Chandra K. Solanki

Pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome

Rationale Acute respiratory distress syndrome (ARDS) affects over 200 000 people annually in the USA. Despite causing severe, and often refractory, hypoxaemia, the high mortality and long-term morbidity of ARDS results mainly from extra-pulmonary organ failure; however the mechanism for this organ crosstalk has not been determined. Methods Using autologous radiolabelled neutrophils we investigated the pulmonary transit of primed and unprimed neutrophils in humans. Flow cytometry of whole blood samples was used to assess transpulmonary neutrophil priming gradients in patients with ARDS, sepsis and perioperative controls. Main results Unprimed neutrophils passed through the lungs with a transit time of 14.2 s, only 2.3 s slower than erythrocytes, and with <5% first-pass retention. Over 97% of neutrophils primed ex vivo with granulocyte macrophage colony-stimulating factor were retained on first pass, with 48% still remaining in the lungs at 40 min. Neutrophils exposed to platelet-activating factor were initially retained but subsequently released such that only 14% remained in the lungs at 40 min. Significant transpulmonary gradients of neutrophil CD62L cell surface expression were observed in ARDS compared with perioperative controls and patients with sepsis. Conclusions We demonstrated minimal delay and retention of unprimed neutrophils transiting the healthy human pulmonary vasculature, but marked retention of primed neutrophils; these latter cells then ‘deprime’ and are re-released into the systemic circulation. Further, we show that this physiological depriming mechanism may fail in patients with ARDS, resulting in increased numbers of primed neutrophils within the systemic circulation. This identifies a potential mechanism for the remote organ damage observed in patients with ARDS.

Side-to-side symmetry of radioprotein transfer from tissue space to systemic vasculature following subcutaneous injection in normal subjects and patients with breast cancer

Quantitative lymphoscintigraphy can be used for investigation of unilateral lymphatic disease of the limbs, such as breast cancer-related lymphoedema (BCRL). Previous studies have compared lymphatic function in the affected limb with that in the unaffected contralateral limb. This study aims to confirm that the assumption of pre-morbid symmetry, never previously demonstrated, is valid. A dual-isotope technique, with bilateral subcutaneous hand injection of polyclonal human immunoglobulin G (HIgG) labelled with either technetium-99m or indium-111, was performed on a total of 37 subjects. The use of two different labels, one for each limb, enabled comparison not only of the rate of clearance from the injection depot, but also of the rate of appearance in venous blood. Results demonstrate clear symmetry between the two arms with respect to both depot clearance and blood appearance rates, as well as the coupling between these two variables. In unilateral lymphatic disease, results of quantitative lymphoscintigraphy should be expressed in relation to the normal arm rather than to an independent control population.

Use of 111-Indium-labelled autologous eosinophils to establish the in vivo kinetics of human eosinophils in healthy subjects

Eosinophils are the major cellular effectors of allergic inflammation and represent an important therapeutic target. Although the genesis and activation of eosinophils have been extensively explored, little is known about their intravascular kinetics or physiological fate. This study was designed to determine the intravascular life span of eosinophils, their partitioning between circulating and marginated pools, and sites of disposal in healthy persons. Using autologous, minimally manipulated 111-Indium-labeled leukocytes with blood sampling, we measured the eosinophil intravascular residence time as 25.2 hours (compared with 10.3 hours for neutrophils) and demonstrated a substantial marginated eosinophil pool. γ camera imaging studies using purified eosinophils demonstrated initial retention in the lungs, with early redistribution to the liver and spleen, and evidence of recirculation from a hepatic pool. This work provides the first in vivo measurements of eosinophil kinetics in healthy volunteers and shows that 111-Indium-labeled eosinophils can be used to monitor the fate of eosinophils noninvasively.

Tissue-to-Blood Transport of Radiolabelled Immunoglobulin Injected into the Web Spaces of the Hands of Normal Subjects and Patients with Breast Cancer-Related Lymphoedema

Aim: The ability to return interstitial protein to central blood is key to the defence against oedema. The aim of this study was to quantify this ability by measuring the rate at which radiolabelled human immunoglobulin (HIgG) accumulated in blood following injection into the subcutis of the hand in normal volunteers and in patients with breast cancer-related lymphoedema (BCRL). Methods: A total of 37 control subjects (healthy normal volunteers or breast cancer patients prior to treatment) and 18 women with BCRL were studied with dual-isotope lymphoscintigraphy. Each received bilateral subcutaneous depot injection in the dorsal web space of HIgG labelled with Tc-99m on one side and In-111 on the other. Activities remaining at the depot and accumulating in blood were measured at regular intervals for 3 h. Clearance from the depot was exponential and expressed as the rate constant kdepot (min–1). Accumulation in blood was essentially linear and, using an estimate of blood volume based on height and weight, was expressed as the linear constant bblood (% administered activity·min–1). The time axis intercept of this linear fit was recorded as an index of the minimum time to arrival of radioprotein in blood. The efficiency with which radioprotein that has left the depot (extra-depot activity) is transported into blood [tissue-to-blood (T-B) transport] was quantified (1) as the quotient bblood/kdepot, and (2) as a function of time after injection by comparing the total amount of radioprotein in blood at any time with the total amount of radioprotein that was no longer in the depot at the same time. Results: Tc-99m-HIgG and In-111-HIgG behaved similarly and are interchangeable. At all times between 60 and 180 min in controls, about 50% of protein that had left the depot was present in blood. T-B transport was reduced to about 20% in BCRL arms in which the hand was involved in swelling (p < 0.001 versus controls), but remained unchanged in patients in whom the hand was spared. The minimum time to arrival of radioprotein in blood was not reduced in BCRL; on the contrary, there appeared to be a small proportion of injected activity that arrived rapidly in blood in BCRL patients but not in controls. Conclusion: We conclude that T-B transport is only impaired in BCRL when radioprotein is injected into swollen tissue. Significant quantities of radioprotein may escape from the arm via local access to blood. Individual variation in this capacity may explain the regional sparing observed in BCRL.

Lymphatic drainage pathways of the breast and the upper limb

ObjectiveTo determine how often the sentinel lymph node (SLN) draining the breast is the same node as the SLN draining the upper limb. A common SLN might increase the risk of upper limb breast cancer-related lymphoedema after SLN biopsy. MethodsPatients with invasive breast cancer, identified as being suitable for axillary lymph node dissection, were injected preoperatively with 40 MBq of technetium-99m (99mTc)-human polyclonal immunoglobulin G intradermally into the ipsilateral breast and 3 MBq of indium-111 (111In)-human polyclonal immunoglobulin G intradermally into the ipsilateral hand, or vice versa. Axillary lymph nodes were removed, separated and assayed in a well counter for 99mTc and 111In. ResultsFifteen patients entered the study. In 13 of 15 patients, the ‘hottest’ lymph node for 99mTc was separate from the ‘hottest’ lymph node for 111In. In two of 15 patients the ‘hottest’ lymph node for 99mTc was also the ‘hottest’ lymph node for 111In, suggesting a common drainage pathway from the ipsilateral breast and upper limb. ConclusionAlthough the majority of patients has different pathways of lymphatic drainage from the ipsilateral breast and upper limb, in a small minority of patients the drainage pathway is through a common SLN. Such patients may be at increased risk of developing upper limb breast cancer-related lymphoedema after SLN biopsy.

Imaging of Lymphatic Vessels in Breast Cancer–Related Lymphedema: Intradermal Versus Subcutaneous Injection of 99mTc-Immunoglobulin

OBJECTIVE The disordered physiology that results from axillary lymph node clearance surgery for breast cancer and that leads to breast cancer-related lymphedema is poorly understood. Rerouting of lymph around the axilla or through new pathways in the axilla may protect women from breast cancer-related lymphedema. The aim of the study was to compare intradermal with subcutaneous injection of technetium-99m ((99m)Tc)-labeled human polyclonal IgG (HIG) with respect to lymphatic vessel imaging. MATERIALS AND METHODS Six women with breast cancer-related lymphedema underwent unilateral upper limb lymphoscintigraphy, using a web space injection of (99m)Tc-labeled HIG, after intradermal and subcutaneous injections on separate occasions. Multiple sequential images were obtained of the affected upper limb and torso over 3 hr on each occasion. Accumulation of activity in blood was quantified from venous blood samples taken from the opposite arm. RESULTS Imaging after intradermal injection clearly showed discrete lymphatic vessels in five of six patients, in contrast to imaging after subcutaneous injection, which did not show any discrete vessels in any patient. Intradermal injection resulted in more rapid visualization of cutaneous lymph rerouting than subcutaneous injection in six of six patients. Recovery of injected (99m)Tc-labeled HIG in venous blood was greater after intradermal injection in six of six patients. CONCLUSION In patients with breast cancer-related lymphedema, lymphatic vessels are more clearly depicted after intradermal than subcutaneous injection as a result of direct access of radiotracer to dermal lymphatics. This finding has implications for imaging lymphatic vessel regeneration and lymph rerouting.

Utility of 111In-labelled leucocyte scintigraphy in patients with fever of unknown origin in an era of changing disease spectrum and investigational techniques.

Background111In-labelled leucocyte, imaging is often used to investigate patients with fever of unknown origin (FUO). Its diagnostic performance, however, has been variable and a broad range of sensitivities and specificities have been reported. The purpose of this investigation was to evaluate the usefulness of 111In-labelled leucocytes scintigraphy in the detection of a cause of FUO in the light of a changing spectrum of diseases causing it and advances in investigational techniques. Materials and methodsSixty-one patients with a clinical diagnosis of FUO underwent whole-body 111In-troponolate-labelled leucocyte scintigraphy in our department over a 2 year period between February 2004 and February 2006. Of these, 54 patients were retrospectively reviewed to identify a cause of FUO. Other parameters such as C-reactive protein (CRP), leucocyte count and radiological findings were also evaluated. ResultsLeucocyte scintigraphy was found to be true positive in 12 patients, true negative in 24 patients, false positive in 10 patients and false negative in eight patients. The overall sensitivity of scintigraphy was 60%, specificity 71%, positive predictive value 55%, and negative predictive value 75%. There was no difference in the scintigraphic sensitivity between patients with spontaneous FUO and those with post-operative FUO although the latter showed a higher specificity and PPV. CRP and leucocyte count did not differ significantly between patients with true positive and true negative scintigrams. Overall, 83% of patients with abnormal radiological examinations had positive findings on scintigraphy and 87% of patients with negative findings on radiology had normal scintigraphy. ConclusionDespite changes in disease spectrum and advances in investigational techniques, our results suggest that 111In-leucocyte scintigraphy is still a useful technique in establishing the cause of FUO. A higher PPV of this test in post-operative situations makes it especially applicable in this category of patients. Equally, the higher NPV in patients with spontaneous FUO virtually excludes infection/inflammation. Finally, a higher pre-test probability based on the radiological tests seems to be important in the optimal use of leucocyte imaging.

Measuring whole-body neutrophil redistribution using a dedicated whole-body counter and ultra-low doses of 111Indium

Eur J Clin Invest 2010; 41 (1): 77–83

A pilot study of dual-isotope lymphoscintigraphy for breast sentinel node biopsy comparing intradermal and intraparenchymal injection

AIMS Identification of sentinel lymph nodes (SLN) may depend on the tissue plane of tracer injection. To explore this, we developed a dual-isotope technique to compare the lymphatic drainage basins accessed by intradermal and parenchymal injections. METHODS Fifteen breast cancer patients had simultaneous parenchymal and intradermal injections of (99m)Tc-labelled human immunoglobulin G (HIG) and (111)In-HIG, respectively, 2-4h before axillary lymph node clearance surgery. All 228 freshly dissected nodes were assayed by well counting and examined for metastatic disease by haematoxylin/eosin staining and immuno-histochemistry. RESULTS Total nodal uptake following intradermal injection was 10 times more than after parenchymal injection. Tracer uptake within the first three draining nodes divided patients into three groups; four (group 1) had identical 1st, 2nd and 3rd echelon nodes, six (group 2) had identical 1st and 2nd echelon nodes and five (group 3) had different 1st echelon nodes. With respect to the first, second and third groups, there was close, moderate and poor correlation (Pearson), respectively, between individual nodal counts accumulated from the two injection sites. Of eight patients with nodal disease, the SLN identified by intradermal and parenchymal injections contained disease in seven and four patients, respectively. CONCLUSIONS Comparison of nodal tracer distributions from the two injection planes allows a functional model to be developed with two possible routes of drainage from the parenchymal plane, one joining the tract from the areolar plexus and the other passing independently to the axilla which builds upon Sappeys original anatomical model. This may explain the variable uptake, discordance and false negative SLN identification.

Measurement of the extraction fractions of nanocolloid and polyclonal immunoglobulin by axillary lymph nodes in patients with breast cancer.

Background99mTc nanocolloid (99mTc-NC) is the most widely used tracer for lymphoscintigraphy, although others have been proposed, including radiolabelled proteins such as human serum albumin and polyclonal human immunoglobulin G (HIG). The extraction fraction of such tracers by individual nodes is clearly important but has not previously been measured in humans. MethodsPatients scheduled for axillary clearance surgery (three groups) received dual-labelled radiotracers 2–4 h before surgery: group 1 (3 patients) received 99mTc-NC (10 MBq) and 111In-HIG (2 MBq) as a mixture (0.2 ml) into the breast parenchyma above the primary tumour; group 2 (3 patients) received 99mTc-HIG (10 MBq) and 111In-HIG (2 MBq) as a mixture (0.2 ml) into the breast parenchyma above the primary tumour; and group 3 (4 patients) received 99mTc-HIG (10 MBq) and 111In-HIG (2 MBq) separately (both 0.2 ml) into the breast parenchyma above the tumour and the intradermal plane at the areola. All resected nodes were counted for 99mTc and 111In in a well-type scintillation counter. In group 1, nodes were ranked according to their 99mTc uptake. In groups 2 and 3, nodes were ranked separately according to their respective 99mTc and 111In uptakes. If nodes are arranged in linear order and each node extracts a constant fraction of incoming tracer, then the activity in the nodes would decrease exponentially with an individual nodal extraction fraction, E, equal to 1−e−k, where k is the rate constant of decrease. ResultsIn the first group, 99mTc-NC and 111In-HIG identified the same sentinel and second echelon nodes. The observed decrease in nodal activity was exponential in all groups, at least for the first five nodes. Average values for E, based on the first five nodes were 0.69 (range 0.57–0.89; n=3) for 99mTc-NC and 0.45 (0.15–0.70; n=17) for HIG (irrespective of label) (Wilcoxon rank sum, P=0.02). With respect to HIG, there was no significant difference in E between 99mTc and 111In or between deep and superficial injections in group 3. ConclusionAlthough HIG has an extraction fraction less than 99mTc-NC, the value of E is still high enough to make HIG a useful tracer for lymphoscintigraphy, especially for identifying second echelon nodes in addition to sentinel nodes and for imaging lymphatic vessels as well as lymph nodes.