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Hematologic and bacteriologic assessment of autologous cord blood for neonatal transfusions
8 10
Brief clinical and laboratory observations
The Journal of Pediatrics November 1~)80
Brief clinical and laboratory observations Hematologic and bacteriologic assessment of autologous cord blood for neonatal transfusions Stephen M. Golden, Commander (MC) USNR,* William F. O'Brien, Lieutenant Commander (MC) USN, Christopher Lissner, Lieutenant Commander (MSC), USN, Robert C. Cefalo, Captain (MC), USN, W. Patrick Monaghan, Lieutenant Commander (MSC) USN, Harold Schumacher, Captain (MC), USN, and Stanford Stass, Lieutenant Commander (MC) USNR, Bethesda, Md.
RESUSCITATION of the newborn infant often requires rapid expansion of the circulatory volume, which is usually accomplished through the use of banked blood or blood components. The use of an infant's autologous cord blood has been recommended for this purpose. ~,~ Autologous cord blood is readily available in the delivery room and does not require the time delay inherent in the use of banked blood. Since ACB is identical to the infant's blood, immunologic reactions and osmolar toxicity are avoided. ACB provides oxygen transport and buffering capacities unavailable in blood substitutes. This report provides bacteriologic and hematologic background for the safety and efficacy of ACB as a method of neonatal volume resuscitation. MATERIALS
AND METHODS
All ACB specimens were obtained from infants delivered at term. Pregnancies were uncomplicated, all deliveries were vaginal, and mothers were afebrile and had received neither antibiotics nor anticoagulants. Following delivery, umbilical cords were clamped and severed in the usual fashion. Bacteriologic studies. The umbilical cord was sprayed with 20% povidone-iodine solution. A sterile syringe with an 18-gauge needle was used to withdraw ACB from the From the Departments of Pediatrics, Obstetrics and Gynecology, and Laboratory Medicine, National Naval Medical Center. Supported in part by CIP No. 0-06-1337, Natiot[al Naval Medical Center. *Reprint address: Department of Pediatrics, Uniformed Services University, School of Medicine, 4301 Jones Bridge Rat., Bethesda, MD 20014.
umbilical vein. Following blood collection, a second sterile needle was placed on the syringe and 5 ml aliquots were immediately inoculated into radiometric aerobic and anaerobic blood culture flasks (BACTEC, Johnston Laboratories, Cockeysville, Md.). After a 72-hour incubation period, all cultures were blindly subcultured aerobically and anaerobically, using chocolate and brucella blood agar, respectively. All cultures suspected of growth by radiometric indices were Gram stained and subcultured. Isolated organisms were identified via standard techniques. See related article, p. 806.
Abbreviations used ACB: autologous cord blood CPD: citrate phosphate dextrose
]
Hematologic studies. Prothrombin, partial thromboplastin time, and thrombin time were obtained on separately drawn specimens of ACB. All coagulation indices were measured by electronically timed changes in optical density. Twenty milliliter aliquots were chosen to determine the efficacy and requirements of heparin anticoagulation. Three heparin dosages were utilized. Method 1 utilized a heparin rinse with 1,000 U/ml, leaving heparin solution only in the hub of the syringe prior to withdrawal of ACB. Method 2 utilized 1 ml of 125 U/ml heparin, to which 20 ml of ACB were added. Method 3 utilized 1 ml of 50 U/ml heparin, to which 20 ml of ACB were added. The requirement for ACB filtration was evaluated by
Volume 97 Number 5
Brief clinical and laboratory observations
8 11
Table. Coagulation measurements in ACB in control and heparinized samples (mean • SEM) Group Control (n = 19) Method 1 (1,000 U/ml) ( u = 27) Method 2 (125 U/ml) (n = 5) Method 3 (50 U/ml) (n = 5)
PT
PTT
TT
PS04
RT
12.5 • 1.2
49 • 11
16.3 • 3.8
--
13/27> 100 14/27 31.5-85
> 100
> 100
> 100
> 100
> 100
> 100
> 100
18-22
1/5> 100 4/5 40-46
> 100
> 100
> 100
18-21
Coagulationtime indicatedin second. PT = Prothrombintime; PTT = kaolinactivatedpartial thromboplastintime; TT = thrombintime; PS04 = protaminesulfate additionto thrombintime; RT = reptilasetime. particle size analysis (Coulter Counter Channelizer C1000, Coulter Electronics, Hialeah, Fla.). Particle counting was performed on specimens of ACB anticoagulated with 50 U / m l heparin (Method 3). Comparisons of particle size distribution were made between filtered and unfiltered ACB and unfiltered CPD anticoagulated bank blood. A 20/~ nylon wool filter was used for filtration. RESULTS Bacteriologic studies. A total of 103 paired aerobic/ anaerobic specimens were obtained. Four aerobic and six anaerobic cultures showed growth, a positive rate of 3.9% and 5.8%, respectively. All positive cultures were in only one member of an aerobic/anaerobic pair. The overall positivity rate was 9.7%. Aerobic organisms isolated were Micrococcus sp and Staphylococcus epidermidis. Anaerobic cultures yielded one each of Clostridium sp, S. epidermidis, Baeteroides ovatus, Streptococcus agalactiae, and Lactobaeillus sp. One anaerobic culture yielding mixed growth lost viability before identification could be completed. Hematologic studies. Coagulation profiles and effects of heparinization are shown in the Table. In order to assess the causality of heparin in the prolongation of thrombin times, reptilase times were performed. All reptilase times were normal, indicating that heparin, not circulating anticoagulants, was responsible for prolongation of coagulation times. No discernible differences were noted in comparisons of particle size analysis of filtered and nonfiltered specimens. The Figure demonstrates representative particle size distribution curves of filtered and nonfiltered ACB along with one-day-old CPD banked blood. Particle size ranged from 29 to 1,t37/z 3. The majority of particles were between 41 and 151/L3. No particles larger than 1,137 ~'~ were detected in any specimen.
DISCUSSION Fifty units of heparin in 20 ml of ACB (2.5 U/ml) are effective for anticoagulation. This volume of blood represents at least 20% of the blood volume Of a 1 kg infant and delivers less heparin dosage than that used for clinical anticoagulation. Paxson, 1 using ACB anticoagulated with approximately 4.5 U heparin/ml, found that 50% of infants transfused with 25 ml/kg of ABC had prolonged PTTs, although this prolongation resolved by 24 hours without any clinical sequelae. Using our lower dosage of heparin, the PT is prolonged three times greater than the control value, and the PTT remains greater than 100 seconds. We have found no differences in microaggregate particle size and distribution between heparinized, 20/~ filtered ACBI and one-day-old CPD blood. The lack of microaggregates supports the above data, suggesting that 2.5U heparin/ml is adequate for anticoagulation. The majority of particle sizes fell between 41 and 151 /~3; this microaggregate distribution is within acceptable limits for transfusion.3 Blood filtration is nonetheless recommended in case of inadvertent inadequate anticoagulation. Filtration will also prevent the possible infusion of large particulate matter such as Wharton jelty. A total of 10 ACB cultures (5.8%) had anaerobic or aerobic growth. Bacteremia has been reported to occur in 3% of infants born with membranes ruptured less than 24 hours and in 17% of infants born with membranes ruptured longer than 24 hours? Kelsell et al) in a prospective study of 200 deliveries, demonstrated inflammatory infiltrates in the umbilical cords of 19.2% of all deliveries and a 39% positive cord blood culture rate. These positive cultures reflected maternal vaginal colonization and were not related to neonatal illness. Similarly, none of our infants with positive ACB cultures developed clinical infections.
8 12
B r i e f clinical and laboratorl; observations
--
\ zoo
nOD
600
---
-
The Journal of Pediatrics November 1980
UNFILTERED CPD BLOOD UNFILTERED CORD BLOOD
............... FILTERED CORD SCALE u
BLOOD
-29
for immediate volume expansion. If cord blood is transfused, we r e c o m m e n d that the ACB be administered as soon as clinically indicated to decrease the time for in vitro bacterial growth. Further studies will be necessary to differentiate cord colonization from fetal bacteremia as the source of growth in cultures of ACB. In summary, ACB anticoagulated with 2.5 U / m l of heparin is hematologically acceptable as an agent for immediate neonatal resuscitation. Bacteriologic studies have revealed a high incidence of positive cultures from ACB. Whether this growth reflects transient neonatal bacteremia or umbilical cord colonization requires further study. The authors acknowledge the technical assistance of HM2 Michael Bilak, HM3 Thomas Baginski and Ann Bachelder, R.N.
800
1obo
izoo
14oo
CUBIC MICRONS
Figure. Particle size distribution curves of fresh, filtered, and unfiltered ACB and one-day-old unfiltered CPD banked blood. The isolates of S. epidermidis and Micrococcus species may represent superficial cord colonization or laboratory contaminants. The growth of Streptococcus agalactiae, Bacteroides ovatus, and Clostridium species cannot be dismissed as laboratory contaminants. Whether these organisms represent fetal bacteremia or cord colonization cannot be determined from this study. T h e lack of clinical disease, especially with respect to the group B streptococcus, supports the probability of umbilical cord colonization. An infant's attending physician must decide whether this potential bacterial colonization is offset by the need
REFERENCES 1. Fischer DE, and Paton JB: Resuscitation of the newborn infant, in Klaus MH, and Fanaroff AA, editors: Care of the high risk neonate, Philadelphia, 1979, WB Saunders Company, p 24. 2. Paxson CL Jr: Collection and use of autologous fetal blood, Am J Obstet Gynecol 134:708, 1979. 3. Miller WV, editor: Technical manual, Wm. V. Miller, Ed, ed 7, Washington, DC 1977, American Association of Blood Banks, pp 230-246. 4. Pomerance W: Chorioamnionitis and maternal sepsis, in Montif GR, editor: Infectious diseases in obstetrics and gynecology, New York, 1974, Harper & Row, Publishers, p 293. 5. Kelsall GRH, Barter RA, and Manessia C: Prospective bacteriological studies in inflammation of the placenta, cord and membrane, J Obstet Gynecol Br Commonw 74:401, 1967.
Transjhsion reaction due to unrecognized ABO hemolytic disease of the newborn infant Casey G. Falterman, M.D., and C. Joan Richardson, M.D.,* Galveston, Texas
THE DIAGNOSIS of ABO hemolytic disease is often difficult, especially in mildly affected infants. Both the small molecular size of the IgG molecule and the relative From the Department of Pediatrics, Division of Perinatal Pediatrics; University of Texas Medical Branch. *Reprint address: Division of Perinatal Pediatrics, Department of Pediatrics, Universityof Texas Medical Branch, Galveston, TX 775509
weakness of fetal red blood cell antigens contribute to the failure of the direct antiglobulin test to detect sensitized cells? The presence of IgG anti-A or anti-B antibodies in the maternal serum by itself has little diagnostic value,
Abbreviations used ABO-HD:ABO hemolytic disease PRBC: packed red blood cells
0022-3476/80/110812+03500.30/0 9 1980 The C. V. Mosby Co.
Brief clinical and laboratory observations
The Journal of Pediatrics November 1~)80
Brief clinical and laboratory observations Hematologic and bacteriologic assessment of autologous cord blood for neonatal transfusions Stephen M. Golden, Commander (MC) USNR,* William F. O'Brien, Lieutenant Commander (MC) USN, Christopher Lissner, Lieutenant Commander (MSC), USN, Robert C. Cefalo, Captain (MC), USN, W. Patrick Monaghan, Lieutenant Commander (MSC) USN, Harold Schumacher, Captain (MC), USN, and Stanford Stass, Lieutenant Commander (MC) USNR, Bethesda, Md.
RESUSCITATION of the newborn infant often requires rapid expansion of the circulatory volume, which is usually accomplished through the use of banked blood or blood components. The use of an infant's autologous cord blood has been recommended for this purpose. ~,~ Autologous cord blood is readily available in the delivery room and does not require the time delay inherent in the use of banked blood. Since ACB is identical to the infant's blood, immunologic reactions and osmolar toxicity are avoided. ACB provides oxygen transport and buffering capacities unavailable in blood substitutes. This report provides bacteriologic and hematologic background for the safety and efficacy of ACB as a method of neonatal volume resuscitation. MATERIALS
AND METHODS
All ACB specimens were obtained from infants delivered at term. Pregnancies were uncomplicated, all deliveries were vaginal, and mothers were afebrile and had received neither antibiotics nor anticoagulants. Following delivery, umbilical cords were clamped and severed in the usual fashion. Bacteriologic studies. The umbilical cord was sprayed with 20% povidone-iodine solution. A sterile syringe with an 18-gauge needle was used to withdraw ACB from the From the Departments of Pediatrics, Obstetrics and Gynecology, and Laboratory Medicine, National Naval Medical Center. Supported in part by CIP No. 0-06-1337, Natiot[al Naval Medical Center. *Reprint address: Department of Pediatrics, Uniformed Services University, School of Medicine, 4301 Jones Bridge Rat., Bethesda, MD 20014.
umbilical vein. Following blood collection, a second sterile needle was placed on the syringe and 5 ml aliquots were immediately inoculated into radiometric aerobic and anaerobic blood culture flasks (BACTEC, Johnston Laboratories, Cockeysville, Md.). After a 72-hour incubation period, all cultures were blindly subcultured aerobically and anaerobically, using chocolate and brucella blood agar, respectively. All cultures suspected of growth by radiometric indices were Gram stained and subcultured. Isolated organisms were identified via standard techniques. See related article, p. 806.
Abbreviations used ACB: autologous cord blood CPD: citrate phosphate dextrose
]
Hematologic studies. Prothrombin, partial thromboplastin time, and thrombin time were obtained on separately drawn specimens of ACB. All coagulation indices were measured by electronically timed changes in optical density. Twenty milliliter aliquots were chosen to determine the efficacy and requirements of heparin anticoagulation. Three heparin dosages were utilized. Method 1 utilized a heparin rinse with 1,000 U/ml, leaving heparin solution only in the hub of the syringe prior to withdrawal of ACB. Method 2 utilized 1 ml of 125 U/ml heparin, to which 20 ml of ACB were added. Method 3 utilized 1 ml of 50 U/ml heparin, to which 20 ml of ACB were added. The requirement for ACB filtration was evaluated by
Volume 97 Number 5
Brief clinical and laboratory observations
8 11
Table. Coagulation measurements in ACB in control and heparinized samples (mean • SEM) Group Control (n = 19) Method 1 (1,000 U/ml) ( u = 27) Method 2 (125 U/ml) (n = 5) Method 3 (50 U/ml) (n = 5)
PT
PTT
TT
PS04
RT
12.5 • 1.2
49 • 11
16.3 • 3.8
--
13/27> 100 14/27 31.5-85
> 100
> 100
> 100
> 100
> 100
> 100
> 100
18-22
1/5> 100 4/5 40-46
> 100
> 100
> 100
18-21
Coagulationtime indicatedin second. PT = Prothrombintime; PTT = kaolinactivatedpartial thromboplastintime; TT = thrombintime; PS04 = protaminesulfate additionto thrombintime; RT = reptilasetime. particle size analysis (Coulter Counter Channelizer C1000, Coulter Electronics, Hialeah, Fla.). Particle counting was performed on specimens of ACB anticoagulated with 50 U / m l heparin (Method 3). Comparisons of particle size distribution were made between filtered and unfiltered ACB and unfiltered CPD anticoagulated bank blood. A 20/~ nylon wool filter was used for filtration. RESULTS Bacteriologic studies. A total of 103 paired aerobic/ anaerobic specimens were obtained. Four aerobic and six anaerobic cultures showed growth, a positive rate of 3.9% and 5.8%, respectively. All positive cultures were in only one member of an aerobic/anaerobic pair. The overall positivity rate was 9.7%. Aerobic organisms isolated were Micrococcus sp and Staphylococcus epidermidis. Anaerobic cultures yielded one each of Clostridium sp, S. epidermidis, Baeteroides ovatus, Streptococcus agalactiae, and Lactobaeillus sp. One anaerobic culture yielding mixed growth lost viability before identification could be completed. Hematologic studies. Coagulation profiles and effects of heparinization are shown in the Table. In order to assess the causality of heparin in the prolongation of thrombin times, reptilase times were performed. All reptilase times were normal, indicating that heparin, not circulating anticoagulants, was responsible for prolongation of coagulation times. No discernible differences were noted in comparisons of particle size analysis of filtered and nonfiltered specimens. The Figure demonstrates representative particle size distribution curves of filtered and nonfiltered ACB along with one-day-old CPD banked blood. Particle size ranged from 29 to 1,t37/z 3. The majority of particles were between 41 and 151/L3. No particles larger than 1,137 ~'~ were detected in any specimen.
DISCUSSION Fifty units of heparin in 20 ml of ACB (2.5 U/ml) are effective for anticoagulation. This volume of blood represents at least 20% of the blood volume Of a 1 kg infant and delivers less heparin dosage than that used for clinical anticoagulation. Paxson, 1 using ACB anticoagulated with approximately 4.5 U heparin/ml, found that 50% of infants transfused with 25 ml/kg of ABC had prolonged PTTs, although this prolongation resolved by 24 hours without any clinical sequelae. Using our lower dosage of heparin, the PT is prolonged three times greater than the control value, and the PTT remains greater than 100 seconds. We have found no differences in microaggregate particle size and distribution between heparinized, 20/~ filtered ACBI and one-day-old CPD blood. The lack of microaggregates supports the above data, suggesting that 2.5U heparin/ml is adequate for anticoagulation. The majority of particle sizes fell between 41 and 151 /~3; this microaggregate distribution is within acceptable limits for transfusion.3 Blood filtration is nonetheless recommended in case of inadvertent inadequate anticoagulation. Filtration will also prevent the possible infusion of large particulate matter such as Wharton jelty. A total of 10 ACB cultures (5.8%) had anaerobic or aerobic growth. Bacteremia has been reported to occur in 3% of infants born with membranes ruptured less than 24 hours and in 17% of infants born with membranes ruptured longer than 24 hours? Kelsell et al) in a prospective study of 200 deliveries, demonstrated inflammatory infiltrates in the umbilical cords of 19.2% of all deliveries and a 39% positive cord blood culture rate. These positive cultures reflected maternal vaginal colonization and were not related to neonatal illness. Similarly, none of our infants with positive ACB cultures developed clinical infections.
8 12
B r i e f clinical and laboratorl; observations
--
\ zoo
nOD
600
---
-
The Journal of Pediatrics November 1980
UNFILTERED CPD BLOOD UNFILTERED CORD BLOOD
............... FILTERED CORD SCALE u
BLOOD
-29
for immediate volume expansion. If cord blood is transfused, we r e c o m m e n d that the ACB be administered as soon as clinically indicated to decrease the time for in vitro bacterial growth. Further studies will be necessary to differentiate cord colonization from fetal bacteremia as the source of growth in cultures of ACB. In summary, ACB anticoagulated with 2.5 U / m l of heparin is hematologically acceptable as an agent for immediate neonatal resuscitation. Bacteriologic studies have revealed a high incidence of positive cultures from ACB. Whether this growth reflects transient neonatal bacteremia or umbilical cord colonization requires further study. The authors acknowledge the technical assistance of HM2 Michael Bilak, HM3 Thomas Baginski and Ann Bachelder, R.N.
800
1obo
izoo
14oo
CUBIC MICRONS
Figure. Particle size distribution curves of fresh, filtered, and unfiltered ACB and one-day-old unfiltered CPD banked blood. The isolates of S. epidermidis and Micrococcus species may represent superficial cord colonization or laboratory contaminants. The growth of Streptococcus agalactiae, Bacteroides ovatus, and Clostridium species cannot be dismissed as laboratory contaminants. Whether these organisms represent fetal bacteremia or cord colonization cannot be determined from this study. T h e lack of clinical disease, especially with respect to the group B streptococcus, supports the probability of umbilical cord colonization. An infant's attending physician must decide whether this potential bacterial colonization is offset by the need
REFERENCES 1. Fischer DE, and Paton JB: Resuscitation of the newborn infant, in Klaus MH, and Fanaroff AA, editors: Care of the high risk neonate, Philadelphia, 1979, WB Saunders Company, p 24. 2. Paxson CL Jr: Collection and use of autologous fetal blood, Am J Obstet Gynecol 134:708, 1979. 3. Miller WV, editor: Technical manual, Wm. V. Miller, Ed, ed 7, Washington, DC 1977, American Association of Blood Banks, pp 230-246. 4. Pomerance W: Chorioamnionitis and maternal sepsis, in Montif GR, editor: Infectious diseases in obstetrics and gynecology, New York, 1974, Harper & Row, Publishers, p 293. 5. Kelsall GRH, Barter RA, and Manessia C: Prospective bacteriological studies in inflammation of the placenta, cord and membrane, J Obstet Gynecol Br Commonw 74:401, 1967.
Transjhsion reaction due to unrecognized ABO hemolytic disease of the newborn infant Casey G. Falterman, M.D., and C. Joan Richardson, M.D.,* Galveston, Texas
THE DIAGNOSIS of ABO hemolytic disease is often difficult, especially in mildly affected infants. Both the small molecular size of the IgG molecule and the relative From the Department of Pediatrics, Division of Perinatal Pediatrics; University of Texas Medical Branch. *Reprint address: Division of Perinatal Pediatrics, Department of Pediatrics, Universityof Texas Medical Branch, Galveston, TX 775509
weakness of fetal red blood cell antigens contribute to the failure of the direct antiglobulin test to detect sensitized cells? The presence of IgG anti-A or anti-B antibodies in the maternal serum by itself has little diagnostic value,
Abbreviations used ABO-HD:ABO hemolytic disease PRBC: packed red blood cells
0022-3476/80/110812+03500.30/0 9 1980 The C. V. Mosby Co.