Table 9.1 Comparison of autologous and allogeneic SCT Autologous Allogeneic Wide age range, generally \211 70 years Age range generally \211 60 years, though higher age limit now that RIC is used No need for donor search if BM clear Sibs have 71 in 4 chance of match Not feasible if BM involved May be used in patients with BM disease Risk of tumor cell re-infusion No tumor contamination of graft Not all patients can be mobilized Donor search may impose delay No GvHD GvHD mortality and morbidity No immunosuppression Graft-versus-disease eff ect (e.g. GvL) Low early treatment related mortality (2 \200\2235%) Higher early treatment-related mortality from GvHD and infection (10 \200\22320%) Risk of long-term MDS from BM injury Lower risk of late MDS 390 CHAPTER 9 Haematopoietic stem cell transplantation Table 9.2 Factors infl uencing outcome of haemopoietic SCT Time Patient Donor Pre-transplant Disease Diagnosis, subtype, stage Age, sex, sex-mismatch, ethnicity, viral status, histocompatibility Major histocompatibilit y antigens Minor histocompatibility antigens ABO Cytokine polymorphisms Individual Age, sex, ethnicity, organ status, viral status, Performance status Environment Geography Economic status Peri-transplant Conditioning GvHD prevention Graft product Intensity: RIC? Prevention method Stem cell source, graft composition Post-transplant Disease Individual Environment Relapse Immune reconstitution Retreatment possibilities Availability for DLI or retransplant Reproduced from Gratwohl, A. (2007). Risk assessment in haemopoietic stem cell transplantat ion. Best Pract Res Clin Haematol, 20, 119 \200\22324, with permission from Elsevier 200 7. Early complications of the transplant procedure Chemo-radiotherapy \200 Nausea/vomiting. \200 Reversible alopecia. \200 Fatigue. \200 Dry skin. \200 Mucositis. \200 Organ failure. \200 VOD (veno-occlusive disease). Infection \200 Bacterial (Gram \210\222ve and +ve). \200 Viral \200\224HZV. \200 CMV (particularly pneumonitis).* \200 Fungal \200\224Candida, Aspergillus*, Mucor*. \200 A typical organisms \200\224Pneumocystis (PCP)*, Toxoplasma*, Mycoplasma*, Legionella*. (*Low risk in autologous SCT; signifi cant to high risk in allogeneic SCT.) 391 HAEMOPOIETIC STEM CELL TRANSPLANTATION (SCT) GvHD May occur in recipients of allogeneic SCT due to tissue incompatibility between donor and recipient undetected by standard tissue-typing tests. Acute and chronic forms. Higher i ncidence of severe GvHD following VUD SCT, and mismatched or haploidentical grafts. Acute o r chronic. Late complications of transplantation \200 Complications of chronic GvHD. \200 Infertil ity (both sexes). \200 Hypothyroidism. \200 2 malignancy. \200 Late sepsis due to h yposplenism (in myeloablative regimens). \200 Cataracts (where TBI used). \200 Psycholo gical disturbance. Follow up and post-transplant surveillance Lifelong follow-up is required. The particular r isks and monitoring required depend on the type of graft and whether TBI was used. Suitable conditioning regimens are outlined in E Stem cell transplant conditioning regimens, p.414. Further reading Apperley, J. et al. (eds.) (2012). The EBMT-ESH Handbook on Haemopoietic St em Cell Transplantation, revised ed. Paris: EBMT. Cant, A.J. et al. (eds.) (2007). Practica l Haematopoietic Stem Cell Transplantation. Oxford: Blackwell Publishing. Gratwohl, A. (ed. ) (2007). Risk assessment in haematopoietic stem cell transpantation. Best Pract Res Clin H aematol, 20(2), 119 \200\22324. 392 CHAPTER 9 Haematopoietic stem cell transplantation Indications for haemopoietic SCT The Accreditation Subcommittee of the European Group for B lood & Marrow Transplantation (EBMT) reports current practice for HSCT in Europe (Tables 9. 3 and 9.4). Table 9.3 EBMT indications for HSCT for adults (recommendation from 2010, adopted and uncha nged 2012)1 Disease Disease status Allo Auto Mismatch unrelated/ >1 antigen mismatched related Sibling donor Matched unrelated /1 antigen mismatched related GNR D CO CO D GNR CO S CO CO GNR GNR D CO S S CO CO CO S S S S S AML CR1 (low risk) CR1 (int risk) CR 1 (high risk) CR2 CR3, incipient relapse APML molecular pe rsistence CO D S CO APML molecular CR2 Relapse/refractory GNR D S GNR D GNR GNR D ALL CR1 (standard/ int risk) CR1 (high risk) CR2, incipient S S S S CO CO D GNR relapse Relapse/ refractory CO D D GNR CML 1st CP, failed imatinib AP, >1st CP Blast crisis S S CO D S CO S CO CO CO D GNR Myelofi brosis 1 , 2 with high/ int Lille score S S D GNR Myelodysplastic syndrome RA, RAEB RAEBt, sAML, CR1, CR2 More advanced stages S S S S S CO CO CO D GNR CO GNR CLL High-risk disease S S D CO CO GNR GNR GNR Diff use large B-cell NHL CR1 (int/high-risk at Dx) S GNR CO CO Chemosensitive relapse; \211 CR2 GNR GNR D D Refractory (continued) 393 INDICATIONS FOR HAEMOPOIETIC SCT Table 9.3 (continued) EBMT indications for HSCT for adults (recom- mendation from 2010, ado pted and unchanged 2012)1 Disease Disease status Allo Auto Sib Matched Mismatched CO CO D D GNR GNR S S Mantle cell lymphoma CR1 Chemosensitive relapse; \211 CR2 Refractory D D GNR GNR CO CO CO CO GNR GNR CO CO CR1 Chemosensitive relapse; Lymphoblastic and Burkitt lymphoma \211 CR2 Refractory D D GNR GNR GNR CO GNR CO GNR D CO S Follicular NHL CR1 (int/high risk at dx) Chemosensitive relapse; \211 CR2 Refractory CO CO D GNR CO CO D CO GNR GNR CO D T-NHL CR1 Chemosensitive relapse; \211 CR2 Refractory D D GNR GNR GNR CO GNR CO GNR CO GNR S HL \200\224classical CR1 Chemosensitive relapse; \211 CR2 Refractory D D GNR CO GNR GNR GNR GNR GNR GNR GNR CO HL \200\224NLPHL CR1 Chemosensitive relapse; \211 CR2 Refractory GNR GNR GNR CO Myeloma CO CO GNR S Amyloidosis (primary; AL) CO CO GNR CO Severe aplastic anemia Newly diagnosed Relapsed, refractory S S CO S GNR CO GNR GNR PNH S CO CO GNR Solid tumors Germ-cell tumors Sensitive relapses 3rd line refractory GNR GNR GNR GNR GNR GNR CO S Breast ca Adjuvant high risk Metastatic respond GNR D GNR D GNR GNR CO D/CO Ovarian ca CR/PR Platinum sensitive relapse GNR D GNR GNR GNR GNR D CO Medulloblastoma Post-surgery GNR GNR GNR D Small cell lung Limited stage GNR GNR GNR D Renal cell ca Metastatic, cytokine refr CO CO GNR CO Soft cell sarcoma Metastatic responsive D GNR GNR D (continued) 394 CHAPTER 9 Haematopoietic stem cell transplantation Table 9.3 (continued) EBMT indications for HSCT for adults (recom- mendation from 2010, ado pted and unchanged 2012)1 Disease Disease status Allo Auto Sib Matched Mismatched Auto immune diseases Immune CO D D CO cytopenias Systemic sclerosis D GNR GNR CO R.A. CO GNR GNR CO Multiple D GNR GNR CO sclerosis Lupus D GNR GNR CO erythematosis Crohn \200\231s disease CO GNR GNR CO CIDP CO GNR GNR D S = standard of care, generally indicated in suitable patients; CO = clinical option, can b e carried out after careful assessment of risks and benefi ts; D = developmental, further t rials are needed; GNR = generally not recommended. NB This classifi cation does not cover patients for whom a syngeneic donor is available. A syngeneic twin donor is generally considered S for all indications except, by defi nition, congenital disorders. Donor type is divided into: HLA identical sibling donor. Matched unrelated: there is no uniformly accepted defi nition. A \211 10/10 antigen matche d donor is unequivocally considered as matched. Mismatched donor: there is no uniformly accepted defi nition. A < 8/8 antigen matched unrelated and a >1 antigen mismatched family donor is unequivocally considered mismatched. 395 INDICATIONS FOR HAEMOPOIETIC SCT Table 9.4 EBMT indications for HSCT for children (recommendation from 2010, adopted and unc hanged 2012)1 Disease Disease status Allo Auto Mismatch unrelated/ >1 antigen mismatched related Sibling donor Matched unrelated /1 antigen mismatched related GNR S S GNR CO S GNR GNR CO GNR S CO AML CR1 (low risk) CR1 (high risk) CR1 (very high risk) CR2 > CR2 S CO S D S D S GNR GNR GNR CO CO GNR CO CO CO GNR S S S GNR S S S ALL CR1 (low risk) CR1 (high risk) CR2 > CR2 CML CP AP S S S S D CO GNR GNR GNR CO CO GNR GNR CO GNR CO S GNR CO S NHL CR1 (low risk) CR1 (high risk) CR2 HL CR1 1st relapse, CR2 GNR CO GNR D GNR GNR GNR S MDS S S D GNR Primary ID S S S N/A Thalassaemia S CO GNR N/A Sickle cell disease High risk S CO GNR N/A Aplastic anemia S S CO N/A Fanconi anemia S S CO N/A Diamond \200\223Blackfan anemia S CO GNR N/A CGD S S CO N/A Kostman \200\231s disease S S GNR N/A MPS-1H Hurler S S CO N/A MPS-1H Hurler- Scheie Severe GNR GNR GNR N/A MPS-VI Maroteaux \200\223Lamy CO CO CO N/A Osteopetrosis S S S N/A Other storage diseases GNR GNR GNR N/A (continued) 396 CHAPTER 9 Haematopoietic stem cell transplantation Table 9.4 (continued) EBMT indications for HSCT for children (recom- mendation from 2010, a dopted and unchanged 2012)1 Disease Disease status Allo Auto Autoimmune disorders GNR GNR GNR CO Germ cell tumor GNR GNR GNR CO Ewing sarcoma High risk or >CR1 D GNR GNR S Soft tissue sarcoma High risk or >CR1 D D GNR CO Neuroblastoma High risk CO GNR GNR S Neuroblastoma >CR1 CO D D S Wilms tumor > CR1 GNR GNR GNR CO Osteogenic sarcoma GNR GNR GNR D Brain tumors GNR GNR GNR CO S = standard of care, generally indicated in suitable patients; CO = clinical option, can b e carried out after careful assessment of risks and benefi ts; D = developmental, further t rials are needed; GNR = generally not recommended. NB This classifi cation does not cover patients for whom a syngeneic donor is available. A syngeneic twin donor is generally considered S for all indications except, by defi nition, congenital disorders. Donor type is divided into: HLA identical sibling donor. Matched unrelated: there is no uniformly accepted defi nition. A \211 10/10 antigen matche d donor is unequivocally considered as matched. Mismatched donor: there is no uniformly accepted defi nition. A < 8/8 antigen matched unrelated and a >1 antigen mismatched family donor is unequivocally considered mismatched. Reference 1. Ljungman, P. et al. (2010). Allogeneic and autologous transplantation for hema tological diseases, solid tumors and immune disorders: current practice in Europe 2009. Bon e Marrow Transplant, 45, 219 \200\22334. 397 INDICATIONS FOR HAEMOPOIETIC SCT 398 CHAPTER 9 Haematopoietic stem cell transplantation Allogeneic SCT Patient and donor selection \200 Recipients should be in good physical condition. Due to the advent of RIC, transplants are being done more and more in older patients with acceptab le toxicity, treatment-related mortality (TRM), and morbidity. Generally \211 70 years old ; \200\230biological \200\231 age is more important than chronological age; the developmen t of reduced-intensity conditioning regimens (RIC) associated with less procedure-related t oxicity, permits an increase in the age-limit for SCT (see Table 9.5). \200 Donor and rec ipient should be fully or closely HLA-matched to reduce the risk of GvHD or graft rejection. Risk of graft failure and GvHD related to degree of donor \200\223recipient HLA-mismatching ; the least mismatched donor should be selected. \200 Greatest chance of full HLA match i s a sibling transplant; each sibling has 71:4 chance of full HLA-match. Matched VUDs may be sought from donor registries (e.g. the Anthony Nolan trust). Haplo-identical sibling or mismatched UD may be considered as a donor for patients in whom no matched sibling or volunteer donor is available and where the i risks of the procedure are acceptable (e.g. poor risk adult AML, Ph-positive adult ALL). Patient and donor CMV status should be considered: with +ve donors preferred for +ve patients and -ve donors preferred for \210\222ve patients. Male donors are preferred for all recipients; VUDs aged \211 40 years preferred to older VUDs; ABO-matched donors preferred for heavily pre-transfused patients. Table 9.5 HLA hierarchy for best choice of donor 1. HLA-identical sibling = phenotypically HLA-matched related donora = HLA-identical related donor umbilical cord bloodb 2. 10/10 molecularly HLA-matched VUD = 6/6 HLA-matched umbilical cord bloodb 3. 9/10 HLA-matched VUD = 5/6 HLA-matched related donor = 4/6 VUD umbilical cord bloodb 4. 8/10 matched VUD = haploidentical related donorc Options 3 and 4 should only be considered in poor risk patients for whom alternative treatm ent is unavailable. Option 4 usually requires in vitro T-cell depletion. Mismatched related donors may be preferred over UDs in certain disorders. a HLA-matched donor where one haplo type is genetypically identical but the other is not. b With nucleated cell dose >3 \227 1 07/kg recipient weight. c Most haploidentical donors for children are parents. Reproduced from Skinner, R. et al. (2007). The transplant. In Cant, A.J. et al. (eds.) Prac tical Haemopoietic Stem Cell Transplantation. Blackwell Publishing, Oxford, pp.23 \200\2234 0, with permission of Blackwell Publishing 2007. 399 ALLOGENEIC SCT Indications for allogeneic SCT See Tables 9.3 and 9.4. Source of stem cells \200 Donors should be allowed to choose between donating BM or PBSCs after non-directive counseling. BM preferred to PB in SCT for non-malignant conditions. Outline of allogeneic SCT procedure \200 Patient receives conditioning therapy with high- dose chemoradiotherapy (traditionally in myeloablative regimens cyclophosphamide (CTX) 120mg/kg + 14Gy fractionated total body irradiation (TBI)) or chemotherapy (e.g. cyclophosphamide 12 0mg/ kg + busulfan 16mg/kg). RIC regimens are increasingly used; moderate doses of chemotherapy and immunosuppression used to achieve engraftment with lower transplant-relate d mortality and morbidity; no TBI used; shifts balance between risks of transplant-related mortality (TRM) and relapse; lower toxicity allows i age range for allogeneic SCT; also all ows SCT in some patients with comorbidities; permits use of adoptive immunotherapy with don or lymphocyte infusion (DLI) for residual disease post-transplant; good results but full-in tensity conditioning remains the treatment of choice in younger patient without co-morbidit ies; RIC discouraged in patients with progressive or refractory disease. In patients receiving UD or haploidentical SCT, alemtuzumab (humanized anti-CD52 monoclonal antibody) is often administered prior to conditioning for T -cell depletion as an immunosuppressant to d the risk of graft rejection. For patients with aplastic anemia less intensive conditioning is used (CTX combined with antithymocyte globulin). Because of sensitivity to alkylating agents in Fanconi anemia, less intensive conditioning is used. In severe combined immunodefi ciency (SCID), it is possible to engraft selected cell lineages without conditioning therapy. 1d after completing conditioning treatment, BM or PBSC are harvested from donor and infused into the patient IV through a central line. After 7 \200\22321d of severe myelosuppression, haematopoietic engraftment occurs. Reverse barrier nursing in a fi ltered air environment, prophylactic anti-infectives (antifungal and aciclovir prophylaxis; consider antibiotic prophylaxis.) re duce the risk of infective complications. Immunosuppression is required to prevent GvHD and graft rejection; generally MTX (in the ea rly engraftment phase) + ciclosporin (for 3 \200\2236 months). 400 CHAPTER 9 Haematopoietic stem cell transplantation Mechanism of cure: evidence for GvL eff ect \200 d risk of relapse in patients with acute and chronic GvHD. \200 i risk of relapse after syngeneic SCT (no GvHD). \200 i risk of relapse after T-lymphocyte-depleted SCT. \200 Delayed clearance of minimal residual dise ase detected post SCT. \200 Induction of remission by DLI after relapse post-SCT. Early complications of allogeneic SCT \200 Overall transplant related mortality for match ed sibling allografts is 75% for VUDs may reach 5-20%. Infection: (severe) myelosuppression together with immune dysfunction from delayed reconstitution or GvHD predisposes to a wide variety of potentiall y fatal infections with bacterial, viral, fungal, and atypical organisms. Both HSV and HZV infections are common \200\224 may present with fulminant extensive lesions. Main causes of infective death post-transplant are: CMV pneumonitis and invasive fungal infections with m olds, e.g. Aspergillus. GvHD: acute GvHD occurs \211 100d of transplant and chronic >100d. \200 Other complicati ons: Endocrine: infertility (both sexes), early menopause, and occasionally hypothyroidism. Cataract (TBI induced) >12 months post-transplant. \200 2 malignancies (esp. skin). \200 EBV-associated lymphoma/PTLD. \200 Psychological disturbances common (serious psych oses rare). Follow-up treatment and post-transplant surveillance Immunosuppression requires careful mon itoring to avoid toxicity. Unlike solid organ transplant recipients, lifelong immunosuppres sion is not required and weaning of ciclosporin is usually started at 3 months post-transpl ant with the aim to discontinue at 6 months. Prophylaxis against pneumococcal sepsis 2 to hyposplenism, CMV reactivation and P. jirovecii infections required. Most patients return to an active, working life without the need for continuing medication. Umbilical cord transplants \200 Umbilical cord blood donation post-delivery is shown to b e safe for mother and child. Cord blood stem cells are immunologically immature and may be more permissive of HLA donor/recipient mismatches. Less risk of GvHD. \200 Successful grafts in children. \200 Due to cell dose required s ingle cord donation often insuffi cient for adult grafts, hence dual cord transplant required with matching between both cords and recipient. Typed 6/6 (due to immunological immaturity). Matching 4/6 between recipient and cords, as well as between 2 cord donations required. Longer time to engraftment, hence i risk of infective complications. \200 Ultimately 1 co rd persists/ \200\231survives \200\231. \200 Due to source of cells no possibility for DL I. 401 ALLOGENEIC SCT 402 CHAPTER 9 Haematopoietic stem cell transplantation Autologous STC Patient selection Patients should be in good physical condition; age range for some pro- ce dures can be extended up to 770 years. BM should be uninvolved or in CR at the time of harv est/mobilization unless disease control rather than cure is the primary intent such as in m ultiple myeloma. Indications E See Tables 9.3 and 9.4, pp.392 \200\223396. Outline of autologous SCT procedure \200 Haematopoietic stem cells harvested are processe d, frozen, and stored in liquid N2. Stem cells are harvested from the PB. SCT may take place within days of harvest or several years later after treatment for recurr ent disease. In some conditions enough stem cells for 2 autografts are collected for possib le relapse planning. Diff erent conditioning chosen for underlying indication, e.g. high-dose melphalan for MM or BEAM for NHL or HL. After completion of conditioning, the stem cell product is thawed rapidly and infused IV (>24h after last chemotherapeutic agent). Bags are thawed by transfe r directly from liquid N2 into a water-bath at 37 \200\22343 C. Product is infused IV rapi dly through indwelling central line. There is a period of myelosuppression (7 \200\22325d) followed by WBC, platelet and RBC engraftment. Early complications of the transplant procedure \200 Overall transplant related mortality is 3 \200\2235%. \200 Morbidity from conditioning regimens: nausea from chemotherapy, mucositis, oral ulceration, buccal desquamation, oesophagitis, gastritis, abdominal pain, a nd diarrhea may all be features. \200 Spectrum of infective organisms seen is similar to allografts but severity and mortality are d. Late complications of autologous SCT \200 Single commonest long-term complication is rela pse of underlying disease. Other late complications similar to allografts, but less frequent and less severe. Higher increase of MDS compared to allograft. Follow-up treatment and post-transplant surveillance \200 Regular hematological follow-up is mandatory and psychological support from the transplant team, family, and friends are important for readjustment to nor mal life. Prophylaxis against specifi c infections required including Pneumococcus, CMV and P. jirovecii. Most patients return to an active, working life without continuing medication. 403 AUTOLOGOUS STC 404 CHAPTER 9 Haematopoietic stem cell transplantation Investigations for BMT/PBSCT Hematology \200 FBC, blood fi lm, reticulocytes, ESR. \200 Serum B12 and red cell folat e, ferritin. \200 Blood group, antibody screen, and DAT. \200 Coagulation screen, PT, A PTT, fi brinogen. \200 BM aspirate for morphology (cytogenetics if relevant). \200 BM t rephine biopsy (not always indicated). Biochemistry \200 U&Es, LFTs. \200 Ca2+, phosphate, random glucose. \200 LDH. \200 Thyroid function tests. \200 Serum and urine Igs. \200 EDTA clearance if renal impairme nt. Virology \200 Hepatitis and HIV serology. \200 CMV IgG, and IgM. \200 EBV, HSV, and V ZV IgG. Immunology \200 Autoantibody screen. \200 HLA type, (if not known) in case HLA matched platelets are subsequently required. HLA and platelet antibody screen (if previously poor increments to platelet transfusions). CRP. Bacteriology \200 Baseline blood cultures (peripheral blood and Hickman line). \200 Rou tine admission swabs: nose, throat, perineum, central line site. \200 MSU, stool cultures if diarrhea. \200 Syphilis serology. \200 Toxoplasma serology. Cardiology \200 ECG. \200 Echocardiogram, to include measurement of systolic ejection f raction. Respiratory \200 Lung function tests. Radiology \200 CXR. \200 Other imaging as indicated Cytogenetics \200 Blood for donor/recipient polymorphisms (allografts only). 405 INVESTIGATIONS FOR BMT/PBSCT Other \200 Consider semen storage/egg harvesting. \200 Dental review. \200 Psychiatri c opinion if previous history of psychological problems. 406 CHAPTER 9 Haematopoietic stem cell transplantation Pretransplant investigation of donors Hematology \200 FBC, reticulocytes, ESR. \200 Blood group, antibody screen, and DAT. \200 Coagulation screen, PT, APTT, fi brinogen. Biochemistry \200 U&Es. \200 LFTs. \200 Thyroid function tests. \200 Urinalysis: di pstick. Virology \200 Hepatitis and HIV serology. \200 CMV IgG. and IgM. \200 EBV, HSV. and V ZV IgG. \200 HTLV-I serology. Immunology \200 Autoantibody screen. \200 CRP. Microbiology \200 Syphilis serology. \200 Toxoplasma serology. Cardiology \200 BP. \200 ECG if indicated. Molecular biology \200 DNA fi ngerprinting for chimerism studies. 407 PRETRANSPLANT INVESTIGATION OF DONORS 408 CHAPTER 9 Haematopoietic stem cell transplantation Bone marrow harvesting Preoperative preparations Elective procedure done under general anesthetic; requires operat or, anesthetist/anaesthesiologist, and available theater. Within 30d before the harvest procedure, arrange the following virological investigations: \200 Hepatitis and HIV serology. \200 HTLV-I serology. \200 Syphilis serology. \200 Spare serum stored. Harvest procedure \200 Check donor FBC and U&E. \200 Cross-match 2 \200\2233U blood (CM V \210\222ve) for donor. If harvest is on healthy donor, off er autologous blood collection to donor. Check consent is in place. \200 Prepare harvest bag adding ACD (acid citrate dextran) wit h a dilution factor of 1:7 \200\22310 for the prospective marrow volume; Ensure the ratio o f marrow to ACD is maintained at 7 \200\22310:1. Attach one 500-micron fi lter, followed by the 200-micron fi lter to the collection bag. Mix 50mL sodium chloride 0.9% with 50,000 units of heparin in a receiver and fl ush several 20mL syringes and the harvest needles with the heparin solution . \200 Donor in prone position. \200 Begin with posterior superior iliac crests, limiti ng the number of skin entry points, the aspirate needle should be maneuvered to collect as much marrow as possible with 5 \200\22310mL maximum from each penetration of the bone. Each aspirate is deposited in the sterile harvest bag and syringe rinsed in the heparinized sal ine prior to re-use. Gently agitate bag at intervals. Midway through harvest (or 500mL) a sample should be sent for FBC to determine the adequacy of the harvest. The aim is to collect between 2.0 and 4.0 \227 108 nucleated cells/kg weight of the recipient. The marrow volume necessary to achieve this is usually between 500 \200\223800mL. The marrow collection should not exceed 1000mL collection (see Box 9.1). 409 BONE MARROW HARVESTING Box 9.1 The volume of marrow required may be calculated as follows: Total volume required for autograft = 2.0 \227 recipient weight (kg)/(collector bag WBC \227 10) e.g. recip. 100kg and the collector bag contains WBC 20 \227109/L then volume req uired: = (2.0 \227100)/(20 \22710) = 1.0L Volume still needed to be harvested = total vol ume \210\222 volume already taken at time of count + 710% Notes 1. The extra 10% compensates for reduced harvesting effi ciency and the ACD. 2. The f ormula works at whatever volume you choose to do the fi rst WBC but is a more accurate prediction at >500mL. 3. If need to harvest >1L, remember to add additional ACD in the same 1 in 7 ratio. 4. For allograft calculations, substitute 3.0 for 2.0 in the formula. If the yield is not adequate from the posterior iliac crests, other sites may be considered (e.g. anterior superior iliac crests and sternum). Review puncture sites the following mor ning for signs of local infection or con- tinuing bleeding. For healthy normal donors, off er out-patient follow-up appointment as additional safeguard and provide access to counseli ng services. 410 CHAPTER 9 Haematopoietic stem cell transplantation Peripheral blood stem cell mobilization and harvesting Properties of stem cells \200 Stem cells are defi ned as the most primitive haemopoietic precursor cell. Unique property is capability of both infi nite self-renewal and diff erentiation to form all mature cells of the haemopoietic and immune systems. In the resting state almost all stem cells reside in the BM although a minority circulate in the peripheral blood. Stem cells in marrow can migrate into the blood after treatment with chemotherapy and/or haemopoietic growth factors. Once circulating, they can easily be harvested using a cell separator machine. Stem cell levels in peripheral blood can be assessed by CD34 immunophenotype analysis. >1d of apheresis may be necessary to achieve required yield. \200 The yield can be assess ed for engraftment potential. Protocols Mobilization and harvesting protocols diff er between diseases. The fol- lowing i llustrate the principal types of schedule: Mobilization after standard chemotherapy \200 G-CSF given at standard time post chemother apy. \200 Harvest times determined by WBC and platelet recovery, and CD34 count. Yields variable. If insuffi cient harvest, further harvest with chemotherapy/growth factor can be done. Suitable for: \200 NHL, e.g. post RCHOP, R-ICE, R-DHAP, Nordic chemotherapy. Mobilization with chemotherapy and haemopoietic growth factors The most common schedule and the best evaluated. Harvest timing and yields more predictable. Typical protocol for NHL \200 Day 0 cyclophosphamide 1.5g/m2 IVI with Mesna. \200 Day + 4 to day +10 G-CSF 5mcg/kg/d SC continued until last day of harvesting. Harvest 7day +10 when CD34 >10 \227 106/L and/or WBC >10 \227 109/L. Typical protocol for myeloma \200 Day 0 cyclophosphamide 4g/m2 IVI with Mesna. \200 Day +1 to day +10 \200\22314, G-CSF 5 \200\22310mcg/kg/d SC. \200 Harvest day +10 to14 when CD34 > 10 \227 106/L and/or WBC >10 \227 109/L. 411 PERIPHERAL BLOOD STEM CELL MOBILIZATION AND HARVESTING Mobilization with haemopoietic growth factor alone \200 Suitable for normal volunteers (a llograft donors). \200 G-CSF 5 \200\22310mcg/kg/d SC for 4 \200\2235d. \200 Harvest d4 \200\2235 (CD34 >10 \227 106/L and/or WBC >10 \227 109/L ideally). Multiple harvest days might be required. Mobilization with plerixafor1,2 \200 Salvage met hod. \200 Immunostimulant used to mobilize haemopoietic stem cells in patients who have not mobilized suffi cient stem cells with standard methods (15 \200\22320 % patients). Suitable for normal volunteers (allograft donors). \200 Approved for use in lymphoma and myeloma patients. \200 Orphan drug status in USA and Europe for the mobilization of haematopoietic stem cells. Plerixafor should be administered approximately 11h prior to initiation of apheresis for up to 4 consecutive days. It is given with preceding G-CSF inje ctions for 4d. Dose: Plerixafor 0.24mg/kg bodyweight SC. The patient \200\231s actual body weight can be u sed. The dose should not exceed 40mg/d. Yield evaluation and target The yield is calculated by the number of CD34+ cells. It is a q uantita- tive assessment of engraftment potential expressed per kg of recipient weight. The daily apheresis and G-CSF should continue until the collec- tion reaches 5 \200\22310 \227 106/kg CD34+ cells. References 1. DiPersio, J. et al. (2009). Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma Blood, 113, 5720 \200\2236. 2. Tanhehco, Y.C. et al. (2013). The evolving role of plerixafor in hematopoietic progenito r cell mobilization. Transfusion, 53(10), 2314 \200\22326. 412 CHAPTER 9 Haematopoietic stem cell transplantation Microbiological screening for stem cell cryopreservation Infective agents, particularly vir uses, can be transmitted through stem cell preparations as through blood products and may c ause signifi cant morbidity and mortality in the recipient. It has been demonstrated that t ransmission of hepatitis B virus has occurred following common storage in a liquid nitrogen tank that contained 1 patient \200\231s hepatitis B surface antigen +ve BM. The following tests should be performed on all patients in whom it is planned to cryopreser ve stem cells \200 Hepatitis B and C serology. \200 HIV Ag and AB testing. \200 HTLV- 1 serology. \200 VDRL = syphilis serology. \200 Additional serum for storage for retros pective analysis. These results must be available to transplant laboratories before cryo- preservation. Since many of these patients will be receiving blood prod- ucts as part of their ongoing treatme nt, they must be performed within 30d of cryopreservation to prevent false \210\222ve anti body tests due to the interval between exposure and seroconversion. In practice these con- straints dictate that samples should be taken between 7 and 30d prior to cryopreservation. Patient samples shown to be \210\222ve for all the above infectious agents should have ste m cells stored in a dedicated liquid nitrogen freezer con- ventionally in the liquid phase. Patient samples shown to be +ve for any of the above agents should be double bagged and sto red in a separate liquid nitrogen freezer in the vapor phase (to reduce transmissibility). Data on all stem cell product samples must be registered in a secure environment on a compu ter- ized database with a logical inventory and retrieval system. No material should be imp orted to the freezers unless a complete negative virolog- ical audit storage trail can be d emonstrated. 413 MICROBIOLOGICAL SCREENING 414 CHAPTER 9 Haematopoietic stem cell transplantation Stem cell transplant conditioning regimens Conditioning is the treatment the patient underg oes immediately prior to a SCT. The purpose is to reduce the burden of residual disease; in allogeneic transplant recipients, it also acts as an immunosuppressant to prevent rejectio n of the graft. Many diff erent protocols using chemo- therapy alone or in combination with TBI have been developed. None has proven to be superior. Fractionation of radiation result s in less tox- icity but the total dose has to be higher to achieve similar immunosup- pres sion. Unrelated transplants might require immunosuppression with anti-T-cell monoclonal ant ibodies or ATG. Reduced intensity regimens can reduce transplant-related mortality and off ers otherwise ineligible patients a potentially curative treatment. Examples See Tables 9.6 and 9.7. Table 9.6 Commonly used transplant conditioning regimens by diseases1,2 Disease Regimen Total dose Days AML and CML Cy/TBI Cyclophosphamide Total body irradiation 120mg/kg 12 \200\22314Gy \210\2226, \210\2225 \210\2223 to \210\2221 Bu/Cy Busulfan Cyclophosphamide 16mg/kg 120mg/kg \210\2227 to \210\2224 \210\2223, \210\2222 ALL Cy/TBI Cyclophosphamide Total body irradiation 120mg/kg 12 \200\22314Gy \210\2226, \210\2225 \210\2223 to \210\2221 TBI/VP Total body irradiation Etoposide 12 \200\22313.2Gy 60mg/kg \210\2227 to \210\2224 \210\2223 Cy/VP-16/TBI Cyclophosphamide Etoposide Total body irradiation 120mg/kg 30 \200\22360 mg/kg 12 \200\22313.2Gy \210\2226, \210\2225 \210\2224 \210\2223 to \210\2221 Severe aplastic anemia Cy/ATG Cyclophosphamide ATG 200mg/kg 90mg/kg \210\2225 to \210\2222 \210\2225 to \210\2223 Thalassaemia Bu/Cy Busulfan Cyclophosphamide 14 \200\22316 mg/kg 200mg/kg \210\2229 to \210\2226 \210\2225 to \210\2222 (continued) 415 STEM CELL TRANSPLANT CONDITIONING REGIMENS Table 9.6 (continued) Commonly used transplant conditioning regi- mens by diseases1,2 Disease Regimen Total dose Days Fanconi anemia Flu/Bu/Cy Busulfan Fludarabine Cyclophosphamide ATG a 6mg/kg 100mg/m2 40mg/kg \210\2229 to \210\2226 \210\2225 to \210\2222 \210\2225 to \210\2222 \210\2224 to \210\2221 140 \200\223200mg/m2 \210\2223, \210\2222 Myeloma High dose melphalan Lymphoma BEAM (LEAM if Lomustin used) Carmustine (BCNU) Etoposide Cytarabine Melphalan 300mg/m2 800mg/m2 800 \200\2231600mg/m2 140mg/m2 \210\2226 \210\2225 to \210\2222 \210\2225 to \210\2222 \210\2221 a ATG dose depends on specifi c brand of ATG. Table 9.7 Examples of reduced intensity conditioning regimens1,2 Regimen Treatment Total dose 90mg/m2 2Gy 150mg/m2 8 \200\22310mg/kg Flu/TBI Fludarabine Total body irradiation Flu/Bu ATG Flu/Bu thiotepa Fludarabine Busulfan ATG a Thiotepa 5mg/m2 150mg/m2 140mg/m2 140 \200\223180mg/m2 150mg/m2 140g/m2 Flu/Mel alemtuzumab Fludarabine Melphalan Alemtuzumab Flu/Cy Fludarabine Cyclophosphamide a ATG dose depends on specifi c brand of ATG. References 1. Aschan, J. (2007). Risk assessment in haematopoietic stem cell transplantatio n: conditioning. Best Practice Res Clin Haematol, 20, 295 \200\223310. 2. Apperley, J. et al. (2012). The EBMT-ESH Handbook on Haemopoietic Stem Cell Transplantation, revised ed. Paris: EBMT. 416 CHAPTER 9 Haematopoietic stem cell transplantation Infusion of cryopreserved stem cells Equipment \200 Dedicated liquid nitrogen freezer con taining stem cells in liquid N2. \200 Water bath heated to 37 \200\22340 C. \200 Tongs . \200 Protective gloves. \200 Patient \200\231s notes. \200 Trolley with: syringes, needles, ampoules of 0.9% saline, blood giving sets, sterile dressing towels and gloves, ch lorhexidine spray, bags of 500mL N/saline. Ensure the patient is consented and con \201 rm identi \201 cation Method 1. Write up the stem cell infusion on the blood product infusion chart. 2. 30min before re-infusion, ensur e water bath is fi lled and heated to 37 \200\22340 C and give (chlorphenamine) 10mg IV an d paracetamol 1g PO. 3. When ready to return the stem cells take the equipment trolley to the patient \200\231s bedside. 4. Check the patient \200\231s vital signs. 5. Set up a standard blood giving set with micr o-aggregate fi lter. Never use additional fi lters. Prime with 500mL 0.9% saline, connect t o the patient and ensure good fl ow before starting to thaw any cells. 6. Check the water b ath is 37 \200\22340 C and using the protective gloves and large tongs remove a bag of cel ls from liquid nitrogen container. Carefully remove from the outer sleeve and place in wate r bath and allow 1min. DMSO (dimethyl sulfoxide) cryopreservative is very toxic to cells on ce thawed so it is important to go straight from rapid thaw to infusion. 7. Remove bag of cells from water bath using the tongs, spray with chlorhexidine and allow it to dry. Check patient identifi cation number and date of birth with the patient and conn ect to the giving set. 8. Cells should be returned as quickly as possible. Each bag con- tains approximately 100 \200\223150mL. Providing the fl ow is good, start thawing the next bag. Only thaw the next bag if you are able to fi nish the previous bag within the next minute. Check the patient \200\231s details on every bag. 9. Check the patient \200\231s observations at 15min intervals. 10. If the patient complain s of abdominal pain, nausea, or feeling faint, slow down the IVI for a short time. If sympt oms persist or patient develops chest tightness or wheezing \200\224stop the infusion. O2 nebulized salbutamol may be required. Anaphylaxis rare. Reinfusion must continue. 417 INFUSION OF CRYOPRESERVED STEM CELLS Special considerations 3 If the bag splits/leaks do not re-infuse \200\224contents will not be sterile. Very rarely, a bag could start to expand rapidly upon thawing if all the air i s not removed from the bag before freezing. A sterile needle may be used to pierce the bag if release of pressure appears essential. 3 Acute anaphylaxis is very rare but epinephrine (adrenaline) (1mL of 1:1000) should be ava ilable in the patient \200\231s room for SC or IM adminis- tration. 3 Central access required for reinfusion. 418 CHAPTER 9 Haematopoietic stem cell transplantation Infusion of fresh non-cryopreserved stem cells Ensure patient is consented and understands procedure. In general, BM will be in a larger volume than an apheresis product. Procedure 1. A medical staff member must be available to start the infusion and stay with the patient for the fi rst 30min. 2. Give paracetamol 1g PO and chlorphenamine 10mg IV 30min prior to start of infusion. 3. Prime blood giving set without an in-line fi lter with 500mL 0.9% saline and connect to the patient \200\224check there is a good fl ow. 4. Check details of patient and donation. 5. Check BP, pulse, and chest auscultation before the infusion Check observations regularly (every 15 \200\22330min). Patient on saturation monitor. 6. Cells are given fresh (not frozen). Give stem cells as slowly as pos- sible for the fi r st 15min, then increase the rate to 100mL in 60min. If after 2h the patient is tolerating i nfusion without problems, increase to 200mL/h until completion. 7. Watch for fl uid overload \200\224give diuretic if necessary. Complications of stem cell infusion \200 Microemboli occasionally cause dyspnoea and cyan osis. O2 should be available. Slow down or stop the stem cell infusion if dyspnoea. Pyrexia, rash, and rigors can occur \200\224treat with further hydrocortisone 100mg IV and chlorphenamine 10mg IV. More frequently seen in blood group mis matched transplant.