proficiency testing experience for viable cd34+ stem cell analysis

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T R A N S P L A N T A T I O N A N D C E L L U L A R E N G I N E E R I N G

Proficiency testing experience for viable CD34+ stem cell analysis

María C. López and David A. Lawrence

BACKGROUND: Successful hematopoietic engraftmentdepends on the number of viable CD34+ stem cells.Therefore, accurate quantification of viable CD34+ stemcells is required.STUDY DESIGN AND METHODS: To evaluate clinicallaboratory performance, the New York State Depart-ment of Health initiated proficiency testing (PT) forviable CD34+ stem cells. Preserved adult peripheralblood was spiked with preserved cord blood CD34+stem cells and was shipped to the participating labora-tories. Three educational and two graded PTs were per-formed by participating laboratories, and their resultswere analyzed for consistency. Comparative analysis ofviability with 7-aminoactinomycin D (7-AAD) andToPro-3 dyes also was performed.RESULTS: Laboratories had to adapt their standardoperating procedures to include a viability dye to quan-tify the number of viable CD34+ stem cells. The major-ity of laboratories chose 7-AAD as their preferredviability dye, but propidium iodide (PI) and ToPro-3were used by two laboratories. Once all laboratoriesstarted to simultaneously analyze viability and stainingfor CD34, graded PTs started. Lower numbers of viableCD34+ stem cells were obtained for ToPro-3 when thedye was compared with 7-AAD.CONCLUSION: It is concluded that ToPro-3 stainsmore cells than 7-AAD and likely includes compromisedcells. The use of new vital dyes, like ToPro-3, that maystain preapoptotic cells could represent an importantadvance to improve the quantification of viable CD34+stem cells, for engraftment purposes. Further studiesare needed to document the benefits of switching to amethod that excludes not only dead cells, but apoptoticcells as well.

CD34+ stem cells are pluripotent hematopoieticstem cells that self-renew and give rise to allcells in the blood, including red cells (RBCs),platelets (PLTs), neutrophils, basophils, mono-

cytes, and lymphocytes.1 During fetal life, CD34+ stemcells populate the liver; at birth they can be found in cordblood, and later on in life, the main source of CD34+ stemcells is the marrow.1-3 Historically, marrow has been theclassic source of CD34+ stem cells that can be used torepopulate the marrow of patients suffering hematopoi-etic malignancies who have received chemotherapy andwhole-body lethal irradiation to eradicate their malignan-cies.4 More recently, two other sources of CD34+ stem cellshave come into use: cord blood and leukapheresisproducts.4-6 Treatment with G-CSF can induce the recircu-lation of marrow CD34+ stem cells to the peripheral blood;this treatment permits the collection of leukapheresisproducts.5,6 Leukapheresis products can be obtained fromthe patient before he or she undergoes lethal irradiation,and stem cells can be isolated and cryopreserved for laterinjection; this strategy avoids the hazards of obtaining ahistocompatible donor.7,8 Cord blood can be obtained atbirth, taking advantage of the existence of cord bloodbanks that collect cord blood from volunteer donors.Some studies suggest that the use of cord blood inducesless adverse symptoms than does the use of marrow, dueto the presence of naïve regulatory T cells in cord blood.9,10

The minimum number of CD34+ stem cells requiredto induce a successful transplantation outcome has beendetermined by several groups4,6,8,11 based on the time

ABBREVIATIONS: 7-AAD = 7-aminoactinomycin D;

PT(s) = proficiency testing (tests).

From the Wadsworth Center, New York State Department of

Health, Albany, New York.

Address reprint requests to: David A. Lawrence, PhD, Wad-

sworth Center, NYS DOH, PO Box 0509, Albany, NY 12201-0509;

e-mail: [email protected].

Supported by New York State.

Received for publication August 16, 2007; revision received

October 26, 2007, and accepted December 3, 2007.

doi: 10.1111/j.1537-2995.2008.01652.x

TRANSFUSION 2008;48:1115-1121.

Volume 48, June 2008 TRANSFUSION 1115

mailto:[email protected]

required to reconstitute the RBCs, PLTs, and neutrophilcompartments of the blood; monocytes and T and B lym-phocytes will appear in circulation afterward.12 Successfulengraftment depends on the number of injected CD34+stem cells and also on their viability.13 Therefore, a suc-cessful engraftment requires accurate quantification ofviable CD34+ stem cells. Several methods have historicallybeen used to analyze CD34+ stem cells; the preferredmethod has undoubtedly been flow cytometry. Moreover,among the various gating strategies developed, the gener-ally chosen method is the ISHAGE (International Societyfor Hematotherapy and Graft Engineering) method.14

Modifications to the original ISHAGE gating procedurehave included the use of a viability dye to determine viableCD34+ stem cells.15 From this information, it can bedecided whether a sample has the minimum number ofCD34+ stem cells with adequate viability to ensure suc-cessful engrafting. On the basis of current knowledge, it isevident that the determination of viable CD34+ stem cellsshould be considered the standard procedure, to ensurethe best chance of survival for the graft-recipient patient.Accordingly, to evaluate clinical laboratory performance,the New York State Department of Health initiated profi-ciency testing (PT) for viable CD34+ stem cells. There are24 to 27 participating laboratories that maintain a NewYork State permit to quantify CD34+ stem cells. This reportsummarizes the results obtained from our PT program.Also described are the shift from determination of totalCD34+ stem cells to determining viable CD34+ stem cellsand the use of a dye that appears to be able to discriminatebetween apoptotic and viable CD34+ stem cells.

MATERIALS AND METHODS

PT sample preparationHuman peripheral blood was collected in blood collectiontubes (Cyto-Chex BCT, Streck Laboratories, La Vista, NE),which contain a preservative reagent for flow cytometry.Frozen CD34+ stem cells from cord blood (AllCells LLC,Berkeley, CA) were thawed, washed with Iscove’s modifiedDulbecco’s medium supplemented with 20 percent fetalbovine serum and 2 mmol per L ethylenediaminetetraac-etate (EDTA), resuspended in the same medium, and pre-served in a BCT tube. Preserved peripheral blood and

CD34+ cells were mixed together and aliquoted (2 mL)into test tubes without additives (Vacutainers, BD Bio-sciences, San Jose, CA). Samples were shipped at roomtemperature; laboratories were expected to analyze thesamples upon receipt of them.

Analysis of CD34+ stem cells with7-aminoactinomycin D and ToPro-3Peripheral blood from 17 adult volunteers was collectedin EDTA Vacutainer tubes. The study was approved bythe Institutional Review Board of the New York StateDepartment of Health. Fluorescein isothiocyanate(FITC)-conjugated anti-CD45 and phycoerythrin (PE)-conjugated anti-CD34 antibodies (BD Biosciences) weredeposited into tubes (TruCount, BD Biosciences), fol-lowed by 50 mL of blood. After mixing, the tubes were incu-bated for 15 minutes in the dark at room temperaturefollowed by addition of 0.15 mol per L ammonium chlo-ride (450 mL) and incubation for an additional 10 minutes.Next, 5 mL of 7-aminoactinomycin D (7-AAD at 1 mg/mL;BD Biosciences) and 10 mL of a 1 mmol per L solutionof ToPro-3 (Molecular Probes, InVitrogen, Carlsbad, CA)were added and incubated for an additional 10 minutes,before samples were run in a flow cytometer (FACSCanto,BD Biosciences) with its accompanying software (FACS-Diva, BD Biosciences). Samples were run in duplicate, andlow- and high-stem-cell controls (BD Bioscience) wererun simultaneously to check for the ability of the reagentsand method to accurately determine sample values.

RESULTS

An example of modified ISHAGE gating with FACSDivasoftware is shown in Fig. 1. The importance of determin-ing viability in all samples is clear from the followingexample: the total number of CD34+ cells and the numberof viable CD34+ cells, in a cord blood sample, were calcu-lated 48 hours after the sample was collected. Stem cellviability, calculated with the number of viable CD34+ cellsand the total number of CD34+ stem cells, was found to belower than 90 percent; the generally accepted cutoff valuefor transplantable specimens is 90 percent viability.

In February 2006, the first educational PT sample wasshipped to all laboratories holding a NYS permit to

�Fig. 1. Description of modified ISHAGE gating of cord blood with FITC-CD45 antibodies, PE-CD34 antibodies, and 7-AAD in a tube.

The sample was run in a flow cytometer and analyzed with its accompanying software. In the first step (A), beads were gated and

removed from further analysis (B-I). Cells were sequentially gated on CD45+ (B), 7-AAD– (viable cells; C), and CD34+ (D) to obtain

the number of viable CD34+ stem cells (E). The total number of CD34+ stem cells was obtained by not gating on 7-AAD- cells (B to F

to G). Lymphocytes also were gated on CD45+ low side light scatter (SSC) (H) and shown on a FSC versus SSC plot (I). The absolute

number of viable CD34+ cells was calculated as the events in the viable CD34+ gate over the number of beads collected multiplied

by the number of beads per tube (provided by the manufacturer) and divided by the sample volume in microliters. (Viable CD34+stem cells = 70/5662 ¥ 53,514/50 = 13.2 cells/mL.) With the same equation, the total number of CD34+ cells was 16.6 cells per mL;

therefore, CD34+ stem cell viability was 79.5 percent.

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1116 TRANSFUSION Volume 48, June 2008

VIABLE CD34+ STEM CELL ANALYSIS IN NYS


analyze CD34+ stem cells. An attestation form to be sub-mitted with the PT results was included in the shipment,and laboratories were asked to report the number of viableCD34+ stem cells. The majority of laboratories werealready using ISHAGE gating and simultaneously deter-mining the viability of the CD34+ stem cells (Table 1).Nonetheless, nearly 40 percent of the laboratories wereusing commercial kits that do not measure viability (Pro-count, BD Biosciences); therefore, these laboratoriesreported the total number of CD34+ stem cells instead ofthe number of viable CD34+ stem cells. The dye mostcommonly used to study viability was 7-AAD, but pro-pidium iodide (PI) and ToPro-3 were also employed. Dyessuch as eosin red or trypan blue are not suitable, becausethey can only be used to microscopically analyze theviability of the entire cell population. After data from thefirst educational PT were analyzed, all participating labo-ratories received a report encouraging them to simulta-neously determine viability and the number of viableCD34+ stem cells and to switch to ISHAGE gating.

At the time when the second educational PT samplewas mailed in June 2006, only a few more laboratories hadswitched to ISHAGE gating and the simultaneous deter-mination of viability (Table 1). Therefore, laboratories

were once again strongly encouraged to change their pro-tocol to achieve measurement of the number of viableCD34+ stem cells. By the time the third educational PT wasshipped, the participating laboratories were prepared tomeasure viability, and they had chosen, in general, to use7-AAD as the viability dye (Table 1). Two laboratories wereusing ISHAGE staining and the Procount kit to double testtheir samples.

The major change in the method used by laboratoriesthat analyzed CD34+ stem cells during this educationalperiod was the inclusion of a viability dye, which obligedlaboratories to move to ISHAGE gating and replace a com-mercial kit with in-house developed kits. The laboratorieswith no-wash processing of the sample outnumbered thenumber of laboratories with washing procedures, and thelaboratories with single platform outnumbered the dual-platform users. These numbers were consistent through-out the educational period as well as for the graded PT.Three educational PTs were shipped.

Once all laboratories began to test for viable CD34+stem cells, it was possible to move to the next step, agraded PT. Two graded PT samples have been completed(Table 1). One laboratory persisted in using an in-housedeveloped kit with ISHAGE gating and a Procount kit fortheir analysis.

The numbers of CD34+ stem cells reported by all par-ticipating laboratories (n = 24-27) are presented in Fig. 2.The lower and upper lines of the box are the 25th and75th percentiles. The line within the box is the medianvalue; the whiskers are the 10th and 90th percentiles. In

TABLE 1. Data from the educational (1-3) andgraded (4 and 5) PTs showing the percentages of

laboratories with single versus dual platform;wash versus no-wash processing; and various

viability dyes, antibody sources, gatingprocedures, and analytical instruments

VariablesPT

1 2 3 4 5

PlatformSingle 66.7 65.4 64.0 70.8 70.8Dual 33.3 34.6 36 29.2 29.2

Processing methodWash 18.5 22.2 12 8.3 8.3No-wash 81.5 77.8 88 91.7 91.7

Viability7-AAD 48.1 55.5 92 91.6 91.7PI 3.7 3.7 4 4.2 4.1ToPro-3 3.7 7.4 4 4.2 4.1Eosin red 3.7 3.7 0 0 0Trypan blue 0 7.4 0 0 0No viability 40.8 22.3 0 0 0

Antibody sourceBC kit 25.9 25.9 20.0 20.0 24.0BD kit 44.4 44.4 8.0 4.0 1.0BC CD45 0 3.7 8.0 8.0 4.0BC CD34 3.7 3.7 12.0 12.0 12.0BD CD45 25.9 29.6 64.0 68.0 68.0BD CD34 25.9 29.6 64.0 64.0 60.0

InstrumentationBD FACSCalibur 63.0 55.6 64.0 62.5 66.7BD FACSCanto 7.4 11.1 12.0 12.5 8.3BD FACScan 14.8 18.5 8.0 8.3 8.3BC XL-MCL 14.8 11.1 8.0 8.3 8.3BC FC500 0 3.7 8.0 8.3 8.3

Gating methodISHAGE 60.7 66.7 92.0 96.0 96.0Procount 39.3 33.3 8.0 4.0 4.0

Fig. 2. Numbers of CD34+ stem cells reported by the laborato-

ries participating in the PTs; data are plotted with Sigma Plot.

The lower and upper lines of the box show the 25th and 75th

percentiles. The line within the box is the median value; the

whiskers show the 10th and 90th percentiles. In the first and

third educational PTs, there was a laboratory reporting a

number of CD34+ stem cells much higher than the 90th per-

centile. The number of laboratories participating in each PT is

shown in parentheses. Black dots represent the outliers.

LÓPEZ AND LAWRENCE


the first and third educational PTs, therewas a laboratory reporting the numberof CD34+ stem cells above the90th percentile; with this one reportremoved, the results still demonstrateda relatively high percentage CV decreas-ing from 133.3 and 285.5 to 50.7 and78.6 for the first and third PT events,respectively.

As mentioned earlier, the majorityof the laboratories used 7-AAD to assessviability; but one laboratory chose PIand another used ToPro-3. There is sub-stantial literature on the use of PI and7-AAD; however, little information isavailable on ToPro-3. Therefore, weneeded to determine whether ToPro-3performs similarly to 7-AAD, withrespect to the accurate determination ofviable CD34+ stem cells. We usedperipheral blood samples to determinethe number of viable CD34+ stem cells.Plots obtained from two individualdonors showed that the staining charac-teristics of the dyes differed. Specimen 1showed no dead cells with 7-AAD; but asmear of negative to positive events wasobserved with ToPro-3 (Fig. 3). Speci-men 2 showed some dead cells close toneutrophils with 7-AAD, whereas withToPro-3, it was possible to distinguisha smear (as for Specimen 1) and alsoa very high positive area with a sidescatter similar to neutrophils. Thenumber of viable CD34+ stem cells permL obtained by gating on either 7-AADor ToPro-3 was calculated for all 17samples and was represented asmean � standard deviation (SD) and asmedian � 25th and 75th percentiles;clearly, ToPro-3 analysis reports signifi-cantly (p = 1.2 ¥ 10-6, t test) fewer viableCD34+ cells than does 7-AAD analysis(Fig. 3).

DISCUSSION

The main goals of the NYS stem cell PTprogram were to ensure that all partici-pating laboratories can successfullyreport the number of viable CD34+ stemcells and to assist any laboratory havingdifficulties with this method. Although ithas been suggested that the predicationof engraftment is not improved with

Fig. 3. Comparison between 7-AAD and ToPro-3. Peripheral blood was stained with

FITC-conjugated anti-CD45 and PE-conjugated anti-CD34, followed by ammonium

chloride treatment to lyse RBCs and simultaneous staining with 7-AAD and ToPro-3

to determine viability. Dot plots from two samples are presented, showing differing

patterns of staining for the two dyes 7-AAD and ToPro-3 (A). The number of viable

CD34+ stem cells was calculated with both dyes, and the results are presented as

mean � SD and as median and 25th and 75th percentiles and 10th and 90th percen-

tiles as described in the legend to Fig. 2. Black dots represent the outliers.



assessment of viable CD34+ cells along with analysis oftheir total number, this conclusion was based on freshlycollected CD34+ cells.16 With frozen preparations, this pre-diction is not reliable. Consistent use of viable cellnumbers provides more safety. Furthermore, once viableCD34+ stem cell numbers necessary for successfulengraftment are determined, collections for engraftmentmay be further optimized. In any case, all flow cytometrylaboratories assisting transplant services should be com-petent to assess viability.

Accurate determination of the number of CD34+ stemcells in a graft sample is crucial to achieve a successfulrepopulation of the patient’s marrow with the stem cellsthat will give rise to all hematopoietic cells in blood. Suc-cessful engraftment of hematopoietic stem cell precursorsis a multistep process that can be analyzed by tracking theappearance in the blood of cells belonging to the variouswhite cell lineages, as well as the RBC and megakaryocyticlineages.17,18 In fact, the success or failure of a transplantcan be estimated from the number of days required fornewly formed PLTs and neutrophils to appear.13 In a suc-cessful engraftment, the patient should generate PLTs byDays 10 to 12, and neutrophils by Days 12 to 14 aftertransplantation.13,18 When RBCs, PLTs, and neutrophilscannot be generated within the first month after trans-plantation, the patient’s life is at risk. The question that israised after a graft failure is always why stem cells were notcapable of repopulating the patient’s marrow and givingrise to the hematopoietic progenitor lineages. Thus, it iscrucial that we have a method able to precisely determinethe number of viable cells in a graft. Even if the number ofviable CD34+ stem cells has been determined to beadequate, at the time of collection, the process of cryo-preservation and thawing can damage a proportion ofstem cells. Recently, discrepancies between the numberof viable cells at the time of collection and the numberafter cryopreservation have been reported; a diminishednumber of viable cells in the grafts after thawing could beassociated with a prolongation of the time for generationof PLTs and neutrophils.13 Another important issue is thatthe determination of viability is by itself insufficient infor-mation to predict the engraftment potential. Cells notstained with 7-AAD are at the time of testing viable, butthey could, for example, be apoptotic. In fact it has beenreported that some CD34+ stem cells can be in early stagesof apoptosis.19 When grafts were thawed for transplanta-tion, the presence of early apoptotic cells was not detect-able by 7-AAD staining and could only be demonstratedafter further staining with Syto 16 and annexin V.19 Suchfindings encourage us to continue to research methodadvances to the analysis of CD34+ stem cells to furtherimprove successful engraftment. The ideal method shouldaccurately quantify viable, nonapoptotic CD34+ stem cellswith use of the fewest possible reagents and manipula-tions. Determination of the number of viable CD34+ stem

cells should be done not only at collection time, but alsoafter thawing, immediately before transplantation. Addi-tionally, we suggest that infusion of minimal numbers ofnonviable cells will reduce any chance for the develop-ment of pathologies resulting from accumulated celldebris in the blood and filtering organs from injection ofdead and dying cells. For that reason, the use of new vitaldyes, like ToPro-3, that appear to stain preapoptotic cells,could represent an important advance to improve thequantification of viable CD34+ stem cells, for engraftmentpurposes. Further studies are needed to document thebenefits of switching to a method that excludes not onlydead cells, but apoptotic cells as well. Finally, it is impor-tant to remember that flow cytometry has been chosenas the preferred method to quantify CD34+ stem cells,because it can provide an answer in 1 hour; in contrast,the older methods based on the ability of viable CD34+stem cells to induce RBC and/or granulocyte–colony-forming units, require more than 1 week.

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proficiency testing experience for viable cd34+ stem cell analysis

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