J Clin Pathol 1992;45:1053-1057

ACP Broadsheet No 134 December 1992

How to harvest bone marrow for transplantation

R Jones, A K Burnett

IntroductionOver the past decade bone marrow trans-plantation (BMT) has graduated from beingan experimental treatment to having an estab-lished role in the first line management ofpatients with life threatening haematologicaldisorders. The therapeutic principle involved isthat the administration of myelotoxic treat-ment (preparative protocol), at doses beyondthe threshold normally set to guarantee haema-tological recovery, will be effective in eradicat-ing the abnormal pathology in the patient. Asecond requirement is that, where an alloge-neic donor is the source of marrow, thepreparative protocol will immunosuppress thehost sufficiently to ensure that the marrow willsuccessfully graft. There are preclinical data,'and some circumstantial evidence in man tosuggest that the mechanisms by which theunderlying disease in the host is eliminated,may not be solely due to the intensity of thepreparative protocol but augmented by animmunologically mediated effect from thedonated marrow, referred to as the "allogeneiceffect".2 3 There are now several conditions towhich bone marrow transplantation has beensuccessfully applied (table).The bulk of this experience has been of

transplantation from an HLA matched, mixedlymphocytic culture (MLC), non-reactive sib-ling donor. The capacity of this form oftreatment to eradicate the underlying diseasemore effectively than conventional approachesis clear and has encouraged investigators toexplore means by which transplantation can bemade available to those who lack a suitablematched sibling. This has led to the use of thepatients' own marrow collected earlier when inremission and, following storage, used as thesource of haematological rescue to supporthigh dose treatment (autologous BMT). Morerecently the organisation of registries of suffi-cient size has permitted a more systematicapproach to using HLA phenotypically mat-ched but unrelated donors as a source of stemcells.

Selection and preparation of the marrowdonorThe availability of an HLA matched, sibling

donor is an important factor in decidingbetween an allogeneic and an autologoustransplant for a given patient. Thus the siblingsof a potential allogeneic transplant recipientmust be screened to identify any who are HLAmatched. In the absence of a related donor asearch of volunteer donor registers may locatean HLA matched unrelated donor. Alterna-tively an autograft may be preferred.When a donor is found, preliminary inves-

tigations constitute a full medical examinationto establish that they are fit for anaesthetic.Routine haematological and biochemicalparameters are checked. The donor mustbe hepatitis B, hepatitis C, and humanimmunodeficiency virus *HIV) negative.If indicated, a chest x ray picture and anelectrocardiogram are taken.

Written informed consent must beobtained.The donor is admitted the night before

harvest and the best method of anaesthesiadiscussed. In our unit we recommend a generalanaesthetic but a regional block by spinal orepidural anaesthesia is an alternative.

Personnel required for bone marrowharvest* Anaesthetist* Operators to harvest marrow* Haematology technician* Scrub sister* Theatre staff

Diseases that may be treated by bone marrow transplant

(1) Bone marrow failureSevere aplastic anaemiaSevere paroxysmal nocturnal haemoglobinuria (PNH)Fanconi s anaemia

(2) LeukaemiaAcute lymphoblastic leukaemiaAcute myeloid leukaemiaChronic myeloid leukaemiaAcute malignant myelosclerosisMyelodysplasia

(3) LymphomaHodgkin's diseaseNon-Hodgkin's lymphoma

(4) Immunodeficiency syndromesSevere combined immunodeficiencyWiskott-Aldrich syndrome

(5) Other genetic disordersThalassaemia Gaucher's diseaseHurler's syndromeInfantile osteopetrosis

This Broadsheet has beenprepared by the authors at theinvitation of the Association ofClinical Pathologists whoreserve the copyright. Furthercopies of this Broadsheet maybe obtained from thePublishing Manager, journalof Clinical Pathology, BMAHouse, Tavistock Square,London WCIH 9JRDepartment ofHaematology, GlasgowRoyal Infirmary,Castle Street,Glasgow, G4 OSFR JonesA K BurnettAccepted for publication24 February 1992

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Equipment required for bone marrowharvest sterile equipment* Jamshidi trephine needle* Two modified Rosenthal marrow harvest

needles or equivalent* One sternal aspiration needle* Eight 30 ml plastic disposable syringes* Two 2 ml plastic disposable syringe

100 ml Waymonth solution (Gibco) withpreservative free heparin (Leo laboratoriesadded at 50 units per ml

* Two blood donor (Tuta) 500 ml bags* Two sampling site couplers (Fenwal)

Sterile drapes, swabs and towel clipsSterile solutions for cleansing operationsiteStand for marrow collection bag

Additional non-sterile equipmentOne light microscopeModified Neubauer cell counting chamber

Technique ofbone marrow harvestThe anaesthetised patient is positioned proneon the operating table with pelvis supported tomake the iliac crests prominent. The skin iscleansed and drapes applied to leave theposterior iliac crests exposed.The exact technique of harvesting varies

between centres but is unlikely to differ greatlyfrom that described by Thomas.4The choice of harvest needle is one of

personal preference as a range are commer-cially available which differ little except in thedesign of the gripping handle. Some have holesalong the lateral aspects of the shafts but theseclog easily and are of little value.The needle with trochar is inserted through

the skin and into the bone, the trochar iswithdrawn, and marrow blood aspirated into a30 ml syringe. The syringe is immediatelydetached and discharged into a collectingreceptacle. The syringes are flushed with Way-mouth solution with the addition of heparinand recycled. We use eight syringes for eachharvest.

Figure 1 Theatre troUley laid up for a bone marrow harvest. (a) aspirating syringes (30ml); (b) needles; (c) flushing fluid (Waymouth); (d) collection bag on stand; (e) bagcap + coupler; (I) syringe for sampling bag.

In some centres donors are anticoagulated toreduce the risk of the aspirated marrow clot-ting, but we have found that flushing thesyringes with anticoagulant between eachaspiration and regular mixing of the marrow inthe collection bag makes this unnecessary.

It is our impression that the yield is opti-mised by inserting the needle superficially, andtwo aspirates taken at that level, and then theneedle advanced and the aspiration repeated.It is usually possible to take marrow at severaldifferent depths from one hole. The needle isthen withdrawn and re-sited, samples beingtaken as widely as possible along the posterioriliac crest. Most marrow will be obtained at theinitial part of "the pull," thereafter the volumewill largely be peripheral blood. We thereforerestrict each pull to 8-10 ml.

If an inadequate cell count is obtained fromthe posterior iliac crests the patient should beturned over and further aspirates taken fromthe anterior iliac crests and the sternum. This issometimes necessary for autologous bone mar-row transplant where the patient has beenheavily pretreated with radiotherapy or cyto-toxic drugs, but in most cases the posteriorcrests are sufficient.

It is quite common for aspirated marrow tobe first discharged through a stainless steelmesh into a sterile beaker to remove fataggregates, clots, or bone spicules. The mar-row may be exposed to airborne contaminationif this technique is used and we prefer a closedsystem where the syringe is emptied directlyinto a 500 ml blood donor bag which containscitrate-phosphate-dextrose (CPD) (Tuta) asanticoagulant. The bag is held in a stand and ismanually agitated.The marrow bag can easily be sampled with

a 2 ml syringe to allow a cell count to beperformed as the harvest proceeds. We nor-mally take 500 to 1000 ml of marrow at aharvest. If inadequate counts are obtained it isrepeated at a later date.

Marrow doseThere are different ways to calculate the doseobtained during a harvest. Some simply take10 ml/kg recipient body weight but in mostcentres a nucleated cell count is performedwhile the harvest is in progress to determinewhen an adequate harvest has been obtained.The marrow cell count can be calculated by

subtracting the donor's peripheral white cellcount from the marrow count but we simplycount the marrow nucleated cells in a Neu-bauer counting chamber and use this as a guideto marrow yield.The absolute amount of marrow needed is

not known and current clinical practice varieswidely, but some principles apply. Successfulre-engraftinent is related to tissue compatibil-ity such that autografts, twins, HLA matchedTreplete siblings; HIA matched unrelated andHILA mismatched donations require a pro-gressively larger graft to increase the chance ofgraft take.The "dose" of nucleated cells is expressed

per kilogram of recipient body weight. An

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historical target figure for matched siblingallografts is 3 x 108 nucleated cells per kg.This was the threshold that ensured engraft-ment in sensitised patients with aplastic anae-mia.5 In unsensitised HLA matched patientswho receive total body irradiation a dose of 1-5- 2-0 x 108/kg is probably sufficient. We aimto have a minimum of 1 x 108/kg for autolo-gous grafts.

Postoperative careThe harvest procedure lasts about 40 minutesand after recovering from the anaesthetic thepatient is transferred back to the ward. It ismandatory to replace volume lost during theprocedure with crystalloids or plasmaexpander and ideally red cells are alsoreplaced. It is desirable to avoid exposing avolunteer donor to the risk of allogeneic bloodtransfusion however slight. Two options exist:either autologous blood is taken a week ormore prior to the procedure or the red cells areseparated from the marrow by centrifugation.

In an autologous harvest replacement from acommunity donor may be required and it isusual to have 2 units of matched irradiatedblood available. Irradiation destroys lympho-cytes that survive in the donated blood and intheory could contaminate aspirated marrow,and when returned to the patient, set up graftversus host disease. Delaying transfusion untilafter the harvest abolishes the risk unless asecond harvest is needed in the future. Weroutinely have 2 units of irradiated bloodavailable but these are very rarely needed.The donor is retained overnight after the

harvest to allow full recovery from the anaes-thetic and red cell replacement.

Complications for the marrow donorPostoperative pain is modest, easily controlled,and transient. With care any risk to the donor isminimal. Serious complications occurred in0-27% of 3000 cases from Seattle and theinternational bone marrow transplant registerreviewed by Bortin and Buckner.6 Five compli-cations related to the anaesthetic, three tosepticaemias secondary to infection at theaspiration site, and one was a cerebral infarc-tion but this occurred many hours after theharvest when the patient had fully recovered.The risks are similar to that for other minoroperative procedures.

In vitro manipulationALLOGENEIC

For allogeneic transplantation the harvestedmarrow can be taken and immediately infusedintravenously into the patient. However, forspecific reasons, some form of in vitro process-ing or treatment may be required before it isgiven. In the autograft setting in vitro treat-ment aimed at reducing occult tumour cellcontamination or preparation for storage maybe needed. It is not the purpose of this articleto discuss the pros or cons of any of thesemanipulations as they affect clinical outcome.

In two circumstances in relation to alloge-neic transplantation it may be necessary toremove selectively the mononuclear cell frac-tion which is known to contain the repopula-tive stem cells. First, density separation, whichresults in a suspension with a very low haema-tocrit, is required where there is a significantblood group incompatibility between donorand host. Second, if removal of T cells isrequired, as a strategy to prevent graft-versus-host disease, then it is convenient to reduce thevolume of the donation before starting incuba-tion with monoclonal antibodies. A convenientway to do this is the automated procedure on aCobe 2991 blood cell washer, as originallydescribed by Gilmore et al.7The Cobe 2991 can provide a buffy coat, or

ficoll-hypaque can be introduced to permit on-line density separation to produce the mono-nuclear cell fraction (MNC). An equivalentresult can be obtained using different cellseparators. Incubation with antibody and com-plement to destroy T cells can be easilyachieved in the system, and the subsequentwashing steps all carried out in a closedsemiautomated system.

Autologous marrowAutologous marrow can be similarly separatedto leave a buffy coat or further processed to amononuclear cell fraction which can be"purged" or stored. The most popular methodaimed at elimination of occult leukaemia fromautologous marrow in acute myeloid leu-kaemia is by incubation with the cyclo-phosphamide metabolite (4hydroxy-per-cyclophosphamide, 4 H-C).8 Animportant variable in the technique of purgingwith this method is the haematocrit of thestarting marrow suspension, so it is helpful tostart with a standard cell preparation. When animmunological method of purging is used,which is most readily applicable in acutelymphoblastic leukaemia, the sequence ofevents is similar to that described above for Tcell depletion of allogeneic marrow. There areseveral different methods of treating marrow invitro with the MNC as the starting point.

Bone marrow storageSome form of storage is always required forautologous BMT. Where the preparative proto-col is short then cryopreservative is notrequired. We have demonstrated satisfactoryreconstitution from a total body irradiation(TBI) based, myeloablative protocol, usingmarrow stored as it was collected in anticoagu-lant, or as an MNC fraction, at 4°C for up to54 hours.9 In vitro survival of committedhaemopoietic precursors (CFU-GM, BFU-E)suggest liquid storage for seven to 10 daysmight be feasible, but this has not been testedclinically in a patient given an ablative proto-col.Where prolonged treatment regimens are

used, or further treatment of the patient isenvisaged autologous marrow must be cryo-preserved. Here the initial preparation of an

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1Jones, Burnett

MNC fraction may also have major advan-tages. The neutrophil content of an MNC issmall and neutrophils freeze poorly which mayresult in clumping. The final volume of anMNC fraction can easily be kept to less than100 ml from a 1 litre harvest, which allowseven freezing through the cell suspension, andreduces the absolute volume of cryoprotectantwhich will be returned to the patient. Thereduction in volume also saves a considerableamount of expense in liquid nitrogen storagecapacity.Having reduced the initial marrow volume

to 50-70 ml resuspended in the patient'sautologous plasma, an equal volume of dime-thyl sulphoxide (DMSO) at 20% in autologousplasma can then be added at 4°C. In theoryglycerol is the best cryoprotectant, but requiresto be eluted from the cells when thawed andthen be washed off, which is impractical.DMSO at a final concentration of 10% hascome to be the accepted cryoprotectant, hav-ing the important advantage that it rapidlydiffuses in and out of the cells. It is, however,toxic to cells at room temperature and it isimportant to introduce it to the marrowsuspension at 4°C, and on thawing, to wasteno time in returning the marrow to the patient.One of the advantages of the low bulk of anMNC is that it is not necessary to wash thesmall amount of DMSO out of the thawedsuspension because it can safely be givendirectly to the patient. Large volumes ofDMSO are toxic when given intravenously.10Any in vitro purging is invariably done beforepreparation for freezing begins. There is noimportant evidence to suggest that marrowswhich have been treated in vitro are any moresusceptible to damage during cryopreserva-tion.There are several factors which are thought

to affect the quality of freezing. These includethe cellular component, the protein content ofthe suspending fluid, the choice and concen-tration of cryoprotectant, the rate of freezingand the storage conditions. These aspects havebeen the subject of a helpful review." It hasbeen generally accepted that a controlledfreezing rate is mandatory (around 1-2°C aminute) at least until the eutectic point, butthis has recently been challenged.'2 Storage inthe liquid phase of nitrogen is probably prefer-able to the vapour phase which is vulnerable tovariation caused by periodic opening of thestorage container.

Assessment of in vitro manipulation andstorageOne of the current difficulties is that there is nodirect laboratory measurement which corre-lates with the regenerative capacity of the graft.In general most storage techniques, partic-ularly when initially introduced, should bemonitored using assays of the committedhaemopoietic precursors (CFU-GM andBFU- E). Marrows can be successfully regen-erated after these populations have beenremoved, but it is reasonably safe to accepttheir presence before and after manipulation as

an indication that the repopulative potentialhas been retained. These precursor popula-tions are not a direct measure of the plur-ipotent stem cell population.We have found that samples taken from the

harvest occasionally yield a positive microbio-logical culture usually with a skin commensal.We have noted no clinical consequences as aresult of this.

Administration of the bone marrowThawing of stored marrow in a water bath at37°C, and the immediate reinfusion to thepatient via the central venous line, usuallypasses uneventfully. From time to time somepatient reaction ranging from nausea andvomiting, to rigors or analphalaxis (rarely) isseen. The frequency of such episodes is not, inour experience, related to whether or not aparticular laboratory manipulation wasinvolved or any blood group disparity existed.Such episodes can be alarming and medicalsupervision of the reinfusion is recommen-ded.

Administration through a standard bloodadministration set ensures that any aggregatesare removed. The small amount of DMSOadministered is expired over the following 24hours without clinical sequelae.

New developmentsUse of stem cells taken from peripheral bloodby leucophoresis is becoming fashionable." 14

It was initially felt that this approach wouldhave the advantage of avoiding any potentialtumour cells which may be contaminating thebone marrow-and in particular would allowan autograft option to be available to patientsknown to have an infiltrated marrow. There isno evidence to support the view that theperipheral blood is a purer source of stem cells,but the thoughtful use of cytokines to mobiliseperipheral blood stem cells (PBSC) has meantthat adequate numbers can be obtained withone or two leucaphereses, making it morepractical. Particularly attractive are the accu-mulating data that the pace of haemopoieticreconstitution-particularly of platelets-ismuch quicker with PBSC alone or with bonemarrow than with marrow alone.

Technical developments now suggest thatpositive selection of a stem cell populationfrom marrow (as defined by a CD34 positivephenotype) is becoming a practical possibility.It has been known for some time that such apopulation can reconstitute irradiated patients,but the techniques involved (column immuno-adsorption) gave poor cell yields. More effi-cient techniques will be validated in the nearfuture. This approach would represent a uni-versal approach to purging and also segregate atarget cell population for in vitro geneticmanipulation.

Dr Jones is a Leukaemia Research Fund Clinical Fellow.

I Boranic M, Tonkovic I. The pattern of the antileukaemiceffect of graft-versus-host reaction in mice. Cancer Res1970;31:1140-7.

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2 Sullivan KM, Weiden PL, Storb R. Influence of acute andchronic graft-versus-host disease on relapse and survivalafter bone marrow transplantation from HIA-Identicalsiblings as treatment for acute and chronic leukaemia.Blood 1989;73:1720-8.

3 Gale RP, Champlin RE. How does bone marrow trans-plantation cure leukaemia? Lancet 1984;ii:28-30.

4 Thomas ED, Storb R. Techniques for human marrowgrafting. Blood 1970:36:507-15.

5 Storb R, Prentice RL, Thomas ED. Marrow transplantationfor treatment of aplastic anaemia. An analysis of factorsassociated with graft rejection. N Engi i Med1977;296:61-6i

6 Bortin MM, Buckner CD. Major complications of marrowharvesting for transplantation. Exp Haematol1983;11:916-21.

7 Gilmore MML, Prentice HG, Blacklock HA. A techniquefor the rapid isolation of bone marrow mononuclear cellsusing ficole-metrizoate and the IBM 2991 Blood CellProcessor. Br Haematol 1982;50:619-26.

8 Kaizer H, Stuart RK, Brookmeyer R, et al. Autologous bonemarrow transplantation in acute leukae,nia: A phase 1

study ot in vitro treatment ot marrow with 4-Hydroxper-oxycyclophosphamide to purge tumor cells. Blood1985;65: 1504-10.

9 Burnett AK, Tansey P, Hills C, et al. haematologicalreconstitution following high dose and supralethal chem-oradiotherapy using stored, non-cryopreserved autolo-gous bone marrow. Br Haematol 1983;54:309-16.

10 O'Donnell JR, Burnett AK, Sheehan T, et al. Safety ofdimethyl-sulphoxide (DMSO). Lancet 1981;ii:498.

11 Gorin NC. Collection Manipulation and freezing ofhaemo-poietic stem cells. Clin Haematol 1986;15:19-48.

12 Clark J, Pati A, McCarthy D. Successful cryopreservation ofhuman bone marrow does not require controlled-ratefreezer. Bone Marrow Transplantation 1991 ;7: 121-5.

13 Juttner CA, To LB, Haylock, et al. Circulating autologousstem cells collected in very early remission from acutenon-lymphoblastic leukaemia produce prompt butincomplete haemopoietic reconstitution after high dosemelphalan or supralethal chemoradiotherapy. Br Jf Hae-matol 1985;61:739-45.

14 Bell AJ, Figes A, Oscier DG, Hamblin TJ. Peripheral bloodstem cell autografting. Lancet 1986;i: 1027.

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