Quantitation of white cell subpopulations by polymerase chain reaction using frozen whole-blood...

I M M U N O H E M A T O L O G Y

Quantitation of white cell subpopulations by polymerase chain reaction using frozen whole-blood samples

T.-H. Lee, N.S. Sakahara, E. W. Fiebig, D.F. Hirschkorn, D.K. Johnson, and M.P. Busch for the Viral Activation Transfusion Study

~

BACKGROUND: Previous methods for processing whole blood (WB) for nucleic acid analyses of white cells (WBCs) required fresh blood samples. A simple protocol that in- volves the freezing of WB for quantitative polymerase chain reaction (PCR) analyses was evaluated. STUDY DESIGN AND METHODS: Controlled studies were conducted in which paired fresh and frozen WB preparations were analyzed.The integrity of WBCs in the frozen WB samples was first assessed by flow cytometry using CD45 fluorescence, and calibration beads to quantitate recovery of WBC subsets. PCR of an HLA-DQ-A sequence was used to quantitate residual WBCs in a double-filtered red cell (RBC) component spiked with serial dilutions of WBCs, as well as in 51 filtered RBCs and 19 filtered platelet concentrates. Y-chromosome-specific PCR was used to quantitate male WBCs in five female WB samples spiked with serial dilutions of male WBCs and in serially collected frozen WB samples from four females transfused with male blood components. RESULTS: By flow cytometry, all major WBC subpopulations in frozen-thawed WB were quantitatively recovered and immunologically intact, although they were nonviable. HLA-DQ-A PCR quantitation of a dilution series from 8 to 16,700 per mL of WBCs spiked into double-filtered RBCs showed linear correlation of the results with both fresh and frozen preparations of the expected WBC concentrations (P= 0.98, p<O.OOOl for both), without significant difference between observed and expected values (p>0.05). Y-chromosome-specific PCR results in female WB samples spiked with male WBCs were not significantly different in fresh and frozen preparations over a 3 log,o range of male cells.The results of WBC survival studies on frozen WB samples were consistent with previous observations in fresh blood samples. CONCLUSION: Direct freezing of WB enables subsequent recovery of WBCs for quantitative PCR analyses, with results comparable to those of fresh preparations.This protocol should facilitate wider implementation of nucleic acid- based analyses for quality control of WBC-reduced components, as well as for prospective clinical studies of microchimerism in transfusion and transolant recioients.

N ucleic acid amplification techniques are in- creasingly employed in research and clinical practice in a wide array of disciplines, including transfusion medicine. lv2 The preparation of

blood samples for these analyses remains relatively cum- bersome and labor-intensive, however, in great part because of the requirement that blood samples be processed shortly after c~ l l ec t ion .~ -~ For example, standard ap- proaches to processing blood for white cell (WC)-associated nucleic acid analyses involve the purification of W C s from fresh blood by using density-gradient red cell (RBC) lysis procedures, followed either by immediate W C lysis and extraction of DNA or RNA or by freezing of W C s (with or without cryoprotectant) for subsequent processing to isolate nucleic acid^.^-^ Although several alternative, rapid protocols for the extraction of nucleic acid from whole blood (W) have recently been described, these have been developed and validated for processing fresh blood samples within 24 hours of ph leb~ to rny .~*~-~ Furthermore, sample preparation methods have generally not been evaluated with respect to quantitative recovery of WBC-associated nucleic acids when the target cell population is present at verylow concentrations in test samples, as is frequently the case in transfusion medicine applications.

ABBREVIATIONS: PCR = polymerase chain reaction; PC(s) = platelet concentrate(s1; RBC(s) = red cell(s); SRY = sex-determining region of the human Y chromosome; WB = whole blood; WBC(s) =white cell(s).

From the Blood Centers of the Pacific, Irwin Center; the Depart- ment of Laboratory Medicine, University of California, San Fran- cisco; and the Clinical Laboratories, San Francisco General Hos- pital, San Francisco, California.

Supported in part by the Viral Activation Transfusion Study, Contract N01-HB-57126 of the National Heart, Lung, and Blood Institute.

Received for publication August 21, 1996; revision received August 30,1997, and accepted September 3,1997.

TRANSFUSION 1998;38:262-270. ~

262 TRANSFUSION Volume 38, March 1998

PCR ON FROZEN WHOLE BLOOD

The requirement of fresh samples for processing is particularly problematic in connection with applications such as quality control of WBC-reduced blood components by blood banks. Although highly sensitive flow cytometry- and polymerase chain reaction (PCR) -based assays have been developed for this purpo~e,~-~ ' the requirement for analysis of fresh samples has prevented wider use of these methods. The development of an approach for batch testing of preserved samples by reference laboratories would over- come this hurdle. A simple preserved WB method would also be useful for prospective transfusion-related studies, in which thousands of samples from donors and recipients at multiple clinical sites must be processed and stored. In many such studies, only a small proportion of collected samples are later determined to warrant testing, and this results in the wasting of resources in processing never- evaluated samples.

In the present study, we describe a simple protocol in which anticoagulated WB samples are directly frozen without cryoprotectant. Samples can be analyzed after short- or long-term frozen storage by a rapid thaw-and-wash protocol that yields aWBC pellet that can be used in subsequent quantitative DNA PCR analyses. This article presents an evaluation of the sensitivity of this approach, in which we compare quantitative PCR results from frozen preparations with those of fresh preparations and show that target WBC recoveries by flow cytometry and DNA amplification effi- ciencies are comparable in fresh and frozen preparations.

MATERIALS AND METHODS Flow Cytometric Analysis of Frozen WB EDTA WB was obtained from two HIV-negative normal blood donors and three HIV-seropositive subjects enrolled in aprospective HIVtype 1 cohort study12 Complete blood counts were performed on an automated hematology analyzer (Baker 9020AX, Serono Diagnos- A

tics, Allentown, PA). Fresh (<l&hour- old) samples were stained with CD3/ CD4/CD45 and CD3/CD8/CD45 monoclonal antibody combinations (TkiTest, Becton Dickinson Immunocytometry Systems [BDIS], Mountainview CAI in tubes containing an internal bead standard (TkuCount Tubes, BDIS) by using a lyselno-wash method as recommended by the manufacturer. These tubes contain a fured number of fluo-

cryopreservative and were stored for an average of 3 months. Frozen samples were allowed to come to room temperature and were gently agitated. The samples were then stained with TkiTest reagents in TkuCount tubes as above. A n aliquot was also stained with CD45-fluorescein isothiocyanate; then 1.0 mL of propidium iodide (20 pgl mL) was added for assessment of WBC viability

Data for each sample were collected on a flow cytometer (FACScan, Becton Dickinson) that had been cali- brated with AutoCOMP software and calibration beads (CaliBrite, Becton Dickinson). List mode files of various sizes were collected with software (LYSIS 11, Becton Dickinson) so that each data file contained 2000hCount bead events. During data acquisition, a fluorescence (FU) triggering threshold was set to exclude most debris and was adjusted between tubes so that all three major WBC populations (lymphocytes, monocytes, and granulocytes) were visible. On frozen samples, side-scatter was adjusted between samples to compensate for degranulation of the granulocytes and to maintain discrete, well-separated cell clusters.

List-mode data files were analyzed with LYSYS I1 software. Each cell population was defined according to CD45 expression (CD45 on granulocytes<monocytes&mpho- cytes) and side scatter (granularity of lymphocytes <monocytes<granulocytes). Analysis regions (gates) were set around each cell cluster and around the bead cluster (Fig. 1). Absolute numbers of eachWBC subset cluster were determined by the formula (# cell events/# bead events) x (bead countlsample volume). Results from two tubes on each frozen sample were averaged and were compared to fresh values. The viability of WBC subpopulations was assessed by using the same gating strategy as described for obtaining the cell counts, with subsequent determination of the presence or absence of propidium iodide staining of nuclear material in the WBC subpopulations.

B

i z

Y 3 rescent beads that enable the CD45 expression CD45 expression quantitation of WBC subset concentrations by flow cytometry analysis of the ratio of cells to beads.

Multiple aliquots of EDTA WB were also frozen at -80°C without

Fig. 1. Flow cytometry dot plots (CD45 expression on X axis VB. side 190" llght] scatter on Y axis) of fresh (A) and frozen-thawed (B) WB samples. R1 gate includes all WBCs; R2 gate = lymphocytes; R3 gate = monocytes; R4 gate = granulocytes; Rs gate = TruCount beads.

Volume 38, March 1998 TRANSFUSION 263

LEE ET AL.

Preparation of Serial Dilutions of Male WBCs into Female WB EDTAWB was collected from 10 blood donors (5 male and 5 female). Total WBC counts were determined in triplicate (Coulter ZM, Coulter Corp., Hialeah FL) and averaged. Se- rial dilutions of the five male samples were made indepen- dently into phosphate-buffered saline to yield suspensions of 2,000,10,000, and 50,000 maleWBCs per mL. Forty pL of each diluted WBC suspension was then separately spiked into a total volume of 0.5 mL of WB from each of the five females. This yielded samples containing 20, 100, and 500 male (Y-chromosome-positive) WBCs in 125 pL of female WB (i.e., the PCR input volume; see below). Four aliquots (0.5 mL each) were prepared for each spiking level, of which two were processed immediately into WBC pellets, while two were frozen at -80°C for subsequent processing.

Preparation of Dilutions of WBCs into Double- Filtered RBCs A 10-mL aliquot of blood was removed from a standard packed RBC unit and mixed thoroughly, and the totalWBC count was determined in triplicate (Coulter ZM) and averaged. The rest of the unit was filtered twice with WBC-reduction filters (BPF4, Pall Biomedical, East Hills, NY), byfol- lowing the manufacturer's directions. Precise volumes of filtered packed RBCs were spiked into aliquots of double- filtered packed RBCs to create samples containing 8, 50, 100, 500, 1,000, 5,000, and 16,700 spiked WBCs per mL. Eight aliquots (0.5 pL each) were prepared from each spiked sample, four of which were processed immediately into WBC pellets, while the other four were frozen at -80°C for subsequent processing.

Filtered RBC and Platelet Components Packed RBCs and platelet concentrates (PCs) were prepared and filtered 2 to 7 days after collection following the manu- facturers' recommended standard procedures. The WBC- reduction filters used for RBC units included BPF4 and RCZL (Pall) and Sepacell R500II (Asahi Sepacell, Tokyo, Ja- pan). PL5AWBC-reduction filters (Asahi) were used for pre- paringWBC-reduced PCs. Aliquots of each filtrate were processed immediately after filtration into WBC pellets by a saponin lysis procedure (see below). Separate aliquots were frozen at -80°C for subsequent processing.

Processing of Fresh WB and RBC Samples for PCR Procedures for preparingWBC DNA from freshWB and RBC samples have been described previou~ly.'~ In brief, 0.5 mL of saponin lysis buffer (0.4% saponin in 0.5% NaCl) was added to 0.5 mL of anticoagulated (EDTA or ACD) WB or RBC sample. After a 5-minute incubation at room temperature with intermittent mixing, the tube was centrifuged at 3000 rpm (600 x g) for 5 minutes. in a microcentrifuge. The

WBC pellet was then washed twice with 1 .O mL of PCR Solu- tion A (100 mmol/L KCl; 10 mmollL Tris HCl, pH 8.3; 2.5 mmol/L MgC1, [Cell Culture Facillity, UCSE San San Fran- cisco, CAI). After removal of supernatant, the dry WBC pellet was either processed immediately for DNA preparation or stored at -20°C.

Processing of Fresh PC Samples for PCR One mL of phosphate-buffered saline with 0.04-percent EDTA was added to 200 pL of freshly filtered PCs; they were mixed by inversion and microcentrifuged at 3000 rpm (600 x g) for 5 minutes. After removal of the supernatant, 1 .O mL of PCR Solution A containing 0.02-percent saponin was added to each cell pellet, and then the mixture was mixed vigorously. The cell suspension was microcentrifuged at 7000 rpm (3500 x g) for 5 minutes; supernatant was removed, and a final wash of the cell pellet was performed with 1 mL of PCR Solution A. After centrifugation, supernatant was aspirated to yield a dry cell pellet, which was then further processed into a DNA preparation or stored at -20°C.

Processing of Frozen WB, RBC, and PC Samples for PCR WB, RBC, and PC aliquots (0.5 mL each) that had been stored at -80°C for 1 to 3 months were thawed at room temperature and inverted several times to mix completely and then microcentrifuged at 5000 rpm (1800 x g) for 5 minutes. After centrifugation, the hemolyzed plasma supernatant was removed and 1 mL of PCR Solution A containing 0.02- percent saponin was added to each pellet: this mixture was then agitated vigorously to resuspend the pellet. The suspension was microcentrifuged at 5000 rpm for 5 minutes, the supernatant was removed, and the pellet was washed with 1 mL of PCR Solution A. After this centrifugation, the dry pellet was either further processed into DNA or stored at -20°C.

One hundred pL of a 1: 1 mixture of PCR Solution A and PCRWBC lysis Solution B (10 mMTris HC1, pH 8.3; 2.5 mM MgCJ; 1%Tkeen-20; 1% Nonidet P-40; and 0.4 mg/mLpro- teinase K [Cell Culture Facility]) was added to eachWBC pellet. This mixture was incubated at 60°C for 1 hour with vig- orous mixing every 20 minutes. The lysates were then incubated at 100°C for 10 minutes to inactivate proteinase K.

A 242-bp region of the HLA-DQ-A gene was amplified by using primers GH26 and GH27.l The PCR reaction mixture consisted of 100 mMKCL20 mMTris HCl (pH 8.3), 3 mM MgCl,, 0.02-percent gelatin, 2 pmol per pL of each primer, and 0.4 U per pL of Amplitaq DNA polymerase (Perkin Elmer). Fifty pL of the PCR reaction mixture was added to 25 pL of each DNA sample (equivalent to 125-pL input blood component) and amplified for 30 cycles. Each cycle was initiated at 95°C for 30 seconds and was followed by 56°C for 1 minute and then 72°C for 1.5 minutes.



A 148-bp region of the male-specific, sex-determining region of the humanY chromosome (SRY) was amplified by using allele-specific primers SA and SD.13 The PCR reaction mixture consisted of 100 mMKCh20 mll.f?tis HCl (pH 8.31, 2.5 mMMgCl,, 0.02-percent gelatin, 1 pmol per pL of each primer, and 0.4 U per pL of Amplitaq DNA polymerase (Perkin Elmer).menty-five pL of DNA lysate (equivalent to 125-UL input blood) was added to 50 pL of PCR reaction mixture; this was then amplified (9600 Thermal Cycler, Perkin-Elmer) for 30 cycles, each consisting of 30 seconds at 95°C and 2 minutes at 70°C.

Specific amplified products were detected by liquid hybridization using 32P-labeled oligomer probes GH64 and SB.11J3 Ten pL of probe mix was added to 30 pL of each postamplification specimen; this step was followed by de- naturation for 5 minutes at 95°C and hybridization for 5 minutes at 59°C. Ten pL of loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol, and 30% glycerol) was added to each hybridized sample, which then under- went 6-percent polyacrylamide gel electrophoresis at 12.5 volts per cm. The gel was exposed to autoradiographic film (XAR-5, Kodak, Rochester, NY) with enhancing screen at room temperature for 2 hours or overnight. Duplicate standard calibration curves (serial I-in-10 dilutions [1@, lo2, lo1, 1001 of male WBCs in lo6 female WBCs [for SRYI or of WBCs in buffer [for HLA-DQ-A]) were analyzed in parallel with experimental samples and used to interpolate target WBC concentration in test samples. Autoradiographs were analyzed by using an electrophoresis analyzer (BioImage, Millipore, Ann Arbor, MI) with whole-band analyzer application software. Image analysis was performed on the dilution (neat or 1-in-10) and film exposure (2 hours or overnight) that was within the linear range of the corresponding standard curve on that film.Values from duplicate determinations were averaged.

Statlstlcal Analysis WBC numbers obtained by flow cytometry on paired fresh and frozen-thawed preparations from HIV-positive patients and controls were compared by Wilcoxon’s signed rank sum test (significance level, 0.05). A nonparametric test was chosen because the values were not normally distrib- uted. Relative bias was calculated according to equation 1:

100 x (frozen - fresh resu1t)lmean of frozen and fresh results (1)

Observed and expected WBC concentrations were compared with paired, two-tailed ttests (significance level, 0.05) and linear regression analysis.

Assuming normal (Gaussian) distribution of the observed WBC concentrations at an expected value of 16,700 WBCs per mL, the 95-percent prediction interval for a new observation at this level was estimated according to Motulsky14 by equation 2:

95% prediction limits = [mean - K x SD] to [mean +K x SD] (2)

where SD is the standard deviation of the mean, and K is a derived f a ~ t o r , l ~ ( p ~ ~ ) which equals 2.78 for n = 6, the number of observed WBC concentrations in the study at an expected level of 16,700 WBCs per mL. Because the number of observations is relatively small, the value for K is greater than 1.96, the value used to calculate 95-percent prediction intervals for a new observation when the number of observations is infinitely large, or 220.

Agreement between WBC concentrations obtained by PCR in filtered blood components on paired fresh and frozen-thawed aliquots was quantified by calculating the mean bias (difference between paired results on fresh and frozen preparations) and 95-percent limits of agreement (mean difference f 1.96 SD), according to Bland and Altman.ls All calculations were performed on a personal computer, using StatView 4.01 software (Abacus, Berkeley, CAI.

RESULTS Flow Cytometric Analysis of Frozen WB Samples We first examined the effect cf freezing and thawing WB on RBC andWBC recovery and integrity as determined by flow cytometry. W samples from five subjects were analyzed fresh and after freezing at -80°C for 1 to 4 weeks, with subsequent thawing, staining, and analysis. As expected, the vast majority of RBCs had lysed during the freezing-thawing process, as evidenced by the loss of intact RBC-related events and increased membrane debris (data not shown). Figure 1 shows a representative comparison of flow cytometry dot plots of W C s from fresh WB and a cone- spondingfrozen-thawed WB sample. In this analysis, CD45 monoclonal antibody was used to stain WBCs to facilitate gating of the three major WBC subsets (lymphocytes, monocytes, granulocytes) by fluorescence and side-scatter properties. The use of CD45 gating was particularly useful with the frozen-thawed WB preparations] because of the slight changes in forward and side scatter that made light scatter-based separation of the WBC subpopulations from each other and from RBC debris more difficult for frozen than for fresh samples. CD45 staining and the light-scatter- ing properties of each of the three major WBC subpopulations were not significantly altered by the freezing-thawing procedures. The membrane integrity of theWBCs in the frozen-thawed WB was drastically altered, however, as evidenced bypropidium iodide nuclear staining of >90 percent of cells in each subpopulation as compared to nuclear staining of e l0 percent with fresh preparations (not shown).

By using an internal bead standard (’ItuCount beads) we were able to evaluate the quantitative recovery of each major WBC subpopulation in the frozen-thawed samples relative to concentrations in fresh WB. The median total WBC count for our five pairs of fresh and frozen-thawed preparations (frozen values represent the mean of duplicate


LEE ET AL.

measurements) was 3400 per pL and 3510 per pL, respectively (p>O.l). The median bias for the frozen preparations was -4.9 percent (range, -7.5 to +3.2%). Significant differences between the median concentrations were not observed for the VariousWBC subpopulations, as well, but the relative bias between fresh and frozen results was more variable, rangingfrom-37.2 to +3.2 percent for lymphocytes and from -33.7 to +2.9 percent for CD3+T cells. The results, including values for granulocytes and monocytes, are sum- marized in Table 1.

HLA-DQ-A Quantitation of WBCs in Filtered Components Double-filtered packed RBC samples were spiked with un- filteredWB to yield theoreticalWBC concentrations of 8,50, 100, 1,000, 5,000, and 16,700 cells per mL. Each spiked preparation was processed fresh and after freezing and thawing, followed by quantitative PCR for HLA-DQ-A to compare WBC-associated DNA recovery. The observed values for repeat determinations (n = 3-6) of WBC concentra-

tions for fresh and frozen-thawedWB were, with one excep- tion, not significantly different from the expected values (p>0.05).The precision of the observed values (CV[%I) was similar for fresh and frozen-thawed preparations but var- ied, depending on the expected cell count levels. At an expected WBC concentration of 16,700 per mL, the CVs for fresh and frozen-thawed WB were 10.4 and 8.6 percent, respectively, while CVs from 26 to 65 percent were calculated at expected values of 1,000 per mL and below (Table 2). At the lowest expected WBC concentrations of 8 per mL, the observed results were not different from zero (data not shown). As shown in Fig. 2, the results for both fresh and frozen-thawed WB were highly correlated and linear over the range of expectedWC concentrations from 8 to 16,700 per mL. Regression analysis yielded r2 = 0.977 for fresh preparations and r2 = 0.982 for frozen-thawed samples (each, p<O.OOOl). Slope coefficients and y-intercepts of the linear regression lines were 0.923 and 227 cells per mL for freshWB and 1.099 and 190 cells per mL for frozen-thawed preparations, respectively.

TABLE 1. Cell counts per pL and relative bias (?h) for palred fresh and frozen-thawed WB allquots* Relative bias (%)

Fresh WB (n = 5) Frozen-thawed WB (n = 5) versus fresh WBt p value* WBCs, total 3400 (2800-6400) 351 0 (2597-631 7) -4.9 (-7.5 to +3.2) NS Granulocytes 1739 (1 632-3624) 1788 (1 655-3288) -0.1 (-9.7 to +4.5) NS Monocytes 327 (236-448) 355 (239-417) -0.5 (-31.1 to 40.3) NS Lymphocytes 1306 (762-2432) 131 3 (523-2507) -1.7 (37.2 to +3.2) NS CD3+ T cells 888 (533-1921) 899 (379-1 978) 1.2 (-33.7 to +2.9) NS

Fresh samples were counted on an automated hematology analyzer; the values for the frozen-thawed aliquots are averaged from duplicate results obtained by flow cytometry using TruCount beads. Values represent medians (range).

t Calculated according to Equation 1. t Fresh versus frozen-thawed weparation. Wilcoxon ranked sum test (NS = nonsianificant: all were D >0.1).

TABLE 2: Spiked WBC concentrations In double-filtered RBC units (expected), compared to observed WBC concentrations determined bv PCR In fresh and frozen-thawed Dremrations

ExDected WBC counts Der mL Preparations 16,700 5,000 1,000 500 100 50

Number 6 3 4 4 4 4 Mean 15,420 6,421 934 760 56 66

6-1 26 95% CI‘ 13,733-17,107 3,794-9,049 Median 14,860 7,016 700 660 60 56 Range 14,000-1 8.280 5,200-7.048 496-1,840 536-1,184 24-80 32-1 20 SD 1,607.3 1,051.8 612.0 289.6 28.5 37.7 cv (“70) 10.4 16.5 65.5 38.1 50.8 57.2 p valuet NS NS NS NS NS NS

Number 6 3 3 4 4 4 Mean 18,280 7.531 947 626 82 38 95% CI’ 16,639-19,921 4,615-10,447 276-1,617 125-1,127 76-88 22-54 Median 18,660 8,168 824 560 80 40 Range 15,800-19,920 6,176-8,248 760-1‘256 328-1.056 80-88 24-48 SD 1,563.6 1,173.9 269.8 315.1 4.0 10.1 cv (“70) 8.6 15.6 28.5 50.3 4.9 26.5 p valuet NS NS NS NS 0.03 NS 95% CI of the mean, assuming Gaussian distribution of the results.

Fresh

0-1,908 299-1,221 56 11-101

Frozen-thawed

t Observed vs. expected WBC concentrations; paired two-tailed t tests, significance level 0.05.



A B shown in Fig. 3, the paired results showed good correlation (? = 0.82, p<O.OOOl) with a very small positive bias for results from frozen-thawed samples relative to those results from fresh samples (mean difference, log,, 0.026 WBClmL, or 1.06 times). The SD of the mean differences, hawever, was quite large, log,,, 0.388 WBCs per

22 mL;therefo=,themtsofagreement (mean difference f 2SD), which de- fine the range for 95 percent of the differences, extended from log,, - 0.750 to log,, +0.802WBCs per mL. In other words, results for 95 percent of frozen-thawed preparations can be expected to be 0.18 times to 6.34

tions. The highest expected value would be 36 times the lowest expected value. This rather wide inter-

val primarilyreflects thevariabilityat lowerWBC concentrations. If only a subset of mean WBC concentrations greater than 5OOO (log ,, 3.7) per mLwas consided (n = 141, the results for the frozen-thawed preparations were onaverage 1.13 times higher than those for the fie& preparations. The limits of agreement were between0.84 and 1.52 times the results for fresh preparations. Thismeans that thehighest expected value is 1.8 times the lowest expected value.

Notably, no observed result with WBC-reduced components exceeded the cutoff for a WBC-reduced compo-

r2-0.977 r2-0.982

0 2 4 6 8 10 12 14 1618 20 22

WBCs expected in samples (x 1OVmL)

% 2 R - a P 0f-d 0- WBcmts bYWd- PcR Q-

pcctsd cdl COnCCntrrtiOM for k S h (A) Md fhEUl-thnwsd @) p w t i O D 8 Of

dauble-mw I I I D P ~ ~PlLed dth -t-tratio-of- (6 50, goo, l,OoO, 5,000, and 16,700 celldml). The number of repeat meuunmcILts rul(led~thrwmorhrrepebapeetsdvrlw.Becrrwromed~mreldm~

meat& The -lid line in thegmplu nprermts thehear regnuion he. the numbes of virlbk rymbob d- not C O m p O d to llumbQ of~eaam- times the results for fresh prep--

Of practical importance is the performance of the assay at the cutoff value for a WBC-reduced blood component, which is 5 x lo6 WBCs for a 300-mL blood unit, or 16,700 WBCs per mL Assuming Gaussian distribution of the observedvalues at this WBC concentration, 95-percent prediction intervals for a new cell count measurement can be calculated according to Equation 2. For a fresh preparation, the new result would be expected to fall between 10,952 and 19,888 per mL, and fora frozen-thawed sample, the new result would be expected to yield a cell count between 13,933and 22,622 permL Assumingfurtherthat the variability of measured cell counts does not increase for expected cell counts that

limitsoftheseintenmls (10,952/mLfora 4 5

fresh sample and 13,933/mL for a frozen-thawed preparation) could serve as p cutoff levels at which 97.5 percent of 2 8 measured cell counts would be expected 3 1 not to exceed a true= concentration % f 1 .s

of 16,700 per mL PCR results on paired fresh and

frozen-thawed preparations, including thesamplesfrom the double-filtered RBC unitsspikedwithknmWBCconcentra- frozen-thawed samples (log,, WBCdmL)

units (n = 51)r and PcS (n = 19)# combmed for and@s Of the a@eement between the P=pmtiona The wBc Obtained fresh and frozen-thawed prep~t iomwere lo@~d~trans - B ) T h e ~ i n n l u e s ~ . , W B W m L i n t h e ~ t l u w e d r u n p k - hg,

Of re-

cilitate comparison of the results. As

are greater than 16,700 per mL, the lower A B

4

c " 3 5

0

1 1 5 2 2 5 3 3 5 4 4 5 0 5 1 1 5 2 2 5 3 3.5 4 4.5 Log,, WBCdmL in the fresh samples Average result for paired fresh and

(n = 24),WBC-filtered PI& 3. AJ The relationship of residual WBC co~ceatratiom in 51 OIteced RBC units and 19 PC units (0) and 24 double-thred RBC units 8piked with known WBC con- cmtrat io~ (.) by quantitative PCR amplitlcatton of EiU-DQ-A gene eequenceon froeen-thawed and liesh prepvatfona An vahreswece pbtted 8fter bg,, truulbnna- tion. The dashed Ilne represents the b e at whlch the correl.ted values are Identical.

WBCdmL in the fresh sunplea) plotted agaimt the mrye log,, WBC per mL of each

np-t the mcllll and 95-percent limits of

to stabilize the s'ltS Over the me Of values and to fa- prlrea fneh pnd --- preparation b depletsd. h m n d


LEE ET AL.

nent (<10,900 WBCslmL and ~13,900 WBCslmL for fresh and frozen-thawed samples, respectively), as calculated on the basis of the spiking studies (see above).

SRY-specific PCR Amplification of Male WBCs Diluted in Female WB To model a transfused donor WBC survival study, samples of WB from five females were spiked with different concentrations of maleWBCs (20,100, and 500 male WBCs/125 pL femaleWB), which was followed by processing as fresh and frozen-thawed WB samples and parallel amplification and hybridization for WBC-associated SRY sequences. Figure 4 shows PCR autoradiographic data comparing WBC-derived SRY DNA in fresh and frozen-thawed preparations. The sensitivity of detecting Y-chromosome-positive W C s by PCR was comparable for fresh and frozen-thawed preparations, with very similar autoradiographic band intensities for fresh and frozen-thawed preparations at each of the three spiking levels in the five independent experiments.

Survival Kinetics of Male Donor WBCs in Female Transfusion Recipients We then used SRY-specific PCR amplification to track the suMval kinetics of transfused male WBCs in four female recipients of fresh RBC components. Pretransfusion and posttransfusion samples from four female patients who received at least 1 unit of packed male RBCs were collected and frozen within 4 hours of phlebotomy. The serial

Preparations Frozen m

Numberof 20 io0500

Fresh m 20 100 500

Fig. 4. Comparison of SRY-speciflc PCR amplification results for fresh and frozen-thawed preparations for flve female W B spedmene, spiked with 20,100, or 500 Y-chromosome-positive male WBCs.

samples were subsequently thawed and processed to- gether; these steps were followed by quantitative SRY PCR analyses. In each case, maleWBCs were undetectable in the pretransfusion specimen, while there was transient detection of donor (male) WBCs during the first week after transfusion and complete clearance by2 weeks after transfusion (Fig. 5). Three of the four patients (Patients 2, 3, and 4) showed an increase in the concentrations of male donor WBCs in their circulation at Day 3 after transfusion (relative to Day 1 levels), which is consistent with our previously published observations in connection with fresh blood ~arnp1es.I~

DISCUSSION We previously reported that WB or buffy coat samples frozen with a cryoprotectant such as dimethyl sulfoxide can be evaluated by flow cytometric and qualitative PCR analyses.16 However, in subsequent studies, we and others found that dimethyl sulfoxide causesWBC degradation, results in modest reductions in W C subpopulations. 17*18 Moreover, RBCs inWB frozen with dimethyl sulfoxide are relativelyre- sistant to subsequent lysis by detergents (e.g., saponin), with consequent “carryover” of hemoglobin into the DNA lysate resulting in partial inhibition of PCR.”

The present studies evaluated a simpler alternative strategy using frozen blood preparations without

Day after transfusion Before I I

transfusion 1 3 5 7 14

Patient 1

Patient 2

Patient 3

Patient 4

a m - *

Fig. 5. Kinetics of male donor WBC survival in four female transfusion recipients, based on analysis of frozen WB samples for SRY-positive WBCs. All patient samples are negative before transfusion, which was followed by transient ap- pearance of male WBCs. Patients 2,3, and 4 show an increase in male donor WBCs in circulation at Day 3 relative to Day 2 after transfusion, which is consistent with transient in vivo proliferation of donor WBCs.



cryoprotectant.The results indicate satisfactory recovery of WBCs from frozen-thawed WB, as determined by flow cytometric analysis and PCR detection of target genes in minor WBC populations. RBCs in WB frozen without cryoprotectant are effectively lysed during the freezing- thawing procedure and therefore hemoglobin does not in- hibit PCR. Theoretically, the freezing-thawing process might destroy WBCs as well as RBCs and therefore would affect the sensitivity of assays intended to quantitate minor populations such as those of interest to transfusion medicine specialists. Indeed, flow cytometric analyses performed on directly frozen WB samples showed that WBCs are damaged during freezing and thawing, such that they fail to exclude vital dye. However, these"deadWBCs are intact with respect to their scatter properties in flow cytometry and their staining by WBC-specific monoclonal antibodies such as CD45 and CD3. Moreover, the cells re- tained their nuclei within the cytoplasmic membrane, as indicated by the results of vital dye staining and the number of WBC events in flow cytometric analyses. This observation is consistent with quantitative recovery of WBC nuclear DNA for PCR analysis using the centrifugation and washing protocol tested in this study.

Despite overall good recovery of WBCs in frozen- thawed WB, the agreement between both flow cytometry and PCR results in fresh and frozen-thawed preparations is quite variable and clearly dependent on the expectedWBC concentration in the sample. Better agreement (smaller relative bias [%I) was consistently observed at higherWBC concentrations, which is an expected finding based on counting statistics alone.l9

As far as the PCR results are concerned, this simple approach for directly freezing WB should have a variety of applications in blood banking and transfusion medicine. In our experience, the protocol works well for quality control of WBC-reduced blood components using PCR, with the advantage that rapid preparation (i.e., freezing) of samples in field laboratories enables more cost-efficient batch testing at a central reference laboratory. A requirement for the use of the assay for quality control of WBC reduction would be the establishment of a cutoff level for the PCR results at which the true WBC concentration would not be expected to exceed the threshold level for a WBC-reduced blood component. Because the variability at the threshold level may vary slightly among different users, it is important that each laboratory establish its own cutoff level by obtaining repeated measurements of blood samples with a known WBC concentration at the threshold level. According to the data reported here, standard 300-mL RBC components, with a mean count of less than 13,900WBCs per mL (4.17 x 1Os/3O0 mL) on duplicate testing of processed frozen- thawed WB samples, could be expected to meet the crite- rion for a WBC-reduced blood component of fewer than 5 x lo6 WBCs per 300-mL unit. Because WBC reduction is

usually performed with high-efficiency filters that generally achieve residualWBC counts less than 1 x 106,20 it is unlikely that workers would encounter filtered component, with PCR results between the lower and upper cutoff limits, that may contain less than 5 x 106WBCs per 300-mL component and therefore could potentially be misclassified as non- WBC-reduced. Our own experience confirms this expecta- tion, in that none of the filtered components in this study exceeded the calculated lower cutoff level for fresh or frozen-thawed samples. For the purpose of determining whether a component meets the requirement for a WBC- reduced component, directly frozenWB should work as well as a fresh sample in the PCR assay. However, given our finding of considerable discrepancy between results for fresh and frozen-thawed preparations at expectedWBC concentrations substantially less than 5000 per mL, it would be advisable to use only values based on either sample type, if comparisons ofWBC concentrations are made at this level (e.g., in filter comparison studies).

We have also successfully used the simple frozen- thawedWB protocol described here to preserve samples for analysis of both intracellular and cell-free (plasma) viral nucleic acids.2l Furthermore, frozen-thawedWB is compatible with flow cytometric enumeration of a variety of lymphocyte subpopulations, including CD3+, CD4+, CD8+, CD45+, and activated lymphocyte subsets.18#22 Therefore, this approach should also have applications in clinical vi- rology, immunology, and transplantation.

The frozen-thawed WB approach described here should be particularly useful for large-scale research studies that store thousands of samples in repositories for subsequent selective analysis. Tho large multicenter studies (the Retrovirus Epidemiology Donor Study [REDS] and the Viral Activation Tkansfusion Study [VATS] have already implemented this protocol and have archived over 100,000 frozen WB samples in reposi tor ie~.~~-~~ These samples are now being accessed for selective flow cytometric and nucleic acid testing. Finally, frozen WB may provide a useful approach for studies conducted in developing countries, where the technical capacity and resources for processing fresh samples in field sites may be lacking.

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AUTHORS

Tzong-Hae Lee, MD, PhD, Associate Director, Molecular Biology Scientific Services, Blood Centers of the Pacific, Irwin Center.

Nancy S. Sakahara, BS, Research Technologist, Scientific Services, Blood Centers of the Pacific, Irwin Center.

Eberhard W. Fiebig, MD, Director, Research/Immunology, Assistant Medical Director, Blood Centers of the Pacific, Irwin Center; Clinical Instructor, Laboratory Medicine, University of California, San Francisco; and Acting Chief, Transfusion Service, San Francisco General Hospital.

Dale F. Hirschkorn, BA, Supervisor, Flow Cytometry, Scien- tific Services, Blood Centers of the Pacific, Irwin Center.

Delene K. Johnson, MS, Assistant Director, Administration, Scientific Services, Blood Centers of the Pacific, Irwin Center.

Michael P. Busch, MD, PhD, Vice-president, Research and Scientific Services, Blood Centers of the Pacific, Irwin Center, 270 Masonic Avenue, San Francisco, CA 941 18. [Reprint re- quests]


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