**Part 3**

**Quality Control for Biotechnology** 

150 Modern Approaches To Quality Control

Wilkinson, Leland; APA Task Force on Statistical Inference (1999). "Statistical methods in

Yau SC, Bobrow M, Mathew CG, Abbs SJ (1996). "Accurate diagnosis of carriers of deletions

analysis". *J. Med. Genet.* 33 (7): 550–558. doi:10.1136/jmg.33.7.550. Zar, J.H. (1984) *Biostatistical Analysis.* Prentice Hall International, New Jersey. pp 43–45

604. doi:10.1037/0003-066X.54.8.594.

psychology journals: Guidelines and explanations". *American Psychologist* 54: 594–

and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage

**8** 

**Establishment and Quality Control Criteria** 

*Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing* 

Animal genetic resources top the list of the most fundamental properties for animal husbandry and medical sciences, play an irreplaceable role in human survival and social prosperity, constitute an invaluable substrate for scientific researches, and have an enormous bearing on sustainable development of economy. Biodiversity takes an underlying part in ecological balance. The havoc people wreaked on nature had been aggravating ever since agricultural civilization, accelerating the extinction of animal species and breed incomparable than gradual natural loss, which became more and more apparent upon the advent of industrial age. The statistics of Food and Agriculture Organization (FAO) in 1995 revealed that approximately 15% of the total 738 registered livestock and poultry breeds in Sub-Saharan Africa were on the brink of extinction. The situation has been aggravating ever since. To date, the proportion of livestock breed in danger has increased from 8% to 19%, while that of poultry has risen from 20% to 34%. Among the 1251 registered breeds in Asia, 10% are severely endangered. From 1995 to 1999, livestock breeds about to be extinct grew from 11% to 14%, and the proportion of poultry was 32% to 37%. Owing to economic pressure, some low yield breeds are being subjected to marketing elimination and shrinkage in population, for instance, the production of poultry and swine depends heavily on only a few breeds. The trend is extremely obvious in Eastern Europe, which is further worsened by political unstability. Similarly, sustaining intensification of animal husbandry makes the food production rely more and more on a small number of high yield breeds, thereby exacerbating the animal diversity crisis. In Latin America, the number of endangered breeds accounts for 20% of the whole. As was reported by the FAO in 2000, livestock and poultry throughout the earth are disappearing at the rate of 2 breeds per week. Worse still, 1350 breeds are next to imminent extinction. For all that matter, animal genetic resources are confronted with a progressive narrowing in diversity. Accordingly, it's

absolutely exigent to protect and preserve them with effective measures.

*Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, PR China* 

1 Weijun Guan, Xiangchen Li, Xiaohong He, Yabin Pu, Qianjun Zhao, Dapeng Jin, Shen Wu, Taofeng

**1. Introduction** 

Lu, Xiaohua Su, Chunyu Bai

**for Population Culture Collection -** 

**Resource Preservation** 

Yuehui Ma et al.1

*PR China* 

**Promising Strategy for Animal Genetic** 

## **Establishment and Quality Control Criteria for Population Culture Collection - Promising Strategy for Animal Genetic Resource Preservation**

Yuehui Ma et al.1

*Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing PR China* 

## **1. Introduction**

Animal genetic resources top the list of the most fundamental properties for animal husbandry and medical sciences, play an irreplaceable role in human survival and social prosperity, constitute an invaluable substrate for scientific researches, and have an enormous bearing on sustainable development of economy. Biodiversity takes an underlying part in ecological balance. The havoc people wreaked on nature had been aggravating ever since agricultural civilization, accelerating the extinction of animal species and breed incomparable than gradual natural loss, which became more and more apparent upon the advent of industrial age. The statistics of Food and Agriculture Organization (FAO) in 1995 revealed that approximately 15% of the total 738 registered livestock and poultry breeds in Sub-Saharan Africa were on the brink of extinction. The situation has been aggravating ever since. To date, the proportion of livestock breed in danger has increased from 8% to 19%, while that of poultry has risen from 20% to 34%. Among the 1251 registered breeds in Asia, 10% are severely endangered. From 1995 to 1999, livestock breeds about to be extinct grew from 11% to 14%, and the proportion of poultry was 32% to 37%. Owing to economic pressure, some low yield breeds are being subjected to marketing elimination and shrinkage in population, for instance, the production of poultry and swine depends heavily on only a few breeds. The trend is extremely obvious in Eastern Europe, which is further worsened by political unstability. Similarly, sustaining intensification of animal husbandry makes the food production rely more and more on a small number of high yield breeds, thereby exacerbating the animal diversity crisis. In Latin America, the number of endangered breeds accounts for 20% of the whole. As was reported by the FAO in 2000, livestock and poultry throughout the earth are disappearing at the rate of 2 breeds per week. Worse still, 1350 breeds are next to imminent extinction. For all that matter, animal genetic resources are confronted with a progressive narrowing in diversity. Accordingly, it's absolutely exigent to protect and preserve them with effective measures.

<sup>1</sup> Weijun Guan, Xiangchen Li, Xiaohong He, Yabin Pu, Qianjun Zhao, Dapeng Jin, Shen Wu, Taofeng Lu, Xiaohua Su, Chunyu Bai

*Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, PR China* 

Establishment and Quality Control Criteria

(Freshney 2000).

term storage (Jenkins, 1999).

**2.2 Trypan Blue exclusion test** 

hemocytometer (Qi et al., 2007).

(PDT) was calculated accordingly.

days as described by Doyle et al. (1990).

**2.5 Karyotyping and chromosomal indices** 

contrast microscopy to detect cytopathogenesis (Hay, 1992).

**2.3 Growth dynamics** 

**2.4 Microbial detection** 

staining.

brought back to the laboratory for further experiments.

for Population Culture Collection - Promising Strategy for Animal Genetic Resource Preservation 155

Modified Eagle's Medium (MEM, Gibco) (for poultry breeds) medium supplemented with ampicillin (100 U/ml) and streptomycin (100 μg/ml). The samples were immediately

The tissues were rinsed and chopped to 1 mm3 pieces, which were then plated onto the bottom of a tissue culture flask in an incubator at 37°C with 5% CO2 for 2 h until the tissue pieces spontaneously adhered to the flask surface, and then DMEM/MEM containing 10% fetal bovine serum (FBS, Gibco) was added. Cells were harvested when they reached 80%–90% confluence and were passaged into more flasks at the ratio of 1:2 or 1:3

After three passages, the cells in logarithmic phase were harvested and resuspended at the concentration of 4 ×106/ml in cryogenic media containing 40% DMEM/MEM, 50% FBS and 10% DMSO (Sigma), aliquoted into cryovials, and kept at 4°C for 20-30 min to equilibrize the DMSO. Then they were put into a programmed cryopreservation system with controllable temperature dropping rate, and finally transferred to liquid nitrogen for long-

Viabilities before cryopreservation and after resuscitation were determined using Trypan blue exclusion test. The cells were plated in 6-well plates at 104/well and counted with a

According to the method of Gu et al. (Gu et al., 2006) and Ikeda Y et al. (Ikeda Y, 1990), cells at the concentration of 1.5×104/ml were plated into 24-well plates. Three wells were counted each day until the plateau phase. Based on the numbers, the mean values of cell density were then calculated and plotted against the culture time. The population doubling time

 Tests for contamination with bacteria, fungi and yeasts: the cells were cultured in antibiotic free media. Bacterial, fungal and yeast contamination was assessed within 3

Test for viruses: the cells were subjected to Hay's hemadsorption protocol using phase-

 Test for mycoplasmas: cells were cultured in antibiotic free media for at least 1 week, and then fixed and stained with Hoechst 33258 (Sigma) according to the method of Masover and Becker (1998) and Freshney (2000). The ELISA Mycoplasma Detection kit (Roche, Lewes, East Sussex, UK) was used to confirm the results of the DNA fluorescent

The cells were harvested upon 80%–90% confluence. Microslide preparation and chromosome staining were performed as described by Suemori et al. (2006). Fifty to 100 spreads were sampled for counting chromosome numbers of diploid cells. There are three important parameters for chromosomal analysis, i.e. relative length, arm ratio, and centromere index, which were determined according to the protocol of Kawarai et al. (2006).

In the context of biodiversity crisis, no country around the world stands indifferent regarding the preservation of animal genetic resources. The contest for genetic resources, the basis for animal husbandry and sustainable development, is nearly incandescent.

Scientists from all around the world have been endeavouring to preserve and to make use of animal genetic resources, which are now stored in terms of individual animals, semen, embryos, genomic libraries, cDNA libraries, etc. Unfortunately, these alternative methods remain problematic for several reasons: i) endangered species and breeds are incredibly diversified, rendering the costs for preservation of individual animals unaffordable; ii) some core techniques for semen and embryos are still immature; iii) genomic DNA or organ preservation are not applicable in long term because of their finite proliferative capabilities; iv) genomic libraries and cDNA libraries are not the basic unit of cellular activities, moreover, their biological function can only be represented in transgenic techniques. Accordingly, preservation of animal genetic resources in terms of somatic cells is essentially an effective and appealing procedure to protect vulnerable mammalian and avian species, as well as all other kinds of animals. In comparison, somatic cell line, by virtue of its low costs, large capacity, convenient application, proliferative potential and so on, is supposed to be a promising strategy for storage of animal genetic resources.

Consistent with this notion, culture collections of animal materials, mainly identified cell lines, have been established and developed over time. American Type Culture Collection (ATCC), for instance, endeavours to isolate, collect, preserve and supply reliable cell lines, with its all identified type culture applicable for register, preservation, instant use, and even commercial provision. European Collection of Animal Cell Culture (ECACC), jointly run by England and Switzerland, has collected and identified some 1600 cell lines. Established in 1986, Kunming Institute of Zoology, Chinese Academy of Sciences has collected cell stains, tissues and germ cells of numerous precious species of wild life.

The lab of Animal Genetic Resources, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, by virtue of its solid technical platform, advantages in animal genetic resources, and persistent efforts in the past decade or so, has established Animal Population Culture Collection of China (APCCC), the most massive animal population culture collection in the world, comprising somatic cell lines with different tissue origins, including ear margin, blood, kidney, heart, brain, muscle, stomach and so on, from 88 animal breeds primarily in China, *e.g.* chicken (*Gallus gallus*), duck (*Anseriformes Anatidae*), goose (*Anser cygnoides orientalis*), sandpiper (*Scolopacidae*), cattle (*Bos taurus*), sheep (*Ovis aries*), goat (*Capra hircus*), pig (*Suidae*), ferret-polecat (*Mustela Pulourius Furot*), raccoon dog (*Nyctereutes procyonoides*), horse (*Equus caballus*), mule, red deer (*Cervus elaphus*), sika deer (*Cervus nippon*), fox (*Vulpinae*), wolf (*Canis lupus*), bactrian camel (*Camelus bactrianus*), tiger (*Panthera tigris*), *etc*., and further endeavours to conserve other animal species and breeds in the world. A well-orchestrated series of standardized technical lines and quality control criteria is steadily ameliorated in this process.

This chapter will introduce the preservation of animal genetic resources in terms of somatic cells and the quality control criteria by detailed experimental description and technical line.

#### **2. Isolation,** *in vitro* **culture and identification of somatic cell lines**

#### **2.1 Sampling and cell culture**

Tissue pieces (about 1 cm3 in size) were sampled from animals and placed into sterile tubes containing Dulbecco's modified Eagle's medium (DMEM, Gibco) (for livestock breeds)/ Modified Eagle's Medium (MEM, Gibco) (for poultry breeds) medium supplemented with ampicillin (100 U/ml) and streptomycin (100 μg/ml). The samples were immediately brought back to the laboratory for further experiments.

The tissues were rinsed and chopped to 1 mm3 pieces, which were then plated onto the bottom of a tissue culture flask in an incubator at 37°C with 5% CO2 for 2 h until the tissue pieces spontaneously adhered to the flask surface, and then DMEM/MEM containing 10% fetal bovine serum (FBS, Gibco) was added. Cells were harvested when they reached 80%–90% confluence and were passaged into more flasks at the ratio of 1:2 or 1:3 (Freshney 2000).

After three passages, the cells in logarithmic phase were harvested and resuspended at the concentration of 4 ×106/ml in cryogenic media containing 40% DMEM/MEM, 50% FBS and 10% DMSO (Sigma), aliquoted into cryovials, and kept at 4°C for 20-30 min to equilibrize the DMSO. Then they were put into a programmed cryopreservation system with controllable temperature dropping rate, and finally transferred to liquid nitrogen for longterm storage (Jenkins, 1999).

## **2.2 Trypan Blue exclusion test**

Viabilities before cryopreservation and after resuscitation were determined using Trypan blue exclusion test. The cells were plated in 6-well plates at 104/well and counted with a hemocytometer (Qi et al., 2007).

## **2.3 Growth dynamics**

154 Modern Approaches To Quality Control

In the context of biodiversity crisis, no country around the world stands indifferent regarding the preservation of animal genetic resources. The contest for genetic resources, the

Scientists from all around the world have been endeavouring to preserve and to make use of animal genetic resources, which are now stored in terms of individual animals, semen, embryos, genomic libraries, cDNA libraries, etc. Unfortunately, these alternative methods remain problematic for several reasons: i) endangered species and breeds are incredibly diversified, rendering the costs for preservation of individual animals unaffordable; ii) some core techniques for semen and embryos are still immature; iii) genomic DNA or organ preservation are not applicable in long term because of their finite proliferative capabilities; iv) genomic libraries and cDNA libraries are not the basic unit of cellular activities, moreover, their biological function can only be represented in transgenic techniques. Accordingly, preservation of animal genetic resources in terms of somatic cells is essentially an effective and appealing procedure to protect vulnerable mammalian and avian species, as well as all other kinds of animals. In comparison, somatic cell line, by virtue of its low costs, large capacity, convenient application, proliferative potential and so on, is supposed to be a

Consistent with this notion, culture collections of animal materials, mainly identified cell lines, have been established and developed over time. American Type Culture Collection (ATCC), for instance, endeavours to isolate, collect, preserve and supply reliable cell lines, with its all identified type culture applicable for register, preservation, instant use, and even commercial provision. European Collection of Animal Cell Culture (ECACC), jointly run by England and Switzerland, has collected and identified some 1600 cell lines. Established in 1986, Kunming Institute of Zoology, Chinese Academy of Sciences has collected cell stains,

The lab of Animal Genetic Resources, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, by virtue of its solid technical platform, advantages in animal genetic resources, and persistent efforts in the past decade or so, has established Animal Population Culture Collection of China (APCCC), the most massive animal population culture collection in the world, comprising somatic cell lines with different tissue origins, including ear margin, blood, kidney, heart, brain, muscle, stomach and so on, from 88 animal breeds primarily in China, *e.g.* chicken (*Gallus gallus*), duck (*Anseriformes Anatidae*), goose (*Anser cygnoides orientalis*), sandpiper (*Scolopacidae*), cattle (*Bos taurus*), sheep (*Ovis aries*), goat (*Capra hircus*), pig (*Suidae*), ferret-polecat (*Mustela Pulourius Furot*), raccoon dog (*Nyctereutes procyonoides*), horse (*Equus caballus*), mule, red deer (*Cervus elaphus*), sika deer (*Cervus nippon*), fox (*Vulpinae*), wolf (*Canis lupus*), bactrian camel (*Camelus bactrianus*), tiger (*Panthera tigris*), *etc*., and further endeavours to conserve other animal species and breeds in the world. A well-orchestrated series of standardized technical lines and quality control criteria

This chapter will introduce the preservation of animal genetic resources in terms of somatic cells and the quality control criteria by detailed experimental description and technical line.

Tissue pieces (about 1 cm3 in size) were sampled from animals and placed into sterile tubes containing Dulbecco's modified Eagle's medium (DMEM, Gibco) (for livestock breeds)/

**2. Isolation,** *in vitro* **culture and identification of somatic cell lines** 

basis for animal husbandry and sustainable development, is nearly incandescent.

promising strategy for storage of animal genetic resources.

tissues and germ cells of numerous precious species of wild life.

is steadily ameliorated in this process.

**2.1 Sampling and cell culture** 

According to the method of Gu et al. (Gu et al., 2006) and Ikeda Y et al. (Ikeda Y, 1990), cells at the concentration of 1.5×104/ml were plated into 24-well plates. Three wells were counted each day until the plateau phase. Based on the numbers, the mean values of cell density were then calculated and plotted against the culture time. The population doubling time (PDT) was calculated accordingly.

### **2.4 Microbial detection**


### **2.5 Karyotyping and chromosomal indices**

The cells were harvested upon 80%–90% confluence. Microslide preparation and chromosome staining were performed as described by Suemori et al. (2006). Fifty to 100 spreads were sampled for counting chromosome numbers of diploid cells. There are three important parameters for chromosomal analysis, i.e. relative length, arm ratio, and centromere index, which were determined according to the protocol of Kawarai et al. (2006).

Establishment and Quality Control Criteria

for Population Culture Collection - Promising Strategy for Animal Genetic Resource Preservation 157

exponential phase. As the cell density increased, proliferation slows down due to contact inhibition. The cells subsequently enter the plateau phase and begin to degenerate. The growth curves before cryopreservation is generally consistent with that after resuscitation.

Fig. 1. Morphology of somatic cells of White Ear Lobe chicken, Luxi cattle, Jingning Black Grey goat, Mongolian horse and Siberian tiger in primary culture, before cryopreservation

and after resuscitation.

## **2.6 Isoenzyme analysis**

Isoenzyme profiles of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) were identified by vertical slab non-continuous polyacrylamide gel electrophoresis (PAGE). The cells were harvested, pelleted and resuspended in protein extraction solution (0.9% Triton X-100, 0.06 mmol/L NaCl:EDTA in volume ratio of 1:15) at the density of 5×107 cells/ml. Then the suspension was centrifuged and the supernatant was stored in aliquots at -70°C. Isovolumic 40% (m/v) sucrose and 2.5 ml loading buffer were added to each sample to get loading solution (Zhongxiao and Shuzheng, 1999). Subsequently the electrophoresis was performed at the voltage of 120 V. When the bromophenol blue migrated into the separation gel, the electrophoresis voltage was modified to 220 V. The electrophoresis was terminated when the bromophenol blue migrated to the bottom (0.5 - 1 cm to the margin). Different mobility patterns were differentiated by the relative mobility front (RFs), which was calculated as the ratio of the migration distances of the isozyme bands to that of the bromophenol blue.

## **2.7 Expression of exogenous genes**

According to the method of Tsuchiya et al. (2002), the fluorescent plasmids pEGFP-C1, pEGFP-N3, pEYFP-N1, pDsRed1-N1,pECFP-N1 and pECFP-mito were transfected into the cells with Lipofectamine™ 2000 transfection reagent (Invitrogen Corp., Carlsbad, CA). The plasmid DNA (μg) to Lipofectamine 2000 (μl) ratio was 1:3. After 8 h, the serum-free transfection media were replaced with complete media. To evaluate the transfection efficiency, the cells were observed under a confocal microscope (Nikon TE-2000-E, Japan)at 24 h, 48 h and 72 h after transfection, respectively. The test data were subjected to multiple comparisons to analyze statistical difference. For each sample, images were captured from ten visual fields, and the total and positive cells were counted in each field to determine the transfection efficiency.

## **3. Results**

## **3.1 Morphological observation**

The somatic cells sprouting from tissue explant pieces grew rapidly and migrated from the tissues with a different time and speed according to the species origin. Fibroblasts were initially mingled with epithelial cells, but the fibroblasts, by virtue of their proliferative superiority, would outgrow the epithelial cells gradually after 2-3 passages, and prevail in the population (Fig. 1). Then, purified fibroblast lines were obtained. The cells had fibrous contour with plump cytoplasm, and during growth they were morphologically fibroblastlike with radiating, flame-like or whirlpool migrating patterns. The cells were then subjected to programmed cryopreservation. Trypan blue exclusion test showed non-significant difference (*P*>0.05) in viability upon proper freezing procedures, and resuscitated cells displayed good morphology and proliferative activities.

## **3.2 Growth dynamics**

The growth curves of the somatic cell lines in APCCC before cryopreservation and after cryopreservation displayed a typical "S" shape (Fig. 2) and the PDT was approximately 24 h to 48 h, which varies from species to species or even between subspecies. There is usually a lag time or latency phase of about 24 h to 48 h after plating, corresponding to the adaptation and recovery of the cells from trypsinization, and then the cells proliferate rapidly and enter

Isoenzyme profiles of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) were identified by vertical slab non-continuous polyacrylamide gel electrophoresis (PAGE). The cells were harvested, pelleted and resuspended in protein extraction solution (0.9% Triton X-100, 0.06 mmol/L NaCl:EDTA in volume ratio of 1:15) at the density of 5×107 cells/ml. Then the suspension was centrifuged and the supernatant was stored in aliquots at -70°C. Isovolumic 40% (m/v) sucrose and 2.5 ml loading buffer were added to each sample to get loading solution (Zhongxiao and Shuzheng, 1999). Subsequently the electrophoresis was performed at the voltage of 120 V. When the bromophenol blue migrated into the separation gel, the electrophoresis voltage was modified to 220 V. The electrophoresis was terminated when the bromophenol blue migrated to the bottom (0.5 - 1 cm to the margin). Different mobility patterns were differentiated by the relative mobility front (RFs), which was calculated as the ratio of the migration distances of the isozyme bands to that of the

According to the method of Tsuchiya et al. (2002), the fluorescent plasmids pEGFP-C1, pEGFP-N3, pEYFP-N1, pDsRed1-N1,pECFP-N1 and pECFP-mito were transfected into the cells with Lipofectamine™ 2000 transfection reagent (Invitrogen Corp., Carlsbad, CA). The plasmid DNA (μg) to Lipofectamine 2000 (μl) ratio was 1:3. After 8 h, the serum-free transfection media were replaced with complete media. To evaluate the transfection efficiency, the cells were observed under a confocal microscope (Nikon TE-2000-E, Japan)at 24 h, 48 h and 72 h after transfection, respectively. The test data were subjected to multiple comparisons to analyze statistical difference. For each sample, images were captured from ten visual fields, and the total and positive cells were counted in each field to determine the

The somatic cells sprouting from tissue explant pieces grew rapidly and migrated from the tissues with a different time and speed according to the species origin. Fibroblasts were initially mingled with epithelial cells, but the fibroblasts, by virtue of their proliferative superiority, would outgrow the epithelial cells gradually after 2-3 passages, and prevail in the population (Fig. 1). Then, purified fibroblast lines were obtained. The cells had fibrous contour with plump cytoplasm, and during growth they were morphologically fibroblastlike with radiating, flame-like or whirlpool migrating patterns. The cells were then subjected to programmed cryopreservation. Trypan blue exclusion test showed non-significant difference (*P*>0.05) in viability upon proper freezing procedures, and resuscitated cells

The growth curves of the somatic cell lines in APCCC before cryopreservation and after cryopreservation displayed a typical "S" shape (Fig. 2) and the PDT was approximately 24 h to 48 h, which varies from species to species or even between subspecies. There is usually a lag time or latency phase of about 24 h to 48 h after plating, corresponding to the adaptation and recovery of the cells from trypsinization, and then the cells proliferate rapidly and enter

**2.6 Isoenzyme analysis** 

bromophenol blue.

transfection efficiency.

**3.2 Growth dynamics** 

**3.1 Morphological observation** 

displayed good morphology and proliferative activities.

**3. Results** 

**2.7 Expression of exogenous genes** 

exponential phase. As the cell density increased, proliferation slows down due to contact inhibition. The cells subsequently enter the plateau phase and begin to degenerate. The growth curves before cryopreservation is generally consistent with that after resuscitation.

Fig. 1. Morphology of somatic cells of White Ear Lobe chicken, Luxi cattle, Jingning Black Grey goat, Mongolian horse and Siberian tiger in primary culture, before cryopreservation and after resuscitation.

Establishment and Quality Control Criteria

**3.4 Karyotyping and chromosomal indices** 

for Population Culture Collection - Promising Strategy for Animal Genetic Resource Preservation 159

Diploid cells of a given species possess a characteristic chromosome number, shape and structure, which remain very stable in normal cells (Fig. 4). Therefore, karyotype analysis is a major method for distinguishing normal cells from mutants. The percentage of diploid cells tends to decrease with increasing passage number. However, the fact that the diploid

proportion is normally higher than 90% warrants the hereditary stability.

Fig. 4. Chromosome at metaphase (left) and karyotype (right).

Fig. 2. Growth dynamics. Growth curves of (A) Siberian Tiger fibroblast line, and (B) Luxi Cattle ear marginal fibroblast line before cryopreservation and after resuscitation. A representative growth curve consist of latency phase, exponential phase, plateau phase and decline phase.

#### **3.3 Microbial detection**

In a sharp contrast with infections by bacteria, fungi and yeasts, characterized by turbidity, colony or hypha, which can be observed by unaided eyes, the mycoplasma contamination, usually undistinguishable, is only accompanied with slightly slower growth and increased cell fragmentation. As a result, Hoechst 33258 staining or molecular assays are required further. Would there be abundant punctiform and filiform blue fluorescence in the nucleoli, it could be concluded that the cells were contaminated by mycoplasmas (Fig. 3B).

In APCCC, all the somatic cells are subjected to microbial detection prior to cryopreservation to ensure they are free of bacterial, fungal and yeast contamination. No microorganisms were observed in the culture media. No viruses were indicated either by the hemadsorption test. Mycoplasma testing by both the ELISA detection kit and Hoechst 33258 staining (Fig. 3A) was negative. Through microbial detection, the safety and reliability of future commercial and experimental applications of the cell lines are to a large extent ensured.

Fig. 3. Hoechst 33258 staining for (A) the detection of mycoplasma in the Siberian tiger fibroblasts; and (B) a positive control of mycoplasma contamination.

## **3.4 Karyotyping and chromosomal indices**

158 Modern Approaches To Quality Control

Fig. 2. Growth dynamics. Growth curves of (A) Siberian Tiger fibroblast line, and (B) Luxi Cattle ear marginal fibroblast line before cryopreservation and after resuscitation. A representative growth curve consist of latency phase, exponential phase, plateau phase and

In a sharp contrast with infections by bacteria, fungi and yeasts, characterized by turbidity, colony or hypha, which can be observed by unaided eyes, the mycoplasma contamination, usually undistinguishable, is only accompanied with slightly slower growth and increased cell fragmentation. As a result, Hoechst 33258 staining or molecular assays are required further. Would there be abundant punctiform and filiform blue fluorescence in the nucleoli,

In APCCC, all the somatic cells are subjected to microbial detection prior to cryopreservation to ensure they are free of bacterial, fungal and yeast contamination. No microorganisms were observed in the culture media. No viruses were indicated either by the hemadsorption test. Mycoplasma testing by both the ELISA detection kit and Hoechst 33258 staining (Fig. 3A) was negative. Through microbial detection, the safety and reliability of future commercial and experimental applications of the cell lines are to a large extent

Fig. 3. Hoechst 33258 staining for (A) the detection of mycoplasma in the Siberian tiger

fibroblasts; and (B) a positive control of mycoplasma contamination.

it could be concluded that the cells were contaminated by mycoplasmas (Fig. 3B).

decline phase.

ensured.

**3.3 Microbial detection** 

Diploid cells of a given species possess a characteristic chromosome number, shape and structure, which remain very stable in normal cells (Fig. 4). Therefore, karyotype analysis is a major method for distinguishing normal cells from mutants. The percentage of diploid cells tends to decrease with increasing passage number. However, the fact that the diploid proportion is normally higher than 90% warrants the hereditary stability.

Fig. 4. Chromosome at metaphase (left) and karyotype (right).

Establishment and Quality Control Criteria

Table 2. Chromosomal parameters of Siberian tiger.

contamination between different cell lines.

**3.6 Expression of exogenous genes** 

**3.5 Isoenzyme analysis** 

for Population Culture Collection - Promising Strategy for Animal Genetic Resource Preservation 161

Chromosome No. Relative lenth (%) Centromere type 1 10.13±0.93 SM 2 9.48±1.05 M 3 8.43±0.92 ST 4 6.65±0.85 M 5 6.31±0.81 SM 6 5.85±0.75 ST 7 5.66±0.70 M 8 5.34±0.67 SM 9 5.22±0.71 SM 10 4.47±0.61 SM 11 4.11±0.75 M 12 3.51±0.66 SM 13 3.54±0.44 M 14 3.34±0.69 T 15 3.18±0.67 T 16 2.84±0.25 SM 17 2.43±0.32 SM 18 2.25±0.64 M X 5.54±0.62 M

Isoenzyme profiles of at least 5 kinds of animals were analysed simultaneously. Each kind of animal has its specific bands. The LDH bands obtained from Siberian tiger, for instance, were compared with those of other species or breeds, and five breed-specific isoenzyme bands (LDH-1, -2, -3, -4, -5) were observed (Fig. 5A). Enzymatic activities were in the order of LDH-3, LDH-2, LDH-4, LDH-5, LDH-1. LDH-2, LDH-3 and LDH-4 were dominant, while LDH-1 and LDH-5 were scarcely observable. In the MDH patterns of Siberian tiger and other breeds, two MDH bands (s-MDH, m-MDH) were observed (Fig. 5B), with the m-MDH band near the cathode and the s-MDH band (comprise two subbands but hardly identified) near the anode (Fig. 5B). Similar activity was seen from both m-MDH and s-MDH. There were significant differences in the isoenzyme patterns of LDH and MDH between the Siberian tiger fibroblasts and other cell lines in APCCC. These animals have their distinctive bands with different relative mobility. These results showed that there was no cross-

Six fluorescent protein genes with stable structures, high expression levels and speciesindependent efficiency (Baird et al., 2000) have been used as marker genes to observe the expression, distribution and function of target proteins in live cells and organisms (Heim et al., 1995; Genyang et al., 2003). At APCCC, the 6 kinds of fluorescent genes were introduced into the preserved cells to evaluate the expressibility of exogenous genes. Positive cells were usually the most abundant and with the strongest fluorescence at 24 h-48 h after transfection. While the transfection efficiency decreased, strong expression levels were observed after a week, indicating that the exogenous genes can be replicated, transcribed,


Note: Relative lenth, 1.0-1.6, Metacentric chromosome (M); 1.7-2.9, Submetacentric chromosome (SM); 3.0-6.0 Subtelocentric chromosome (ST); ≥7.0 Telocentric chromosome (T).

Table 1. Chromosomal parameters of White ear lobe chicken(♀).

The chromosome number of Luxi cattle was 2n = 60, comprising 58 autosomes and two sex chromosomes, XY or XX. All the autosomes are acrocentric, and only the two sex chromosomes (XY) were submetacentric (Table 1). The chromosome numbers were counted for 100 spreads of passages 1, 3 and 4 respectively, and the frequencies of cells with 2n = 60 were 92.2%, 91.6% and 90.7% accordingly.

The chromosome number of Siberian tiger is 2n=38, consisting of 36 autosomes and two sex chromosomes, XY or XX. The karyotype composition of the Siberian tiger is 12 (M) + 16 (SM) + 4 (ST) + 4 (T), XY (M, M) (Table 2). The chromosome numbers were counted for 100 spreads of passages 1, 3 and 4 respectively, and the frequencies of cells with 2n=38 were 91.6%, 91.2% and 90.2% accordingly.


Table 2. Chromosomal parameters of Siberian tiger.

## **3.5 Isoenzyme analysis**

160 Modern Approaches To Quality Control

Chromosome number Relative length (%) Centromere type 1 5.58 ± 0.26 T 2 5.12 ± 0.16 T 3 4.68 ± 0.34 T 4 4.49 ± 0.41 T 5 4.23 ± 012 T 6 4.05 ± 0.45 T 7 3.87 ± 0.38 T 8 3.86 ± 0.57 T 9 3.81 ± 0.04 T 10 3.76 ± 0.22 T 11 3.61 ± 0.11 T 12 3.56 ± 0.19 T 13 3.41 ± 0.33 T 14 3.36 ± 0.20 T 15 3.27 ± 0.41 T 16 3.26 ± 0.32 T 17 3.01 ± 0.09 T 18 2.97 ± 0.19 T 19 2.97 ± 0.06 T 20 2.71 ± 0.31 T 21 2.70 ± 0.24 T 22 2.60 ± 0.12 T 23 2.58 ± 0.27 T 24 2.21 ± 0.19 T 25 2.14 ± 0.22 T 26 2.09 ± 0.53 T 27 2.07 ± 0.10 T 28 1.85 ± 0.35 T 29 1.75 ± 0.32 T X 4.47 ± 0.11 SM Note: Relative lenth, 1.0-1.6, Metacentric chromosome (M); 1.7-2.9, Submetacentric chromosome (SM);

3.0-6.0 Subtelocentric chromosome (ST); ≥7.0 Telocentric chromosome (T). Table 1. Chromosomal parameters of White ear lobe chicken(♀).

were 92.2%, 91.6% and 90.7% accordingly.

91.6%, 91.2% and 90.2% accordingly.

The chromosome number of Luxi cattle was 2n = 60, comprising 58 autosomes and two sex chromosomes, XY or XX. All the autosomes are acrocentric, and only the two sex chromosomes (XY) were submetacentric (Table 1). The chromosome numbers were counted for 100 spreads of passages 1, 3 and 4 respectively, and the frequencies of cells with 2n = 60

The chromosome number of Siberian tiger is 2n=38, consisting of 36 autosomes and two sex chromosomes, XY or XX. The karyotype composition of the Siberian tiger is 12 (M) + 16 (SM) + 4 (ST) + 4 (T), XY (M, M) (Table 2). The chromosome numbers were counted for 100 spreads of passages 1, 3 and 4 respectively, and the frequencies of cells with 2n=38 were Isoenzyme profiles of at least 5 kinds of animals were analysed simultaneously. Each kind of animal has its specific bands. The LDH bands obtained from Siberian tiger, for instance, were compared with those of other species or breeds, and five breed-specific isoenzyme bands (LDH-1, -2, -3, -4, -5) were observed (Fig. 5A). Enzymatic activities were in the order of LDH-3, LDH-2, LDH-4, LDH-5, LDH-1. LDH-2, LDH-3 and LDH-4 were dominant, while LDH-1 and LDH-5 were scarcely observable. In the MDH patterns of Siberian tiger and other breeds, two MDH bands (s-MDH, m-MDH) were observed (Fig. 5B), with the m-MDH band near the cathode and the s-MDH band (comprise two subbands but hardly identified) near the anode (Fig. 5B). Similar activity was seen from both m-MDH and s-MDH. There were significant differences in the isoenzyme patterns of LDH and MDH between the Siberian tiger fibroblasts and other cell lines in APCCC. These animals have their distinctive bands with different relative mobility. These results showed that there was no crosscontamination between different cell lines.

#### **3.6 Expression of exogenous genes**

Six fluorescent protein genes with stable structures, high expression levels and speciesindependent efficiency (Baird et al., 2000) have been used as marker genes to observe the expression, distribution and function of target proteins in live cells and organisms (Heim et al., 1995; Genyang et al., 2003). At APCCC, the 6 kinds of fluorescent genes were introduced into the preserved cells to evaluate the expressibility of exogenous genes. Positive cells were usually the most abundant and with the strongest fluorescence at 24 h-48 h after transfection. While the transfection efficiency decreased, strong expression levels were observed after a week, indicating that the exogenous genes can be replicated, transcribed,

Establishment and Quality Control Criteria

at 72 h after transfection.

**4. Conclusion** 

for Population Culture Collection - Promising Strategy for Animal Genetic Resource Preservation 163

Fig. 7. The expression and distribution of pEGFP-C1, pEGFP-N3, pDsRed1-N1 and pEYFP-N1 in White Ear Lobe chicken fibroblasts (×40). A, B, C and D are the expression of pEGFP-C1, pEGFP-N3, pDsRed1-N1 and pEYFP-N1 at 24 h; E, F, G and H at 48 h; and I, J, K and L

Animal resources, a fundamental respect of agriculture and industry in close correlation with production and social stability, supply human beings with meat, eggs, milk, furs, medicinal materials, products for athletic and ornamental purposes, etc. In most developed countries, scalization of animal husbandry has restricted animal feeding to high yield breeds or crossbreeds with an intensified operating system, greatly compromising the diversity of local animal breeds. In the meanwhile, despite the abundance of animal genetic resources in developing countries, the lack of efficient preservation strategies and blind introduction of exotic breeds for hybridization also has reduced the animal variety. Emerging evidence has revealed that owing to the interference from human activities, species extinction has sped up for about 1000 fold, 100 million times faster than speciation, or in other words, 1 species per day. The total 7176 livestock and poultry breeds throughout the earth are disappearing

Species extinction signifies a perpetual loss of the precious hereditary information, and will be an irreparable defeat of world genetic resources and biological theoretical repositories. Haven't the genetic resources been preserved in any forms before their extinction, not only the genetic resources will be lost evermore, but also it becomes impossible to investigate the

at the rate of 2 per week, and 690 are on the edge of extinction.

Fig. 5. LDH zymotype and MDH zymotype of several cell lines. A, SDS–PAGE electrophoresis of LDHs, from up to down, there were LDHs-1, 2, 3, 4 and 5. Panel A: 1,2 Simmental cattle, 3,4 Zhiwei goat, 5,6 Jining black goat, 7,8 Mongolian horse, 9,10 Bengal tiger, and 11,12 Siberian tiger; Panel B, MDHs from up to down were mMDH and sMDH. 1 Siberian tiger, 2 Bengal tiger, 3 Large white pig, 4 Songliao Black pig, 5 Jining Black goat, 6 Mongolian sheep, 7 Saf sheep, 8 Simmental cattle.

translated and modified within the cells. The transfected cells were not significantly less viable than the control cells (*P*>0.05), showing that the expression of fluorescent proteins had no obvious effect on the growth and proliferation of the transfected cells.

Fig. 6. Comparative figures of six fluorescent proteins in White Ear Lobe chicken fibroblasts at 24 h after transfection (×10). A, B, C, D, E and F were the transfection results of pEGFP-C1, pEGFP-N3, pEYFP-N1, pDsRed1-N1, pECFP-N1 and pECFP-mito, respectively.

Establishment and Quality Control Criteria for Population Culture Collection - Promising Strategy for Animal Genetic Resource Preservation 163

Fig. 7. The expression and distribution of pEGFP-C1, pEGFP-N3, pDsRed1-N1 and pEYFP-N1 in White Ear Lobe chicken fibroblasts (×40). A, B, C and D are the expression of pEGFP-C1, pEGFP-N3, pDsRed1-N1 and pEYFP-N1 at 24 h; E, F, G and H at 48 h; and I, J, K and L at 72 h after transfection.

## **4. Conclusion**

162 Modern Approaches To Quality Control

Fig. 5. LDH zymotype and MDH zymotype of several cell lines. A, SDS–PAGE

had no obvious effect on the growth and proliferation of the transfected cells.

Mongolian sheep, 7 Saf sheep, 8 Simmental cattle.

electrophoresis of LDHs, from up to down, there were LDHs-1, 2, 3, 4 and 5. Panel A: 1,2 Simmental cattle, 3,4 Zhiwei goat, 5,6 Jining black goat, 7,8 Mongolian horse, 9,10 Bengal tiger, and 11,12 Siberian tiger; Panel B, MDHs from up to down were mMDH and sMDH. 1 Siberian tiger, 2 Bengal tiger, 3 Large white pig, 4 Songliao Black pig, 5 Jining Black goat, 6

translated and modified within the cells. The transfected cells were not significantly less viable than the control cells (*P*>0.05), showing that the expression of fluorescent proteins

Fig. 6. Comparative figures of six fluorescent proteins in White Ear Lobe chicken fibroblasts at 24 h after transfection (×10). A, B, C, D, E and F were the transfection results of pEGFP-C1, pEGFP-N3, pEYFP-N1, pDsRed1-N1, pECFP-N1 and pECFP-mito, respectively.

Animal resources, a fundamental respect of agriculture and industry in close correlation with production and social stability, supply human beings with meat, eggs, milk, furs, medicinal materials, products for athletic and ornamental purposes, etc. In most developed countries, scalization of animal husbandry has restricted animal feeding to high yield breeds or crossbreeds with an intensified operating system, greatly compromising the diversity of local animal breeds. In the meanwhile, despite the abundance of animal genetic resources in developing countries, the lack of efficient preservation strategies and blind introduction of exotic breeds for hybridization also has reduced the animal variety. Emerging evidence has revealed that owing to the interference from human activities, species extinction has sped up for about 1000 fold, 100 million times faster than speciation, or in other words, 1 species per day. The total 7176 livestock and poultry breeds throughout the earth are disappearing at the rate of 2 per week, and 690 are on the edge of extinction.

Species extinction signifies a perpetual loss of the precious hereditary information, and will be an irreparable defeat of world genetic resources and biological theoretical repositories. Haven't the genetic resources been preserved in any forms before their extinction, not only the genetic resources will be lost evermore, but also it becomes impossible to investigate the

Establishment and Quality Control Criteria

PDT - population doubling time

**6. References** 

8140.

4827.

2427.

5050.

PAGE - polyacrylamide gel electrophoresis

pp. (11984-9). ISSN 0027-8424

ISBN 0199637962,Oxford.

0896035476, New Jersey.

6516, pp. (663-4).ISSN 1476-4687.

Liss,Inc., ISBN 0471348899, New York.

for Population Culture Collection - Promising Strategy for Animal Genetic Resource Preservation 165

Baird GS, Zacharias DA, & Tsien RY. (2000). Biochemistry, mutagenesis, and oligenerization

Doyle A., Hay R., & Kirsop B.E. (1990). Animal Cells (Living Resources for Biotechnology).

Freshney R.I. (2000). Culture of animal cells: a manual of basic technique(4th ed). Wiley-

Genyang Cheng, Xiangmei Cheng, & Xueyuan Bai. (2003). The gene construction and

Gu Y.P., Li H.Z., & Mik J. (2006). Phenotypic characterization of telomeraseimmortalized

Hay R.I., (1992). Cell line preservation and characterization. In: Animal Cell Culture: A

Heim R, Cubitt AB, & Tsien RY. (1995). Improved green fluorescence. Natur. Vol. 373, No.

Jenkins N. (1999). Animal cell biotechnology methods and protocols. Humana Press, ISBN

Kawarai S., Hashizaki K., & Kitao S. (2006). Establishment and characterization of primary

Ikeda Y, Ezaki M, Hayashi I, Yasuda D, Nakayama K, & Kono A. (1990). Establishment and

Masover G.K., Becker F.A., (1998). Detection of mycoplasmas in cell cultures by cultural

Qi Yitao, Tu Yiding, Yang Di, Chen Qian, Xiao Jun, & Chen Yiqiang. (2007). Cyclin A but not

Suemori H., Yasuchika K., Hasegawa K., Fujioka T., Tsuneyoshi N., & Nakatsuji N. (2006).

Res. Commun. Vol. 345, No. 3, pp. (926–932), ISSN 0006-291X.

(207–215, 217–226). Humana Press Inc., Totawa NJ.

210, No. 1, pp. (63-71). ISSN 1097-4652.

Cambridge University Press, ISBN 0521352231, Cambridge, UK.

of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci. Vol. 97, No. 22,

location in tubular epithelial cells of fused by green fluorescence protein and human kidney gene NaDC3. J Cell Biol. Vol. 25, No. 3, pp.(170-3). ISSN 1540-

primary non-malignant and malignant tumor-derived human prostate epithelial cell lines. Experimental Cell Research. Vol. 312, No. 6. Pp. (841–843). ISSN 0014-

Practical Approach (2nd ed). Freshney R.I. pp. (104–135), Oxford University Press,.

canine hepatocellular carcinoma cell lines producing alpha-fetoprotein. Vet. Immunol. Immunopathol. Vol. 113, No. 1-2, pp. (30–36), ISSN 0165-

characterization of human pancreatic adenocarcinoma cell line in tissue culture and the nude mouse. Jpn J Cancer Res. Vol. 81, No. 10, pp. (987-93). ISSN 0910-

methods. In: Methods in Molecular Biology, Miles R.J., Nicholas R.A.J., et al. pp.

cyclin D1 is essential in c-myc-modulated cell cycle progression. J Cell Physiol. Vol.

Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage. Biochem. Biophys.

unknown cell and molecular mechanisms, let alone to regenerate corresponding species via cloning technique. Therefore, it is exigent to employ practical measures to conserve endangered animal species.

Therefore, the APCCC has as yet preserved, for each cell line, 45-1250 cryovials of somatic cells from 30-212 individuals using primary explantation, serial passage and programmed cryopreservation. Each vial contains approximately 1.5×106 cells. The cells are cryopreserved within 3 passages, and are subjected to evaluation in terms of morphology, growth kinetics, viability, microbial detection, karyogram and isoenzyme analyses according to quality control standards of ATCC. The purified fibroblasts are fusiform, displaying flame-like or swirl-like patterns. The growth curves are sigmoidal with characteristic PDTs. Trypan blue exclusion test suggests that programmed preservation exerts a non-significant effect (*P*>0.05) on cell viability compared with that before freezing. Tests for bacteria, fungi, viruses and mycoplasmas are unanimously negative. Karyograms of peripheral blood lymphocytes and the in vitro cultured cells are photographed, according to which the mode of chromosome numbers are determined as that of the diploid cells, and indices including relative length, arm ratio and centromeric index and kinetochore type are calculated or dertermined. Comparison between peripheral blood lymphocytes and the in vitro cultured cells in respects of chromosome number and non-banding karyotype reveals no perceptible differences, manifesting the genetic stability of the cell lines established. Isoenzyme patterns of LDH and MDH are detected using vertical slab non-continuous PAGE assay, the breed specific bands of which rule out cross-contamination amongst the cell lines, and in the meanwhile further evince the hereditary stability.

Aforementioned results indicated that the APCCC conforms to all the ATCC criteria for somatic cell lines. In addition, plasmids of pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1, and pEYFP-N1 encoding the corresponding fluorescent proteins are transfected into the cells using lipofectin mediated protocol to study the expression of exogenous genes. By observation or detection of spatiotemporal expression of the fluorescent proteins, proliferation and growth of positive cells, apoptotic rate and viability, the ability and characteristics to accommodate exogenous genes are initiatively adopted as a constitutional index for cell line quality control.

The establishment of the APCCC is technically and theoretically conducive to preserve genetic resources of animals at somatic cell level, and definitively has a profound and longlasting influence on biological and biomedical research in the future. The quality control standards it's been adopting will definitely provide insights for future development of culture collections.

## **5. Abbreviations**

APCCC - Animal Population Culture Collection of China ATCC - American Type Culture Collection DMEM - Dulbecco's modified Eagle's medium ECACC - European Collection of Animal Cell Culture FAO - Food and Agriculture Organization LDH - lactic dehydrogenase MDH.- malic dehydrogenase MEM - modified Eagle's medium

PAGE - polyacrylamide gel electrophoresis PDT - population doubling time

## **6. References**

164 Modern Approaches To Quality Control

unknown cell and molecular mechanisms, let alone to regenerate corresponding species via cloning technique. Therefore, it is exigent to employ practical measures to conserve

Therefore, the APCCC has as yet preserved, for each cell line, 45-1250 cryovials of somatic cells from 30-212 individuals using primary explantation, serial passage and programmed cryopreservation. Each vial contains approximately 1.5×106 cells. The cells are cryopreserved within 3 passages, and are subjected to evaluation in terms of morphology, growth kinetics, viability, microbial detection, karyogram and isoenzyme analyses according to quality control standards of ATCC. The purified fibroblasts are fusiform, displaying flame-like or swirl-like patterns. The growth curves are sigmoidal with characteristic PDTs. Trypan blue exclusion test suggests that programmed preservation exerts a non-significant effect (*P*>0.05) on cell viability compared with that before freezing. Tests for bacteria, fungi, viruses and mycoplasmas are unanimously negative. Karyograms of peripheral blood lymphocytes and the in vitro cultured cells are photographed, according to which the mode of chromosome numbers are determined as that of the diploid cells, and indices including relative length, arm ratio and centromeric index and kinetochore type are calculated or dertermined. Comparison between peripheral blood lymphocytes and the in vitro cultured cells in respects of chromosome number and non-banding karyotype reveals no perceptible differences, manifesting the genetic stability of the cell lines established. Isoenzyme patterns of LDH and MDH are detected using vertical slab non-continuous PAGE assay, the breed specific bands of which rule out cross-contamination amongst the

Aforementioned results indicated that the APCCC conforms to all the ATCC criteria for somatic cell lines. In addition, plasmids of pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1, and pEYFP-N1 encoding the corresponding fluorescent proteins are transfected into the cells using lipofectin mediated protocol to study the expression of exogenous genes. By observation or detection of spatiotemporal expression of the fluorescent proteins, proliferation and growth of positive cells, apoptotic rate and viability, the ability and characteristics to accommodate exogenous genes are initiatively adopted as a

The establishment of the APCCC is technically and theoretically conducive to preserve genetic resources of animals at somatic cell level, and definitively has a profound and longlasting influence on biological and biomedical research in the future. The quality control standards it's been adopting will definitely provide insights for future development of

cell lines, and in the meanwhile further evince the hereditary stability.

constitutional index for cell line quality control.

APCCC - Animal Population Culture Collection of China

ATCC - American Type Culture Collection DMEM - Dulbecco's modified Eagle's medium ECACC - European Collection of Animal Cell Culture

FAO - Food and Agriculture Organization

LDH - lactic dehydrogenase MDH.- malic dehydrogenase MEM - modified Eagle's medium

culture collections.

**5. Abbreviations** 

endangered animal species.


**9** 

*USA*

**Genomic Microarray Quality Assurance** 

The use of microarray technology is revolutionizing the field of clinical cytogenetics. This new technology has transformed the cytogenetics laboratory by the adaptation of techniques that had previously been the province of molecular geneticists. Proficiency with these techniques is now a must for the modern cytogeneticist. This chapter will focus on quality assurance principles associated with microarray analysis for the diagnosis of copy number

Microarrays consist of a glass slide or other solid support on which small amounts of DNA ("probes" or "targets") are deposited and immobilized in an ordered fashion (DeRisi et al., 1996; Schena et al., 1995). Probes vary in size from oligonucleotides manufactured to represent genomic regions of interest (25-85 base pairs [bp] of DNA) to large genomic clones such as bacterial artificial chromosomes (BACs, 80-200 thousand base pairs [kb]). Analysis methodology for microarray-based comparative genomic hybridization (aCGH) is consistent regardless of the probe content. First, DNA is extracted from a test sample (e.g., blood, skin, cells from pregnancy). The patient DNA is labeled with a fluorescent dye, while a DNA from a normal control (reference) sample or pooled control samples is labeled with a different-colored fluorescent dye. The two genomic DNAs, test and reference, are then mixed together and applied to the array. Because the DNAs have been denatured, they are single strands; when applied to the array, the single-strand DNAs hybridize with the arrayed single-strand probes. Using a dual-color scanner, digital images are captured, and the relative fluorescence intensities of the hybridized labeled DNA probes are quantified. The fluorescence ratio of the test and reference hybridization signals is determined at different positions along the genome and provides information on the relative copy number of sequences in the test genome compared to the normal diploid genome, enabling the detection of submicroscopic chromosomal deletions and duplications at an unprecedented

Launching a new assay in the clinical setting requires an effective validation of the assay, clear protocols for use at the bench and clearly defined quality assurance (QA) and quality control (QC) procedures prior to the launch. Every laboratory must develop a strong Quality Management System (QMS) that is coordinated with the defined policies under regulatory bodies, such as CLIA '88 (Schwartz, 1999), College of American Pathologists and state regulating agencies. These agencies perform rigorous inspections and verify that a diagnostic laboratory follows defined principles to ensure quality patient care and correct diagnosis. This chapter covers many of the QA and QC principles identified and monitored

**1. Introduction** 

changes associated with genetic disease.

level (Beaudet & Belmont, 2008; Shaffer & Bejjani, 2009).

for laboratories offering microarray-based diagnostics.

Catherine D. Kashork, Lisa G. Shaffer and Kyle S. Sundin

*Signature Genomics Laboratories* 

Tsuchiya R., Yoshiki F., Kudo Y., & Morita M. (2002). Cell type-selective expression of green fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures. Brain Res. Vol. 956, No. 2, pp. (221–229), ISSN . 0006- 8993.

Zhongxiao He, & Shuzheng Zhang. (1999). Electrophoresis. Scientific Press; Beijing.

## **Genomic Microarray Quality Assurance**

Catherine D. Kashork, Lisa G. Shaffer and Kyle S. Sundin *Signature Genomics Laboratories* 

*USA*

#### **1. Introduction**

166 Modern Approaches To Quality Control

Tsuchiya R., Yoshiki F., Kudo Y., & Morita M. (2002). Cell type-selective expression of green

Zhongxiao He, & Shuzheng Zhang. (1999). Electrophoresis. Scientific Press; Beijing.

8993.

fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures. Brain Res. Vol. 956, No. 2, pp. (221–229), ISSN . 0006-

> The use of microarray technology is revolutionizing the field of clinical cytogenetics. This new technology has transformed the cytogenetics laboratory by the adaptation of techniques that had previously been the province of molecular geneticists. Proficiency with these techniques is now a must for the modern cytogeneticist. This chapter will focus on quality assurance principles associated with microarray analysis for the diagnosis of copy number changes associated with genetic disease.

> Microarrays consist of a glass slide or other solid support on which small amounts of DNA ("probes" or "targets") are deposited and immobilized in an ordered fashion (DeRisi et al., 1996; Schena et al., 1995). Probes vary in size from oligonucleotides manufactured to represent genomic regions of interest (25-85 base pairs [bp] of DNA) to large genomic clones such as bacterial artificial chromosomes (BACs, 80-200 thousand base pairs [kb]). Analysis methodology for microarray-based comparative genomic hybridization (aCGH) is consistent regardless of the probe content. First, DNA is extracted from a test sample (e.g., blood, skin, cells from pregnancy). The patient DNA is labeled with a fluorescent dye, while a DNA from a normal control (reference) sample or pooled control samples is labeled with a different-colored fluorescent dye. The two genomic DNAs, test and reference, are then mixed together and applied to the array. Because the DNAs have been denatured, they are single strands; when applied to the array, the single-strand DNAs hybridize with the arrayed single-strand probes. Using a dual-color scanner, digital images are captured, and the relative fluorescence intensities of the hybridized labeled DNA probes are quantified. The fluorescence ratio of the test and reference hybridization signals is determined at different positions along the genome and provides information on the relative copy number of sequences in the test genome compared to the normal diploid genome, enabling the detection of submicroscopic chromosomal deletions and duplications at an unprecedented level (Beaudet & Belmont, 2008; Shaffer & Bejjani, 2009).

> Launching a new assay in the clinical setting requires an effective validation of the assay, clear protocols for use at the bench and clearly defined quality assurance (QA) and quality control (QC) procedures prior to the launch. Every laboratory must develop a strong Quality Management System (QMS) that is coordinated with the defined policies under regulatory bodies, such as CLIA '88 (Schwartz, 1999), College of American Pathologists and state regulating agencies. These agencies perform rigorous inspections and verify that a diagnostic laboratory follows defined principles to ensure quality patient care and correct diagnosis. This chapter covers many of the QA and QC principles identified and monitored for laboratories offering microarray-based diagnostics.

Genomic Microarray Quality Assurance 169

molecule, and the promoter molecule is incorporated into the genomic DNA. As with preanalytic assessment of DNA, the quantity assessment of the labeling product is performed by assessing the 260/280 nm readings from the spectrophotometer. Laboratories should define post-labeling quantity requirements that indicate labeling efficiency. In addition to labeling efficiency as a quality indicator, visual assessment of the set-up of labeling product on the array should be considered. In the event that there are air bubbles or non-complete contact of array product to the hybridization area (Fig. 2), the quality of the microarray

Fig. 2. Microarray after hybridization with an air bubble. The air bubble creates an area of incomplete contact of array product to the hybridization area, which compromises the array. Post-analytic assessments may include average standard deviation (SD), intensity and background values. The SD value is the standard deviation of the normalized log2 intensity ratios for autosomal regions (excluding large copy number imbalances) and provides a measure of quality for aCGH experiments (Vermeesch et al., 2005). The SD value provides a quantitative metric that is relative to the overall noise on an array. As the overall noise of an aCGH experiment increases, so does the SD value. Our laboratory has established SD values

result may be compromised and should be documented.

## **2. Quality systems with strong monitoring for quality metrics**

Regulatory bodies require diagnostic laboratories to build a strong QMS (Deming, 2000). A robust QMS integrates the organization's processes, policies and procedures for total quality management. In the diagnostic laboratory industry, CAP and other accrediting bodies require defined metrics throughout all phases of testing, including pre-analytical, analytical and post-analytical.

For microarray technology, pre-analytical metrics may include assessment of DNA quality and yield. Each laboratory must define the ideal quality of DNA prior to implementing the assay into clinical testing. In our experience, a gel assessment that indicates clean genomic DNA free of RNase and degradation should lead to quality microarray results. If a DNA specimen has artifacts or appears to have degraded (Fig. 1), the laboratory should inform the client that results may be compromised because of DNA quality or obtain a new specimen from which to perform the analysis. In addition to a visual assessment of the DNA via gel electrophoresis, the laboratory should assess the DNA yield post-extraction. The quantity of DNA required in the analytic labeling phase of microarray analysis determines DNA yield requirements. Spectrophotometric assessment of DNA offers two indicators of quality DNA, including quantity and purity. A 260/280 nm reading indicates quantity, and a 260/230 nm reading indicates purity. These measurements are imperative for the downstream labeling process. Insufficient DNA quantity and quality (purity) will compromise successful microarray analysis. The spectrophotometer measures optical density (OD), which is the physical process of absorbing light. The OD, or absorbance, is calculated as a mathematical quantity. OD readings for pure DNA should measure at 1.8 (Sambrook & Russell, 2001). Our laboratory uses OD measurements from 1.4 to 1.8, although quality of labeling product can be compromised at the lower OD readings. There are many causes of poor yield, including compromised technique during extraction and poor sample quality (e.g., from increased age or exposure).

Fig. 1. Gel electrophoresis for the assessment of DNA degradation. Lane 1 has the molecular mass standard. Lanes 2 & 5 show high molecular weight samples that do not exhibit any signs of degradation. The two samples in lanes 3 & 4 show lower molecular weight DNA below the main high molecular weight bands in the other lanes. Degraded DNA typically leads to compromised array results.

Assessment of quality should be implemented throughout all phases of testing including the analytic phase. For aCGH, analytical metrics may include, but are not limited to, spectrophotometric assessment of the labeling product and the identification of labeling efficiency, which has an impact on results. Microarray analysis usually requires a dye incorporation using a random priming method. The dyes are tagged to a promoter

Regulatory bodies require diagnostic laboratories to build a strong QMS (Deming, 2000). A robust QMS integrates the organization's processes, policies and procedures for total quality management. In the diagnostic laboratory industry, CAP and other accrediting bodies require defined metrics throughout all phases of testing, including pre-analytical, analytical

For microarray technology, pre-analytical metrics may include assessment of DNA quality and yield. Each laboratory must define the ideal quality of DNA prior to implementing the assay into clinical testing. In our experience, a gel assessment that indicates clean genomic DNA free of RNase and degradation should lead to quality microarray results. If a DNA specimen has artifacts or appears to have degraded (Fig. 1), the laboratory should inform the client that results may be compromised because of DNA quality or obtain a new specimen from which to perform the analysis. In addition to a visual assessment of the DNA via gel electrophoresis, the laboratory should assess the DNA yield post-extraction. The quantity of DNA required in the analytic labeling phase of microarray analysis determines DNA yield requirements. Spectrophotometric assessment of DNA offers two indicators of quality DNA, including quantity and purity. A 260/280 nm reading indicates quantity, and a 260/230 nm reading indicates purity. These measurements are imperative for the downstream labeling process. Insufficient DNA quantity and quality (purity) will compromise successful microarray analysis. The spectrophotometer measures optical density (OD), which is the physical process of absorbing light. The OD, or absorbance, is calculated as a mathematical quantity. OD readings for pure DNA should measure at 1.8 (Sambrook & Russell, 2001). Our laboratory uses OD measurements from 1.4 to 1.8, although quality of labeling product can be compromised at the lower OD readings. There are many causes of poor yield, including compromised technique during extraction and poor sample quality (e.g., from increased age

Fig. 1. Gel electrophoresis for the assessment of DNA degradation. Lane 1 has the molecular mass standard. Lanes 2 & 5 show high molecular weight samples that do not exhibit any signs of degradation. The two samples in lanes 3 & 4 show lower molecular weight DNA below the main high molecular weight bands in the other lanes. Degraded DNA typically

Assessment of quality should be implemented throughout all phases of testing including the analytic phase. For aCGH, analytical metrics may include, but are not limited to, spectrophotometric assessment of the labeling product and the identification of labeling efficiency, which has an impact on results. Microarray analysis usually requires a dye incorporation using a random priming method. The dyes are tagged to a promoter

**2. Quality systems with strong monitoring for quality metrics** 

and post-analytical.

or exposure).

leads to compromised array results.

molecule, and the promoter molecule is incorporated into the genomic DNA. As with preanalytic assessment of DNA, the quantity assessment of the labeling product is performed by assessing the 260/280 nm readings from the spectrophotometer. Laboratories should define post-labeling quantity requirements that indicate labeling efficiency. In addition to labeling efficiency as a quality indicator, visual assessment of the set-up of labeling product on the array should be considered. In the event that there are air bubbles or non-complete contact of array product to the hybridization area (Fig. 2), the quality of the microarray result may be compromised and should be documented.

Fig. 2. Microarray after hybridization with an air bubble. The air bubble creates an area of incomplete contact of array product to the hybridization area, which compromises the array.

Post-analytic assessments may include average standard deviation (SD), intensity and background values. The SD value is the standard deviation of the normalized log2 intensity ratios for autosomal regions (excluding large copy number imbalances) and provides a measure of quality for aCGH experiments (Vermeesch et al., 2005). The SD value provides a quantitative metric that is relative to the overall noise on an array. As the overall noise of an aCGH experiment increases, so does the SD value. Our laboratory has established SD values

Genomic Microarray Quality Assurance 171

Fig. 3. (A-B) Microarray plots from two subjects showing identical single-copy gains of 17 oligonucleotide probes from the terminal long arm of chromosome 3, approximately 189 kb in size (chr3:199,067,024-199,255,755, hg18 coordinates). Probes are ordered on the x-axis according to physical mapping positions, with the most proximal 3q29 probes to the left and the most distal 3q29 probes to the right. Values along the y-axis represent log2 ratios of patient:control signal intensities. Results are visualized using Genoglyphix (Signature

One disadvantage of FISH visualization of microarray results, particularly when using highdensity oligonucleotide arrays, is that high-density oligonucleotide arrays can detect abnormalities well below the size of the smallest FISH probes, which are 100-350 kb in size. However, PCR-based methodologies such as MLPA and Q-PCR can visualize small gains and losses. MLPA targets the region of interest with two oligonucleotide probes, one probe containing a forward primer sequence and the second probe containing the reverse primer sequence (Schouten et al., 2002). The oligonucleotide probes are allowed to hybridize to the DNA followed by a ligation step. If the two probes are adjacent to each other the ligation will combine the two probes into a single probe with a fluorescently tagged forward primer on one end and reverse primer on the other end. The probe is amplified by PCR and only the ligated probe is amplified. The amplified product is dependent on the number of target sites present in the DNA. The forward primer is fluorescently labeled, which allows a comparison of the ratio of the fluorescent intensity between reference sample and the test

Q-PCR amplifies and simultaneously quantifies the relative amount of DNA when compared against a reference. Two Q-PCR methods have been developed. The first method uses fluorescent dyes that intercalate nonspecifically with the double-stranded DNA which produces fluorescent signals relative the quantity of DNA present (VanGuilder et al., 2008). The ratio is compared against a normal reference to confirm the relative quantity of the sample to the control. The second method uses a fluorescently labeled probe that is targeted to the region of interest. The fluorescently labeled probe has a fluorescent reporter and a

Genomics, Spokane, WA).

subject to determine the relative quantity of the probe.

that indicate whether an aCGH experiment is optimal, suboptimal, or failed. In our laboratory the average SD is used daily to monitor the collective SD values for all patients. Daily monitoring of the average SD value allows for establishment of a system to monitor the average SD over time. If a shift in the average SD value is observed, the laboratory processes can be evaluated to determine the potential cause and potentially prevent a system-wide failure. Each laboratory will need to define and validate a quality metric to measure the quality of the array and implement a system to track the performance of the metric.

In addition to monitoring the SD value of the array, the signal intensity of the two fluorescent dyes relative to the background can be monitored. These two values can be tracked independently or together by monitoring the signal-to-noise ratio (SNR). The SNR is the signal intensity divided by the background noise. Low signal intensity or high background noise will result in a low SNR value. A low SNR is an indicator for poor-quality array data (Basarsky et al., 2000). Low signal intensities can result from several factors, including poor fluorescent dye incorporation in labeling, inadequate denaturation of the probe, inadequate quantity of the probe, and suboptimal hybridization. Several factors can result in high background noise, including labeling reaction impurities, drying of the array during hybridization or during the post-hybridization washes, and incorrect Cot-1 to probe ratio. Constant monitoring of these metrics allows the laboratory staff to anticipate potential system failures leading to failed or inaccurate findings.

#### **3. Verification of array results**

In addition to the microarray assessments, there are other post-analytical assessments of quality that validate the microarray findings and lead to a complete, quality result used by the clinician for the diagnosis of the patient. Methods for confirmation of array results may include fluorescence *in situ* hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), quantitative PCR (Q-PCR) and other PCR techniques.

FISH is an established technique that is used to identify numerical and structural chromosome abnormalities by using fluorescently labeled DNA probes to detect the presence or absence of the DNA in the interphase nucleus or in metaphase, the stage of active cell division when the chromosomes are visibly condensed and can be observed in a microscope (Kashork et al., 2010). FISH commonly uses unique-sequence BAC probes; depending on the specific probe used, the resolution of metaphase FISH is ~80-200 kb (Shaffer et al., 2001). In our experience, deletions are easy to visualize by FISH. However, tandem duplications represent a challenge to any laboratory using confirmatory FISH because the duplicated material is not of sufficient distance from the original genomic location to generate a distinct fluorescent signal to allow detection by interphase or metaphase analysis. In some cases the intensity of the signal may be twice as intense on the duplicated homolog compared to the normal homolog, but this is not always the case.

In addition, although microarray analysis can detect DNA copy number changes, it does not identify the provenance of the abnormality; seemingly identical array results may be caused by distinct molecular mechanisms. Complete understanding of the rearrangement so that accurate genetic counseling can be provided requires visualization of the rearrangement, which can be accomplished with FISH. For example, a copy-number gain identified by microarray analysis (Fig. 3) may be a duplication, an insertion, a marker chromosome or an unbalanced translocation (Fig. 4).

that indicate whether an aCGH experiment is optimal, suboptimal, or failed. In our laboratory the average SD is used daily to monitor the collective SD values for all patients. Daily monitoring of the average SD value allows for establishment of a system to monitor the average SD over time. If a shift in the average SD value is observed, the laboratory processes can be evaluated to determine the potential cause and potentially prevent a system-wide failure. Each laboratory will need to define and validate a quality metric to measure the quality

In addition to monitoring the SD value of the array, the signal intensity of the two fluorescent dyes relative to the background can be monitored. These two values can be tracked independently or together by monitoring the signal-to-noise ratio (SNR). The SNR is the signal intensity divided by the background noise. Low signal intensity or high background noise will result in a low SNR value. A low SNR is an indicator for poor-quality array data (Basarsky et al., 2000). Low signal intensities can result from several factors, including poor fluorescent dye incorporation in labeling, inadequate denaturation of the probe, inadequate quantity of the probe, and suboptimal hybridization. Several factors can result in high background noise, including labeling reaction impurities, drying of the array during hybridization or during the post-hybridization washes, and incorrect Cot-1 to probe ratio. Constant monitoring of these metrics allows the laboratory staff to anticipate potential

In addition to the microarray assessments, there are other post-analytical assessments of quality that validate the microarray findings and lead to a complete, quality result used by the clinician for the diagnosis of the patient. Methods for confirmation of array results may include fluorescence *in situ* hybridization (FISH), multiplex ligation-dependent probe

FISH is an established technique that is used to identify numerical and structural chromosome abnormalities by using fluorescently labeled DNA probes to detect the presence or absence of the DNA in the interphase nucleus or in metaphase, the stage of active cell division when the chromosomes are visibly condensed and can be observed in a microscope (Kashork et al., 2010). FISH commonly uses unique-sequence BAC probes; depending on the specific probe used, the resolution of metaphase FISH is ~80-200 kb (Shaffer et al., 2001). In our experience, deletions are easy to visualize by FISH. However, tandem duplications represent a challenge to any laboratory using confirmatory FISH because the duplicated material is not of sufficient distance from the original genomic location to generate a distinct fluorescent signal to allow detection by interphase or metaphase analysis. In some cases the intensity of the signal may be twice as intense on the duplicated homolog compared to the normal homolog, but this is not always the case. In addition, although microarray analysis can detect DNA copy number changes, it does not identify the provenance of the abnormality; seemingly identical array results may be caused by distinct molecular mechanisms. Complete understanding of the rearrangement so that accurate genetic counseling can be provided requires visualization of the rearrangement, which can be accomplished with FISH. For example, a copy-number gain identified by microarray analysis (Fig. 3) may be a duplication, an insertion, a marker chromosome or an

amplification (MLPA), quantitative PCR (Q-PCR) and other PCR techniques.

of the array and implement a system to track the performance of the metric.

system failures leading to failed or inaccurate findings.

**3. Verification of array results** 

unbalanced translocation (Fig. 4).

Fig. 3. (A-B) Microarray plots from two subjects showing identical single-copy gains of 17 oligonucleotide probes from the terminal long arm of chromosome 3, approximately 189 kb in size (chr3:199,067,024-199,255,755, hg18 coordinates). Probes are ordered on the x-axis according to physical mapping positions, with the most proximal 3q29 probes to the left and the most distal 3q29 probes to the right. Values along the y-axis represent log2 ratios of patient:control signal intensities. Results are visualized using Genoglyphix (Signature Genomics, Spokane, WA).

One disadvantage of FISH visualization of microarray results, particularly when using highdensity oligonucleotide arrays, is that high-density oligonucleotide arrays can detect abnormalities well below the size of the smallest FISH probes, which are 100-350 kb in size. However, PCR-based methodologies such as MLPA and Q-PCR can visualize small gains and losses. MLPA targets the region of interest with two oligonucleotide probes, one probe containing a forward primer sequence and the second probe containing the reverse primer sequence (Schouten et al., 2002). The oligonucleotide probes are allowed to hybridize to the DNA followed by a ligation step. If the two probes are adjacent to each other the ligation will combine the two probes into a single probe with a fluorescently tagged forward primer on one end and reverse primer on the other end. The probe is amplified by PCR and only the ligated probe is amplified. The amplified product is dependent on the number of target sites present in the DNA. The forward primer is fluorescently labeled, which allows a comparison of the ratio of the fluorescent intensity between reference sample and the test subject to determine the relative quantity of the probe.

Q-PCR amplifies and simultaneously quantifies the relative amount of DNA when compared against a reference. Two Q-PCR methods have been developed. The first method uses fluorescent dyes that intercalate nonspecifically with the double-stranded DNA which produces fluorescent signals relative the quantity of DNA present (VanGuilder et al., 2008). The ratio is compared against a normal reference to confirm the relative quantity of the sample to the control. The second method uses a fluorescently labeled probe that is targeted to the region of interest. The fluorescently labeled probe has a fluorescent reporter and a

Genomic Microarray Quality Assurance 173

National ozone standards have been established by the EPA to protect public health. The established standard peak ozone level set by the EPA is 80 ppb, which is based on the annual fourth maximum 8-hour average (EPA, http://www.epa.gov/air/ozonepollution, last accessed May 9, 2008). The EPA has also established an air quality index system for monitoring the daily pollution levels. The "good" air quality range is 0 to 60 ppb. The fluorescent dyes commonly used in aCGH are sensitive to ozone levels as low as 5 to 10 ppb (Branham et al., 2007; Fare et al., 2003). Thus, ozone levels considered normal for environmental standards are well above those ranges, demonstrating sensitivity of the

Ozone has been shown to strongly affect dyes that are commonly used in aCGH, including cyanine 5 (Cy5) and Alexa dyes (Alexa Fluor 647 and Alexa Fluor 5) and, to a lesser extent, cyanine 3 (Cy3) and the Alexa equivalents (Alexa Fluor 555 and Alexa Fluor 3) (Branham et al., 2007; Byerly et al., 2009; Fare et al., 2003). Several studies have identified the posthybridization washes as the most sensitive period for exposure to ozone (Branham et al., 2007; Byerly et al., 2009; Fare et al., 2003). These studies have demonstrated the difficulty with which laboratories identify the source of ozone-related problems, especially considering the extremely low levels of ozone (5 to 10 ppb) that cause these problems, the duration of exposure (as little as 10 to 30 seconds), and the seasonal emergence of ozone itself. The effects of ozone must be addressed when aCGH is performed, particularly in a clinical diagnostic setting, where it is critical to have consistent high quality and reproducible results. Failure to protect the fluorescent dyes from ozone during the posthybridization washes will result in considerable negative impact on the array data. The implementation of quality control measures such as ozone reduction and monitoring to ensure high-quality aCGH results is mandatory for any aCGH laboratory. There are many commercially available enclosures and scrubbers designed to protect the dyes during the post-hybridizations washes. Some laboratories have gone as far as developing ozone-free rooms where post-hybridization washes and the subsequent scanning and analysis are

In addition to ozone degradation, the dyes are also photosensitive and often must be used in a reduced-light environment. Systems should be implemented to prevent photobleaching of the fluorescent dyes. To mitigate against the effect of photobleaching, the dyes should be protected from the light whenever possible. This can be done by using indirect lighting in the work area, using amber tubes, covering the samples with tin foil or placing the samples

Normalization, which aims to separate biologically relevant signal from experimental artifacts, is a crucial step of microarray analysis (Neuvial et al., 2006). Each laboratory must identify a system for normalization. Most microarray vendors offer software with built-in normalization methods optimized for their own platforms. Laboratories can use a normalization package that is developed by the microarray vendor or can develop their own package. One normalization method that is used in the laboratory is the locally weighted polynomial regression (LOESS) (Cleveland, 1979). This normalization applies a spatial correction to correct position-dependent non-uniformity of signals across the

performed. The latter is most desirable but may not always be feasible.

in areas with little or no light when not being directly handled.

**5. Normalization** 

array.

dyes.

quencher to hide the fluorescent signal until the region is amplified (Udvardi et al., 2008). During each round of the PCR process, the exonuclease activity of the polymerase releases the fluorescent reporter, unquenching the signal and allowing detection.

Additional PCR-based methods such as polymorphic microsatellite analysis have also been used as a confirmatory assay for aCGH. Although these molecular assays can confirm a copy number gain or loss, they cannot reveal the chromosomal rearrangement or mechanism giving rise to the copy number variant (CNV). Each laboratory must determine the appropriate confirmatory assay to meet its needs.

Fig. 4. (A) FISH visualization of the gain shown in Fig. 3A revealed an unbalanced translocation of the 3q29 material to 1p. BAC clone RP11-23M2 from 3q29 is labeled in red, and BAC clones RP11-9A9 from 3q11.2 and RP11-438F14 from 1q44 are labeled in green as controls. The presence of one red signal on one of the chromosome 1 homologues indicates translocation of 3q29 onto 1p (arrow). (B) FISH of the gain shown in Fig. 3B. BAC clone RP11-159K3 from 3q29 is labeled in red, and chromosome 3 centromere probe D3Z1 is labeled in green as a control. The presence of two red signals on metaphase FISH rules out an unbalanced translocation, while the additional finding of three red signals on interphase FISH (inset) suggests a duplication. The patient shown in Fig. 3A may have inherited the unbalanced translocation from a parent with a balanced translocation, whereas the duplication in the patient shown in Fig. 3B may have arisen *de novo*, although parental testing is necessary to confirm the inheritance.

## **4. Environmental controls**

Over time, specific environmental controls have been developed and implemented within the laboratory to ensure quality microarray diagnostics. For aCGH, ozone is an important environmental factor to control. Ozone is a common pollutant found in the lower atmosphere and is the primary component of smog. Ozone is formed when nitric oxides and volatile organic compounds (VOCs) react in the presence of sunlight (US Environmental Protection Agency [EPA], http://www.epa.gov/air/ozonepollution, last accessed May 9, 2008). Nitric oxides and VOCs are emitted by motor vehicle exhaust, industrial emissions, gasoline vapors, chemical solvents, and natural sources. Consequently, ozone levels are higher in urban and industrial areas, especially during the summer months.

quencher to hide the fluorescent signal until the region is amplified (Udvardi et al., 2008). During each round of the PCR process, the exonuclease activity of the polymerase releases

Additional PCR-based methods such as polymorphic microsatellite analysis have also been used as a confirmatory assay for aCGH. Although these molecular assays can confirm a copy number gain or loss, they cannot reveal the chromosomal rearrangement or mechanism giving rise to the copy number variant (CNV). Each laboratory must determine

Fig. 4. (A) FISH visualization of the gain shown in Fig. 3A revealed an unbalanced

translocation of the 3q29 material to 1p. BAC clone RP11-23M2 from 3q29 is labeled in red, and BAC clones RP11-9A9 from 3q11.2 and RP11-438F14 from 1q44 are labeled in green as controls. The presence of one red signal on one of the chromosome 1 homologues indicates translocation of 3q29 onto 1p (arrow). (B) FISH of the gain shown in Fig. 3B. BAC clone RP11-159K3 from 3q29 is labeled in red, and chromosome 3 centromere probe D3Z1 is labeled in green as a control. The presence of two red signals on metaphase FISH rules out an unbalanced translocation, while the additional finding of three red signals on interphase FISH (inset) suggests a duplication. The patient shown in Fig. 3A may have inherited the unbalanced translocation from a parent with a balanced translocation, whereas the duplication in the patient shown in Fig. 3B may have arisen *de novo*, although parental

Over time, specific environmental controls have been developed and implemented within the laboratory to ensure quality microarray diagnostics. For aCGH, ozone is an important environmental factor to control. Ozone is a common pollutant found in the lower atmosphere and is the primary component of smog. Ozone is formed when nitric oxides and volatile organic compounds (VOCs) react in the presence of sunlight (US Environmental Protection Agency [EPA], http://www.epa.gov/air/ozonepollution, last accessed May 9, 2008). Nitric oxides and VOCs are emitted by motor vehicle exhaust, industrial emissions, gasoline vapors, chemical solvents, and natural sources. Consequently, ozone levels are

higher in urban and industrial areas, especially during the summer months.

the fluorescent reporter, unquenching the signal and allowing detection.

the appropriate confirmatory assay to meet its needs.

testing is necessary to confirm the inheritance.

**4. Environmental controls** 

National ozone standards have been established by the EPA to protect public health. The established standard peak ozone level set by the EPA is 80 ppb, which is based on the annual fourth maximum 8-hour average (EPA, http://www.epa.gov/air/ozonepollution, last accessed May 9, 2008). The EPA has also established an air quality index system for monitoring the daily pollution levels. The "good" air quality range is 0 to 60 ppb. The fluorescent dyes commonly used in aCGH are sensitive to ozone levels as low as 5 to 10 ppb (Branham et al., 2007; Fare et al., 2003). Thus, ozone levels considered normal for environmental standards are well above those ranges, demonstrating sensitivity of the dyes.

Ozone has been shown to strongly affect dyes that are commonly used in aCGH, including cyanine 5 (Cy5) and Alexa dyes (Alexa Fluor 647 and Alexa Fluor 5) and, to a lesser extent, cyanine 3 (Cy3) and the Alexa equivalents (Alexa Fluor 555 and Alexa Fluor 3) (Branham et al., 2007; Byerly et al., 2009; Fare et al., 2003). Several studies have identified the posthybridization washes as the most sensitive period for exposure to ozone (Branham et al., 2007; Byerly et al., 2009; Fare et al., 2003). These studies have demonstrated the difficulty with which laboratories identify the source of ozone-related problems, especially considering the extremely low levels of ozone (5 to 10 ppb) that cause these problems, the duration of exposure (as little as 10 to 30 seconds), and the seasonal emergence of ozone itself. The effects of ozone must be addressed when aCGH is performed, particularly in a clinical diagnostic setting, where it is critical to have consistent high quality and reproducible results. Failure to protect the fluorescent dyes from ozone during the posthybridization washes will result in considerable negative impact on the array data. The implementation of quality control measures such as ozone reduction and monitoring to ensure high-quality aCGH results is mandatory for any aCGH laboratory. There are many commercially available enclosures and scrubbers designed to protect the dyes during the post-hybridizations washes. Some laboratories have gone as far as developing ozone-free rooms where post-hybridization washes and the subsequent scanning and analysis are performed. The latter is most desirable but may not always be feasible.

In addition to ozone degradation, the dyes are also photosensitive and often must be used in a reduced-light environment. Systems should be implemented to prevent photobleaching of the fluorescent dyes. To mitigate against the effect of photobleaching, the dyes should be protected from the light whenever possible. This can be done by using indirect lighting in the work area, using amber tubes, covering the samples with tin foil or placing the samples in areas with little or no light when not being directly handled.

## **5. Normalization**

Normalization, which aims to separate biologically relevant signal from experimental artifacts, is a crucial step of microarray analysis (Neuvial et al., 2006). Each laboratory must identify a system for normalization. Most microarray vendors offer software with built-in normalization methods optimized for their own platforms. Laboratories can use a normalization package that is developed by the microarray vendor or can develop their own package. One normalization method that is used in the laboratory is the locally weighted polynomial regression (LOESS) (Cleveland, 1979). This normalization applies a spatial correction to correct position-dependent non-uniformity of signals across the array.

Genomic Microarray Quality Assurance 175

Fig. 5. Different multiplex array formats. (A) Agilent 4-plex, (B) NimbleGen 6-plex, and (C)

guidelines distinguishes between different levels of validation depending on platform type (e.g., FDA-approved, investigation-use-only/research-use-only, or home-brew microarrays) and requires a demonstration of expertise of array performance and analysis through defined validation criteria for new microarray platforms, new versions of existing microarrays, and new lots of the same microarray. Some states have additional validation requirements. For example, New York has the Clinical Laboratory Evaluation Program (CLEP). Accreditation through CLEP requires additional quality assurances and validation criteria to which laboratories must adhere. Outside of accreditations, laboratories need to consider state regulations when considering offering their testing services nationwide. Some states have regulations that not only impact laboratories that reside in that state but also

When defining the control specimen of choice, a laboratory can choose to use same-sex or sex-mismatched controls from the patient. Same-sex controls offer detection of autosomal gains and losses, and complex sex chromosome abnormalities are more easily visualized. Sex-mismatched controls offer the same detection of autosomal gains and losses but can be

impact laboratories that test samples from that particular state.

NimbleGen 12-Plex.

**9. Control samples** 

Another normalization system used in the laboratory is the Qspline fit normalization (Workman et al., 2002). This normalization compensates for the inherent differences in signal intensities between the two dyes. There are many normalization methodologies available; each laboratory will have to define its method of choice.

## **6. Automation**

One of the key elements for any clinical assay is reproducibility. By replacing manual processes with automation, a laboratory can substantially improve the consistency and reproducibility of its daily operations. In addition, automation can increase throughput, which is often an advantage for a growing laboratory and helps to reduce the dependency on staffing levels. The laboratory protocols that have been successfully automated include DNA isolation, labeling and hybridization, washing and analysis.

However, automation can present several challenges. Because it is based on a plate format, if the input materials are flawed or a technical issue occurs (e.g., labeling master mix or an automation failure) it will impact the entire plate, which may consist of 48 or more patients. The consequences of a failure of this magnitude are substantial in terms of cost and the potential loss of the sample.

## **7. Multiplexing of microarray platforms**

As laboratories become more accustomed to using microarrays, the demand for the assay may increase. In addition to automation, multiplexed array formats can help the laboratory satisfy the increased demand. Multiplexed array formats allow for the simultaneous hybridization of 2 to 12 or more samples depending on the probe coverage of the array and the array manufacturer. The multiplex design (Fig. 5) has many positive features including decreased costs and higher throughput. As this technology continues to advance, higher multiplexed formats are likely to be developed.

Although increased throughput has its advantages, it also creates challenges for any quality system. The laboratory must ensure that there is no cross contamination between each subarray, which would affect patient samples. Some manufacturers have included QA/QC features into the development of multiplexed arrays, such as tracking controls that can be spiked into the experiments that identify unique positions on the array such that each position of the array can have a unique tracking control. Unique tracking controls are added to each sample before they are introduced to the array, which allows the laboratory to monitor each sub-array for cross contamination. In the event of cross contamination or leaking between the sub-arrays, the laboratory can determine the sample involved in the cross-contamination event based on the unique tracking controls involved.

## **8. Validation**

As laboratories begin to adopt microarray technology within their facility, they should identify the validation criteria which they are responsible for meeting. The American College of Medical Genetics (ACMG; Shaffer et al., 2007) and other US state guidelines have been developed to ensure laboratories have thoroughly tested and reviewed the capacity and expectation of the assay prior to clinical release. These validations include testing known abnormal specimens to verify the expected outcomes. Section E13.2 of the ACMG

Another normalization system used in the laboratory is the Qspline fit normalization (Workman et al., 2002). This normalization compensates for the inherent differences in signal intensities between the two dyes. There are many normalization methodologies

One of the key elements for any clinical assay is reproducibility. By replacing manual processes with automation, a laboratory can substantially improve the consistency and reproducibility of its daily operations. In addition, automation can increase throughput, which is often an advantage for a growing laboratory and helps to reduce the dependency on staffing levels. The laboratory protocols that have been successfully automated include

However, automation can present several challenges. Because it is based on a plate format, if the input materials are flawed or a technical issue occurs (e.g., labeling master mix or an automation failure) it will impact the entire plate, which may consist of 48 or more patients. The consequences of a failure of this magnitude are substantial in terms of cost and the

As laboratories become more accustomed to using microarrays, the demand for the assay may increase. In addition to automation, multiplexed array formats can help the laboratory satisfy the increased demand. Multiplexed array formats allow for the simultaneous hybridization of 2 to 12 or more samples depending on the probe coverage of the array and the array manufacturer. The multiplex design (Fig. 5) has many positive features including decreased costs and higher throughput. As this technology continues to advance, higher

Although increased throughput has its advantages, it also creates challenges for any quality system. The laboratory must ensure that there is no cross contamination between each subarray, which would affect patient samples. Some manufacturers have included QA/QC features into the development of multiplexed arrays, such as tracking controls that can be spiked into the experiments that identify unique positions on the array such that each position of the array can have a unique tracking control. Unique tracking controls are added to each sample before they are introduced to the array, which allows the laboratory to monitor each sub-array for cross contamination. In the event of cross contamination or leaking between the sub-arrays, the laboratory can determine the sample involved in the

As laboratories begin to adopt microarray technology within their facility, they should identify the validation criteria which they are responsible for meeting. The American College of Medical Genetics (ACMG; Shaffer et al., 2007) and other US state guidelines have been developed to ensure laboratories have thoroughly tested and reviewed the capacity and expectation of the assay prior to clinical release. These validations include testing known abnormal specimens to verify the expected outcomes. Section E13.2 of the ACMG

cross-contamination event based on the unique tracking controls involved.

available; each laboratory will have to define its method of choice.

DNA isolation, labeling and hybridization, washing and analysis.

**6. Automation** 

potential loss of the sample.

**8. Validation** 

**7. Multiplexing of microarray platforms** 

multiplexed formats are likely to be developed.

Fig. 5. Different multiplex array formats. (A) Agilent 4-plex, (B) NimbleGen 6-plex, and (C) NimbleGen 12-Plex.

guidelines distinguishes between different levels of validation depending on platform type (e.g., FDA-approved, investigation-use-only/research-use-only, or home-brew microarrays) and requires a demonstration of expertise of array performance and analysis through defined validation criteria for new microarray platforms, new versions of existing microarrays, and new lots of the same microarray. Some states have additional validation requirements. For example, New York has the Clinical Laboratory Evaluation Program (CLEP). Accreditation through CLEP requires additional quality assurances and validation criteria to which laboratories must adhere. Outside of accreditations, laboratories need to consider state regulations when considering offering their testing services nationwide. Some states have regulations that not only impact laboratories that reside in that state but also impact laboratories that test samples from that particular state.

## **9. Control samples**

When defining the control specimen of choice, a laboratory can choose to use same-sex or sex-mismatched controls from the patient. Same-sex controls offer detection of autosomal gains and losses, and complex sex chromosome abnormalities are more easily visualized. Sex-mismatched controls offer the same detection of autosomal gains and losses but can be

Genomic Microarray Quality Assurance 177

This chapter highlights many of the quality assurance principles that impact a laboratory setting up or using aCGH. This is not an exhaustive set of challenges to implementation as there may be lab-, region-, environment- and vendor-specific variations. Each laboratory should perform initial quality verification at the time of test development. Post development, a thorough validation must be performed, which may uncover variation that should be controlled prior to launching the clinical assay. A laboratory's role in developing new assays should include an established, documented and maintained quality system that ensures that the test conforms to specified requirements and ultimately leads to accurate

We thank Aaron Theisen (Signature Genomics, Spokane, WA) for his critical review of our

Basarsky T., Verdnik D., Willis D. & Zhai J. (2000). An overview of a DNA microarray

*Microarray biochip technology*, Schena M. (Ed.). Eaton Publishing, Natick, MA Beaudet A.L. & Belmont J.W. (2008). Array-based DNA diagnostics: let the revolution begin.

Branham W.S., Melvin C.D., Han T., Desai V.G., Moland C.L., Scully A.T. & Fuscoe J.C.

Byerly S., Sundin K., Raja R., Stanchfield J., Bejjani B.A. & Shaffer L.G. (2009). Effects of

Cleveland W.S. (1979). Robust locally weighted regression and smoothing scatterplots. *J* 

Deming W.E. (2000). *The New Economics For Industry, Government & Education* (2nd), The MIT

DeRisi J., Penland L., Brown P.O., Bittner M.L., Meltzer P.S., Ray M., Chen Y., Su Y.A. &

Fare T.L., Coffey E.M., Dai H., He Y.D., Kessler D.A., Kilian K.A., Koch J.E., LeProust E.,

Kashork C.D., Theisen A. & Shaffer L.G. (2010). Diagnosis of cryptic chromosomal

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(2007). Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements. *BMC Biotechnol*, Vol.

ozone exposure during microarray posthybridization washes and scanning. *J Mol* 

Trent J.M. (1996). Use of a cDNA microarray to analyse gene expression patterns in

Marton M.J., Meyer M.R., Stoughton R.B., Tokiwa G.Y. & Wang Y. (2003). Effects of atmospheric ozone on microarray data quality. *Anal Chem*, Vol. 75. No. 17, (Sep 1),

syndromes by fluorescence in situ hybridization (FISH). *Curr Protoc Hum Genet*,

**10. Conclusion** 

results.

manuscript.

**12. References** 

**11. Acknowledgments** 

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more challenging when assessing sex chromosome changes. However, sex-mismatched controls offer the laboratory an internal assessment of hybridization success because of the expected deviations when comparing a male against a female. The deviations that are inherent to sex-mismatched controls are a result of the copy number variation of the X/Y chromosome ratio. When a female genome is compared to a male genome, there is an apparent gain of chromosome X (two copies in the female against the single copy in males) and a loss of chromosome Y (Fig. 6).

Fig. 6. Microarray plots from three different gender parings: male/male, female/male, and female/female. The probes are ordered on the x-axis according to physical mapping positions starting with chromosome 1 on the left and the X and Y chromosomes on the right. Values along the y-axis represent log2 ratios of patient:control signal intensities. (A) A samesex male/male comparison showing identical dosage at the X and Y regions. (B) An opposite-sex female/male comparison showing a gain of the X chromosome (two copies in the female versus a single copy in the male) and a loss of Y (no copies in the female versus one copy in the male). (C) A same-sex female/female comparison showing identical dosage at the X region and no hybridization at the Y region. Results are visualized using Genoglyphix (Signature Genomics, Spokane, WA).

In addition to determining the ideal sex of the control, the laboratory must decide if a single control or pooled controls will be used in the facility. Often, these decisions are made based on available controls. Some institutions have used a consistent male and female control for easy identification of known CNVs and for monitoring the performance of arrays. However, in the absence of consistent candidates for controls, laboratories can create or purchase a pooled DNA control. All variations of these controls must be assessed with any change in the pool so that they will not significantly impact reporting. In addition, CNVs present in the control DNA can be used as a positive indicator of assay performance when using same-sex controls. However, pooled control samples will have diluted CNVs, which may not be apparent on the microarray results or may appear as mosaics or background noise.

## **10. Conclusion**

176 Modern Approaches To Quality Control

more challenging when assessing sex chromosome changes. However, sex-mismatched controls offer the laboratory an internal assessment of hybridization success because of the expected deviations when comparing a male against a female. The deviations that are inherent to sex-mismatched controls are a result of the copy number variation of the X/Y chromosome ratio. When a female genome is compared to a male genome, there is an apparent gain of chromosome X (two copies in the female against the single copy in males)

Fig. 6. Microarray plots from three different gender parings: male/male, female/male, and female/female. The probes are ordered on the x-axis according to physical mapping

positions starting with chromosome 1 on the left and the X and Y chromosomes on the right. Values along the y-axis represent log2 ratios of patient:control signal intensities. (A) A same-

In addition to determining the ideal sex of the control, the laboratory must decide if a single control or pooled controls will be used in the facility. Often, these decisions are made based on available controls. Some institutions have used a consistent male and female control for easy identification of known CNVs and for monitoring the performance of arrays. However, in the absence of consistent candidates for controls, laboratories can create or purchase a pooled DNA control. All variations of these controls must be assessed with any change in the pool so that they will not significantly impact reporting. In addition, CNVs present in the control DNA can be used as a positive indicator of assay performance when using same-sex controls. However, pooled control samples will have diluted CNVs, which may not be apparent on the microarray results or may appear as

sex male/male comparison showing identical dosage at the X and Y regions. (B) An opposite-sex female/male comparison showing a gain of the X chromosome (two copies in the female versus a single copy in the male) and a loss of Y (no copies in the female versus one copy in the male). (C) A same-sex female/female comparison showing identical dosage

at the X region and no hybridization at the Y region. Results are visualized using

Genoglyphix (Signature Genomics, Spokane, WA).

mosaics or background noise.

and a loss of chromosome Y (Fig. 6).

This chapter highlights many of the quality assurance principles that impact a laboratory setting up or using aCGH. This is not an exhaustive set of challenges to implementation as there may be lab-, region-, environment- and vendor-specific variations. Each laboratory should perform initial quality verification at the time of test development. Post development, a thorough validation must be performed, which may uncover variation that should be controlled prior to launching the clinical assay. A laboratory's role in developing new assays should include an established, documented and maintained quality system that ensures that the test conforms to specified requirements and ultimately leads to accurate results.

#### **11. Acknowledgments**

We thank Aaron Theisen (Signature Genomics, Spokane, WA) for his critical review of our manuscript.

## **12. References**


**Part 4** 

**Planning for Quality Control** 

