Needlestick injury: Blood, mononuclear cells, and acquired immunodeficiency syndrome

BRIEF REPORTS

Needlestick injury: Blood, mononuclear cells, and acquired immunodeficiency syndrome Djamshid Shirazian, PhD Barry C. Herzlich, MD Foroozan Mokhtarian, PhD Giuseppe Spatoliatore, MD David Grob, MD Brooklyn, New York

Introduction: Because transmission of HIV to health care workers after needlestick injury has occurred mainly a result of deep insertion of large gauge needles, blood and viable mononuclear cells transferred after needlestick injury were measured. Methods: Needles of 20 to 27 gauge were filled with HIV-1 seropositive blood and inserted through extracorporeal human skin or parafilm covering physiologic saline solution modified Drabkin's solution, or culture medium, or inserted directly into one of these fluids, to a depth of one third of the needle length (0.5 inch) for I second. Volume of blood transferred was measured by both modified Drabkin's method and by chromium 5 l labeling of red blood cells. Transfer of viab]e mononuclear cells was measured by growth in culture medium containing autologous feeder cells. Results: The volume of blood transferred from a needle passed through skin varied from 312 -+ 69 nl from a 20-gauge needle to 14 ± 4 nl from a 27-gauge needle, as measured by modified Drabkin's technique, and from 404 ± 80 n] to 12 ± 3.1 nl, as measured by chromium 51 labeling of red blood cells. The volume of blood transferred from a needle passed through parafilm was twice that transferred through skin. The volume of b]ood transferred through skin was 40% that transferred directly into fluid not covered by any barrier; blood transferred through parafilm was 80% of that transferred directly. When needles containing blood were inserted into culture medium for 1 second in the absence of a barrier, at least one viable mononuclear cell was almost always transferred to fluid from all gauges of needle tested. Insertion of needles through skin prevented transfer of all viable mononuclear cells from only 3% to 5% of 20- to 23-gauge needles, and from 12% to 32% of 26- and 27-gauge needles. Parafilm was an even less effective barrier than skin. Insertion of needles through parafilm completely prevented transfer of viable mononuclear ceils from no 20- to 23-gauge needles and from only 5% to 10% of 26- and 27-gauge needles. Conclusion: The volume of blood transferred after needle insertion through skin for 1 second varied with the gauge of the needle and was 30-fold higher from a 20-gauge than from a 27-gauge needle. Variable mononuclear cells were transmitted after insertion through skin from more than 95% of 20- to 23-gauge needles and from 68% to 88% of 26- and 27-gauge needles. Parafilm was less effective than skin in reducing transmission of blood and viable mononuclear cells. (AJIC AM J INFECTCONTROL1992;20:133-7)

From the Division of Immunology, Department of Medicine, Maimonides Medical Center, and the Departments of Medicine, and Microbiologyand Immunology,SUNYHealthScienceCenterat Brooklyn. Supported by a grant from the Maimonides Research and Development Foundation. Human skin supplied by Dr. Mehran

Manouel, Orthopedic Biomechanics Laboratory, Department of Orthopedic Surgery, Maimonides Medical Center. Reprint requests: Djamshid Shirazian,PhD, Immunologyresearch, Department of Medicine, Maimonides MedicalCenter, 4802 Tenth Ave., Brooklyn, NYo11219.

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Fig. 1. Drabkin's method was modified to achieve greater sensitivity for hemoglobin quantitation. Modification resulted in a sharp absorption band at 410 nm (A).

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Fig. 2. For each determination, a representative standard curve for HIV-infected blood was used in calculating volume by Drabkin's modified method.

Hospital workers are exposed to numerous occupational hazards. As the prevalence of HIV infection increases, there is growing concern among this population regarding potential exposure and infection. As of January 1991, a total 5815 health care workers in the United States were reported to have acquired HIV infection; 22 of these transmissions occurred through occupational exposure. 1 Concern over the occupational risk of AIDS increased 4 years ago, when the Centers for Disease Control reported that four of 1500 health care workers experienced seroconversion after being stuck by needles. The rate of HIV infection after needlestick with blood known to have HIV antibodies is relatively low (0.4%). 2'3 There appears to be a positive correlation between seroconversion and

the amount of infected blood received by healthy persons. Eighty-nine percent of recipients of a large volume of HIV antibody-positive blood had seroconversion; exposure to a small volume of HIV-contaminated blood through accidental percutaneous injury was associated with a much lower risk of seroconversion. We investigated the quantity of blood and viability of lymphomononuclear cells transmitted by needlestick injury. These are important factors in the possibility of HIV seroconversion in health care workers after needlestick or other exposure to blood containing the virus. METHODS

Heparinized blood was collected from five consenting HIV-infected patients. HIV antibody was

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T a b l e 1. The amount of blood (in nanoliters) transmitted from needle to media with and without barriers by needle gauge 20

22

23

26

27

Barrier

Hb*

51Crt

Hb*

SlCrt

Hb*

51Crt

Hb*

SlCrt

Hb*

51Crt

None Parafilm Human skin

312 ___ 69 287 _+ 81 198 +- 62

404 + 80 340 -- 63 233 -+ 119

294 +- 98 203 + 94 83 -+ 41

289 --+ 132 269 + 85 213 -+ 104

172 -+ 56 143 + 41 71 - 8

183 -+ 97 175 _+ 34 81 -+ 43

51 -+ 18 39 -+ 21 18 -+ 8

53 -+ 1 41 -+ 11 15 _ 11

14 --+ 4 7 -+ 4 4 - 5

12 - 3 9 _+ 2 3 -- 4

All data mean _+ SD. *Volume measured by modified Drabkin's method (quantitation of hemoglobin). l-Volume measured by means of ~Cr-labeled red blood cells.

determined by enzyme-linked immunosorbent assay and Western blot analysis.

and then discarded. For blood from each patient, five replications were performed with each gauge of needle and each barrier.

M e a s u r e m e n t of v o l u m e of blood

Determination by red blood cells labeled with chromium 51. Five ml citrated blood from an HIV

Modified Drabkin's method. Drabkin's method 4

quantifes hemoglobin by spectrophotometry. To improve the sensitivity of this method, reagent concentrations were changed to 2% sodium bicarbonate, 0.11% potassium cyanide, and 0.91% potassium ferricyanide. Maximum absorption with a Gilford Response 11 UV/VIS spectrophotometer (Ciba Corning Diagnostic Corp. Gilford System, Oberlin, Ohio) occurred at 410 nm. This modification of Drabkin's method gave a sharp band for the maximum absorption at 410 nm (Fig. 1). A standard curve was constructed with varying amounts of blood ranging from 1 ~1 to 1 nl. The resulting color was read at 410 nm with a microplate reader (MR 700; Dynatech Laboratories, Inc, Chantilly, Va.) and the standard curve was constructed for each subject (Fig. 2). Human skin and parafilm as barriers. Models consisting of human skin, parafilm (0.13 mm thick), or no barriers, covering microtiter plates filled with Drabkin's solution or vials filled with phosphate-buffered saline (PBS) solution were employed. Human skin was obtained from amputated limbs. D e t e r m i n a t i o n of t r a n s m i t t e d blood Hemoglobin assay. Blood from an HIV antibody-positive subject was drawn into syringes by means of 20-gauge through 27-gauge needles. Only one third of the needle was inserted into the blood, to simulate the usual length of needle insertion into a vein to withdraw blood. Each needle was then separated from the syringe, passed through skin or parafilm, and immersed in 50 ~1 of Drabkin's modified fluid to a depth of 3 mm (to simulate the depth of penetration through the skin seen in accidental needle injury) for less than 1 second. Each needle was used once

antibody-positive subject was mixed with 0.3 ml chromium 51 solution and incubated for one hour at room temperature. To construct a standard curve, amounts of 5~Cr-labeled blood ranging from 10 ~1 to 1 nl were mixed with 1.5 ml of PBS solution, and radioactivity was measured with a gamma counter. Labeled blood was drawn into syringes by means of 20-gauge through 27-gauge needles. Each needle was passed through the barrier as described. For each patient, five replications were performed with each gauge of needle and each barrier. Quantity of blood transmitted to PBS solution was then calculated relative to the standard curve. Determination of mononuclear cells. Mononuclear cells (MNCs) were isolated from normal peripheral blood by Lymphoprep density gradient centrifugation (Accurate Chemical & Scientific Corp., Westbury, N.Y.). The MNCs were washed twice in Hanks' balanced salt solution (HBSS) and once in RPMI- 1640 medium containing 10% heatinactivated fetal calf serum (FCS). Samples of 107 cells were suspended in 10 ml culture medium and irradiated with 500 rads/min gamma radiation for 10 minutes to kill all the cells, enabling them to be used as feeder cells. A further 104 irradiated cells were placed in each well of a 96-well, roundbottomed microtiter plate in 100 I~I complete culture medium consisting of RPMI-1640 medium supplemented with 10% FCS, 2 mmol/L glutamine, sodium pyruvate, nonessential amino acids, 25 mmol/L N-(2-hydroxyethyl)piperazineN'-2-ethanesulfonic acid (HEPES), 5 × 10-5 mo]/L 2-mercaptoethanol, 100 p,g/ml gentamycin, and recombinant interleukin-2 (15 U/ml) and incubated at 37 ° C in 5% carbon dioxide, 95% air. 5' 6 Human skin or a layer of paraflm was placed on top of microtiter plates.

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T a b l e 2. Percentage reduction of volume of blood transmitted to media through barriers; by needle gauge 20

22

23

26

27

Barrier

Hb*

SlCrt

Hb*

SlCrt

Hb*

SlCrt

Hb*

slCrt

Hb*

slCrt

Parafitm Human skin

9 36.6

16 43

31 71

7 27

17 69

4.4 64

24 65

22.7 72

50 71

15 75

The data are calculated from Table 1. There are statistically significant differences between the volume of blood reduced by parafilm vs. human skin (/3 = 0.001) when data for each barrier are pooled (Student's t test). *Volume measured by modified Drabkin's method (quantitation of hemoglobin). tVolume was measured by means of slCr-labeled red blood cells,

T a b l e 3. Frequency of transmission of at least one viable lymphomononuclear cell to media with various barriers, by needle gauge Barrier

20

22

23

26

None Parafilm Human skin

280/280 (100%) 273/274 (100%) 250/259 (96%)

280/280 (100%) 276/276 (100%) 276/284 (97%)

282/282 (100%) 281/281 (100%) 243/255 (95%)

276/276 (100%) 283/283 (100%) 241/271 (88%)

27 281/283 (98.9%) .271/274 (98.9%) 168/244 (68%)

Each denominator represent the number of needlesticks and each numerator represents the number of times at least one lymphomononuclear cell was transferred. Percentage of viable transmissions is given in parentheses.

Normal blood was drawn into syringes by means of sterile 20-gauge through 27-gauge needles, with one third of needle length inserted into the blood. Each needle was then removed from the syringe, passed through skin or parafilm, and immersed in 100 I~1 culture medium containing autologous feeder cells (104 cells/well) for less than 1 second. Contro] procedures employed sterile needles inserted into wells containing feeder cells and blood-contaminated needles inserted into wells with no feeder cells. Experiments were performed in triplicate. Microtiter plates were incubated at 37 ° C in 5% carbon dioxide, 95% air for 14 days. The cultures were fed on day 7 by replacing 0.1 ml culture medium with fresh m e d i u m containing interleukin-2. After 14 days of culture the microwells were scored microscopically for growth. There was no growth in control experiments. All reagents were sterilized by filtration through a 0.22 &m m e m b r a n e and all experiments were carried out in a biosafety level II sterile cabinet. RESULTS A m o u n t of blood transmitted by passage of needle through skin or parafllm Modified Drabkin's method. As measured by the

modified Drabkin's method, the amount of blood transferred when a needle containing blood was passed into the media ranged from 312 _+ 69 nl for a 20-gauge needle to 14 _ 4 nl for a 27-gauge needle. When parafilm or h u m a n skin was used as a barrier, however, the amount of blood transmitted ranged from 287 _+ 81 with parafilm or

1 9 8 _ 62 nl with human skin for a 20-gauge needle to 7 -+ 4 with parafilm and 4 _ 5 nl with h u m a n skin for a 27-gauge needle (Table 1). The difference between the amounts of blood transmitted to fluid with and without human skin as a barrier may have represented the volume of blood trapped in the skin; alternatively, this amount of blood may have been trapped in the needle. This volume ranged from 114 to 10 nl for 20- through 27-gauge needles. The percentage of blood blocked by h u m a n skin was 36% for a 20-gauge needle, but ranged from 60% to 70% for 22through 27-gauge needles. Red blood cells labeled with chromium 51. As measured by chromium 51 assay, the amount of blood transferred by needlestick when human skin was used as a barrier ranged from 233 _ 119 nl to 3.5 - 4.5 nl w h e n 20- through 27-gauge needles were used (Table 1). H u m a n skin reduced the volume of blood transferred by 43% and 27% for 20- and 22-gauge needles, respectively, and by 60% to 75% for 23- through 27-gauge needles (Table 2). A slightly higher volume of blood was found to have entered the PBS solution when volume was determined by 51Cr-labeled red blood cells than when it was determined by Drabkin's method, but the difference was not statistically significant. Frequency of transmission of viable MNCs

We were able to demonstrate cell growth on day 14 by culturing blood transferred from needle to media containing irradiated autologous mononuclear feeder ce]ls. Wells containing control prep-

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arations (needlestick blood without feeder cells or feeder cells without blood) had no growth. When blood-contaminated needles of gauges 20 through 27 were inserted into complete culture media for 1 second, at least one viable MNC could be cultured (Table 3). After insertion of needles through h u m a n skin, viable MNCs were transferred to culture m e d i u m in 95% to 97% of trials with 20- through 23-gauge needles and in 88% and 68% of trials with 26- and 27-gauge needles. Parafilm was less effective in blocking transfer of MNCs to media. In 99% to 100% of trials, MNCs were transferred from 20- through 27-gauge needles to the media. DISCUSSION

The incidence of acquisition by health care workers of HIV infection after accidental percutaneous needlestick with HIV-infected blood is approximately 0.4%. Although the risk of occupational infection is low, the risk of progression to clinical AIDS after seroconversion may be 100%. Since most instances of seroconversion after needlestick injury have occurred after deeper penetration of large-bore needles and since seroconversion occurs almost invariably after transfusion of HIV infected blood, it appears likely that the volume of blood transmitted by needlestick injury strongly influences and may determine the transfer of infection. Therefore we have studied the volume of blood transferred from needles of various sizes during simulated needlestick. Previously we demonstrated, with 51Cr-labeled red blood cells, that residual volume of blood remaining in needles after drawing blood varied from 183 - 50 ~1 for a 20 gauge needle to 7.8 1 ~1 for a 27-gauge needle. 7 In this study using two different methods, we demonstrated that only 1/500 of the blood in the needle was transferred to fluid when the needle containing blood was dipped into fluid for less than 1 second. Approximately 0.35 and 0.01 I~1 are transmitted to fluid from 20-gauge and 27-gauge needles, respectively. When parafilm or h u m a n skin was used as a barrier over the fluid, the volume of blood transmitted was reduced by 27% to 75% by skin and 9% to 50% by parafilm (Table 2). The 20-gauge needle transferred 30 to 50 times the volume of blood transferred by a 27-gauge needle, so that injury from the large-bore needle is m a n y times more likely to transfer enough blood to cause infection than injury from a small-bore needle. The volume of blood transmitted to m e d i u m after a 22-gauge needlestick with parafilm as a

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barrier (0.2 ~1) is less than previously reported (1.4 ~1).s This difference may be due to several factors. In our current study, only one third of the needle was dipped into blood when blood was d r a w n into the syringe and only 3 m m of the needle was then inserted into fluid, where it remained for less than 1 second. It is likely that these conditions simulate those of a needlestick injury more closely. In most needles (more than 60%) tested after exposure to normal blood, at least one viable MNC was transmitted to fluid through even the smallest gauge of needle. Blood from patients with AIDS has reduced number of MNCs and may therefore transfer fewer of these cells during needlestick injury. Our model consisted of wells or vials covered by either h u m a n skin or parafilm. We plan to investigate further the volume of blood and n u m b e r of MNCs and of viable HIV particles transferred from solid needles used in suturing. We believe that more accurate risk assessment can be obtained by developing a model that would include volume of blood transmitted, stage of HIV infection or HIV titer, and use and duration of antiretroviral therapy. We gratefullyacknowledgethe assistance of Hamid Mohammadi, Karen Kodsi, and Ada Lee in preparation of this article. References

1. Metier R, Ciesielski C, Marcus R, Berkelman R. Occupationally acquired HIV infection, United States [Abstract]. Abstract number WD 4178. Florence, Italy: Seventh International Conferenceon AIDS, June 16-21, 1991. 2. McCray E, Cooperative Needlestick Surveillance Group. Occupational risk of the acquired immunodeficiencysyndrome among health care workers. N Engl J Med 1986; 314:1127-32. 3. Marcus R, Cooperative Needlestick Surveillance Group. Surveillance of health care workers exposed to blood from patients infected with human immunodeficiencyvirus. N Engl J Med 1988;319:1118-23. 4. Nelson DA. Basic methodology: clinical diagnosis and management by laboratory methods. 16th ed. WB Saunders, 1979:858-918. 5. Ofosu-AppiahW, MokhtarianF, MillerA, Grob D. Characterization of in vivo-activatedT-cellclones from peripheral blood of multiple sclerosis patients. Clin Immunol Immunopathol 1991;58:46-55. 6. Mokhtarian F, Pino M, Ofosu-AppiahW, Grob D. Phenotypic and functional characterization of T cells from patients with myasthenia gravis. J Clin Invest 1990;86: 2099-108. 7. ShirazianD, HerzlichBC, MokhtarianF, Grob D. Detection of human immunodeficiencyvirus antibody and antigen (p24) in residual blood on needles and glass. Infect Control Hosp Epidemiol 1990;11:180-4. 8. Napoli VM, McGowan Jr JE. How much blood is in a needlestick? [Letter] J Infect Dis 1987;155:828.