PREPARATION OF BODY FLUID SAMPLE FOR RNA ANALYSIS

Abstract: The invention relates to a method for the treatment of a body fluid sample containing eosinophils, so as to derive an RNA therefrom that is suitable for analysis especially RT-PCR. The method according to the invention is characterised in that eosinophils are removed from said sample before the RNA is subjected to analysis. This method prevents degradation of RNA by ribonuclease derived from eosinophils.

Description: PREPARATION OF BODY FLUID SAMPLE FOR RNA ANALYSIS This invention relates to a method for treating a body fluid sample in order to derive an RNA therefrom suitable for further analysis. BACKGROUND OF THE INVENTION The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference. Analyses of RNA from body fluid samples are useful for many purposes. As examples can be mentioned the detection of RNA viruses, the detection of mutations in the DNA of cells where said DNA comprises unknown intron sequences. In this case the problem is avoided by using the transcription product mRNA for the detection. A further reason for the analysis of RNA may be to study the expression of any gene. Good recovery and high-quality RNA from the samples are prerequisites for accurate analyses. The isolation of RNA from a sample is very difficult compared with the isolation of DNA because the samples comprise enzymes, ribonucleases, which are effective in the cleavage of RNA. Such enzymes are present in many cells and tissues and they tend to cause the degradation of the RNA during the isolation process. Various methods have been developed in order to avoid degradation of RNA during the isolation. The commercialised acid-guanidiniumthiocyanate-phenol-chloroform (AGPC) RNA extraction first developed by Chomczynski and Sacchi (1987) is widely used. This method is, however, very tedious when a number of samples should be analysed. An important clinical application of reverse transcription-polymerase chain reaction (RT-PCR) is the analysis of residual leukaemia cells after bone marrow transplantation (BMT). For a single residual disease RT-PCR, mRNA or total RNA from about 100 000 leukocytes is usually analysed. Extraction of RNA from ribonuclease-rich leukocyte preparation is tedious, unreproducible and difficult to apply to an automated RT-PCR assay for clinical laboratory. We have earlier shown that RNA extraction caused 77.7 % of the total analytical variation in our Philadelphia chromosome (bcr/abl translocation) assay from leukocytes of chronic myelogenous leukaemia patients (Eskola et al 1994). To apply a leukocyte lysate directly to RT-PCR after the treatment of diethyl pyrocarbonate by the method of Kawasaki (1990) proved unsuccessful in the assay. A lysate of 10 5 leukocytes entirely inhibited reverse transcription, whereas PCR remained almost uneffected. The addition of the placental RNase inhibitor (Blackburn et al 1977) to leukocyte lysate did not improve the assay. The other published methods use a lysate directly from low number of leukocytes or cultured cells (Fung and Fung 1991, Shi and Liu, 1992, O'Brien et al 1994, Klebe et al 1997), or from boiled cell samples (Ferre & Garduno 1989). Low number of cells can be used without RNA extraction if proper RNase inhibitors are added and if there is universal expression of the studied mRNA. The heating of the sample may result in the partial destruction of mRNA demising the intact transcript of the studied gene below the detection limit. In the study disclosed below we characterised the origin of RT-inhibitory activity of a leukocyte lysate and found out that it is mainly due to eosinophil-derived neurotoxin, EDN. OBJECT AND SUMMARY OF THE INVENTION The object of the present invention is to provide a method for treating a body fluid sample containing eosinophils so as to obtain an RNA therefrom which is suitable for analysis, i.e. an RNA in which the degradation caused by ribonuclease of eosinophils in the sample has been substantially prevented. The present invention thus concerns a method for the treatment of a body fluid sample so as to derive an RNA therefrom that is suitable for analysis. The method is characterised by the removal of eosinophils from said sample before RNA is derived and subjected to analysis. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the inhibition by human leukocytes on the Ph'-chromosome RT-PCR of K562 cells. Five hundred Ph 1 -positive K562 cells were lysed with varying number of leukocytes in 50 μl of water and 5 μl of the lysate (50 K562 cells) was used to RT-PCR assay. Reverse transcription was performed using either M-MLV or AMV reverse transcriptase. The PCR product was quantitated by hybridisation with an Eu 3+ -labelled probe and time-resolved fluorometry. Figure 2 shows cation exchange chromatography of the purification of the reverse transcription-inhibiting factor from leukocytes. A lysate from 3 x 10 9 leukocytes was applied to a 5 ml HiTrap SP cation exchange column (Pharmacia Biotech). Unbound proteins were eluted by 20 mM Tris-HCl, pH 7.4 at 2 ml/min, collecting 5 ml fractions. Bound basic proteins were eluted by the same buffer containing 2 mol/1 NaCl. Dilutions of the fractions (1 :50) were used to measure the inhibition of RT-PCR assay of 50 Ph 1 chromosome-positive K562 cells as described in Fig. 1. Figure 3 shows gel filtration chromatography of the purification of RT-inhibiting factor from leukocytes using Superdex 200HR 10/30 column (Pharmacia Biotech). The sample was 1 ml of the concentrated fractions #15-16 from cation exchange chromatography (Fig. 2). The proteins were eluted with 0.1 M Tris-HCl, pH 7.2, 0.15 M NaCl at 0.6 ml/min collecting 1 ml fractions. The RT-inhibiting activity of the 1 :50 diluted fractions was measured as expressed in Fig. 1. The inhibition peak at 19 ml elution volume suggests a 18 000 kDa protein when compared to the elution volumes of standard proteins shown on the upper side of x-axis. Figure 4 shows reverse phase chromatography of the purification of RT-inhibiting factor from leukocytes using Vydac C4 column (2.1 x 150 mm, The Separations Group). The sample was 0.5 ml of the concentrated gel filtration fractions #19-20 (Fig 3). The solvent system was A: 0.1 % trifluoroacetic acid/H 2 O; B: 0.08 % trifluoroacetic acid/acetonitrile. The solvent gradient was 0-30 % B (0-63 min), 30-60 % B (63-95 min), 60-80 % B (95-105 min), 80 % B (105-110 min) with a flow rate of 0.15 ml/min. Twenty fractions were collected according to the protein UV 28 o nm absorption chromatogram (inserted in the figure). The RT-inhibiting activity was measured from 1 :11 diluted fractions as given in Figure 1. Eosinophil- derived neurotoxin (EDN) was measured from 1:100 diluted fractions by RIA. DETAILED DESCRIPTION OF THE INVENTION The method according to the present invention can be used for deriving of total RNA, rRNA or mRNA from a body fluid sample containing eosinophils. The sample can comprise cells from which RNA shall be derived. The RNA can be mRNA useful for gene expression analysis. As a particular use of this method can be mentioned leukocytes which shall be screened for the presence of Philadelphia (Ph 1 ) chromosome which relates to haematological malignancies. The method of the invention is also useful for deriving of RNA from a sample comprising an RNA-virus or for deriving of RNA from a sample containing a bacterium. The RNA can be rRNA useful for the analysis of bacteria species. According to one specific embodiment of the invention the removal of the eosinophils is carried out by incubating the sample with an antibody specific for an eosinophil surface antigen to form an antibody-antigen complex, and removing the antibody-antigen complex. The RNA of the remaining sample is collected for analysis. According to an alternative method the sample containing cells the RNA of which shall be analysed and eosinophils, is incubated with an antibody specific for a surface antigen of said material to form an antibody-antigen complex, followed by withdrawing the antibody-antigen complex from the sample, and subjecting the RNA of the withdrawn cells to analysis. In case it is desired to analyse the RNA of bacteria or viruses and the aforementioned separation method is used, a prerequisite is that said bacteria or viruses are encompassed in the withdrawn cells. The antibody can be a polyclonal antibody, a monoclonal antibody, a mixture of several monoclonal antibodies, a mixture of several polyclonal antibodies, or a mixture of monoclonal and polyclonal antibodies. According to yet another embodiment of the invention, the eosinophils are removed from the sample by centrifuging a composition comprising the sample and a centrifugation medium, the density of the centrifugation medium and the force of centrifugation being selected to cause a substantial portion of the eosinophils to gather in a specific fraction, and removing the eosinophil-rich fraction. The RNA from the remaining sample is collected for analysis. According to a preferred embodiment, the density of the centrifugation medium is in the range 1.075 to 1.082 g/ml (preferentially 1.077 g/ml) made up from known solutions such as Ficoll-Paque R or Percoll R (Pharmacia Biotech) or Lymphoprep R (Nycomed AS) or from an analogous solution. The separation methods using antibodies or gradient centrifugation mentioned above can be used for deriving of RNA from cells as well as RNA from viruses and bacteria. In the centrifugation method, the heavy granulocyte layer contains the main part of the eosinophils. The RNA is derived from the lighter, mononuclear cells containing layer. In case the sample comprises cells from which RNA shall be derived, this is lysed so as to release the RNA from the cells before the analysis thereof. The RNA can be amplified by known methods such as RT-PCR, amplification by reverse transcriptase and T7 RNA polymerase, or RNA-directed RNA polymerase, before the detection of the nucleotide sequence. According to a preferred embodiment, the RNA of the sample from which the eosinophils have been removed, is directly used in the subsequent amplification and analysis steps without foregoing isolation of said RNA. The invention will be described in more detail by the following study of the reverse transcription-inhibiting activity of leukocytes in the analysis of residual disease in CML. Summary of the study The extraction of RNA from leukocytes of clinical samples for RT-PCR is time- consuming, causing most of the variation in the analysis of Philadelphia (Ph 1 ) chromosome of chronic myelogenous leukaemia (CML) by RT-PCR. To detect a residual disease of CML after bone marrow transplantation, mRNA from about 10 5 leukocytes is usually analysed. Due to the RNase activity of the cells simple lysis of leukocytes is applicable only to RT-PCR of small number of cells. We identified the main source of reverse transcription-inhibiting activity of leukocytes as eosinophil-derived neurotoxin (EDN) by a three-step chromatographic process and amino acid sequence analysis. Since EDN is localised almost entirely to eosinophils we removed eosinophils from a leukocyte preparation using magnetic particle-bound CD 15 or biotinylated CD 16 antibodies or by fractionating the cells by Ficoll-Paque R density gradient. Depletion of eosinophils reduced the EDN content of leukocyte lysate to about one tenth allowing a 77.4 % signal from a lysate of 50 K562 Ph 1 -chromosome-positive cells and 100 000 CD 16+ granulocytes compared with a leukocyte free control. The mononuclear cell fraction obtained from Ficoll-Paque density gradient allowed a 52.8 % signal compared with the control whereas RNA extraction produced only 24.4 % of the signal of leukocyte free control. The removal of myeloid cells with CD 15 antibodies gave inconsistent results showing large variation in the signal level between separations. The depletion of eosinophils during the preparation of sample for RT-PCR efficiently reduces the risk of mRNA degradation by ribonucleases. Material and Methods Determination of reverse-transcription inhibiting activity A RT-PCR assay designed to detect Philadelphia-chromosome of CML patients (1) was used to study reverse transcription inhibitors. Briefly: A 5 μl aliquot of leukocyte lysate or diluted chromatography-purified fraction was added to 15 μl of RT-mixture containing a specific downstream primer (2), 50 U of M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD), 20 U of RNasin ® (Promega Corp., Madison, WI) and 50 to 100 Philadelphia chromosome-positive K562 cells. The tubes were incubated for 30 min at 37 °C. In one experiment AMV reverse transcriptase instead of M-MLV enzyme was used at 42°C. After incubation the tubes were heated for 5 min at 100°C and PCR-mixture (80 μl) with 1 U of AmpliTaq DNA-polymerase (Perkin-Elmer Corp., Foster City, CA) was added. PCR conditions were: 50 s at 95 °C; 1 min at 55 °C; 2 min at 72 °C for 30 cycles. The amplification products were quantified by a sensitive solution hybridisation assay using streptavidin-coated microtiter wells and biotin-labelled and europium-labelled probes and by measuring the time-resolved fluorescence of liberated europium chelate (Hurskainen et al 1991). We observed that the leukocyte lysate or its chromatography-purified fractions did not inhibit the PCR or hybridisation phases of the assay (data not shown). Total RNA extractions were made with the method of Chomczynkski and Sacchi (1987) using the commercial TRIzol ® reagent (Gibco BRL) Inhibition of reverse transcription by leukocyte lysate Number of leukocytes vs RT -inhibition. Two million leukocytes from a healthy human donor were lysed in 100 μl of sterile water containing 4 units of Inhibit- ACE RNase inhibitor (5 Prime-3 Prime, Inc, West Chester, PA) for 5 minutes. After centrifugation at 13 000 x g for 2 min the supernatant was serially diluted with water to a final concentration of 200 lysed leukocytes /ml. Five microlitres of the dilutions were used in RT-PCR assay with 50 Philadelphia chromosome-positive K562 cells. Inhibition tests were run using both M-MLV and AMV reverse transcriptase enzymes. Use of RNase inhibitors in leukocyte lysate. To find an efficient RNase inhibitor to prevent ribonuclease of leukocyte lysate six commercial RNase inhibitors were tested. Two million leukocytes were lysed in 50 ml of water containing 1000 K562 Philadelphia chromosome-positive cells and one of the following human placental RNase inhibitors with 5 mmol/1 dithiothreitol (DTT): RNasin, 100 U (Promega, Madison, WI), RNAguard, 100 U (Pharmacia, Uppsala, Sweden), RNase inhibitor 50 U (Perkin Elmer), or the protein RNase inhibitors: Rnase Block II, 2 U (Stratagene, LaJolla, CA), or Inhibit-ACE, 2 U, (5 prime-3 prime) or Ribonucleoside-Vanadyl Complex, 10 mmol/1 (New England Biolabs, Beverly, MA). Five microlitres of the centrifuged lysate (100 K562 cells and 200 000 leukocytes) were taken to the RT-PCR assay. Purification and identification of reverse transcription inhibiting factor from leukocytes Leukocyte lysate. Leukocytes were collected by centrifugation from the buffy coats of expelled whole blood bags after sedimentation of the erythrocytes with 1.2 % dextran. The composition of leukocytes was: lymphocytes 31.3 %; neutrophils 56.5 %; basophils 0.8 %; eosinophils 2.6 %; monocytes 6.4 ; and large unstained cells 2.5 %. The cells were rewashed once by phosphate buffered saline (PBS) and 3-10 9 leukocytes were lysed in 8 ml of 20 mM Tris-HCl buffer, pH 7.4, for 5 min at room temperature. After centrifugation at 5000 x g f or 10 min at 4°C the pellet was washed with 6 ml of the same buffer and centrifuged again. The volume of combined supernatants was 14 ml and protein concentration 29.9 mg/ml, as determined by the Folin-phenol reagent (Lowry et al 1951). Ion exchange chromatography. Leukocyte lysate (13.5 ml) was applied to a 5 ml HiTrap SP cation exchange column (Pharmacia Biotech, Uppsala, Sweden), conditioned by 20 mM Tris-HCl, pH 7.4. The column was washed with 70 ml of the buffer at a flow rate of 2 ml/min, collecting 5 ml fractions. The proteins attached to the column were eluted with 40 ml of the same buffer containing 2 M NaCl. The UV absorption of proteins at 280 nm was recorded. The inhibition of reverse transcription was determined from 1 :50 diluted fractions. Active fractions 15-16 were concentrated to 1.7 ml using Centricon 30 concentrator (Amicon, Inc., Beverly, MA). The reverse transcription inhibiting activity was totally retained by the 30 000 molecular weight (Mwt) sieve. The protein concentration of the preparation was 1.25 mg/ml. Gel filtration. One millilitre of the active preparation obtained by ion exchange chromatography was applied to Superdex 200HR 10/30 gel filtration column (Pharmacia Biotech, Uppsala, Sweden). The column was eluted with 50 mM Tris-HCl buffer containing 0.9% NaCl, pH 7.4 at a flow rate of 0.6 ml/min, collecting 1 ml fractions. The reverse transcription inhibiting fractions 19-20 were combined and concentrated to 0.6 ml by a 3000 Mwt sieve (Centricon 3, Amicon). The protein concentration of the preparation was 0.31 mg/ml. Reverse phase chromatography. Half a millilitre of the concentrated active fraction from gel filtration was loaded onto a Vydac C4 (2.1 x 150 mm) reverse phase column, protected with a C4 guard column (The Separations Group, Hesperia, CA). The elution solvents were A: 0.1 % trifluoroacetic acid/H 2 O; B: 0.08 % trifluoroacetic acid/acetonitrile. The solvent gradient used was 0-30 % B (0-63 min), 30-60 % B (63-95 min), 60-80 % B (95-105 min), 80 % B (105-110 min) with a flow rate of 0.15 ml/min. Twenty fractions showing different protein peaks at UV 280 were collected and their reverse transcription inhibiting activity were determined from 1:11 dilutions. Sequence analysis. Amino terminal amino acid sequence analyses of the reverse phase chromatography fractions 4 and 5 were performed with an Applied Biosystems model 477A protein sequencer equipped to Applied Biosystems model 120 A phenylthiohydantoin (PTH) amino acid analyser. The samples were applied to a polybrene-coated and precycled fiber filter. Standard cycle parameters provided by the manufacturer were used. Mass analysis. The 0.5 μl sample from fraction 5 of RP chromatography for mass spectrometry, mixed with 0.5 μl of sinapinic acid (10 mg/ml) in 60% acetonitrile, was analysed by a matrix assisted laser desorption mass spectrometer (MALDI- MSLASERMAT ® , Thermo Bioanalysis Ltd., Hemel Hempstead, UK. Determination of eosinophil-derived neurotoxin from leukocyte lysates EDN content of leukocytes. Eosinophil-derived neurotoxin, EDN (also known as nonsecretory ribonuclease precursor and as eosinophil protein X, EPX) was determined by the EPX radioimmunoassay kit of Pharmacia Diagnostics (Uppsala, Sweden). The polyclonal antiserum of the kit was raised in rabbits against purified human eosinophil protein X (Peterson & Venge 1983). Lysed leukocytes were centrifuged at 13 000 g for 2 min, the supernatant was diluted to contain 10 5 to 10 7 lysed cells/ml and EDN content of the lysate was determined by the EPX/EDN RIA. Fractionation of leukocytes by Ficoll-Paque R density gradient. The leukocytes from 10 ml of normal blood were separated from erythrocytes with 1.2 % dextran. The leukocytes (2.5 % eosinophils) were washed with PBS and fractionated to mononuclear cell and granulocyte fractions using Ficoll-Paque density gradient solution (Pharmacia Biotech). One million mononuclear cells were lysed with 500 K562 cells in 50 ml of water, containing 80 U RNasin ® and 5 microlitres of centrifuged lysates were used in Ph-chromosome RT-PCR assay. Fractionation of leukocytes by CD antibodies. Leukocytes, separated from blood with 1.2 % dextran, were washed with Hank's buffered saline solution (HBSS) and suspended into same solution with 0.1 % foetal calf serum at 4 x 10 7 /ml. Four million cells (2.5 % eosinophils) were incubated for 30 min at 4 °C with primary antibody-coated Dynabeads ® M-450 CD 15 particles (DYNAL A.S. Oslo, Norway) After separation of the magnetic particle-attached cells, the unbound (CD15-) cells were washed 3 times with PBS, containing 0.1 % BSA and divided into one million cells per tube. The granulocyte-associated antigen, CD 15 has been found on neutrophil and eosinophil granulocytes and on monocytes, although to a varying degree. Monoclonal anti-CD 16 (clone LNK16, HyTest Ltd. Turku, Finland) was first biotinylated as described previously (Matinlauri et al 1994). Stre tavidin-coated microtitration strips (Wallac Co., Turku, Finland, cat no C122-105) were incubated with the biotinylated anti-CD 16 (30 μg/ml, 70 μL/well) for 1.5 h at room temperature. The wells were washed three times with PBS and 200 000 leukocytes (separated from blood with 1.2 % dextran) were added in 50 μL of PBS containing 0.2 % human serum albumin. Leukocytes were incubated for 30 min at room temperature after which unbound cells were washed out three times using the same buffer. Anti-CD 16 -bound cells were treated with chymopapain (ChymoCell-T, The Boots Company, PLC, Nottingham, Poole, UK) at 20 KAT units/well for 30 min at room temperature to remove the cells from antibody. The cells were collected, counted and centrifuged to obtain one million cells per tube. The Fc gamma receptor III, CD 16, is expressed on neutrophils and basophils but not on eosinophils. The eosinophil depleted cells were lysed in 50 μL of autoclaved water containing 500 K562 cells and the centrifuged cell lysates were used for RT-PCR assay as described above. EDN content of the lysates was determined by the EPX/EDN RIA. Results Inhibition of reverse transcription by leukocyte lysate The number of lysed leukocytes needed to inhibit reverse transcription by 50 % was about 7000 when M-MLV reverse transcriptase enzyme was used and 4000 with the AMV enzyme (Figure 1). In both cases about 25 000 lysed leukocytes totally inhibited RT reaction. Table I shows that the RNase inhibitors were unable to prevent reverse transcription inhibition when the reaction mixture contained a lysate of 200 000 leukocytes. The only slightly positive signal was obtained when Rnase block II was present in the lysate. In addition, ribonucleoside-vanadyl complex without leukocytes showed a 78 % inhibition of the assay. Table I. Effect of RNase inhibitors in leukocyte lysate to protect bcr/abl transcript for RT-PCR RNase inhibitor Concentration Time-resolved Fluorescence, CPS K562 cells only 82636 + RVC 10 mM 10036 + RVC + leucosytes 10 mM 593 + Rnasin 2000 U/ml 83947 + Rnasin + leukocytes 2000 U/ml 662 + Rnasin guard 2000 U/ml 75601 + Rnasin guard + leukocytes 2000 U/ml 665 + Placental RI (Perkin-Elmer) 1000 U/ml 68583 + Placental RI + leukocytes 1000 U/ml 687 + Rnase block II 40 U/ml 67375 + Rnase block II + leukocytes 40 U/ml 2260 + Inhibit ACE 40 U/ml 69131 + Inhibit ACE + leukocytes 40 U/ml 726 500 K562 cells were lysed with or without 2 million leukocytes in 50 μl of water with a ribonuclease inhibitor. Five micrometers of the lysate were applied to RT-PCR. Abbreviations: RVC, Ribonucleoside-vanadyl complex; RI, Ribonuclease inhibitor. Purification and identification of the reverse transcription inhibiting factor form leukocytes The three chromatographic steps in the purification of RT-inhibiting factor from leukocytes are shown in Figures 2 to 4. In the first ion exchange step (Figure 2) about 20 % activity was lost with the main protein fraction, not attached to the column. The elution volume (19 ml) of gel filtration fraction 19 with highest RT-inhibitory activity indicates a globular protein of about 18 kDa (Figure 3). The reverse phase chromatography revealed at least 20 fractions absorbing UV-light at 280 nm (Figure 4). The highest RT-inhibitory activity was in the fraction 5. Sequence analysis. The .amino acid sequence of protein from RP-chromatographic fraction 4 was: DIPEVVVSLAWDES matching human neutrophil 56 amino acid prodefensins (Mwt 6306 for prodefensins 1 and 2, Mwt 6351 for prodefensin 3) of SWISS-PROT Protein Sequence Databank (University of Geneva and Europan Bioinformatics Institute, Geneva, Switzerland). The sequence obtained from fraction 5 was: KPPQFTWAQWFET matching nonsecretory ribonuclease precursor, also known as eosinophil-derived neurotoxin (EDN) or eosinophil protein X (EPX). The molecular weight of EDN without the 27 amino acid secretory peptide is 15463. Theoretical pis of 9.2 (SWISS-PROT databank) or 8.9 (Barker et al 1989) have been calculated for the molecule. The molecular wt of mature granule protein depends on the degree of glycolysation, a Mwt range from 18 to 25 kDa has been reported with the major band at around 18 kDa in SDS- PAGE electrophoresis (Gleich et al 1986, Rosenberg and Tiffany 1994). Mass analysis. The mass analysis of fraction 5 of reverse phase chromatography showed three peaks: one at mass/charge ratio (m/z) of 17444, denoting eosinophil- derived neurotoxin; a double peak of the former at m/z of 8823.6 and 8589.2, and the third peak at m/z of 6337.9, denoting a mixture of 56 amino acid prodefensins 1 through 3 (Valorie and Ganz 1992). Effect of eosinophil removal Table II shows that 77.4 % of the maximum signal were obtained when 100000 eosinophil-depleted granulocytes (CD 16+ cells) were used together with 50 K562 cells in Ph-chromosome RT-PCR, and 52.8 % of the maximum signal when mononuclear cells were used in the assay. The EDN content was less than 6 ng/10 6 cells in these leukocyte fractions. The depletion of myeloid cells with CD 15 antibodies was not successful since eosinophils showed incomplete binding to anti-CD 15 -coated magnetic beads in some separations. 7 b/e //. The effect of depletion of eosinophils on the analysis of bcr/abl transcript of leukocytes using RT-PCR a Signal from 50 K562 cells EDN concentration Tr-fluorescence, CPS % ng/10 6 cells RT-PCR sample N Mean ± SD Control 0 , no leukocytes 10 100 ND Leukocytes' 1 , no selection 10 0.4 ± 0.8 36.2 ± 8.2 Total RNA extract 6 6 24.4 ± 3.4 ND Mononuclear cells 6 52.8 ± 11.8 3.3 ± 0.9 CD 16+ cells 6 77.4 ± 18.5 5.3 ± 1.3 CD 15- cells 6 20.3 (1.6-64) f 3.5 (3.3-60) f a After cell separation 10 6 leukocytes were lysed in the presence of 500 K562 cells and 80 units of RNasin ® in 50 μL of water, and 5uL of the centrifuged lysate was used for RT-PCR. b Eosinophil-derived neurotoxin (EDN) was determined with RIA. ND=not determined. c Lysates of 500 K562 cells served as a control samples. d Leukocytes were extracted from blood by dextran sedimentation. e RNA was extracted by the method of Chomczynski and Sacchi (1987). f Mean and range. Discussion We identified the main reverse transcriptase inhibiting factor of leukocytes as eosinophil-derived neurotoxin by the 3-step chromatographic procedure and amino acid sequencing. The binding of EDN to cation-exchange resin at neutral pH enabled a high first step purification. During this step about 20 % of the activity was lost with the main protein. Most of this RT-inhibiting activity was probably EDN and other cellular ribonucleases (Egesten et al. 1997) which were associated to other proteins and was removed during the wash of column. The purification procedure resulted in EDN with some contamination with defensins. Mass analysis showed a molecular wt of 17444 for the glycosylated EDN which is slightly less than those determined earlier by SDS-PAGE (Gleich et al 1986, Rosenberg and Tiffany 1994) but identical to the Mwt of kidney non-secretory ribonuclease (Lawrence et al 1993). However, the unique amino acid sequences of the proteins confirm their identities. Figure 4 strongly suggests that EDN of the two proteins is responsible of the inhibition of reverse transcription. EDN belongs to the ribonuclease superfamily and has high ribonuclease activity in addition to its reported neurotoxic and helminthotoxic effects (Gullberg et al 1986, Slifman et al 1986, Sorrentino et al 1992). An anti-retroviral activity of eosinophils has been connected to the ribonuclease activity of EDN (Domachowske and Rosenberg 1997). It was somewhat surprising to get RNase as a purification product because we carefully tried to exclude the ribonucleases out from candidates of RT-inhibitors by testing various ribonuclease inhibitors in leukocyte lysate (Table I). Although the placental ribonuclease inhibitor binds tightly to placental RNase (identical to EDN, Shapiro and Vallee, 1991) we have observed that several minutes are needed for full RNase effect at room temperature (unpublished result from this laboratory). During this period EDN is able to degrade RNA. The same concerns both the use of EDN antiserum (Sorrentino et al 1992) and heating the sample as means to inhibit RNase-activity (Sznajd and Naskalski 1973, Zale and Klibanow 1986). It has been shown earlier that one of the tested RNase inhibitors, Inhibit ACE, has been shown ineffective against EDN ribonuclease activity (Sorrentino et al 1992). Our experiments demonstrate that inhibitors are ineffective against high amounts of RNases in such applications as RT-PCR for detection of minimal residual disease in leukaemia. Because EDN is expressed almost exclusively in eosinophils (small amounts has also been found in neutrophils, Venge 1994, and monocytes, Egesten et al. 1997), we wanted to remove eosinophils with antibodies against cell surface markers or by Ficoll-Paque density gradient. Depletion of eosinophils with antibodies against CD 16 reduced EDN content to a level where a 77.4 % of the RNase-free signal could be obtained. We found that only 24.4 % of the signal was saved when total RNA was extracted from unfractionated leukocytes by the method of Chomczynski and Sacchi (1987). Depletion of eosinophils with CD15 antibodies gave inconsistent results, probably due to an unstable binding of myeloid cells to the antibody-coated particles. When residual disease of CML is studied, it is not possible to deplete myeloid cells which could be considered in the case of lymphoblastic leukaemia's. Separation of granulocytes from mononuclear cells by Ficoll-Paque density gradient produced a 52.8 % signal level compared with the leukocyte free sample. We think that the key point of the method of Kawasaki (1990) of using leukocyte lysate to RT-PCR without RNA extraction is the initial selection of mononuclear cells for the assay. There is still a need for an immediate RNase-inhibitor without interfering reverse transcriptase. At the moment the deactivation of RNases by guanidinium thiocyanate or by its derivatives and the subsequent extraction of RNA is the method of choice to get mRNA for the determination of residual haematological malignancies by RT-PCR. Recently, a modification of the method by Chomczynski and Sacchi (1987) has been introduced for extraction of RNA from eosinophil- infiltrated lung (Kodavanti et al 1996). This supports the results of this study that eosinophil granulocytes are the main source of ribonuclease activity of leukocytes. The removal of eosinophils from leukocyte population substantially reduces the risk of mRNA destruction by eosinophil-derived ribonuclease. It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention. 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Claims: CLAIMS 1. A method for the treatment of a body fluid sample containing eosinophils, so as to derive a RNA therefrom that is suitable for analysis, characterised in that eosinophils are removed from said sample before the RNA is subjected to analysis. 2. The method according to claim 1, characterised in that the sample comprises cells from which said RNA is derived. 3. The method according to claim 2, characterised in that the RNA is mRNA useful for gene expression analysis. 4. The method according to claim 2, characterised in that the RNA is rRNA useful for analysis of bacteria species. 5. The method according to claim 2, characterised in that the cells are leukocytes. 6. The method according to claim 1, characterised in that the sample comprises an RNA-virus from which said RNA is derived. 7. The method according to claim 1 , characterised in that the sample comprises a bacterium from which said RNA is derived. 8. The method according to any of the claims 1 to 7, characterised by the steps of incubating the sample with an antibody specific for an eosinophil surface antigen to form an antibody-antigen complex, - removing the antibody-antigen complex, and subjecting the RNA of the remaining sample to analysis. 9. The method according to any of the claims 1 to 7, characterised by the steps of incubating the sample containing eosinophils with an antibody specific for a surface antigen of the cells the RNA of which shall be analysed, withdrawing the antibody-antigen complex from the sample, and subjecting the RNA of the withdrawn cells to analysis. 10. The method according to any of the claims 1 to 7, characterised by the steps of centrifuging a composition comprising the sample and a centrifugation medium, the density of the centrifugation medium and the force of centrifugation being selected to cause a substantial portion of the eosinophils to gather in a specific fraction, removing the eosinophil-rich fraction, and deriving the RNA from the remaining sample for analysis. 11. The method according to claim 8, 9 or 10, characterised in that the sample, which comprises cells from which RNA shall be derived, is lysed so as to release the RNA from the cells before the analysis thereof. 12. The method according to any of the claims 1 to 11 characterised in that the RNA is before the analysis amplified by reverse transcription polymerase chain reaction (RT-PCR). 13. The method according to any of the claims 1 to 11 characterised in that the RNA is before the analysis amplified by reverse transcriptase and T7 RNA polymerase. 14. The method according to any of the claims 1 to 11 characterised in that the RNA is before the analysis amplified by RNA-directed RNA polymerase. 15. The method according to any one of the claims 1 to 11 characterised in that the RNA of the sample from which the eosinophils have been removed is directly used in the subsequent amplification and analysis steps without foregoing isolation of said RNA.

Inventors: ESKOLA JARKKO, HAEMAELAEINEN MAURI, HELLMAN JUKKA, ESKOLA, JARKKO, HAEMAELAEINEN, MAURI, HELLMAN, JUKKA
Applicants: ESKOLA JARKKO, HAEMAELAEINEN MAURI, HELLMAN JUKKA, ESKOLA, JARKKO, HAEMAELAEINEN, MAURI, HELLMAN, JUKKA
Application year: 1999
Main classification: C12Q1
IPC codes: C12N15, C12Q1