the Universal Postal Union (UPU), the International Aviation Organization (ICAO) and

fluids, etc.). Obtained for diagnostic or investigational (41).

International Air Transport Association (IATA) (42-44).

developmental rates.

(40) more difficult.

**2.3 Contamination in transport**

number of concepts (17).

animals or humans.

never be found in the commercial LN2.

bacterium Deinococcus radiodurans is inactivated (>- 4log) by administe-ring 400.000 µWs/cm² per each sterilization cycle. An adequate amount of UV radiation deactivates the growth of all kinds of micro-organisms, from viruses like Hepatitis (which require an 8.000 UV dose) to fungi like Aspergillus Niger (330.000 UV dose) (33). At CRYO 2011 Dr. Parmegiani spoke about a new dispositive of UV sterilization of the common containers with cells or tissues inside but the scientific community thinks that is too dangerous biological samples exposed to UV rays without any protection. Although his group is proposing special canisters "not transparent" I think they have to do many more tests to rule out damage to the samples because the common view is confirmed that UV light is harmful, even if used just overnight decreased embryo

The requirements to be met for local transport are as follows:

You must ensure perfect coordination of transport between the sender, carrier and recipient to ensure delivery. Thus, each party involved should carry out its part perfectly and

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 115

Generally, using open devices the achieved cooling rates are approximately 20.000-30.000 ° C / min which favor good vitrification of the sample. The problem is that being in direct contact with the cooling solution there is a risk of pathogen transmission to the biological sample at the time of freezing and a high risk of cross contamination in the common cooling containers. They have been prohibited in many countries for this high risk of contamination and the global trend is to ban them for use with human samples. Recent microbiological studies indicate a 45% pathogen contamination (Pseudomonas and E-coli) with a simple 10-second contact with the open device (Cryotop) with contaminated

In the OPS method, 0.25 mm standard insemination plastic mini-straws were heat-softened over a hot plate and pulled manually, as originally described by Vajta et al. (48). The inner diameter and the wall thickness of the pulled part of the straw are approximately 0.8 and 0.07 mm respectively. Cells are load into the pulled straws by placing the narrow end of the pulled straw in the third droplet of medium and aspirating oocytes within a 2–3 mm long liquid column (1–1.5 μl) using capillarity. The straws are then cooled by being plunged directly into LN2 and stored briefly. For warming, the open end of the straw is immersed vertically into 4.5 ml of the warming solution at 37°C. The solidified vitrification solution became liquid within

Fig. 3. A) The 0.25 ml conventional straw is loaded with 1 cm of vitrification medium, 0.5 cm

vitrification medium using a syringe. (B) The open pulled straw is loaded with vitrification solution (1–2 μl) containing oocytes by means of the capillary effect by a simple touch.

The Cryoloop (Hampton Research, Aliso Viejo, CA, USA ), used as a vessel in vitrification, is a thin nylon loop used to suspend a film of cryoprotectant containing the oocytes and directly immerse them in LN2. Vitrification of oocytes using the Cryoloop has advantages over conventional vitrification procedures in that the open system lacks a thermo insulating

of air, 2 cm of vitrification medium containing oocytes, 0.5 cm of air, and 3.5 cm of

cooling solution (28).

**Open pulled straw**

**Cryoloop**

The most known and used open devices are the following:

1–2 s. A cooling rate of 16.700ºC/min is obtained with this device (49).

appropriately. So stand out from other actions that the sender must ensure the proper identification, packaging, labeling and documentation according to established biosafety guidelines in the "Recommendations of the Committee of Experts of the United Nations Transport of Dangerous Goods" transporting must be kept in appropriate conditions (temperature, light ...) the material from which the sender receives it until it is delivered to your destination and have the appropriate licenses to perform this type of transport, and finally, the recipient must confirm with national authorities that the material can be legally imported.

According to AEBT (21), the possibility of returning a material that hasn't been used should be avoided, as a rule, the return of the semen that has been provided by the Bank, as it will only accept the return of the displayed when you meet the following 3 conditions:


**6. Conclusions**

In conclusion, Maluku have long practiced traditional '*dusung*' agroforestry management patterns through local wisdom known as "*Sasi*" and "*Masohi.*" The role of agroforestry can be seen from its contribution to the economic, ecological, sociocultural functions of people, as well as the mitigation and adaptation of greenhouse gases through carbon sequestration. Land management through agroforestry, actually increases the absorption of CO in the air. According to studies, there is a possible decrease in primary forest in the world due to designation for other functions. Therefore, agroforestry is one of the safety valves for forest sustainability where people can take advantage of agricultural products to meet their basic needs and in handling climate change. Furthermore, based on a cluster determination study in accordance with the land area and composition, there are various types of monoculture, semi-monoculture and mixed farming crops grown from the coast to the mountains. These crops are used to analyze the cluster variables, with the villages on the small island of Maluku classified as good (the average score for the sustainability index was 89.2) and moderate (the average score for the sustainability index was 69.7), with none in the poor category.


*2.4. Statistical Analyses*

One-way analysis of variance (ANOVA) tests with a subsequent Games–Howell test evaluated differences in parameter distributions. Levene's tests were used to investigate homoscedasticity (Table S2). Data are presented as means ± standard deviations (SD), and statistical significance was determined at *p* < 0.05. The statistical results are shown in Tables S3–S5.

To assess the effects of the mixed treatment, EC50-72 h values were obtained from each compound, and the mixture treatments were used. Concentration addition (CA) and independent addition (IA) models were used to evaluate the interaction patterns of nickel and BED-47 mixtures, followed by an analysis with the MIXTOX tool [33]. Calculation details were described in a previous study [33]. These models evaluate putative interaction patterns for combinations of chemicals. CA models are generally used to evaluate combinations of chemicals that share the same mechanism of action. In contrast, IA models are generally used for those that exhibit different mechanisms of action. Nevertheless, it is difficult to confirm the model that should be used in many cases. The MIXTOX model can be used with the additional parameter '*a*' to evaluate potential interaction patterns [34,35]. Briefly, the '*a*' parameter was used to evaluate the synergism or antagonism of two chemical interactions, wherein *a* < 0 indicates synergism and *a* > 0 indicates antagonism.


3.1.1. Spectral Entropy

Power Spectral Density (PSD) estimation is commonly used in signal-processing literature. By transforming a given time series *xt* from the time domain to the frequency domain using the discrete Fourier transform, the latter provides information about the power of each frequency component. These frequencies describe a spectral probability distribution which can be used to assess the uncertainty about a future prediction, namely spectral entropy *Hspct* (a cartoon of this process is shown in Figure 1B). To calculate this from a TS (assumed to be stationary), we first require its Autocovariance Function (ACVF). This is defined as

$$\gamma\_x(k) = \mathbb{E}[(\mathbf{x}\_t - \mu\_x)(\mathbf{x}\_{t-k} - \mu\_x)], \qquad k \in \mathbb{Z}, \tag{4}$$

where *μx* is the TS mean value and *k* corresponds to the lag. With the ACVF, the spectrum of the TS is obtained through the Fourier transform such as

$$S\_{\mathbf{x}}(\lambda) = \frac{1}{2\pi} \sum\_{j=-\infty}^{\infty} \gamma\_{\mathbf{x}}(j) e^{ij\lambda}, \qquad \qquad \lambda \in [-\pi, \pi], \tag{5}$$

where *i* = √−1 and *Sx* : [−*π*, *π*] → R+. To understand the implications of Equation (5) consider a white noise TS *ω*. In such case, *γω*(*k*) = 0 for *k* = 0, thus, the spectrum is constant for all *λ* ∈ [−*π*, *π*] [24]. Then, if we define *σ*2*x* = *π*−*π Sx*(*λ*) for *k* = 0, the spectral density of *xt* is

$$f\_{\mathbf{x}}(\lambda) = \frac{S\_{\mathbf{x}}(\lambda)}{\sigma\_{\mathbf{x}}^2} = \frac{1}{2\pi} \sum\_{j=-\infty}^{\infty} \rho\_{\mathbf{x}}(j) e^{ij\lambda},\tag{6}$$

where *ρ*(*k*) = *γ*(*k*) *γ*(0) corresponds to the Autocorrelation Function (ACF). The *fx*(*λ*) can be used as a probability density function of a random variable such that it is ascribed to the unit circle. For instance, in the case of *ω*, *fx*(*ω*) = 12*π* , which corresponds to the spectral density of the uniform distribution [24].

Using Equation (6), the spectral entropy *Hspct* is defined as

$$H\_{\rm spcf} = \int\_{-\pi}^{\pi} f\_{\mathbf{x}}(\lambda) \log g\_{\mathbf{z}} f\_{\mathbf{x}}(\lambda) d\lambda. \tag{7}$$

If the value obtained by Equation (7) is relatively small, *xt* is more *forecastable* since it contains more signal than noise, whereas a larger value stands otherwise [18]. The previous analysis can be simplified by normalizing *Hspct* within 0 ≤ *Hspct* ≤ 1 by dividing it by the uniform distribution entropy i.e., *loga*(2*π*) (which has the maximum entropy for a finite support). In this sense, the uncertainty about a required prediction *xt*+*h* is given by the characteristics of the process itself [24].


**3.3 LAD velocity profiles**

Early systolic flow reversal did not occur with eptifibatide while it was noticed in 6 (17%) in those without, p<0.05 (table 2).

Table 2. LAD Systolic Flow Reversal

discharge was normal. In figure 7, an example of a patient with unfavorable LAD blood velocity profile after primary angioplasty and bare metal stent implantation in a patient with acute anterior STEMI. Short diastolic deceleration time (less than 600msec) and early systolic flow reversal are demonstrated. In this patient left ventricular ejection fraction was

Fig. 6. Favourable LAD blood velocity profile with prolonged diastolic deceleration time

Fig. 7. Unfavourable LAD blood velocity profile with reduced diastolic deceleration time

Early systolic flow reversal did not occur with eptifibatide while it was noticed in 6 (17%) in

ESFR 48 Hr. ESFR 5 Days

reduced at admission and did not improve later.

and forward systolic flow.

and early systolic flow reversal.

those without, p<0.05 (table 2).

Table 2. LAD Systolic Flow Reversal

ESFR

Early

EPT-Yes 0 0 0 EPT-No 6 4 2

**3.3 LAD velocity profiles**

Diastolic deceleration time of LAD flow averaged 629±238 msec in patients treated with eptifibatide and 593±344 msec in those without, p=0.7 (figure 8). Short (<600msec) diastolic deceleration time occurred in 6 (40%) of those treated with eptifibatide, compared to 12 (39%) in those not treated, p=ns (figure 8).

Fig. 8. Histogram of diastolic deceleration time and pressure half time of LAD blood velocity profiles in patients with and those without eptifibatide treatment.

Patients treated with eptifibatide had higher diastolic velocities, 39±11 cm/sec, vs 31±9 cm/sec, p=0.043 and tended to have higher diastolic LAD flows, 49±26 ml/min, vs 35±17 ml/min, p=0.09 (figure 9).

Fig. 9. Histogram of diastolic and systolic velocities of LAD blood velocity profiles in patients with and those without eptifibatide treatment.

Effects of Eptifibatide on the Microcirculation After Primary Angioplasty in

in both treatment groups, early after PCI and pre-discharge.

**4.4 Validity of LAD blood velocities and flow calculations**

**4.2 Applicability of sampling of LAD blood velocities**

units.

et al, 1993).

not needed.

Acute ST-Elevation Myocardial Infarction: A Trans-Thoracic Coronary Artery Doppler Study 69

evidenced by absence of early systolic flow reversal in subjects treated with epitifibatide; however, less severe dysfunction of the microcirculation was not different between the groups since diastolic deceleration times by Doppler, and myocardial blush grades were similar. Moreover, diastolic maximal blood velocities early after PCI were higher in patients treated with eptifibatide but similar later on. Eptfibatide treatment was associated with a tendency of larger diastolic blood flow through the LAD but not later. All these changes with eptifibatide treatment did not affect left ventricular systolic function which was similar

Transthoracic Doppler sampling of coronary blood velocities is not mentioned or not stressed sufficiently in most textbooks of echocardiography. Thus, the importance of this study is related not only to the treatment of patients with acute STEMI. We found that sampling of LAD blood velocities was possible in all the patients and at all occasions. We believe that sampling of LAD blood velocities should be applied widely and repeated when needed and in all echocardiographic studies. In fact, electrocardiographic recording is performed when patients have chest pain, and in a similar fashion, echocardiography and sampling of coronary blood velocity may be performed in such patients in coronary care

**4.3 Transthoracic Doppler sampling of LAD blood velocities and other methods**

Measurement of coronary flow velocities using Doppler wire and pressure recordings to assess severity of coronary artery and microcirculation are invasive procedures in addition to other disadvantage (Iliceto et al 1991; Erbel et al, 1991; Kozakova et al, 1994; Donohue et al, 1993; Miller et al, 1994; Di Carli et al, 1995). Normal peak diastolic velocities in the present study were similar to those reported previously by invasive Doppler flowires (Ofili

Trans-esophageal echocardiography visualizes only the proximal coronary arteries and Doppler sampling is feasible in less than 70% of patients (Joye et al, 1994; Kern et al 1995; Abizaid et al, 1998). Recent technologic advances in trans-thoracic echocardiography made Doppler sampling of coronary artery velocities possible (Voci et al, 1998; Caiati et al, 1999; Hildick-Smith et al, 2000; Higashiue et al, 2001; Pizzuto et al, 2001; Takeuchi et al, 2001). Contrast agents may enhance the detection rate of coronary velocities (Abizaid et al, 1998; Caiati et al, 1999), however, with increasing experience of the operator contrast agents are

The range of the value of LAD blood velocities and time velocity integrals (Sharif et al, 2010) is similar to those found through cardiac valves with similar reproducibility and applicability. The diameter of the LAD and of the colour jet of blood flow through the vessel is in the range of diameter of vena contracta of regurgitant jets through cardiac valves. Moreover, LAD-colour jet diameter is similar to that of proximal iso-velocity surfaces of regurgitant jets through the mitral valve, so it can be applied in a similar fashion and