Broz L.¹, Valová M.¹, Vajtr D.², Adámek T² Tomásová H. ³, Mociková H.⁴

1 Burn Centre, and ²lnstitute for Forensic Medicine, Faculty Hospital Královské Vinohrady and 3^rd Medical Faculty, Charles University, Prague,
³ lnstitutes for Medical Chemistry and Biochemistry, Faculty Hospital and 2nd Medical Faculty, Charles University, Prague,
⁴ Department of Clinical Hematology, Faculty Hospital Královské Vinohrady and 3rd Medical Faculty,
Charles University, Prague, Czech Republic

SUMMARY. We have followed in a pilot study a group of patients for cytological and biochemical changes of lavage in the upper and lower respiratory system. Into the study patients with respiratory burns confirmed by bronchoscopy and skiagraphy were included. We divided patients according to the Lung Injury Scores (LIS). We obtained the values in our sample group between intubation and last swab (before extubation). Listed are the risk factors, probability of survival. and lung histology results are listed for patients who died.

ZUSAMMENFASSUNG
Inhalationstrauma bet verbrannten Patienten - zytologische and biochemische Befunde.

Im Rahmen einer Pilotstudie haben wir bet vier Patienten die zytologische and biochemische Veränderungen in den Lavagen der oberen and unteren Atemwege beobachtet. Patienten mit Inhalationstrauma, das durch Bronchoskopie and Skiagraphie bestätigt wurde, wurden in die Studie eingegliedert. Die Patienten wurden nach der LIS (Lung Injury Score) klassifiziert. Die Daten werden zu einer Zeit zwischen der Intubation and der letzten Abnahme (nach der Extubation) erworben. Die Risikofaktoren, Überlebenswahrscheinlichkeit, and Ergebnisse der histologischen Lungenuntersuchung der Verstorbenen, werden angefůhrt.

Key words: inhalation trauma, ARDS-BAL, cytology, cytochemistry

   Burn shock is followed by a complex circulatory and microcirculatory reaction. It is a combination of hypovolemic and distribution shock and a release of local and systemic mediators. After burn injury, the patient's body reacts with multiple organ failure on several levels. Acute Phase Reaction (APR) is the first reaction with an activation of the neuroendocrine system. If the burn injury is extensive and lasts longer, APR progresses to a total change of homeostasis which lasts 1-2 weeks. This phenomenon was defined as a Systemic Inflammatory Response Syndrome (SIRS). At this point vascular endothelium and microcirculation plays an important part. SIRS can progress to an organ microcirculation and functional failure called Multi-Organ Dysfunction Syndrome (MODS). The symptoms of MODS are in the respiratory system defined as an Acute Respiratory Distress Syndrome (ARDS) with diffuse neutrophil edema. Ausbach et al. first described this syndrome in 1967. In 1994, the AmericanEuropean Consensus Conference described a milder form of the ARDS as Acute Lung Injury (ALI). Inhalation trauma is a serious complication in patients with burn injuries, worsening the patient's prognosis by 50%, causing 50100%r of the mortality. The damage to the protective epithelial barrier is a gateway to infection. There is a risk of a decubital ulcer formation in the respiratory mucus membrane with the possibility of a tracheal-esophageal fistula formation. The fistula can, in this phase of healing, cause pseudo-membranous inflammation and narrowing of the trachea. Edema in the larynx and accumulation of the secretions lead to worsening ventilation and development of atelectasis. According to Monafo et al., healing of the respiratory burn injury can take approximately 3 weeks. Respiratory burn is often complicated by inhalation of a carbon monoxide that decreases chance of survival and can cause death at the site of fire. With the inflammatory reaction, proteinases act as effective instruments for lysis of cellular and intercellular components. Neutrophil elastase and proteinase 3 in the lungs can induce apoptosis of the endothelial cells, keratinocytes, and neutrophils. Elastase further induces production of leukotrienes; macrophages and proteinase in the nonphagocyte cells directly increase intracellular reactants of the oxidation process (ROS). Combined action of proteinase and oxidative products cause damage to the permeability of the alveolocapillary membrane, and in conjunction with the metalloproteinase destroy the basal membrane.

SAMPLES AND METHODOLOGY

   Our group consisted of 17 patients from the Burn Centre (Faculty Hospital Kralovské Vinohrady) with burn injuries. We considered risk factors allowing us to evaluate probable mortality according to Ryan and Schoenfeld: the respiratory burn, age over 60 and a burn of more than 40% of the body surface. We confirmed respiratory burn in 14 patients in our group by bronchoscopy. Clinically we classified patients by the Lung Injury Score (LIS).

   During the respiratory system lavage, we have instilled 5 ml of homogenized saline. We have performed bronchoalveolar lavage within 24 hours of admission and then weekly until extubation or death of the patient. We have centrifugated cellular detritus and used it for cytological swab. We diluted cellular sediment with 1 ml of blood plasma and placed the swab on a slide. We have used a counterstain according to Papanicolaou, Mey-Grunwald-Gimsey. In nine patients, we have used cytochemical method of counterstain RNA and nucleoli according to Smetana. We have observed quantitative changes of inflammatory elements in the visual field under magnification of 400 and 1000 (per 100 neutrophil granulocytes). We dyed histological slices with hematoxilin-eosin and established the value of total protein in supernatant in seven patients by the method defined by Lowry. We determined serene proteinase and activity of elastase by spectrophotometry.

RESULTS

Clinical progression

   Six out of 17 patients in our group died. Two patients had three risk factors, where the cited mortality is 90%r. Both of these patients survived. Five out of eight patients who died had two risk factors with the cited mortality of 30%. One out of ten patients died who had one risk factor. Ten patients suffered ARDS (Fig. 1); others suffered ALI, a milder form of ARDS. All patients with ALI survived, except of one patient, who died secondary to heart failure. This patient also suffered from ischemic disease of the coronary arteries and was 91 years old. Five patients, with two risk factors who suffered ARDS died. None of the patients had symptoms of pulmonary fibrosis or chronic lymphocyte inflammation. Survival time was from 3 to 5 weeks. Nine patients were extubated the first or the second week. Two patients were extubated after the third week and six patients died. The healing course of respiratory burn was confirmed by bronchoscopy, skiagraphy and by the inflammatory signs.

Histological survey in patients who died

   Autopsy was performed in five patients after death and histological survey did not prove the presence of neutrophil granulocytes. In four out of the five patients, granulating tissue or hyaline matter was found (Fig. 2). Siderophils and macrophages were also found in four patients.

Cytochemical method of RNA and nucleoli counterstain

   We used cytochemical method of RNA and nucleoli counterstain in nine patients. Three patients were in the ALI group and six in the ARDS group. Our set consisted of non-active cells with micronucleoli with permanent RNA barrier, all segmental polynuclears. We found annular nucleoli in lymphocytes, compact nucleols in macrophages and compact nucleoli as well as micronucleoli in epithelials. Both compact and annular nucleoli in agranulocytes, identified as immature monocytes were identified in one patient, who died secondary to deterioration of condition and sepsis (Fig. 3). In addition, we found predominantly macrophage-like cells without signs of non-phagocytosis with a compact nucleolus as well as a compact nucleolus in activated lymphocytes. Sporadically, we identified immature blood elements (metamyelocytes) (Fig. 4).

Cytology and hematology

   Since the neutrophil granulocytes were in almost the entire visual field, we listed the number of other cells per hundred neutrophils in multiple visual fields. The increase of macrophages above 5 cells per 100 neutrophils was proved in 10 patients, in 3 patients that died and in 7 patients that were extubated. In one patient, who later died, accumulation of macrophages was associated with improvement of ventilation parameters to the extent that the patient was extubated within 18 hours. After that, we had to re-intubate this patient again secondary to hypoventilation and respiratory failure. In 14 patients, we found the ratio of macrophages to monocytes greater than one. Accumulation of lymphocytes greater than 10 over 100 neutrophils was exhibited in three patients. In one patient, during the fourth week was the increase of lymphocytes in lavage to 20%o CD3 positive cells. We used flow cytometry to detect Th lymphocytes CD3' , CD4' (total of 15/0 of positive cells), Tc lymphocytes CD3', GDS' (total 4.8%), B-lymphocytes CD19+ with negative findings and NK cells to CD16+, CD56+ (total 1.57%). Eosinophiles were sporadic, less than one over 100 in 13 patients. Only in 4 patients was the ratio greater. Epithelia were, according to the level of respiratory burn, usually cylindrical, occasionally tabular cells if larynx was involved. In three patients without inhalation trauma was the number of ephithelia smaller than 5 cells per 100 neutrophils. We have found Candida only after 3 weeks of antibacterial therapy.

Biochemical results

   In all evaluated patients (5 with ARDS and 2 with ALI) the total protein in lavage of upper and lower respiratory system was smaller than 22g/1. Activity of serine proteinase increased in the first week and reached 88 nkat/l. At the time of extubation in 5 patients was the level under 15 nkat/I and in 2 patients over 50 nkat/l. Activity of elastase of the neutrophil elements in all patients prior to extubation was less than 3 nkat/l, 230 nkat/I in patients who died.

DISCUSSION

   Inhalation injury is a serious complication in patients after burn injury. In a retrospective study C.M. Ryan described it as one of the risk factors decreasing chance of survival. Neutrophil exudation edema confirmed by histopathology is characteristic of ARDS. U. Pison et al. presents following distribution of inflammatory elements: ARDS without Ina contusion 5070 of neutrophils 0-2"d day, 77% 3-6" day and 60%n 7-14`x' day after injury. CH. Liatcos et al. indicates that bronchoalveolar lavage in a healthy person is comprised predominantly of macrophages (83.7%), neutrophils (approx 2.58%) and 10% lymphocytes. In a septic patient, the ratio is inversed with the increase of neutrophils to 36.3%, decrease of macrophages to 53.4% and decrease of lymphocytes to 7.3%. In our data set, the autopsy of dead patients did not prove any neutrophil exudation. According to Dakin, Griffith and Steinberg the accumulation of macrophages is a factor of positive prognosis. S. Rosseau et al. divided patients into two groups after two to three weeks from the beginning of ARDS therapy. The first group had no monocyte-like alveolar macrophages; instead they had accumulation of the mature macrophages and a good survival prognosis. The second group had increased number of monocytes with predominant immature alveolar macrophages and had increased mortality. Presence of macrophages with compact and annular nuclei is a positive factor for subsidence of acute inflammation. In our set of patients with the increased amount of macrophages to more than 5 per 100, 7 patients survived and 4 died. In the group with the lesser amount of macrophages the ratio of survived to died patients was 1:1. Macrophages were present in lavage after 2-3 weeks of treatment. W. W. Monafo et al. indicate that the length of the respiratory system healing is approximately three weeks.

   The damage to the alveolocapillary membrane determines the ratio of proteins in bronchoalveolar lavage and in the plasma. Total protein in secretions is a result of the production of the proteins in situ and a proteins transuding from plasma. Total activity of serine proteinases includes activity of a large group of enzymes that lyse for example coagulation and fibrimolysis factors including activation of metaloproteinases. During the inflammatory process, their activity increases secondary to alveomacrophages, polymorphonuclears, and pneumocytes of type 11. Elastase of the polymorphonuclears and alveolar macrophages is released from cellular granules during microbial activity, adherence, cellular migration, and phagocytosis. In all inflammatory processes, we can prove its increased activity. In control groups, the values of serine proteinase are up to 5nkal/I and activity of elastase PMN in not evident at all.

CONCLUSION

   There were 17 patients in our pilot study where respiratory burn was confirmed by bronchoscopy in 14 of these patients. We have classified these patients by LIS, used skiagraphy, and supported them by artificial ventilation. In the first 2 weeks, in all of the patients' lavage was a prevalence of polymorphonuclears. After 2-3 weeks, gradual increase of macrophages in 11 patients was proved. The presence of dislodged pseudo-membranes and epithelia in lavage in the first weeks after intubation indicated healing of the injured areas. Flow cytometry proved in one case to increase CD3' cells to 20%n. In all patients the total protein in lavage in upper and lower respiratory system was lower than 22g/í. Activity of the serine proteinase was elevated in the first week. At the time of extubation, the level in 5 patients was less than 15nkal/l Activity of elastase neutrophil elements was, at the time of extubation in all patients, less than 3nkat/l. In none of the patients, the inflammatory process became chronic or progressed to diagnosis of pulmonary fibrosis. Out of six patients that died, five had ARDS.

CONCLUSION

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