The debate about the ideal blood volume or inoculant size for blood culture in neonates continues. It is common knowledge that a higher inoculant blood volume has a greater chance for pathogen isolation.1,2,3 However, neonatologists face the unique challenge of obtaining adequate blood volume in the smallest sized individuals for laboratory testing in order to yield a positive test result. The need to obtain just enough blood volume in very low birth weight (VLBW) infants for laboratory testing purposes must be balanced against comprising the hemodynamic and cardiorespiratory status of already vulnerable neonates.

In the article “Neonatal Blood Culture Inoculant Volume: Feasibility and Challenges” by Woodford et al.,4 the author’s primary aim was to determine the inoculant volume sent for neonatal sepsis evaluation. The authors demonstrate that ≥1 ml total blood inoculant size was used (as per blood inoculant volume by weighing culture bottles) for blood culture evaluation in 93.4% of neonatal sepsis evaluations at their center. Contrary to the popular belief of providing inadequate inoculant volume for neonatal blood culture, the authors demonstrate that adequate inoculant volume could be achieved in the neonatal intensive care unit (NICU) by ensuring adequate clinician documentation of blood volume drawn for blood culture in the electronic medical record system and by additionally weighing blood culture bottles before and after inoculation. The secondary objective was to identify areas of improvement in obtaining recommended inoculant volumes in all neonates including very low birth weight (VLBW) infants. The authors state that the current recommendations for adequate inoculant volume for blood cultures were met and that this can be achieved by incorporating monitoring techniques with periodic ongoing education about measuring inoculant volumes. While they achieved higher inoculum size (≥1 ml inoculant in 96.9% evaluations as per clinician report and 93.4% by weighing culture bottles) for blood culture testing in the overall population for sepsis evaluation, the majority of the blood culture specimens for sepsis evaluations were performed ≤24 h after birth (71.2%) with blood samples that were either drawn by phlebotomy or from a central line (82.2%) in cultures that were obtained ≤7 days after birth. In addition, significantly higher proportions of bacteria (either contaminants or Coagulase-negative Staphylococci) were isolated with lower blood volume (<1 ml) inoculant size from phlebotomy compared to inoculant volume of >1 ml (6/36 vs. 29/508, p = 0.009) in VLBW infants >7 days after birth. Specifically, low inoculant volume for late-onset sepsis evaluation was attributed to clinical instability and a sicker population.

So, what do these findings imply and why does this matter? Both, the absolute requirement for optimal blood volume for sepsis evaluation and caution for pathogen contaminants from phlebotomy samples despite strict surface sterilization protocols remain!

Ideal inoculant volume

There are several studies that reviewed the optimal blood volume for sepsis evaluation in the neonatal patient population2,5,6,7,8 (Table 1). Despite several published recommendations for pediatric blood collection, there is no consensus regarding the recommended blood volumes for routine clinical practice. Ohnishi et al.5 noted that the positive detection rate did not increase significantly even when the collected blood volume increased. Guidelines from the 2018 update by the Infectious Diseases Society of America and the American Society for Microbiology recommend collecting 4, 3, 2.5, and 1.8–2.7% of the total blood volume into a single aerobic blood culture bottle in patients under 2, 2.1–12.7, 12.8–36.3, and over 36.3 kg, respectively.9 Woodford et al. mentions that their local guidelines recommend sending an aerobic–anaerobic paired bottle with 1 ml inoculant in each, in accordance with the 2019 American Academy of Pediatrics recommendation for sending an additional anaerobic bottle.10 The potential benefit of adding anaerobic culture bottle for early-onset sepsis evaluations could result in a higher yield of facultative anaerobes but will require samples of larger blood volume to accommodate a paired bottle.

Table 1 Overview of studies of blood volume inoculant size for blood culture and sepsis evaluation in infants and children.
Full size table

While it is crucial to obtain optimal blood volume for blood culture to aid with a rapid and reliable diagnosis of bloodstream infections, there are numerous challenges to a blood draw in neonates, especially among those without central lines. One major factor is the technical difficulty of obtaining a large enough inoculum size by bleeding in sick VLBW infants. Due to inadequacy of sampling, Harewood et al. reported over one-third of blood culture samples from infants <1 month of age have negligible blood volume for blood culture.8 Moreover, low-volume cultures were more likely to yield contaminants.8 NICU providers are especially reluctant to bleed larger blood volumes for laboratory testing as this can exacerbate anemia of prematurity.11 Dedicated phlebotomy personnel with specialized training to serve the neonatal population are an absolute requirement to avoid multiple painful, unsuccessful attempts. While accounting for risk assessment approaches to improve the efficiency of sepsis evaluation, the authors rightly point to efforts to implement diagnostic stewardship of sepsis evaluations and thereafter minimize overuse of antibiotics. NICU providers must stop viewing sterile culture results with skepticism and refrain from prolonged and unnecessary antibiotic treatment for culture-negative sepsis.12

Up and coming modalities such as polymerase chain reaction (PCR)-based assays performed directly on whole blood may complement the conventional blood culture method. While the diagnostic accuracy of the blood culture may be hampered by longer wait times, concomitant antibiotic treatment, low levels of circulating bacteria (low-colony forming bacteremia), and poor sensitivity for slow-growing, intracellular, and fastidious microorganisms, the molecular-based assays, in theory, could markedly improve pathogen detection by detecting the presence of low-colony forming bacteremia, yielding quicker results and decrease the burden on NICU population. Despite promising advances by more modern, advanced, and sophisticated molecular-based tests for assessing infection, there remains one distinct advantage of the standard culture method—antibiotic susceptibility testing.13,14 PCR diagnostic testing that uses smaller blood volumes with rapid turnaround time and avoiding inconclusive results from prior antimicrobial treatment seems to be a promising alternative to standard blood culture technique.15 However, this promising molecular-based technology needs to be further refined to account for contamination, improve sensitivity, decrease cost, increase the range of pathogen identification and provide antibiotic sensitivity reports. While some resistance markers can now be detected by PCR or similar genetic testing, there are still many resistance determinants for which such markers are not yet available.

Cautionary tale for pathogen contamination

Pathogen contamination must be reduced by paying extra attention to meticulous hand hygiene and one must apply sterile techniques while harvesting blood from either peripheral phlebotomy blood samples or from central lines. While blood culture from peripheral arterial or venous sampling remains the gold standard for diagnosis for sepsis with excellent sensitivity and adequate inoculum size,16 umbilical cord blood obtained at delivery could be used as an alternative source for blood culture testing, especially when a majority of the sepsis evaluations are performed ≤24 h after birth.17 Woodford et al.4 did not use placental/umbilical cord blood, but instead used blood samples that were either drawn by phlebotomy or from a central vascular catheter for blood culture.

Umbilical cord blood could prove to be an effective relatively noninvasive alternative to peripheral arterial or venous blood culture and can be performed by a less skilled member of the perinatal team and provide blood volumes far greater than that from a peripheral artery or vein of the VLBW infant. The procedure of umbilical cord blood collection when performed by trained providers can ensure extraction of higher cord blood volume. Timing of the clamping of the umbilical cord after birth greatly influences the amount of blood that the newborn can receive from placental transfusion and the amount of blood that can remain in the cord post-clamping. The practice of immediate clamping of the umbilical cord soon after birth, (often 10–15 s after birth) has largely given way to delayed cord clamping (DCC). DCC facilitates placental transfusion and aid in the increase of blood volume in the newborn and prevents iron-deficiency anemia in infants.18 In addition, placental transfusion from delayed cord clamping and use of umbilical cord blood for neonatal admission laboratory testing, including blood culture, have both resulted in reduced blood transfusion rate and improved outcomes in premature infants.17,19 NICU providers should be aware that DCC does not preclude harvesting blood for laboratory testing despite a significant decrease in the volume of blood remaining in the umbilical cord post-DCC.

While many studies support the feasibility of using cord blood for blood cultures,20,21 other studies exert caution for the possibly higher number of contaminants, despite strict surface sterilization protocols especially with vaginally delivered placentas22 although the risk of contamination is no worse than with peripheral cultures.23 Several methods have been reported to adequately achieve a sterile cord/placenta. Contamination can be reduced by adhering to meticulous and fastidious umbilical cord blood collection techniques including drying the placental surface prior to initiation of sterilization technique in order to eliminate the pooled fluid that remain in depressions between vessels and the use of alcohol, betadine, and tincture of iodine to adequately achieve sterility of a sterile umbilical cord/placenta. A placental drying procedure may not be necessary when a cord segment is used for obtaining a cord blood sample.

We cannot reverse prematurity once an infant is born earlier than expected, but we certainly can mitigate risk by objective assessments and limiting blood culture tests to the infants at the highest risk of infection based on clinical presentation and the course of illness. Therefore, it is of the utmost importance that research continues to focus on optimizing modern molecular methods for the identification of pathogens in small volume blood culture samples to shorten the response times in diagnosis, optimize the antibiotic treatment, and facilitate stewardship programs. Until then, one must continue with the important role of educational intervention in improving the blood culture volume in newborn infants, consider using umbilical cord blood culture for early-onset sepsis evaluations, address important causes of inadequate volume collection and identify, prevent, and judiciously treat VLBW infants with antibiotics.