JOURNAL OF HEMATOTHERAPY 5:145-148 (1996)Mary Ann Liebert, Inc.

Cord Blood Stem Cells: How to Get Them and What to Dowith Them

ROBERT A. DRACKER

ABSTRACT

This article reviews the means of obtaining cells from the available reservoirs of cord blood, in-tended as sources of immature hematopoietic stem cells that ultimately could be useful for trans-plantation, gene therapy, and research. Various issues must be considered when collecting umbili-cal cord blood regardless of the method employed. One must regard the basic fetal-placentalphysiology and hemodynamic characteristics prior to and at the time of procurement. Additionalconcerns exist with the mother, not only at the time of collection but also prenatally, including in-formed consent, health history, and psychosocial issues. Collection methods may be characterizedas either ex utero or in utero, employing either open or closed collections methods. Each of thesevariables presents limitations and offers specific advantages over the others. Once collected, the cellsmust be appropriately tested, processed, and prepared for cryopreservation if not used immediately,using good manufacturing practices and acceptable standards of operation. An ideal collectionmethod has yet to be defined that fulfills the need for reliability, reproducibility, and ease of use.

INTRODUCTION

Prior to birth, the circulation of the fetus is con-

tinuous within the fetal-placental unit via the umbili-cal cord. The placenta, serving as the prenatal site ofgaseous, substrate, and waste exchange with the support-ing maternal circulation, represents a significant reservoirof fetal blood up until the time of birth. Fetal blood, cir-culating throughout the cord and placenta, has also beenrecognized as a significant source of hematopoietic stemcells, existing in numbers far in excess of those found inthe postnatal circulation (1,2). Previously, the residualblood in the detached cord and placenta was routinely dis-carded following birth. Cord blood (CB), however, hasbeen routinely sampled for newborn testing purposes andoccasionally collected, using a syringe technique, for ad-ministration to a moribund newborn (3-5). More recently,efforts have been made to collect and preserve the blood

for autologous transfusion, particularly in the preterm in-fant commonly in need of multiple small-volume transfu-sions (6).

To effectively collect sufficient volumes of CB, in-tended as a source of transplantable stem cells, the nor-mal blood volume distribution within the fetal-placentalcirculation and the hemodynamic changes that occur atthe time of birth must be appreciated.

FETAL-PLACENTAL HEMODYNAMICSAND PHYSIOLOGY

As reviewed earlier, the fetal circulation at the time ofbirth consists of the fetus or delivered newborn, the um-bilical cord, and the fetal side of the placenta. The gelati-nous or spongy texture of the placenta is primarily re-lated to its vascular structure, representing its sinusoidal

Biocyte Corporation and Departments of Pediatrics and Pathology, SUNY Health Science Center at Syracuse, Syracuse, NY13210.

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blood flow. In the term infant, one third to one half ofthe blood within the fetal circulation is contained on thefetal side of the placenta. For example, in the average 3.5kg newborn having an estimated blood volume of 300ml, the placental blood volume would range between 100and 150 ml. However, this does not include the residualvolume within the umbilical cord and does not take intoaccount other hemodynamic characteristics of fetal-pla-cental blood flow occurring at the time of birth. In a terminfant, the blood volume may vary significantly, from 50to 100 ml/kg, with a mean value of 85 ml/kg (7). Thepremature infant generally has an increased blood vol-ume, ranging from 85 to 105 ml/kg, primarily due to an

increased plasma volume (8). The time of cord clampingafter birth has a marked effect on the newborn blood vol-ume. Late cord clamping, generally occurring more than1 min after birth, results in an average blood volume of98-100 ml/kg as compared with 78-80 ml/kg found af-ter early cord clamping (9). Obviously, the increasedblood volume of the infant associated with late clampingresults in a decreased placental volume.

Within the first 15 sec of childbirth, the placental trans-fusion process occurs, whereby the umbilical arteriesconstrict, limiting fetal to maternal flow. The umbilicalvein remains dilated, permitting continued, gravity-basedfetal blood flow. At birth, the infant to placental blooddistribution is approximately 2:1, as compared with 4:1at 1 min of age and 6.7:1 at 2 min of age (10). Consideringthe dynamic nature of placental and fetal blood flow atthe time of birth, it follows that the effectiveness of thecollection technique depends on the method employedand the timing of the procedure.

THE COLLECTION PROCEDURE

Although reviewed elsewhere, a number of issues mustbe considered before the actual collection of CB. Foremostis the informed consent procedure, whereby all aspects ofthe testing, collection, processing, storage, and potentialultimate uses of the stem cell source should be discussedwith the parents of the unborn child. Performance of a CBcollection at the time of birth now modifies the experienceso that there is a two-part birth process in the parent's mind.The delivery and condition of the placenta following thebirth of the child now takes on new significance, havingthe associated hopes for a successful collection of a val-ued commodity, the CB. Failed collections are associatedwith significant disappointment in some parents, requiringthose involved with the collection procedure to providesatisfactory explanations and information in a timely man-ner. Associated with this procedure, the prenatal history ofthe mother must be obtained, as well as maternal testingfor infectious disease markers. Prenatal issues that mayhave an impact on the successful collection of sufficient

CB samples include maternal medications, prenatal andperinatal infections, hypertension, poor fetal growth,abruption, and abnormal placentation.

When preparing to collect CB, one must consider a va-

riety of issues: how to perform the collection, when tostart the collection and how long to do so, what maneu-vers are going to be used to minimize the risks of cont-amination, how to maximize the collection volumes, andhow effective anticoagulation can be maintained for ablood product that will vary unpredictably in volume. Thecollection procedure should also include provisions forthe inspection of the placenta and the cord, which willassist with potential modifications in the collection pro-cedure or suggest reasons for failed or difficult collec-tions. With regard to the placenta, attention should bepaid to the presence of clots or fragments, calcifications,or lacerations sustained during the birthing procedure.

In defining the collection method, it can be either openor closed in nature, specifically referring to the collec-tion of free flowing blood from a transected area of thecord in the open method, or the cannulation of the um-bilical vein with either a needle or catheter-type device,representing a more closed approach. Although the openmethod was initially the one predominantly employed,most collections currently use some variation of a closedmethod. Intrinsic problems associated with an openmethod include the release of tissue factors and collec-tion of free cord fragments, difficulty in the preparationand maintenance of sterility near the end of the collec-tion, and difficulty in preventing fluids on the outsideof the cord from contaminating the collected CB.Additionally, the draining blood is collected into an openreceptable of some type, exposed to the environment. Inperforming a closed collection, the venipuncture deviceis typically connected to a segment of tubing leading intoa contiguous receiving bag. The entire device, includingthe venipuncture device, tubing, and bag, is sterilized be-fore collection. Most closed collections rely on gravitydrainage of the placental and cord blood, although otherapproaches, including both positive placental pressureand negative bag pressure, have been investigated withinitial success. An additional advantage of the venipunc-ture approach is that it affords the opportunity to iden-tify the venipuncture site and to perform adequate sitepreparation with antiseptics, such as providone-iodine oralcohol.

Regardless of the collection method used, the protocolshould delineate the duration of the collection procedureand the form and amount of anticoagulant. Generallyspeaking, the open collection method takes longer thanthe closed method, potentially increasing the risk of con-tamination and clot formation. Collections should beginas soon as possible following separation of the infantfrom the placenta. An upper limit for the collection timeshould be determined through experience and evaluation

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of data associated with the protocol employed. The most

widely used anticoagulant at the present time is citratephosphate dextrose (CPD), a commonly used wholeblood anticoagulant. Ideally, a blood/CPD ratio of near

10:1.4 should be maintained, although practically itshould not exceed 12:1 (11). Acid citrate dextrose (ACD)solution has also been shown to be an effective antico-agulant for CB samples. Heparin can be used as a CB an-

ticoagulant, having the advantages of a much broaderblood/heparin range necessary to ensure adequate anti-coagulation. This advantage may be significant when one

considers the potentially wide variation in the final CBvolumes, which may be as little as 30 ml to excess of200 ml. Obviously, provisions and procedures must be de-fined to allow the addition of more CPD as the collectedCB volume increases. Data, although limited, have so fardemonstrated no clear superiority of either citrate-based or

heparin anticoagulation with respect to the short-term,room temperature stability of CB stem cells (12,13).

Finally, there should be flexibility with regard to whenthe collection procedure is performed, characterized as

either in utero, or before placental delivery, versus ex

utero, or after placental delivery. Although some anec-

dotal studies report increased collection volumes for inutero collections, others suggest that there are no signif-icant differences. The belief that uterine contractions fa-cilitate placental blood collection through a squeezing or

compression effect is not valid, in that uterine contrac-tions following childbirth contribute mainly to placentaldetachment and subsequent expulsion. The more likelyexplanation for reports of increased blood volume col-lections with in utero placentas is probably the shortertime period from newborn detachment to start of the col-lection, avoiding CB clotting, which progresses withtime. Obviously, the in utero collection method is more

convenient for the physician or midwife performing a col-lection. However, the collection is being performedwithin a semisterile field and can be considered to be in-vasive, since the placenta is still within the uterus of thewoman. The ex utero collection offers the advantages ofbeing noninvasive and free of risks to either the motheror child, allows for collection of the CB in a defined, pre-pared space, and permits collection to be performed bypersonnel other than a physician or midwife. The obvi-ous disadvantage, however, is that there may be signifi-cant delay in collection of the sample following deliveryof the placenta, ultimately affecting the final collectionvolume.

PREPROCESSING CONCERNS

Once the CB is collected, proper identification of theunit using at least two identifiers must be performed.Additionally, details of the collection, including blood

volume, collection time, and any variation from proto-col, must be recorded. Obviously, the parents should beinformed that the collection was either successful and towhat extent or that the collection was insufficient. Detailsof a failed or insufficient collection should be made avail-able to both the parents and the health care provider in-volved at the time of birth. This information may requirethe assistance of the medical staff from the collection ser-

vice to ensure satisfactory answers to all questions thatmay arise.

Besides the CB sample intended for processing and ul-timate storage, additional sample aliquots of blood mustbe obtained for testing and to keep for future access.Minimal testing of the CB sample should include cellcounts, mononuclear cell enumeration, viability determi-nation, and blood cultures. Additional samples for cellmarker studies, colony-forming unit assays, and bloodand HLA typing may or may not be obtained dependingon the protocols and requirements related to the intendeduse of the product. Once collected, the labeled productshould be contained in such a way that it will sustain han-dling or shipping. If shipped, parameters for the mainte-nance of temperature range and stability and the time in-terval between collection and when it will be processedmust be established. If the product is shipped to a pro-cessing laboratory, blood safety and security must be as-

sured by selection of a reliable means of transportation.

DISCUSSION

A final concern for the collection of CB is to establisha method that demonstrates ease of use while being bothreliable and reproducible, regardless of who is performingthe collection procedure. Such an ideal collection systemshould also ensure the maintenance of product integrity,even during transportation and shipping. As we have ex-

perienced in other forms of hematopoietic transplantation,a great deal of variation in methods has existed both inprocurement and processing of the stem cell products andin their methods of use. CB stem cell transplantation, rep-resenting a new and rapidly developing field of hema-totherapy, provides us with the opportunity to develop con-

sensus and excellence in an emerging health care

technology that will benefit many individuals worldwide.

REFERENCES

1. Broxmeyer, H.E., Douglas, G.W., Hangoc, G., et al. Humanumbilical cord blood as a potential source of transplantablehematopoietic stem/progenitor cells. Proc. Nati. Acad. Sei.USA 86:3828, 1989.

2. Dracker, R. Hematopoietic stem cells, form—method—characteristics. Immunol. Invest. 24:443, 1995.

3. Bloom, R.S. Delivery room resuscitation of the newborn.

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In Neonatal-Perinatal Medicine, eds. Fanaroff, A.A. &Martin, R.J. C.V. Mosby Co., St. Louis, MO, 1992, p. 301.

4. Paxson, C.L. Collection and use of autologous fetal blood.Am. J. Obstet. Gynecol. 134:708, 1979.

5. Golden, S.M., O'Brien, W.F., Lissner, C, et al. Héma-tologie and bactériologie assessment of autologouscord blood for neonatal transfusions. J. Pediatr. 97:810,1980.

6. Bifano, E., Dracker, R., Lorah, K., et al. Collection and 28day storage of human placental blood. Pediatr. Res. 36:90,1994.

7. Usher, R., Shepard, M., Lind, J., et al. The blood volumeof the newborn infant and placental transfusion. ActaPaediatr. 52:497, 1963.

8. Usher, R. & Lind, J. Blood volume of the newborn pre-mature infant. Acta Paediatr. Scand. 54:419, 1965.

9. Günther, M. The transfer of blood between baby and pla-centa in the minutes after birth. Lancet 1:1277, 1957.

10. Yao, A.C., Lind, J., Tiisala, R., & Michelsson, K. Placental

transfusion in the premature infant with observation on

clinical course and outcome. Acta Paediatr. Scand. 58:561,1969.

11. Beutler, E. Preservation of liquid red cells. In: Principlesof Transfusion Medicine, eds. Rossi, E., Simon, T. & Moss,G. Williams & Wilkins, Baltimore, MD, 1991, p. 47.

12. Winkelstein, A., Rybka., W., Roscoe, R., et al. Effects ofanticoagulants on survival of cord blood stem cells. 46thAnnual Meeting of the AABB, October 1993.

13. Rybka, W., Hamilton, P., Roscoe, R., et al. Comparison ofshort-term storage procedures for umbilical cord bloodstem cells. International Society of Blood Transfusion,Amsterdam, July 1994.

Address reprint requests to:Robert A. Dracker, M.D., M.H.H.

6846 Buckley RoadNorth Syracuse, NY 13212

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