for the 53 selected cases , 28 right and left bone marrow samples were allocated to the edta ( sigma - aldrich , st .
louis , mo , usa ) protocol and hcl ( calci - clear rapid , national diagnostics , atlanta , ga , usa ) protocol , respectively .
samples from the 25 remaining cases were assigned to the rdo gold ( rdo ) group ( apex engineering products corporation , aurora , il , usa ) protocol and hcl protocol .
concentration , processing time , and temperature are summarized in table 1 . to test dna quality ,
five cases were randomly selected from each of the three groups ( edta , hcl , and rdo ) .
dna was extracted , and the quantity and quality were confirmed using nanodrop nd-2000 spectrophotometer ( thermo fisher scientific inc . , waltham , ma , usa ) .
the braf pna clamping method ( panagene , daejeon , korea ) and comparison with ct values of internal controls were used to evaluate the efficacy of polymerase chain reaction ( pcr ) dna amplification . to evaluate the efficacy of dna in situ hybridization , her2 dual color silver in situ hybridization ( ventana , tucson , az , usa )
rna in situ hybridization and immunohistochemical studies were performed prospectively . according to the potential differential diagnoses for suspicious lesions observed within bone marrow , appropriate rna probes or protein antibodies were applied . for immunohistochemistry ,
the following primary antibodies were used and the details are summarized in table 2 : cyclin d1 , ki67 , bcl2 , bcl6 , tdt , cd138 , cd20 , cd79a , cd3 , cd5 , cd23 , cd10 , cd30 , and myeloperoxidase . to state the process , after deparaffinization and rehydration
, the sections were incubated in benchmark xt automated slide stainer ( ventana ) for 16 minutes at 37c and then counterstained with hematoxylin reagent .
two pathologists reviewed the hematoxylin and eosin ( h&e ) , immunohistochemistry , and in situ hybridization slides .
the quality of immunohistochemistry and rna in situ hybridization were assessed using a 3-tiered grading scale : good , equivocal , or poor .
her2 silver in situ hybridization was assessed by detecting two signals of her2 and cep17 per nucleus from the normal hematopoietic cells of bone marrow .
all statistical analyses were carried out by spss ver . 20.0 for windows ( ibm co. , armonk
dna quantity , purity , and ct values of internal controls after real - time pcr in the edta versus hcl group and rdo versus hcl group are depicted in table 3 and fig .
1 . although differences were not statistically significant , the dna yield of the edta protocol was about 2 times higher than the hcl protocol .
in addition , the ct value of the former protocol was significantly lower than that of the latter ( p<.001 ) with the estimated difference being about 7 . furthermore , the ct values of edta processed samples were lower than 30 , demonstrating that the amount of intact dna feasible for pcr with the edta protocol is better preserved by a factor of 2   than the hcl protocol .
there were no significant differences between dna yield and ct values of rdo and hcl methods .
the ct values of both protocols were above 33 , indicating that the amount of intact dna feasible for pcr was very small .
the morphological comparison and quality assessment of h&e stain , her2/cep17 dual color silver in situ hybridization , kappa / lambda in situ hybridization , and immunohistochemistry studies were analyzed .
the rates of high quality staining for each study were compared between the three protocols .
there was no difficulty in microtome dissection of 4 m or less in thickness in any of the three methods .
the morphological quality of h&e slide was similar in all three protocols , showing well - preserved histological features of the bone marrow ( fig .
all five cases in the rdo versus hcl group had severe dna breakdown on her2/cep17 dual color silver in situ hybridization , revealing no her2 or cep17 nuclear signal in bone marrow hematopoietic cells .
however , in the edta versus hcl group , all 5 cases in the edta protocol showed two her2 and cep17 nuclear signals from almost all of the hematopoietic cells , whereas nearly no nuclear signal was detected in samples from the hcl protocol ( fig .
bone marrow specimens from the edta and rdo protocols that underwent kappa / lambda rna in situ hybridization showed well preserved rna signal in the nuclei of plasma cells , while those in the hcl group had severe breakdown of rna signals ( fig .
4 ) . nuclear proteins such as ki67 , cyclin d1 , and tdt were relatively better preserved on immunohistochemistry with both edta and rdo protocols , while samples from the hcl protocol showed breakdown and lower quality of nuclear protein staining ( fig .
immunohistochemistry targeting the cytoplasmic membrane or cytoplasmic cd markers was well preserved in all three protocols ( fig .
dna quantity , purity , and ct values of internal controls after real - time pcr in the edta versus hcl group and rdo versus hcl group are depicted in table 3 and fig .
1 . although differences were not statistically significant , the dna yield of the edta protocol was about 2 times higher than the hcl protocol .
in addition , the ct value of the former protocol was significantly lower than that of the latter ( p<.001 ) with the estimated difference being about 7 . furthermore , the ct values of edta processed samples were lower than 30 , demonstrating that the amount of intact dna feasible for pcr with the edta protocol is better preserved by a factor of 2   than the hcl protocol .
there were no significant differences between dna yield and ct values of rdo and hcl methods .
the ct values of both protocols were above 33 , indicating that the amount of intact dna feasible for pcr was very small .
the morphological comparison and quality assessment of h&e stain , her2/cep17 dual color silver in situ hybridization , kappa / lambda in situ hybridization , and immunohistochemistry studies were analyzed .
the rates of high quality staining for each study were compared between the three protocols .
there was no difficulty in microtome dissection of 4 m or less in thickness in any of the three methods .
the morphological quality of h&e slide was similar in all three protocols , showing well - preserved histological features of the bone marrow ( fig .
all five cases in the rdo versus hcl group had severe dna breakdown on her2/cep17 dual color silver in situ hybridization , revealing no her2 or cep17 nuclear signal in bone marrow hematopoietic cells .
however , in the edta versus hcl group , all 5 cases in the edta protocol showed two her2 and cep17 nuclear signals from almost all of the hematopoietic cells , whereas nearly no nuclear signal was detected in samples from the hcl protocol ( fig .
bone marrow specimens from the edta and rdo protocols that underwent kappa / lambda rna in situ hybridization showed well preserved rna signal in the nuclei of plasma cells , while those in the hcl group had severe breakdown of rna signals ( fig .
nuclear proteins such as ki67 , cyclin d1 , and tdt were relatively better preserved on immunohistochemistry with both edta and rdo protocols , while samples from the hcl protocol showed breakdown and lower quality of nuclear protein staining ( fig .
immunohistochemistry targeting the cytoplasmic membrane or cytoplasmic cd markers was well preserved in all three protocols ( fig .
some retrospective studies compared the morphology of in situ hybridization or immunohistochemistry by using stored tissues containing bone .
other studies extracted nucleic acid from stored tissues containing bone and compared the quantity , purity , and ct value of the dna and/or rna after real - time pcr [ 1 - 4 ] .
we investigated the quality of nucleic acid and protein in decalcified bone marrow tissue employing several types of genetic tools , immunohistochemistry , and morphological assessments .
in addition , we compared the effect of decalcification protocol while limiting bias variance , which may be caused by sampling from different patients and cell degeneration from long - term storage .
we prospectively investigated pairs of bone marrow biopsy specimens from the same patients sampled for diagnostic purposes in a clinical setting . to our knowledge , this is one of the first studies utilizing clinical samples to assess decalcification protocols .
we compared the conventional protocol using hcl , the well - known alternative protocol using edta , and the new protocol using rdo .
all three methods had equally good performance with respect to microtome dissection and preservation of cytomorphologic and histomorphologic features .
the edta protocol was superior in preserving nucleic acid ( dna and rna ) , allowing for the feasibility of genetic studies , such as real time pcr and in situ hybridization .
the edta protocol would be an appropriate option for genetic studies in bone - contained tissues .
the present study also confirmed that the hcl protocols are inappropriate for genetic studies due to the severe damage of genetic material .
the quality of nucleic acids in the rdo group was equivocal , but this method also did nt seem to be suitable for genetic studies . in immunohistochemistry targeting nuclear protein ,
edta protocols seem to be the most appropriate in that comprehensive immunohistochemical stains can be applied to the specimens processed by this protocol .
the feasibility of immunohistochemistry using specimens processed with the hcl protocol would be limited in many cases , especially when immunostaining for nuclear proteins is necessary .
immunohistochemical markers can be more widely applied in samples processed with the rdo protocol than with the hcl protocol , but diagnostic options are still more limited than the edta protocol . in this study ,
the edta protocol was the most feasible method for several types of genetic studies and immunohistochemistry .
such advantages of edta decalcification protocol are already widely known [ 1,5 - 7 ] .
the present study confirms again the superiority of the edta protocol in a wide range of ancillary tests for pathologic diagnosis .
the potential of the rdo protocol is higher than the conventional hcl protocol ; however , it is not superior to the edta protocol and thus , it can not be considered an alternative to edta protocols , particularly when genetic studies are needed . when choosing decalcification agents , cost - effectiveness and turn - around time from biopsy to final pathologic diagnosis are important issues .
small bone tissues like bone marrow take about three to twenty four hours for decalcification in edta protocol , but most of the other bone specimens need more time when using the same protocol .
rdo protocols can shorten decalcification time , but this agent is also expensive . in many cases , bone biopsies are needed for diagnosis of hematologic cancer , metastatic tumors , and primary bone sarcoma ; these types of cancers usually need further gene - based diagnosis .
bone marrow biopsy is widely performed in patients with hematologic malignancies and pediatric sarcomas or blastomas .
therefore , preserving genetic materials is critical in handling bone marrow tissues , and cost and turn - around time may be less important in such cases .
preserving intact nucleic acid , as well as intact proteins , is critical to providing not only accurate pathologic diagnosis but also diverse therapeutic options to the patients .
considering this , in this study the edta protocol was the most appropriate method for handling bone marrow specimens .
rdo may also be useful in that it requires less decalcification time and it enables more ancillary tests than hcl , but its usefulness is limited by less potential for genetic studies in processed samples than edta . in this era of expanding genetic molecular study , better tissue handling methods are needed .
the present study suggests an appropriate approach in handling bone marrow tissues , and this approach should be expanded to other types of tissue specimens .
 background : the conventional method for decalcification of bone specimens uses hydrochloric acid ( hcl ) and is notorious for damaging cellular rna , dna , and proteins , thus complicating molecular and immunohistochemical analyses . a method that can effectively decalcify while preserving genetic material 
 is necessary.methods:pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups : edta versus hcl and rdo gold ( rdo ) versus hcl . 
 pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the edta versus hcl comparison group , and 25 cases to the rdo versus hcl comparison group . 
 the decalcification protocols were compared with regards to histomorphology , immunohistochemistry , and molecular analysis . for molecular analysis 
 , we randomly selected 5 cases from the edta versus hcl and rdo versus hcl groups.results:the decalcification time for appropriate histomorphologic analysis was the longest in the edta method and the shortest in the rdo method . 
 edta was superior to rdo or hcl in dna yield and integrity , assessed via dna extraction , polymerase chain reaction , and silver in situ hybridization using dna probes . 
 the edta method maintained intact nuclear protein staining on immunohistochemistry , while the hcl method produced poor quality images . 
 staining after the rdo method had equivocal results . 
 rna in situ hybridization using kappa and lambda rna probes measured rna integrity ; the edta and rdo method had the best quality , followed by hcl.conclusions:the edta protocol would be the best in preserving genetic material . 
 rdo may be an acceptable alternative when rapid decalcification is necessary . 
MATERIALS AND METHODS
RESULTS
Isolated DNA quality
Morphological comparison of DNA, RNA, and protein expression
DISCUSSION