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Ann Thorac Surg 2000;70:229-233
© 2000 The Society of Thoracic Surgeons

Original articles: Cardiovascular

Inhibition of needlestick-induced simulated viremia by local measures

Francis Robicsek, MD, PhDa, Alexander A. Fokin, MD, PhDa, Thomas N. Masters, PhDa, Joseph W. Cook, MDb

a Heineman Medical Research Laboratory, Carolinas Medical Center, Charlotte, North Carolina, USA
b Department of Thoracic and Cardiovascular Surgery, Carolinas Medical Center, Charlotte, North Carolina, USA

Address reprint requests to Dr Robicsek, Carolinas Medical Center, 1000 Blythe Blvd, Charlotte, NC 28203


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. The possibility of confinement of simulated retrovirus to the inoculation site after needlestick injuries to enhance chances of local intervention and function of lymphaticovenous communications was investigated.

Methods. Using the canine model, technetium-99 m sulfur colloid particles were injected subcutaneously and into the vein and lymphatics. Blood and lymph were collected at a higher level from the femoral vein and the major lymphatic. Flow rates, particle arrival times, concentrations, and other variables were evaluated for 45 minutes by {gamma} counting. A tourniquet was used to slow dissemination after subcutaneous injection.

Results. After subcutaneous inoculation, particles arrived in the blood at 2.81 ± 0.54 minutes and in the lymph at 6.0 ± 1.47 minutes. Application of a tourniquet delayed appearance in the blood to 7.11 ± 1.5 minutes and in the lymph to 40.0 ± 5.1 minutes. Concentration of particles in lymph was 1,000 times higher than in the blood. Flux values were comparable in both pathways, but accumulation patterns were different. After intravenous injection, particles arrived in lymph at 25.4 ± 6.44 minutes. After intralymphatic injection particles arrived in the blood within 4 seconds.

Conclusions. There are functional lymphaticovenous communications at the peripheral level. The period between virus inoculation and blood and lymph invasion may be extended by application of a tourniquet; therefore, time could be gained for local intervention.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
The chance of infection after a single contaminated needlestick or sharp instrument injury is estimated to be 0.3% for human immunodeficiency virus and from 6% to 30% in cases of hepatitis B virus [1–4].

Needlestick injuries occur three times higher in cardiac as compared to general thoracic surgery and are usually caused by needles or by sternal wires [5]. Because of the harmful, often tragic consequences of such injuries, every effort should be made to prevent them and neutralize the infection if it occurs despite precautionary measures.

In the 1991 volume of this Journal [6], we reported that in experimental animals with simulated accidental injuries, as they may occur in health care and research facilities, seroconversion may be prevented by rapid infiltration of the inoculation site with 0.2% povidone–iodine. The reason for the present study was: (1) to further investigate the mechanism of tissue–lymph–blood and lymph-to-blood transport of viral-size particles, and (2) to examine whether additional steps, such as application of a tourniquet may contain the particles at the inoculation site and thus prolong the time during which viruses may be rendered inactive by topical measures such as local infiltration with povidone–iodine.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
The dog, as a large mammal, was chosen for the experiments because its circulation is comparable in many aspects to that of humans and because its size also allowed us to create a reliable, repeatable, and steady lymph flow pattern, providing the potential for the collection of a substantial amount of lymph. Accidental percutaneous injuries with retro- or adenovirus (human immunodeficiency virus and hepatitis B)-contaminated instruments, which occur in daily health care and research practice, were simulated by subcutaneous introduction of virus-sized particles. The animals were anesthetized with pentobarbital sodium (33 mg/kg), restrained in a supine position, and artificially ventilated. The principal lymph vessel at the groin was ligated proximally, and distally cannulated with a No. 24 angiocatheter. All visible lymphatic collaterals were tied to inhibit particle escape. Prior injection of dye to visualize the lymphatics was not used, as not to alter lymph transport by overloading and stretching the lymphatics and to minimize the possibility of injury to the microvessels at the inoculation site. The femoral vein was cannulated with a 16-gauge angiocatheter and snared just above the collection site. The internal aspect of the knee was chosen for subcutaneous inoculation. The site for intravenous and intralymphatic inoculation was at the paw, where a peripheral vein and lymphatic were exposed.

The experiments were done in four groups. In group A (7 animals), an inflatable tourniquet was placed on the thigh and inflated immediately to 250 mm Hg after the knee-level subcutaneous inoculation. In group B (7 animals), subcutaneous injection of microparticles was used, but without tourniquet occlusion, as the control. In group C (5 animals), injection into the peripheral vein and in group D (7 animals) injection into the lymphatic was carried out at the paw.

The animals were maintained and handled according to the National Research Council, National Academy Press, Washington, DC, 1996 "Guide for Care and Use of Laboratory Animals."

Particle inoculation was carried out by slow injection using a 1.0-mL syringe with a 25-gauge needle for subcutaneous inoculation and a 30-gauge needle for intravenous and intralymphatic injection. One milliliter of 500 µCi active technetium-99 m sulfur colloid microparticles (Mallinckrodt Inc, St. Louis, MO) was used for subcutaneous injection and 0.5 mL for intravenous and intralymphatic injection. Using double filtering with Millex-GV 0.22 µm and Millex-VV 0.1 µm filter from Millipore Corporation (Bedford, MA), the size of the particles was narrowed to the 100 to 200 nm range, identical to that of human immunodeficiency virus. The radioactivity of all samples was determined using auto {gamma}-counter model Cobra II (Packard Instrument Co, Meriden, CT) for 30 minutes and expressed in counts per minute (cpm) with background correction. Before each experiment, the baseline of the {gamma} counter was normalized. The counter stability and reproducibility was assured by constancy measurements.

Depending on the amount of flow, lymph samples were collected in 1- to 5-minute intervals. Venous blood samples were obtained continuously for the first 15 minutes, then every 5 minutes for up to 45 minutes. Flow rates, particle arrival time, concentration, and accumulation were monitored.

"Arrival time" for particulate matter was defined as the period between inoculation and the initial detection. "Particle flux" was expressed in counts per minute, per minute of collection time (cpm/min) and was a product of concentration over flow rate and used as a denominator to account for differences in volume and speed. "Particle accumulation" was expressed in total counts per minute ({Phi}) to represent at any period of time the total amount of particles escaped through the blood vessels and lymphatics.

Values were expressed as mean ± standard error. Student’s t test was applied for comparison of mean values (SigmaPlot for Windows 95, version 4.0, SPSS Inc, Chicago, IL), p less than 0.05 was considered significant. The SAS System for Windows, version 6.12, was used to complete repeated measures analysis of variance for group time interaction comparisons.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
The mean value of venous flow was 23.13 ± 0.61 mL/min without, versus 17.46 ± 0.61 mL/min with the application of a tourniquet (p < 0.05). Mean values of lymph flow throughout a time period of 45 minutes was comparable in no-tourniquet and tourniquet conditions (30.38 ± 2.79 µL/min versus 39.16 ± 15.86 µL/min, respectively). It is notable that after the application of a tourniquet, initially there was an increase in lymph flow because of lymph being forced in both directions from the point of application, followed by a statistically significant reduction (mean, 8.23 ± 1.19 µL/min) about 4.7 minutes after the tourniquet was tightened (p < 0.001). This original "surge" contributed to the increase in mean flow value, and disguised the consequential reduction. The release of the tourniquet induced a second, sharp increase in lymph flow.

After subcutaneous injection, the mean arrival time of the particulate matter in venous blood without a tourniquet was 2.81 ± 0.54 minutes and 7.11 ± 1.50 with the application of a tourniquet (p < 0.05). The mean particle arrival time in lymph was 6.0 ± 1.47 minutes without a tourniquet versus 40.0 ± 5.10 minutes with a tourniquet (p < 0.001). After intravenous injection, the mean arrival time of the particulate matter in lymph was 25.4 ± 6.44 minutes. After intralymphatic injection, the particulate matter arrived in the blood within 4 seconds.

The mean concentration of radioactivity in venous blood with no tourniquet was 1.11 x 103 ± 1.46 x 102 cpm/mL. Application of a tourniquet reduced concentration to less than half, 4.69 x 102 ± 48 cpm/mL (p < 0.001). The mean concentration value in lymph was 4.06 x 106 ± 1.70 x 106 cpm/mL with no tourniquet and 2.71 x 105 ± 1.42 x 105 after the application of a tourniquet and remained higher by a magnitude of 1,000 than in venous blood. The application of a tourniquet did not change this proportion. Comparison of lymph concentration values between the two groups by repeated measures analysis of variance showed that the values in the no-tourniquet group changed differently over time than those in the tourniquet group (p < 0.0001). There were no significant differences within the two groups in venous blood concentrations.

The particle flux value in blood under normal conditions was 1.25 x 104 ± 1.22 x 103 cpm/min and 1.55 x 104 ± 2.56 x 103 cpm/min in lymph. The application of the tourniquet reduced particle flux in the blood to 5.34 x 103 ± 5.50 x 102 cpm/min and in the lymph to 3.34 x 103 ± 1.54 x 103 cpm/min, respectively. In the presence of high concentration but low flow in lymph on one hand, and low concentration and high flow in blood on the other, the flux of viral size particles in both situations was comparable. Repeated measures analysis of variance indicated a lack of differences in particle flux in blood in both groups, but revealed significant differences between the two groups in particle flux in lymph (p < 0.0001).

The data on particle accumulation are presented in Figures 1 and 2. The mean value of viral size particle accumulation in the blood at 15 minutes with no tourniquet was 1.42 x 105 ± 6.72 x 104 total counts/min and 3.68 x 104 ± 1.53 x 104 total counts/min if a tourniquet was applied. The accumulation value in lymph at 15 minutes with no tourniquet was 9.45 x 104 ± 4.07 x 104 cpm, significantly less than in the blood at the same time period. The value of accumulation in lymph at 45 minutes with no tourniquet was 7.78 x 105 ± 1.51 x 105 cpm and 1.82 x 104 ± 1.36 x 104 cpm if a tourniquet was applied (p < 0.05). Repeated measures analysis of variance of lymph accumulation data showed that the no-tourniquet group values changed differently over time in comparison with tourniquet group values (p < 0.0001); however, there were no such differences in the blood. The analysis of the accumulation curves revealed an increase in blood earlier and faster than that in lymph. The lymph accumulation curve increases later and steeper. Neither curve plateaued during the experiment. The tourniquet was more efficient in reducing accumulation of viral size particles in the lymph than in the blood.



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  		Fig 1. Accumulation of viral size particles in blood and lymph without tourniquet application.

 


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  		Fig 2. Accumulation of viral size particles in blood and lymph with tourniquet application.

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
In most cases of accident-induced retro- and adenovirus infections occurring in the healthcare environment, the source–patient is known. Injections, drawing of blood samples, and suturing are among the most common procedures that lead to such mishaps [1, 4, 7]. The average volume of inocula because of needlestick injury by an 18- to 22-gauge needle is up to 1 µL [8, 9]. Univariate analysis of human immunodeficiency virus transmission revealed that it was related to the size of the needle, the depth and extent of the injury, visible blood on the device, as well as to the stage of the disease in the source–patient [1]. The left hand was the body part most often injured, and latex gloves proved to be poor barriers; however, they may reduce the transferred blood volume by more than 50% [4, 10, 11]. Wearing a thicker glove on a nondominant hand may provide some protection, but is certainly not foolproof [12]. In search of a way to prevent seroconversion caused by needlestick or knife cut accidents, we have used the feline model to examine whether seroconversion may be prevented by local measures. We found that this was indeed feasible, but only if these measures were applied soon (within 20 seconds) after inoculation. Our experiments, in which the inoculation site was infiltrated with povidone–iodine diluted with 1% xylocaine, also demonstrated that injuries, such as knife cuts, which were associated with more tissue damage than needlesticks, are more difficult to control with topical intervention [6]. The observation that exposure to blood from patients with florid acquired immunodeficiency syndrome is associated with a higher risk of transmission and that a larger inoculum is more likely to cause seroconversion and acquired immunodeficiency syndrome suggests that the development of viremia is quantum dependent [13, 14]. Even in situations where the viral inoculum is not rendered totally inactive by topical measures and a few viruses escape, the chances of a generalized infection are still decreased. Documented failures of postexposure zidovudine prophylaxis indicate that any protection afforded by this treatment is not infallible [1, 2] and may be time dependent, thus favoring a prompt intervention [15].

The results of our past experiments have already shown that after subcutaneous inoculation, particulate matter appears in blood faster than in lymph and that this difference is statistically significant [16]. Besides confirming the same, our present observations also indicate that when a tourniquet is applied this difference becomes even more pronounced and that the tourniquet definitely slowed the arrival of particles into both blood and lymph, but especially in lymph.

Lymph flow is approximately 1,000 times slower than venous blood flow. Tourniquet application reduced the flow of both lymph and blood, with a more abrupt and more substantial decline in the lymph flow.

The considerable difference in concentration indicates that lymph pathways are primarily responsible for collecting a substantial amount of viral-sized particles after subcutaneous inoculation.

The flux values, however, show that although the blood pathway carries less particulate matter, it appears much sooner than in the lymph pathway. The latter transports a larger amount, but at a slower speed. In addition, the blood accumulation increases faster and earlier than in the lymph, but does not attain values as high as in lymph, in which the accumulation steadily increases throughout the experiment. These findings suggest that the release of viruses from the subcutaneous inoculation site will be maintained for prolonged periods. Clinically important is the finding that initially (2 hours after subcutaneous inoculation), extracellular transport dominates over intracellular, regardless of tissue damage by the needle, or acute local inflammation [17]. If, however, because of the nature of the injury circumventing the interstitial route, part of the inoculum directly enters the bloodstream or lymph, then the viral spread will depend mainly on the flow and can be altered by tourniquet application.

These postulates, drawn from these observations, are also supported by scintigraphic studies in which the colloid particle clearance from the skin and subcutis was done by the isotope method and it was found that clearance by blood accounts for a mere 1.5% in comparison with the lymph clearance, and that the volume of injection did not significantly influence the half-clearance time [18]. Massage, however, significantly enhanced lymph flow and transport of particulate matter [19].

An intriguing result of the described experiments was the almost immediate arrival of the particulate matter in the venous blood after intralymphatic injection. This could be explained by the presence of functional direct and extensive lymphovenous communications at the microlevel even under physiologic conditions. These shunts are beneficial by allowing drainage of any overload in the lymph system, but at the same time they may also increase the speed of infection. The prevalent notion is that such communications normally appear to be closed and shunting may be temporary [20].

Besides the universal precautions for the handling of sharp and contaminated instruments, the following have been recommended as part of hospital protocol:

  1. Wear a thicker or doubled glove on the nondominant hand.
  2. Limit the amount of participants at the final stage of operations when mental concentration is reduced [21].
  3. Minimize the number of sharp instruments used and pass them to a table instead of hand-to-hand.
  4. Use blunt instruments wherever possible (blunt tip or retracting scalpels, blunted suture needles, staplers, and so on).

To the above we add the following recommendations when an operation is performed on a known or suspected virus carrier:

  1. Use experienced scrub nurses and assistants.
  2. All sharp instruments should be stored on a separate stand.
  3. When the scrub nurse hands a sharp instrument to the surgeon, she or he is to say "sharp" and assure that the surgeon indeed looks at the instrument to be passed.
  4. Have a syringe loaded with antiviral agent, such as 0.5% povidone–iodine, as well as different sizes of tourniquets ready on the table. In case of injury, a tourniquet should be applied immediately to the injured extremity and the site of injury infiltrated with povidone–iodine in the speediest possible manner.
  5. Immobilize and avoid massaging the injured hand.

We also believe that the onset of systemic treatment after accidental injury should be in direct proportion to exposure, tissue damage, and the severity of the source–patient’s condition.

In conclusion, there are functional lymphaticovenous shunts at the microlevel even under physiologic conditions that may readily enhance the spread of virus infection. Our results also suggest that the time between accidental retrovirus inoculation and invasion of the blood and lymph may be extended by rapid immobilization of the limb and application of a tourniquet above the site of injury. This may enhance the opportunity to destroy the virus inoculum locally and prevent seroconversion.


   Acknowledgments
 
We acknowledge the support by Dr Geert Schmid-Schönbein of the Department of Bioengineering, University of California San Diego, La Jolla, California. This work was supported by grants from the Heineman Foundation of Research, Education, Charitable, and Scientific Purposes, Inc, of New York.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

  1. Cardo D.M., Culver D.H., Ciesielski C.A., et al. Center for Disease Control, and Prevention Needlestick Surveillance Group. A case control study of HIV seroconversion in the health care workers after percutaneous exposure. N Engl J Med 1997;337:1485-1490.[Abstract/Free Full Text]
  2. Gerberding J.L., Limiting the risks of health care workers. In: Sande M.A., Volberding P.A., eds. The medial management of AIDS, 5th ed. Philadelphia: WB Saunders, 1997:75-85.
  3. Tokars J.I., Marcus R., Culver D.H., et al. Surveillance of HIV infection and zidovudine use among health care workers after occupational exposure to HIV-infected blood. Ann Intern Med 1993;118:913-919.[Abstract/Free Full Text]
  4. Exposure Prevention Information network (EPINet) Data Reports. International health care worker safety center, University of Virginia. Current working estimates. 1999:1–3.
  5. Kjaergard H.K., Thiis J., Willimberg N. Accidental injuries and blood exposure to cardiothoracic surgical teams. Eur J Cardiothoracic Surg 1992;6:215-219.[Abstract]
  6. Robicsek F., Duncan G.D., Black J.W., Masters T.N., Robicsek S.A., Rice H.E. Prevention of retrovirus infection after injury with contaminated instruments. Ann Thorac Surg 1991;52:74-77.[Abstract]
  7. Morgan DR. HIV and needle stick injuries [Letter]. Lancet 1990;May 26;335(8700):1280.
  8. Napoli V.M., McGowan J.E. How much blood is in a needlestick?. J Infect Dis 1987;155:828.[Medline]
  9. Gaughwin M.D., Gowans E., Ali R., Burrel C. Bloody needles. AIDS 1991;5:1025-1027.[Medline]
  10. Johnson G.K., Nolant T., Wuh H.C., Robinson W.S. Efficacy of glove combinations in reducing cell culture infection after glove puncture with needles contaminated with HIV type 1. Infect Control Hosp Epidemiol 1991;12:435-438.[Medline]
  11. Mast S.T., Woolwine J.D., Gerberding J.L. Efficacy of gloves in reducing blood volumes transferred during simulated needlestick injury. J Infect Dis 1993;168:1589-1592.[Medline]
  12. Hussain S.A., Latif A.B., Chaudhary A.A. Risks of surgeons. Br J Surg 1988;75:314-315.[Medline]
  13. Ho D.D. Viral counts count in HIV infection. Science 1996;272:1124-1125.[Medline]
  14. Mellors J.W., Rinaldo C.R., Gupta P., White R.M., Todd J.A., Kingsley L.A. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 1996;272:1167-1170.[Abstract]
  15. Beckman S.E., Vlahov D., Koziol D.E., McShalley E.D., Schmitt J.M., Henderson D.K. Temporal association between implementation of universal precautions and a sustained, progressive decrease in percutaneous exposures to blood. Clin Infect Dis 1994;18:562-569.[Medline]
  16. Robicsek F., Duncan G.D., Masters T.N., Robicsek S.A., Rice H.E. Can AIDS be prevented after injury with contaminated instruments?. Ann Thorac Surg 1990;49:984-986.[Abstract]
  17. Ikomi F., Hanna G.K., Schmid-Schönbein G.W. Mechanism of colloidal particle uptake into the lymphatic system; basic study with percutaneous lymphography. Radiology 1995;196:107-113.[Abstract/Free Full Text]
  18. Mortimer P.S., Simmonds R., Rezvani M., Robbins M., Hopewell J.W., Ryan T.J. The measurement of skin lymph flow by isotope clearance—reliability, reproducibility, injection dynamics, and effect of massage. J Invest Dermatol 1990;95:677-682.[Medline]
  19. Ikomi F., Schmid-Schönbein G.W. Lymph pump mechanics in the rabbit hind leg. Am J Physiol 1996;271(1 Pt 2):H173-H183.[Abstract/Free Full Text]
  20. Schmid-Schönbein G.W. Microlymphatics and lymph flow. Physiol Rev 1990;70:987-1028.[Abstract/Free Full Text]
  21. Kantu S., Lee D., Nash M., Lucente F.E. Safety awareness for the otolaryngologist caring for the HIV-positive patient. Laryngoscope 1996;106:982-986.[Medline]
Accepted for publication December 30, 1999.




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