CN104498431B - A kind of method of slide Culture in situ amniocyte and caryogram Treatment Analysis - Google Patents
A kind of method of slide Culture in situ amniocyte and caryogram Treatment Analysis Download PDFInfo
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Abstract
The invention provides a kind of slide Culture in situ amniocyte and the method for caryogram Treatment Analysis, the present invention is further improved on the basis of previous studies, using the hypotonic medium of certain concentration and species, pre-fix liquid, the temperature and humidity of decentralized environment during strict control Chromosome spread, to obtain abundant high-quality split coil method, this technology will provide the foundation technology platform for high flux automatically scanning capture system, set up automation diagosis flow, lift clinical position efficiency.
Description
Technical field
The present invention relates to a kind of amniotic fluid cell culture field, and in particular to a kind of slide Culture in situ amniocyte and core
The method of type Treatment Analysis.
Background technology
Amniotic fluid cell culture and karyotyping are the key technologies of pre-natal diagnosis, and the technology is needed after amniocyte patch
Wall, harvest（It is hypotonic, pre-fix）, film-making（Chromosome spread）, clap under microscope and take all multi-steps such as caryogram and karyotyping,
To complete final clinical report.As Prenatal Screening is constantly popularized and pre-natal diagnosis demand rapidly increased situation, tradition
Amniotic fluid cell culture（Time-consuming, effort）Clinical demand can not be met, and traditional amniotic fluid cell culture step is various, film-making matter
Amount is unstable, is unsuitable for pre-natal diagnosis quality control.In order to shorten incubation time, operating efficiency is improved, researcher successfully grinds
Make automatic caryogram scanner, i.e., microscope be connected with the track of automatic threading, with realize 24 hours it is unattended full-automatic
Change piece, take piece, scanning, oil dripping, collection caryogram.The instrument shows quick, labour-saving pole in peripheral blood and the capture of FISH caryogram
Good effect, but amniotic fluid in situ culture cell attachment in 4 × 6cm2Plastic culture bottle, the out-of-flatness of plastic bottle egative film, thickness are too
Height, microscope can not automatically adjust the capture of focal length automatically scanning.Foreign countries generally take coverslip method, and the method is by cell attachment in lid
Slide, cover glass is removed using culture dish as carrier, after harvesting on slide, then with it is gluing it is attached realize karyotyping,
But due to adhesive relation, thickness is not suitable for microscope regulation and focused on, or with the automatic diagosis of reverse side, effect limitation.Slide is former
Position culture amniocyte can solve the deficiency of above two method.Tabor etc. is entered using the slide culture dish of NUNC companies
Row amniocyte Culture in situ, but analysis cost is too high, and the country is difficult popularization.
The content of the invention
In order to solve the deficiencies in the prior art, it is an object of the invention to provide a kind of slide Culture in situ amniocyte
And the method for caryogram Treatment Analysis.
The technical solution that the present invention is used is：A kind of method of slide Culture in situ amniocyte, including it is following
Step：
（1）Inoculation：Each amniocyte sample is taken into 18-22ml, the sterile conical centrifuge tube 2 that is divided into is managed, 9-11ml/ pipes,
Equilibrium centrifugation 10min, rotating speed is 1200rpm, and each pipe leaves and takes 0.5ml cell suspensions, each to add 1.5ml amniotic fluid culture mediums, fully
Mix, the cell attachment face of slide is laid in, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, 2 days after in slide again
3ml amniotic fluid culture mediums are slowly added, the culture medium newly added is well mixed with old culture medium, is replaced in 37 DEG C, 5%CO2's
Under the conditions of cultivate 5 days；
（2）Change liquid：Amniotic fluid cell culture is treated to 7-8 days, micro- Microscopic observation growth progress is inverted in, treats clone cell group
Propagation, old culture medium is replaced with 2ml/ bottles of fresh amniotic fluid culture mediums, treats that second day i.e. amniotic fluid of the harvest comprising Culture in situ is thin
Born of the same parents' slide.
A kind of method of described slide Culture in situ amniocyte, described step（1）Middle cell suspension is amniotic fluid
Cell precipitation and amniotic fluid.
A kind of method of amniocyte caryogram Treatment Analysis, comprises the following steps：
（1）Harvest, film-making：Colchicine is added in the amniocyte slide containing Culture in situ, is shaken up, then insert
5%CO2, 37 DEG C of incubator 25min, abandoning supernatant；
（2）It is hypotonic：The hypotonic medium 6ml of 37 DEG C of pre-temperature, room temperature horizontal 25min are slowly added in amniocyte slide；
（3）Pre-fix：The slow supernatant for extracting slide, abandons it, rear slow plus after pre-fixing liquid 6ml, 7min, abandons
Supernatant, adds 4ml Fresh fixatives, rinses cell membrane, abandons supernatant；
（4）It is fixed：The Fresh fixative 6ml in slide plus such as, is stored at room temperature 10min, abandons supernatant again；
（5）Repeat the（4）Rapid 2 times step by step, time of repose shorten to 1min；
（6）Slide is taken out with tweezers, unnecessary fixer is sucked with gauze, is put into Chromosome spread instrument, is set
Temperature is 21 DEG C, and humidity is 30 or 33%, treats that slide is completely dried；
（7）Slide is placed at 65 DEG C of baking oven and bakes piece 2h, is taken out after slide, 1mg/ml pancreatin immersion treatments 16-
20s, Giemsa dye liquor immersion treatment 10min；
（8）Bat takes caryogram：Clapped using microscope and take amniocyte slide：, the automatic caryogram scanners of GLS-120, which are captured, to be adopted
Collect karyotype.
A kind of method of described amniocyte caryogram Treatment Analysis, it is characterised in that：Described colchicine concentration is
20ug/ml。
A kind of method of described amniocyte caryogram Treatment Analysis, it is characterised in that：Described hypotonic medium is concentration 1%
Sodium citrate.
A kind of method of described amniocyte caryogram Treatment Analysis, it is characterised in that：Described pre-fixes liquid for concentration
5% glacial acetic acid.
A kind of method of described amniocyte caryogram Treatment Analysis, it is characterised in that：Described fixer is methanol：Ice
Acetic acid=3：1 Kano fixer.
A kind of method of described amniocyte caryogram Treatment Analysis, it is characterised in that：Described Giemsa dye liquor compositions
For 1：10 Giemsa stostes and PH6.8 phosphate buffer.
The beneficial effect that the present invention is obtained is：At a kind of slide Culture in situ amniocyte and caryogram
The method for managing analysis, the present invention is further improved on the basis of previous studies, using the hypotonic medium of certain concentration and species, low
Decentralized environment when oozing the time, pre-fixing liquid, pre-fix time, fixer, set time and strict control Chromosome spread
Temperature and humidity, to obtain abundant high-quality split coil method, this technology will provide the foundation for high flux automatically scanning capture system
Technology platform, sets up automation diagosis flow, lifts clinical position efficiency, the beneficial effects are mainly as follows：（1）This
Pre-natal diagnosis program is not disturbed in invention, does not increase amniocentesis number of times and amniotic fluid volume, in the absence of ethics problem；（2）Institute of the present invention
The reagent being related to is Self-made reagent, and raw material domestic can be bought, and without extra increase reagent, does not increase cost；（3）The present invention
The amniotic fluid in situ cultural method of foundation can be used for micrometron scanning, solve pre-natal diagnosis automation key technology, and
Formal description is set up, reaches that slice-making quality is stable, culture is obtained and clones abundant, split coil method high-quality, the features such as success rate is high；
（4）The present invention can use domestic slide, obtain number of cell clones and effectively division number of phases ability is no less than import slide, and
Domestic slide price is low.
Brief description of the drawings
Fig. 1 is domestic slide sterile culture box of the invention.
Fig. 2:For slide schematic diagram of the present invention.
Fig. 3 is that amniocyte of the present invention grows figure.
Fig. 4 is that karyotype dust dispersion quality assesses compares figure.
In wherein Fig. 2：A is slide mark；B is slide cell attachment face.
Embodiment：
With reference to specification drawings and specific embodiments, the present invention will be further described.
This technology is described further with reference to specific embodiment（The present invention reagent unless otherwise specified, state
Produce slide and be purchased from Hangzhou Bao Rong companies；Import slide is Italy Euroclone；Amniotic fluid culture medium is Israel
BIOAMF-2 culture mediums）：
Embodiment 1：Domestic slide culture amniocyte synchronous with import slide
（1）Inoculation：10 amniotic fluid samples are chosen, each sample takes 18-22ml.The sterile conical centrifuge tube 2 that is divided into is managed, 9-
11ml/ is managed, and equilibrium centrifugation 10min, rotating speed is 1200rpm, and each pipe leaves and takes 0.5ml cell suspensions（Amniocyte precipitation+a little sheep
Water）, it is each to add 1.5ml amniotic fluid culture mediums, domestic slide, the cell attachment of the original-pack slide blake bottle of import are laid in respectively
Face（Fig. 2 b）, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, minimum mechanical disturbance, in the mark of slide after 2 days（Figure
2a）3ml amniotic fluid culture mediums are added again, and the culture medium newly added is well mixed with old culture medium, is replaced in 37 DEG C, 5%CO2's
Under the conditions of cultivate 5 days.
（2）Change liquid：Amniotic fluid cell culture is treated to 7-8 days, micro- Microscopic observation growth progress is inverted in, treats clone cell
Group increases, and area is big, when bright cell is more, replaces old culture mediums with 2ml/ bottle fresh amniotic fluid culture mediums, treats to receive for second day
Obtain the amniocyte slide for including Culture in situ.Culture passage originally continues to give birth into correspondence tissue cultuer bottle
It is long, after report is gone out, abandon it.
（3）Harvest, film-making：Colchicine is added in the amniocyte slide containing Culture in situ, is shaken up, then insert
5%CO2, 37 DEG C of incubator 25min, abandoning supernatant；
（4）It is hypotonic：Mark in amniocyte slide（Fig. 2 a）The hypotonic medium 6ml of 37 DEG C of pre-temperature is slowly added to, it is low
Sepage is the sodium citrate of concentration 1%, room temperature horizontal 25min；
（5）Pre-fix：In the mark of slide（Fig. 2 a）The slow supernatant for extracting slide, abandons it, rear slowly to add
Liquid 6ml is pre-fixed, pre-fixes after the glacial acetic acid that liquid is concentration 5%, 7min, abandons supernatant, add 4ml Fresh fixatives, rush
Cell membrane is washed, supernatant is abandoned；
（6）It is fixed：Add such as Fresh fixative 6ml in slide again, fixer is methanol：Glacial acetic acid=3：1 Kano
Fixer, is stored at room temperature 10min, abandons supernatant；
（7）Repeat fixing step 2 times, time of repose shorten to 1min；
（8）Slide is taken out with tweezers, slide edge is leaned against on gauze, sucks unnecessary fixer, be put into chromosome point
Dissipate in instrument, design temperature is 21 DEG C, and humidity is 30 or 33%, treats that slide is completely dried；
（9）Slide is placed at 65 DEG C of baking oven and bakes piece 2h, is taken out after slide, 1mg/ml pancreatin immersion treatments 16-
20s, Giemsa dye liquor immersion treatment 10min, Giemsa dye liquor composition are 1：10 Giemsa stostes and PH6.8 phosphoric acid buffer
Liquid；
（10）Bat takes caryogram：Using GLS-120 automatically scannings capture system capture karyotype.Chromosome spread quality
Using digit score：1) disperse（Not all chromosome is all in a visual field）；2) high-quality（All chromosomes are all regarded at one
Yezhong, and chiasma is less equal than 5）；3) it is poor（Chiasma is more than 5）.
（11）Clinical report：Analyze karyotype, 15 caryogram/people.
Colchicine concentration used is 20ug/ml in above-mentioned steps.
3) number of cell clones and high-quality split coil method adherent on slide is calculated.Cell clone grows in flakes on slide,
Cell is calculated as 60.
As shown in figure 3, Fig. 3：a：Cell clone grows on domestic slide；b：High power lens（100 times of amplifications）In seeing largely
The split coil method that phase disperses；c：See high-quality division phases.
Compared by culture synchronous with import slide, number of cell clones P=0.128（P>0.05）, high-quality division number of phases P=
0.555（P>0.05）No difference of science of statistics（Table 1）, it is believed that it obtains cell attachment clone and effectively split coil method ability is no less than
Import slide, and domestic slide is cheap.
Embodiment 2：Influence of the different hypotonic mediums to split coil method during domestic slide culture amniotic fluid harvest
1) it is inoculated with：24 amniotic fluid samples are taken, each sample takes 5ml, is mixed（Eliminate each body difference between sample）, divide 12 bottles,
Each 2 bottles of condition.10ml/ is managed, and equilibrium centrifugation 10min, rotating speed is 1200rpm, and each pipe leaves and takes 0.5ml cell suspensions（Amniotic fluid is thin
Born of the same parents' precipitation+a little amniotic fluid）, it is each to add 1.5ml amniotic fluid culture mediums, domestic slide, the original-pack slide training of import are laid in respectively
Support the cell attachment face of bottle（Fig. 2 b）, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, minimum mechanical disturbance is carrying glass after 2 days
The mark of piece（Fig. 2 a）3ml amniotic fluid culture mediums are added again, and the culture medium newly added is well mixed with old culture medium, is replaced in
37 DEG C, 5%CO2Under conditions of cultivate 5 days.
2) liquid is changed：Amniotic fluid cell culture is treated to 7-8 days, micro- Microscopic observation growth progress is inverted in, treats clone cell group
Increase, area is big, when bright cell is more, replaces old culture mediums with 2ml/ bottle fresh amniotic fluid culture mediums, treat to harvest for second day
Amniocyte slide comprising Culture in situ.
3) harvest：Colchicine is terminated after division, is respectively added slowly to the different hypotonic mediums of 37 DEG C of pre-temperature（Specific species is shown in
Table 2）Each 6ml, room temperature horizontal, 25min is pre-fixed, fixed, scattered afterwards（Be the same as Example 1）.
Film making（Be the same as Example 1）.
High-quality split coil method is chosen according to Chromosome spread quality, it is as a result as follows（Table 2）：
As a result display uses 1% sodium citrate hypotonic medium, and the high-quality split coil method that every slide is obtained is more.
Embodiment 3：Difference pre-fixes influence of the liquid to split coil method during domestic slide culture amniotic fluid harvest
1) it is inoculated with：20 amniotic fluid samples are taken, each sample takes 5ml, is mixed（Eliminate each body difference between sample）, divide 10 bottles,
Each 2 bottles of condition.10ml/ is managed, and equilibrium centrifugation 10min, rotating speed is 1200rpm, and each pipe leaves and takes 0.5ml cell suspensions（Amniotic fluid is thin
Born of the same parents' precipitation+a little amniotic fluid）, it is each to add 1.5ml amniotic fluid culture mediums, domestic slide, the original-pack slide training of import are laid in respectively
Support the cell attachment face of bottle（Fig. 2 b）, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, minimum mechanical disturbance, after 2 days carry
The mark of slide（Fig. 2 a）3ml amniotic fluid culture mediums are added again, and the culture medium newly added is well mixed with old culture medium, put again
In 37 DEG C, 5%CO2Under conditions of cultivate 5 days.
2) liquid is changed（Be the same as Example 1）
3) harvest：Colchicine terminate division after, the hypotonic 25min of 1% sodium citrate, be slowly added to difference pre-fix liquid and
（Specifically it is shown in Table 3）After 7min, supernatant is abandoned, it is fixed, scattered afterwards（Be the same as Example 1）.
4) make film（Be the same as Example 1）
High-quality split coil method is chosen according to Chromosome spread quality, it is as a result as follows（Table 3）：
As a result display pre-fixes liquid using 5% glacial acetic acid, and the high-quality split coil method that every slide is obtained is more.
Embodiment 4：Influence of the temperature and humidity of decentralized environment to split coil method during Chromosome spread
1) it is inoculated with：60 amniotic fluid samples are chosen, each sample takes 5ml, is mixed（Eliminate each body difference between sample）, divide 30
Bottle, each 2 bottles of condition.10ml/ is managed, and equilibrium centrifugation 10min, rotating speed is 1200rpm, and each pipe leaves and takes 0.5ml cell suspensions（Amniotic fluid
Cell precipitation+a little amniotic fluid）, it is each to add 1.5ml amniotic fluid culture mediums, domestic slide, the original-pack slide of import are laid in respectively
The cell attachment face of blake bottle（Fig. 2 b）, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, minimum mechanical disturbance, after 2 days
The mark of slide（Fig. 2 a）3ml amniotic fluid culture mediums are added again, and the culture medium newly added is well mixed with old culture medium, again
It is placed in 37 DEG C, 5%CO2Under conditions of cultivate 5 days.
2) liquid is changed（Be the same as Example 1）
3) harvest, film-making：Optichrome Chromosome spread instrument is put into after harvest, different temperature are selected, humidity is done
It is dry.Temperature range：19 ~ 21 DEG C, it is spaced 2 DEG C；Humidity range：30 ~ 42%, interval 3%.
4) microscope is clapped and takes caryogram：GLS-120 gathers caryogram picture, calculates karyotype and disperses integrity degree, chromosome
Overlapping number, judge caryogram satisfaction（Table 4）.
Remarks：*15/40（38%）40 complete karyotypes of analysis are represented, wherein the Chromosome spread quality quilt of 15 caryogram
" excellent " is assessed as, 38% is accounted for.
Judgment criteria is as shown in figure 4, a:Karyotype dust dispersion quality is evaluated as " excellent "；b：Karyotype dust dispersion quality
It is evaluated as " disperseing "；c：Karyotype dust dispersion quality is evaluated as " poor ", chiasma number>5.
Select the film-making condition compared with thermophilic degree, humidity combination：T=21,H=30；T=21,H=33.Can have>In 60% high-quality
Phase split coil method is used for chromosome karyotype analysis.
Embodiment 5：Domestic slide and common plastics bottle Culture in situ clinical practice Comparison Study
1) it is inoculated with：100 clinical pre-natal diagnosis amniotic fluid samples are chosen, each sample takes 20ml.Centrifugation, each pipe is left and taken
0.5ml cell suspensions（Cell precipitation+a little amniotic fluid）, fully mix, wherein a pipe plus 1.5ml amniotic fluid culture mediums, are mixed, tiling
In the cell attachment face of slide（Fig. 2 b）, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, minimum mechanical disturbance, after 2 days
In the mark of slide（Fig. 2 a）3ml culture mediums are added again.Another pipe adds 4ml culture mediums, mixes, and is laid in histocyte training
Support bottle, 37 DEG C, 5%CO2Under conditions of cultivate.
2) liquid is changed：Culture 7 ~ 8 days, observation growth progress under inverted microscope, clone cell group is more, and area is big, bright
When cell is more, replaces culture medium with 2ml/ bottles of fresh amniotic fluid culture mediums and harvest for second day.Correspondence is arrived in culture passage originally
In tissue cultuer bottle, continued growth after report is gone out, abandons it.
3) harvest, film-making, be put into Chromosome spread instrument（Ditto）.
4) microscope is clapped and takes caryogram.Compare culture success ratio, effectively number of cell clones, the division of two kinds of cultivating systems of analysis
Phase, caryogram coincidence rate.
5) clinical report.Analyze karyotype, 15 caryogram/people.
Domestic slide is compared such as with common plastics bottle cells in situ cultivation 100 amniotic fluid of synchronous culture, both results
Under：
As a result it is 100% to show two methods culture success ratio, number of cell clones P=0.497（P>0.05）, effectively division
Number of phases P=0.759（P>0.05）, it is believed that no difference of science of statistics（Table 5）.Karyotype result coincidence rate is up to 100%.Concept clinical
Plastic bottle cells culture in situ effect same can be obtained using display slide Culture in situ amniocyte technology.
Summarize：
（1）The present invention does not increase amniocentesis number of times and amniotic fluid volume, and in the absence of ethics problem, pre-natal diagnosis journey is not influenceed
Sequence；
（2）Reagent involved in the present invention is mainly Self-made reagent, and raw material domestic can be bought, without extra increase examination
Agent, does not increase cost；
（3）The present invention use domestic slide, its obtain number of cell clones and effectively division number of phases ability be no less than into
Mouth slide, and domestic slide price is low；
（4）Preliminary Clinical of the present invention shows that domestic slide Culture in situ amniocyte technology can obtain plastic bottle
Cells culture in situ effect same；
（5）The amniotic fluid in situ cultural method that the present invention is set up can be used for micrometron scanning, solve pre-natal diagnosis
Key technology is automated, possesses culture and clones abundant, slice-making quality stabilization, split coil method high-quality, the features such as success rate is high；
In summary, this technology makes amniocyte realize cell attachment on domestic slide, obtain abundant clone and
High-quality split coil method, and it is cheap.Key technology is solved on pre-natal diagnosis automatic flow, it will significant increase clinic work
Make efficiency.
Claims (8)
1. a kind of method of slide Culture in situ amniocyte, it is characterised in that comprise the following steps：
（1）Inoculation：Each amniocyte sample is taken into 18-22ml, the sterile conical centrifuge tube 2 that is divided into is managed, 9-11ml/ pipes, balance
10min is centrifuged, rotating speed is 1200rpm, and each pipe leaves and takes 0.5ml cell suspensions, it is each to add 1.5ml amniotic fluid culture mediums, fully mix,
The cell attachment face of slide is laid in, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, 2 days after it is slow again in slide
3ml amniotic fluid culture mediums are added, the culture medium newly added is well mixed with old culture medium, is replaced in 37 DEG C, 5%CO2Condition
It is lower to cultivate 5 days；
（2）Change liquid：Amniotic fluid cell culture is treated to 7-8 days, micro- Microscopic observation growth progress is inverted in, clone cell group's increasing is treated
Grow, old culture medium is replaced with 2ml/ bottles of fresh amniotic fluid culture mediums, treat to harvest the amniocyte for including Culture in situ in second day
Slide.
2. a kind of method of slide Culture in situ amniocyte according to claim 1, it is characterised in that：Described step
Suddenly（1）Middle cell suspension is that amniocyte is precipitated and amniotic fluid.
3. a kind of method of amniocyte caryogram Treatment Analysis, it is characterised in that comprise the following steps：
（1）Harvest, film-making：Colchicine is added in the amniocyte slide of described Culture in situ in claim 1, is shaken
It is even, then insert 5%CO2, 37 DEG C of incubator 25min, abandoning supernatant；
（2）It is hypotonic：The hypotonic medium 6ml of 37 DEG C of pre-temperature, room temperature horizontal 25min are slowly added in amniocyte slide；
（3）Pre-fix：The slow supernatant for extracting slide, abandons it, rear slow plus after pre-fixing liquid 6ml, 7min, abandons supernatant
Liquid, adds 4ml Fresh fixatives, rinses cell membrane, abandons supernatant；
（4）It is fixed：Fresh fixative 6ml is added in slide again, 10min is stored at room temperature, abandons supernatant；
（5）Repeat the（4）Rapid 2 times step by step, time of repose shorten to 1min；
（6）Slide is taken out with tweezers, unnecessary fixer is sucked with gauze, is put into Chromosome spread instrument, design temperature
For 21 DEG C, humidity is 30% or 33%, treats that slide is completely dried；
（7）Slide is placed at 65 DEG C of baking oven and bakes piece 2h, is taken out after slide, 1mg/ml pancreatin immersion treatment 16-20s,
Giemsa dye liquor immersion treatments 10min；
（8）Bat takes caryogram：Clapped using microscope and take amniocyte slide, the automatic caryogram scanner capture collection dyes of GLS-120
Colour solid caryogram.
4. a kind of method of amniocyte caryogram Treatment Analysis according to claim 3, it is characterised in that：Described autumn waters -- limid eyes
Celestial element concentration is 20 μ g/ml.
5. a kind of method of amniocyte caryogram Treatment Analysis according to claim 3, it is characterised in that：Described is hypotonic
Liquid concentration is 1% sodium citrate.
6. a kind of method of amniocyte caryogram Treatment Analysis according to claim 3, it is characterised in that：Described is pre- solid
Determine the glacial acetic acid that liquid concentration is 5%.
7. a kind of method of amniocyte caryogram Treatment Analysis according to claim 3, it is characterised in that：Described fixation
Liquid is methanol：Glacial acetic acid=3：1 Kano fixer.
8. a kind of method of amniocyte caryogram Treatment Analysis according to claim 3, it is characterised in that：Described
Giemsa dye liquors composition is 1：10 Giemsa stostes and pH6.8 phosphate buffer.
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