the progression of hiv disease involves various stages characterized by changes in lymphocyte subsets and viral load .
after initial infection , an acute viremic stage occurs , which is accompanied by a rapid decline in the number of cd4 + t lymphocytes and a slow increase in cd8 counts . during the clinical latency phase ,
cd4 counts gradually decline , and hiv load is lower than during the acute stage , although virus is still present . finally , the late stage of hiv disease is reached , where cd4 + t - cells fall severely , and the number of viral particles surge . in the case of the hiv infected patient , emphasis is placed on cd4 counts because when the number of cd4 + t - cells falls below a critical level , prophylaxis against opportunistic infection is initiated .
flow cytometry is not useful for samples that have been frozen or stored , therefore retrospective studies involving hiv - infected patients ' samples are unable to view the data in the light of information regarding the immune status at the time the samples were frozen .
we also perceived that studies of hiv-1 status , particularly in infants , could be facilitated by a technique using smaller amounts of blood , such as that from a heelstick or finger - prick .
quantitative methods for reverse transcriptase polymerase chain reaction ( rt - pcr ) are rapidly surpassing all other methods of quantifying mrna levels .
we therefore set out to develop an rt - pcr based method to quantitate cd4 and cd8 mrna in the hopes that this could be used to predict cell counts .
although it has long been known that hiv-1 down - regulates cd4 cell surface expression , the loss of cd4 is not complete and the hiv - infection does not reduce cd4 mrna levels .
thus , a method based on quantitating cd4 and cd8 mrna could provide a means of assessing immune status without requiring repetitive and large blood draws , and could be exquisitely sensitive .
this method may also be useful in assessing the immune status of patients retrospectively from archived or frozen samples .
qc - rt - pcr , and related methodologies , however , did not prove useful as substitutes for assessment of cd4 and cd8 cell numbers in hiv - infected persons .
samples were acquired from special hematology , louisiana state university medical center , after exempt status was obtained from tulane institutional review board . the relationship between
cd4 and cd8 cell number by flow cytometry , and the amount of mrna as quantitated by qc - rt - pcr , was determined .
flow cytometry was performed as previously described   along with white blood cell and differential counts , including percent lymphocytes , in order to determine numerical values .
intact mrna was successfully extracted from as little as 400 l of whole blood , comparable amounts to that obtained from infants via heelstick .
this mrna was used to quantify the amount of cd4 , cd8 , as well as gapdh , mrna that is being transcribed in t lymphocytes within the blood .
the corrected quantity of cd4 and cd8 mrna as determined by qc - rt - pcr and the corresponding cd4 and cd8 counts of that patient as determined by flow cytometry are depicted in table 1 .
the patients with unknown status all have inverted cd4:cd8 cell ratios and are therefore likely to be hiv infected ( the majority of the samples sent for flow cytometry are hiv - positive ) .
the relationship between the mrna and cell number for cd4 and cd8 are depicted ( fig .
the relationship between cell counts and the quantity of mrna as determined by qc - rt - pcr .
( a ) cd4 , and ( b ) cd8 . the hiv status of each sample is depicted
.   represents hiv positive status ,  represents hiv negative status , and  reepresents unknown status .
our results indicate that there is no correlation between cd4 and cd8 mrna levels and numbers of cells expressing these proteins on their surface .
the formation of cd4-gp160 complexes in intracellular compartments may contribute to cell surface cd4 down - modulation during hiv-1 structural protein expression . these and other mechanisms may explain the lack of correlation between message and surface expression in hiv - positive individuals .
hiv infection may also interfere with the translation or surface expression of cd8 , although it is not used as a viral receptor .
whatever the mechanism , no correlation between cd4 and cd8 cell counts and mrna levels of cd4 and cd8 could be determined by qc - rt - pcr .
there was also no correlation between cd4 and cd8 mrna levels and the number of cells expressing these proteins on their surface .
given the magnitude of the variations observed by qc - rt - pcr it is unlikely that other methods to quantitate these mrnas , e.g. northern blotting or real - time pcr , would reveal a correlation either .
qc - rt - pcr and related techniques such as real - time pcr are not useful as substitutes for assessment of cd4 and cd8 cell status in hiv - infection by flow cytometry .
the strategy for development of competitor templates for quantitative - competitive rt - pcr ( qc - rt - pcr ) was similar to that of lipman and coworkers .
oligonucleotide primers ( clontech , palo alto , ca ) were chosen that span introns of cd4 and cd8 genes in order to avoid amplification of cellular dna contaminating rna preparations .
products are generated from cdna and deletional mutants are constructed by restriction endonuclease digestion and religation .
the deletional mutants were then subcloned using the ta cloning vector pcr2.1 ( invitrogen , carlsbad , ca ) , and sequenced for confirmation .
the method used to generate a competitor for cd8 was slightly different than previously reported methods .
the wild type pcr product was inserted into the ta cloning vector before digesting with bbsi , an enzyme with no recognition sites inside the vector and only one site in the insert .
the linearized plasmid was gel purified , the free ends of the plasmid were then shortened using bal31 nuclease .
the ends were filled using the klenow fragment of dna polymerase , and then religated .
the competitor template for glyceraldehyde phosphate dehydrogenase ( gapdh ) , used to control the amount of rna in each extraction , have already been constructed by lipman et al . , ( 1994 ) , and were kindly provided by dr .
known concentrations of the competitor cdna was amplified in the same reaction tube as unknown amounts of sample derived rt - cdna .
total rna is extracted from 400 l to 1 ml of whole blood using a similar method to zhang and yunis .
first strand cdna is synthesized using oligo dt primers and maloney murine leukemia virus reverse transcriptase ( promega , madison , wi ) , diluted 1 to 3 , then 3 l is used as template in a 25 l pcr .
the remainder of the reactions were made up by 5 l pcr buffer ( gibco brl , grand island , ny ) , 0.25 g of each primer , 6 mm mgc12 in each cd4 reaction and 4 mm mgc12 for cd8 reactions , 2 mm total dntp 's , and 0.5 u of taq polymerase . for cd4 and cd8 reaction conditions
were : 94 for 3 mins , then 33 cycles of 94 for 45 secs , 55 for 45 secs , and 72 for 2 mins on a perkin elmer thermocycler .
33 cycles was chosen as this was the largest number still within the linear range of the pcr ( data not shown ) . in each assay
we performed at least six different reactions for each sample using increasing amounts of competitor dna .
the amounts of sample were quantitated by extrapolating across at least three reactions in which approximately equal amounts of sample and competitor products were produced .
. quantity of sample derived mrna can be determined when the intensity of its pcr band is equal to that of the competitor 's upon co - amplification . staining with ethidium bromide
the standard curve plots the log cdna / competitor band intensity ( as determined by analysis of pict files with nih image ) on the x - axis versus the log of incremental dilutions of competitor dna on the y - axis . solving for the antilog of x
when y = 0 will give the concentration of unknown amount of dna  that of cd4 , cd8 or gapdh .
the success of the rna extraction and reverse transcription is determined by quantification of gapdh mrna by qc - rt - pcr . each value for quantity of cd4 or cd8
the strategy for development of competitor templates for quantitative - competitive rt - pcr ( qc - rt - pcr ) was similar to that of lipman and coworkers .
oligonucleotide primers ( clontech , palo alto , ca ) were chosen that span introns of cd4 and cd8 genes in order to avoid amplification of cellular dna contaminating rna preparations .
products are generated from cdna and deletional mutants are constructed by restriction endonuclease digestion and religation .
the deletional mutants were then subcloned using the ta cloning vector pcr2.1 ( invitrogen , carlsbad , ca ) , and sequenced for confirmation .
the method used to generate a competitor for cd8 was slightly different than previously reported methods .
the wild type pcr product was inserted into the ta cloning vector before digesting with bbsi , an enzyme with no recognition sites inside the vector and only one site in the insert .
the linearized plasmid was gel purified , the free ends of the plasmid were then shortened using bal31 nuclease .
the ends were filled using the klenow fragment of dna polymerase , and then religated .
the competitor template for glyceraldehyde phosphate dehydrogenase ( gapdh ) , used to control the amount of rna in each extraction , have already been constructed by lipman et al . , ( 1994 ) , and were kindly provided by dr .
known concentrations of the competitor cdna was amplified in the same reaction tube as unknown amounts of sample derived rt - cdna .
total rna is extracted from 400 l to 1 ml of whole blood using a similar method to zhang and yunis .
first strand cdna is synthesized using oligo dt primers and maloney murine leukemia virus reverse transcriptase ( promega , madison , wi ) , diluted 1 to 3 , then 3 l is used as template in a 25 l pcr .
the remainder of the reactions were made up by 5 l pcr buffer ( gibco brl , grand island , ny ) , 0.25 g of each primer , 6 mm mgc12 in each cd4 reaction and 4 mm mgc12 for cd8 reactions , 2 mm total dntp 's , and 0.5 u of taq polymerase . for cd4 and cd8 reaction conditions
were : 94 for 3 mins , then 33 cycles of 94 for 45 secs , 55 for 45 secs , and 72 for 2 mins on a perkin elmer thermocycler .
33 cycles was chosen as this was the largest number still within the linear range of the pcr ( data not shown ) . in each assay
we performed at least six different reactions for each sample using increasing amounts of competitor dna .
the amounts of sample were quantitated by extrapolating across at least three reactions in which approximately equal amounts of sample and competitor products were produced .
. quantity of sample derived mrna can be determined when the intensity of its pcr band is equal to that of the competitor 's upon co - amplification . staining with ethidium bromide
the standard curve plots the log cdna / competitor band intensity ( as determined by analysis of pict files with nih image ) on the x - axis versus the log of incremental dilutions of competitor dna on the y - axis . solving for the antilog of x
when y = 0 will give the concentration of unknown amount of dna  that of cd4 , cd8 or gapdh .
the success of the rna extraction and reverse transcription is determined by quantification of gapdh mrna by qc - rt - pcr . each value for quantity of cd4 or cd8
heather jaspan conceived of the study , performed all the molecular analyses , and wrote the manuscript .
 backgrounda polymerase chain reaction ( pcr)-based method for quantitating cd4 and cd8 mrna could provide a means of assessing immune status of aids patients and other immunologically compromised persons without requiring large blood draws , and could be exquisitely sensitive . 
 such a method would also be useful in assessing the immune status of patients retrospectively.resultsquantitative competitive reverse transcription pcr ( qc - rt - pcr ) assays were developed for measurement of cd4 and cd8 mrna . 
 samples were obtained from hiv - positive and negative patients whose cd4 and cd8 counts had been determined via flow cytometry . 
 the quantity of cd4 ( n = 13 ) and cd8 ( n = 28 ) mrna standardized according to gapdh mrna quantities , all determined by qc - rt - pcr , were compared to cell number as determined by flow cytometry . 
 there was no correlation between cd4 and cd8 cell counts and mrna levels of cd4 and cd8 as determined by qc - rt - pcr . 
 there is no correlation between cd4 and cd8 mrna levels and the number of cells expressing these proteins on their surface.conclusionqc-rt-pcr , and related methodologies are not useful substitutes for assessment of cd4 and cd8 cell numbers in hiv - infected persons . 
Background
Results
Discussion
Conclusions
Methods
QC-RT-PCR
Authors' contributions
Acknowledgements