Autologous umbilical cord blood transfusion

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					Archives of Disease in Childhood 1995; 73: F181-F183                                                                            F181

                               Autologous umbilical cord blood transfusion
                               A Ballin, E Arbel, G Kenet, M Berar, D Kohelet, A Tanay, H Zakut, D Meytes

                               Abstract                                             been published elsewhere.3 The salient
                               The purpose of this study was to examine             features of the preliminary work done before the
                               some aspects of umbilical cord blood                 autologous umbilical cord blood transfusion
                               collection for autologous transfusion in             (AUCBT) was performed are summarised
                               premature infants. All 120 microbacterial            briefly below.
                               cultures (aerobic and anaerobic) of cord                The collection of umbilical cord blood was
                               blood samples as well as 30 cultures of              initiated immediately following delivery. After
                               mycoplasma were treated. Cord pro-                   clamping the cord a distal portion of the cord
                               thrombin fragment (F 1+2) concentra-                was cleansed with betadine and alcohol 70%
                               tions were quantified at one and 10                  for five seconds (the same procedure was per-
                               minutes after clamping of the cord. F 1+2           formed following caesarean sections). A 21
                               concentrations assessed on 25 newborn               gauge needle of a quadruple blood collection
                               infants were similar and no linear associa-          set (Travenol Co) was introduced into the
                               tion with time of clamping could be                 umbilical vein. The collecting bag contained
                               drawn. This means that cord blood                    CPDA-1 in the recommended ratio of about
                               thrombosis is not activated for at least 10          1:7 with blood. Cord blood filled the bag by
                               minutes following clamping of the cord.             gravity and by uterine contractions.
                               As far as is known, the first newborn                   Sterility was tested by culturing the blood in
                               infant to benefit from this method of               a pair of Bactec NR 730 bottles (aerobic and
                               transfusion is reported here. The prema-            anaerobic), incubating it for seven days at
                               ture infant received two portions of autol-         35°C, and checking it daily.4 Other samples
                               ogous blood (on days 5 and 7). No                   were cultured for genital mycoplasma
                               untoward effects were noted.                         (Mycoplasma homonis and Ureaplasma
                                 Blood, collected from the umbilical               urealyticum). To investigate the coagulation
                               cord, is a safe source for autotransfusion,         profile of the frozen plasma, fresh cord plasma
                               provided that bacteriological testing has           and plasma stored at -29°C for 45 days were
                               been carried out.                                   tested. Prothrombin time (PT), partial throm-
                               (Arch Dis Child 1995; 73: F181-F183)                boplastin time (PTT), thrombin time (TT),
                                                                                   fibrinogen, and factors V, VII, VIII, IX, X, XI,
                               Keywords: anaemia of prematurity, cord blood,       XII, and XIII were determined (reagents were
                               autologous transfusion.                             from Sigma Chemical Co, USA). All tests
                                                                                   were performed using the automated coagula-
                                                                                   tion timer (Electra 800 USA) and Dade
                               Studies have shown that very low birthweight        (Switzerland). The coagulation screening tests
                               infants receive about 50 ml of blood per kg         (PT, PTT, and TT) were performed using
                               from as many as eight to 10 adult donors            routine microtechniques.5
                               during the first month of life.1 The use of adult      Factor assays were based on mixing known
                               donor blood poses risks for these infants,          human deficient plasma with 10 Rl to 30 ,ul of
                               including transmission of viral agents such as      each infant's cord plasma. Von Willebrand
                               hepatitis B, cytomegalovirus, and HIV, as well      factor and plasminogen activities were also
The Paediatric                 as graft versus host disease and other compli-      determined.6 To verify that thrombin activity
Haematology Unit               cations.' 2 Placental vessels contain a quarter     had not been generated following clamping,
A Ballin
                               to a third of the newborn blood volume - blood      concentrations of prothrombin fragments (F
The Departments of             which is currently discarded. Following pre-        1+2) were determined as follows: at one and
Gynecology                     liminary studies on collection and storage of       10 minutes after clamping a 21 gauge needle
G Kenet
M Berar                        cord blood,1 3 we have further expanded the         was inserted into the umbilical vein and a 2 ml
H Zakut                        experiments on sterility of the collected blood     blood sample collected. The syringe contained
                               and its coagulation system.                         0-4 ml anticoagulant solution consisting of 38
Clinical Immunology
A Tanay                           To examine the possibility of thrombin           mmol/l citric acid, 75 mmolIl sodium citrate,
                               activation caused by clamping of the cord, we       136 mmolIl dextrose, 6 mmol/l EDTA, 6
Newborn Infants                quantified the concentration of prothrombin         mmol/l adenosine and 25 ,u/ml heparin.7 The
E Arbel                        fragments (F 1+2) one and 10 minutes after
D Kohelet                                                                          ratio of anticoagulant to blood was 0 2: 10
                               clamping the cord. We report the first prema-       (vol/vol). The blood was centrifuged and the
Haematology                    ture infant who benefited from cord blood           plasma frozen at - 20°C until batch analysis of
D Meytes                       transfusion following storage of the blood.         the specimens. Concentrations of prothrombin
Edith Wolfson Medical                                                              F 1 + 2 were determined with the ELISA
Center, Holon, Israel                                                              Thrombonostica F 1+2 (Organon, Teknika
Correspondence to:             Methods                                             Corporation) using a spectrophotometer
Dr Ami Ballin, The Institute   All the studies were carried out in the Edith (Molecular devices Co) at 450 nm.
of Haematology, Edith
Wolfson Medical Center,        Wolfson Medical Center, Holon, Israel, with       Comparison of the results of the coagulation
Holon 58100, Israel.           the approval of the institutional committee of tests and of the cord blood and adult F 1+2 con-
Accepted 8 August 1995         ethics. Some of the preliminary results have centrations was determined using Student's t test.
F182                                                               Ballin, Arbel, Kenet, Berar, Kohelet, Tanay, Zakut, Meytes

       Results                                                       a     1.6
       The following results are based on 120                        CN4
                                                                     +     1.4
       collections: the mean volume of the cord blood
       obtained was 86 ml (range 62-105) from full                   en 1.2
       term and 44 ml (range 31-58) from premature                   CD
                                                                           1 .0
       infants (31-34 weeks' gestation).                                   0.8
          All microbacterial cultures (aerobic and                   m 0.6
       anaerobic) were negative, as were all 30
       cultures examined for the presence of                         : 0.4
       mycoplasma.                                                   E
                                                                     o 042
          All the blood coagulation results, performed               0
                                                                     -       0
       on 10 samples either before or after storage                 X             1 2 3 4 5 6 7 8 9 10                     15
       were within normal limits for this age group                                    Minutes after clamping
       (data not shown). There were no significant                  Figure 2 F 1+2 concentrations at one and 10 minutes
       differences in all the paired variables tested.              after clamping.
       The mean (SD) concentrations of Von
       Willebrand factor (n=26) and plasminogen                     mechanically ventilated. On day 5, the haemat-
       (n=26) were 131-8 (32-9)% and 57 (10-7)%,                    ocrit dropped to 35% and the haemoglobin to
       respectively - that is in the normal range for               1 7 g/l. The collected umbilical blood was com-
       this age group.                                              patible with the mother's serum, and the cul-
          Thirty three samples of cord blood were                   tures were negative. Following approval by
       examined for F 1+2 concentrations. The                       the committee of ethics and the mother's
       samples were obtained from 25 cords of full                  written consent having been obtained, 10 ml of
       term infants (14 boys and 11 girls) whose birth-             the packed cells were transfused into the infant.
       weights ranged from 3030 to 3970 g. Seventeen                No untoward effects were observed. Blood gas
       samples were collected at varying times after                analysis, as well as concentrations of sodium,
       clamping while another eight sample pairs were               potassium, calcium and glucose were all within
       withdrawn at one and 10 minutes after clamp-                 normal limits. The packed cell volume (PCV)
       ing. The results of F 1+2 determinations are                 increased to 42%. Two days later, the PCV
       shown in fig 1. The mean (SD) cord blood F                   dropped to 37%, so an additional 10 ml of
       1 +2 concentration was 0 53 (0 35) nM. The F                 packed cells were transfused. Again, vital signs,
       1+2 concentrations at one and 10 minutes after               blood gas analygis, and all biochemical tests
       clamping were statistically similar, and no linear           were normal. Two days later, the patient was
       association with time of clamping could be                   extubated. At the time of writing, the infant was
       drawn (r=0.29) (fig 2). All these results encour-            3 months old and healthy.
       aged us to apply AUCBT in the infant reported
                                                                   Premature infants are frequently subjected to
       CASE REPORT                                                 blood transfusions. Most premature infants
       The third in a set of triplets born by caesarean            born at less than 32 weeks of gestational age will
       section at 31 weeks' gestation, the baby                    receive more than two transfusions after 2 weeks
       weighed 1250 g. Following delivery, the pla-                of age.' Serious medical complications may
       centa was placed on a sterile surface and cord              accompany the transfusions. They include
       blood was collected from the umbilical vein, as             infections such as AIDS,8 cytomegalovirus,9
       described. Thirty five millilitres of venous                and hepatitis'0; sensitisation to plasma, red cell,
       blood were collected within two minutes. A                  or HIA antigensI 1; errors in blood group or
       sample of the blood was sent for bacteriological            patient identification and graft-versus-host
       testing; the remainder was stored in the blood              disease.12 Placental vessels contain 75 to 125 ml
       bank in three separate bags.                                of blood at birth or nearly one quarter to one
          Subsequently, the infant, whose haematocrit              third of the fetal blood volume.'3 This study
       and haemoglobin concentrations at delivery had              shows that the fetal blood left in the
       been 46% and 153 g/l, respectively,                         placental vessels may serve as a source of
       developed hyaline membrane disease and was                  blood for autotransfusion. Horn et al found that
                                                                   cord blood cells can be stored in a CPDA-1
       2 2.5                                                       medium for at least 35 days. 14 Bifano et al exam-
                                                                   ined the feasibility of collecting and storing
        +                                                          placental blood.' The authors concluded that
       _- 2
                                                                   placental blood can be used for autologous
                                                                   transfusion for the sick neonate. Apart from
        E 15                                                       being 'self donation, thus avoiding the above
                                                                   mentioned homologous transfusion associated
        C 1-0                            .                         complications, AUCBT has other advantages:
                                                                   (a) immediate availability; (b) high levels of
        E 0-5*               .U                                    haematopoietic progenitor cells, such as colony
                                                                   forming units - granulocyte macrophage
       &        1 2 345 67 89 10                           15 16   (CFUJ-GM), as well as high levels of granulo-
                        Minutes after clamping                     cyte machrophage-colony stimulating factor
       Figure 1 F 1+2 concentrations in 33 samples of cord         (GM-CSF) and granulocyte-colony stimulating
       blood.                                                      factor (G-GSF7). It has already been shown that
Autologous umbilical cord blood transfusion                                                                                                            F183

                               cord blood stem and progenitor cells, may                       1 Bifano EM, Dracker RA, Lorah K, Palit A. Collection and
                                                                                                    28-day storage of human placental blood. Pediatr Res
                               reconstitute marrow haematopoiesis.15-19                             1994; 36: 90-4.
                               Umbilical cord blood, enriched with self-                      2 Camielli V, Montini G, Da Riol R, Dall'Amico R,
                                                                                                    Cantarruti F. Effect of high dose of human recombinant
                               haematopoietic growth factors and progenitor                        erythropoietin on the need for blood transfusions in
                               cells, may be beneficial to premature infants                       preterm infants.JPediatr 1992; 121: 98-102.
                                                                                              3 Ballin A, Kenet G, Gutman R, Samra Z, Zakut H,
                               who frequently suffer from leucopenia and                           Meytes D. Autologous cord blood transfusion (ACBT).
                               thrombocytopenia caused by lack of marrow                           Acta Paediatrica 1994; 83: 700-3.
                                                                                              4 Reller LB, Murray PR, MacLowry JO. Blood cultures II.
                               reserves.                                                           Cumitech 1A. Washington, DC: ASM, 1982.
                                  Our study has shown that umbilical cord                     5 Johnson M, Zipursky A. Microtechnology for the study of
                                                                                                   the blood coagulation system in newbom infants. Can Jf
                               blood can be withdrawn without bacterial con-                       Med Technol 1980; 42: 159.
                               tamination. The coagulation assays performed                   6 Andrew M, Paes B, Milner R, Johnston M, Mitchell L,
                                                                                                   Tollefsen DM, et al. Development of the human coagula-
                               in this study have proved the efficacy of stored                    tion system in the full-term infant. Blood 1987; 70:
                               cord plasma as a source of coagulation factors.                      165-77.
                                                                                              7 Bauer KA, Brockmans AW, Bertina RM, Conard J,
                               We have also demonstrated that the clotting                         Horrelon M-H, Samama MM, et al. Hemostatic enzyme
                               system is not significantly activated for at least                  generation in the blood of patients with hereditary protein
                                                                                                   C deficiency. Blood 1988; 71: 1418-26.
                               10 minutes after clamping.                                     8 Nicholas SW, Sondheimer DL, Willoughby AD, Yaffe SJ,
                                  The premature infant described here is the                       Katz SL. Human immunodeficiency virus infection in
                                                                                                   childhood, adolescence and pregnancy: A status report
                               first (as far as is known) reported newborn to                      and national research agenda. Pediatrics 1989; 83:
                               benefit from AUCBT following storage of the                         293-308.
                                                                                              9 Kin HC. Red blood cell transfusion in the neonate. Semin
                               blood. The collected umbilical cord blood                           Perinatol 1983; 7: 159-74.
                               served for autologous transfusion twice. No side              10 Esteban JL, Gonzalez A, Hemandez JM, Viladomiu L,
                                                                                                   Sanchez C, Lopez-Talavera JC, et al. Evaluation
                               effects were seen. The idea of using placental                      of antibodies to hepatitis C virus in a study of trans-
                               blood for transfusion was initially proposed by                     fusion-associated hepatitis. N Engl J Med 1990; 323:
                               Halbrecht.20 He reported 220 cord blood trans-                11 Class FMJ, Lagaay EL, Van Rood JJ. Immunological conse-
                               fusions (obtained from 520 placentas) given to                      quences of blood transfusion. Schweiz Med Wochenschr
                                                                                                   1991; 121 (suppl 43): 70.
                               anaemic children and adults. In recent years                  12 Anderson KC, Weinstein HJ. Transfusion-associated-
                               there has been renewed interest in the use of                       graft-versus-host disease. N Engl J Med 1990; 323:
                               cord blood as a source of stem and progenitor                 13 Oski FA. Normal blood values in the newborn period. In:
                               cells for marrow reconstitution.9                                   Oski FA, Naiman JL, eds. Hematological problems in the
                                                                                                   newborn. Philadelphia: WB Saunders Co, 1988: 8.
                                  Recombinant human erythropoietin (EPO)                     14 Horn S, Mazor D, Zmora E, et al. Storage induced changes
                               administration to premature infants is a rela-                      in human newborn erythrocytes. Transfusion 1982; 27:
                               tively new strategy to reduce the risks of blood              15 Laver J, Duncan E, Abboud M, Gasparetto C, Sahdev I,
                               transfusion in nurseries.21 This mode of treat-                     Warren D, et al. High levels of granulocyte and granulo-
                                                                                                   cyte-macrophage-colony-stimulating factors in cord blood
                               ment has some disadvantages and side effects.                       of normal full-term neonates. J Pediatr 1990; 116:
                               Treatment with EPO, given subcutaneously,                           627-32.
                                                                                             16 Broxmeyer HE, Douglas GW, Hanjoc G, Cooper S, Bard J,
                               may increase the risk of infections, it depletes                    English D, et al. Human umbilical cord blood as a poten-
                               iron stores, the infants gain less weight than                      tial course of transplantable hematopoietic stem/progeni-
                                                                                                   tor cells. Proc NatlAcad Sci USA 1989; 86: 3828-32.
                               their controls and it is expensive.                           17 Gluckman E, Broxmeyer HE, Auerbach AD, Friedman HS,
                                  We believe that blood, collected from the                        Douglas GW, Devergie A, et al. Hematopoietic reconsti-
                                                                                                   tution in a patient with Fanconi's anemia by means of
                               umbilical cord, is a safe source for auto-                          umbilical cord blood from an HLA-identical sibling. N
                               transfusion, provided that bacteriological test-                   EnglJ3Med 1989; 321: 1174-8.
                                                                                             18 Broxmeyer HE, Kurtzberg J, Gluckman E, Auerbach AD,
                               ing has been carried out. Furthermore,                              Douglas GW, Cooper S, et al. Umbilical cord blood
                               any autologous transfusion programme should                        hematopoietic stem and repopulating cells in human
                                                                                                   clinical transplantation. Blood Cells 1991; 17: 313.
                               incorporate secure methods and safeguards in                  19 Mayanai H, Landsdorp PM. Thy-i expression is linked to
                               identification for collecting, storing, and dis-                    functional properties of primitive hematopoietic progeni-
                                                                                                   tor cells from human umbilical cord blood. Blood 1994;
                               pensing the harvested blood. More clinical                          83: 2410-7.
                               experience is warranted to consolidate all these              20 Halbrecht J. Fresh and stored placental blood. Lancet 1939;
                                                                                                  ii: 1263-8.
                               concepts.                                                     21 Maler RF, Obladen M, Scigalla P, Lindercamp 0, Duc G,
                                                                                                  Hieronimi G, et al. The effect of Epoetin beta (recombi-
                               We thank Dr S Bar-Shany and Dr E Shinar from the Israeli           nant human erythropoietin) on the need for transfusion in
                               Blood Bank for their professional assistance and Mrs D Ben-        very low-birth-weight infants. N Engl J Med 1994; 330:
                               Porath for her cooperation.                                         1173-8.

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