Assessing circulatory failure

Abstract

The present invention relates to a method of identifying or monitoring circulatory failure in a subject, which method comprises assessing the subject's microcirculation in respect of the following parameters: (a) functional capillary density (FCD); (b) heterogeneity of the FCD; (c) capillary flow velocity; (d) heterogeneity of capillary flow velocity; (e) oxygen saturation of microvascular erythrocytes (SmvO.sub.2); and (f) heterogeneity of SmvO.sub.2; wherein parameters (a) to (d) are assessed visually by microscopy and parameters (e) and (f) are assessed by diffuse reflectance spectroscopy (DRS); well as apparatus and software designed for performance of such a method.

Claims

1. A method of identifying or monitoring circulatory failure in a subject using a microscope and a spectrometer for measuring diffuse reflectance, said method comprising: (i) applying a microscope emitting white unpolarized light and a light emitting probe connected to the spectrometer to the skin of a subject, and measuring: (a) functional capillary density (FCD); (b) heterogeneity of the FCD; (c) capillary flow velocity; (d) heterogeneity of capillary flow velocity; (e) oxygen saturation of microvascular erythrocytes (SmvO.sub.2); and (f) heterogeneity of SmvO.sub.2; wherein parameters (a) to (d) are measured using white unpolarized light microscopy image(s) yielded by white unpolarized light microscopy, and parameters (e) and (f) are measured by diffuse reflectance spectroscopy (DRS); (ii) comparing measurements obtained in respect of parameters (a) to (f) in the subject with measurements of the same parameters in a healthy control; and (iii) determining the deviation between the measurements of parameters (a) to (f) in the subject and the healthy control, wherein a statistically significant deviation indicates circulatory failure in said subject; and further comprising the step of ceasing, continuing or altering a therapeutic intervention or regimen based on the outcome of said determination step (iii).

2. The method claim 1, wherein the measurement comprises use of a video light microscope, images obtained therefrom, or a combination thereof.

3. The method of claim 1, wherein one or more of parameters (a) to (d) are measured using computer assisted video microscopy (CAVM), images obtained therefrom, or a combination thereof.

4. A method of making a prognosis for a subject with circulatory failure, the method comprising: (i) applying a microscope emitting white unpolarized light and a light emitting probe connected to a spectrometer to the skin of a subject, and measuring: (a) functional capillary density (FCD); (b) heterogeneity of the FCD; (c) capillary flow velocity; (d) heterogeneity of capillary flow velocity; (e) oxygen saturation of microvascular erythrocytes (SmvO.sub.2); and (f) heterogeneity of SmvO.sub.2; wherein parameters (a) to (d) are measured using white unpolarized light microscopy image(s) yielded by white unpolarized light microscopy, and parameters (e) and (f) are measured by diffuse reflectance spectroscopy (DRS); (ii) comparing measurements obtained in respect of parameters (a) to (f) in the subject with measurements of the same parameters in a healthy control; and (iii) determining the deviation between the measurements of parameters (a) to (f) in the subject and the healthy control, wherein a statistically significant deviation indicates circulatory failure in said subject; and further comprising the step of ceasing, continuing or altering a therapeutic intervention or regimen based on the outcome of said determination step (iii).

5. The method of claim 1, further comprising measurement of oxygen extraction by the microvasculature.

6. The method of claim 1, wherein the subject: (i) is being considered for or undergoing intensive care therapy, such as extra-corporeal membrane oxygenation (ECMO) or extra-corporeal life support treatment (ECLS); or (ii) is suffering from pre-eclampsia; or (iii) is suffering from sepsis; or (iv) is suffering from chronic or acute heart failure; or (v) has a chronic skin wound; or (vi) is asphyxiated; or (vii) has acute or chronic respiratory failure; or (viii) has acute or chronic limb ischaemia; (ix) has had an organ transplant; (x) has erythromelalgia.

7. The method of claim 4, further comprising measurement of oxygen extraction by the microvasculature.

8. The method of claim 1, wherein the method is computer implemented.

9. The method of claim 4, wherein the method is computer implemented.

10. The method of claim 4, wherein the subject: (i) is being considered for or undergoing intensive care therapy, such as extra-corporeal membrane oxygenation (ECMO) or extra-corporeal life support treatment (ECLS); or (ii) is suffering from pre-eclampsia; or (iii) is suffering from sepsis; or (iv) is suffering from chronic or acute heart failure; or (v) has a chronic skin wound; or (vi) is asphyxiated; or (vii) has acute or chronic respiratory failure; or (viii) has acute or chronic limb ischaemia; (ix) has had an organ transplant; (x) has erythromelalgia.

11. The method of claim 5, further comprising measurement of oxygen extraction by heterogeneity of said extraction.

12. The method of claim 7, further comprising measurement of oxygen extraction by heterogeneity of said extraction.

Description

(1) An embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings:—

(2) FIG. 1 is a schematic illustration of an apparatus for assessing a neonate's microcirculation by microscopy;

(3) FIG. 2 is a schematic illustration of an apparatus for assessing a neonate's microcirculation by DRS;

(4) FIGS. 3 (a) and (b) are Bland-Altman plots showing inter-observer (a) and intra-observer (b) reliability of functional capillary density measurements;

(5) FIGS. 4 (a) and (b) are graphs showing capillary flow categories in the chest (a) and head (b).

(6) With reference to FIG. 1, there is shown a part of a patient's body 1, a hand-held video microscope head 2, video microscope controller 3, computer 4, keyboard 5 and display unit 6.

(7) The microscope head 2 is shown in contact with the patient's body 1 as it would be when acquiring images therefrom under the control of microscope controller 3. Once images have been obtained, the microscope head 2 is removed from the patient's body 1.

(8) Images are passed via the microscope controller 3 to the computer 4 for processing. This involves analysis of the images to identify and measure/quantify the following:— (a) optionally pericapillary bleedings and/or dark haloes (number per unit area); (b) functional capillary density (FCD) (number per unit area); (c) heterogeneity of the FCD (coefficient of variation); (d) CFV or MFCV; (e) heterogeneity of CFV.

(9) In one variant of the embodiment, these characteristics are displayed on a screen for identification/analysis by a human operator who then makes appropriate entries of representative values via the keyboard 5. In another variant, image recognition software identifies capillaries (and associated bleedings and haloes) and speed of blood flow therein and assigns values automatically

(10) The computer then calculates a weighted sum of these values and outputs this to the display 6, along with the values on which it is based. This score, together with scores for Smv0.sub.2 is indicative of the degree of pathology of the neonate's microcirculation.

(11) With reference to FIG. 2, there is shown a part of a patient's body 1, a light source 7, a probe 8, a spectrometer 9, computer 4, keyboard 5 and display unit 6.

(12) The probe 8 is shown in contact with the patient's body 1 as it would be when it is emitting light received by an optical fibre from the light source 7 and receiving reflected light from the body, this reflected light being transmitted via an optical fibre to the spectrometer 9. Once the reflected light has been processed by the spectrometer 9, the probe 8 is removed from the patient's body 1.

(13) Data from the spectrometer 9 are passed to the computer 4 for recordal and processing. The spectrometer 9 generates data in the form of reflectance spectra, decomposition of the spectra is performed by the computer 4 to estimate Smv0.sub.2 and the heterogeneity of Smv0.sub.2. The computer then calculates a weighted sum of these values and outputs this to the display 6, together with the values on which it is based. This score, together with the score based on visual analysis is indicative of the degree of pathology of the neonate's microcirculation.

(14) In one embodiment the spectrometer 9 and microscope controller 3 are connected to the same computer 4. The computer 4 may analyse the collected frames/films and the spectra. In another embodiment, the frames/films and spectra may be transferred to another computer for analysis and computer(s) 4 act only to receive the data from the microscope controller 3 and spectrometer 9.

(15) A clinical study based on a further embodiment, which further comprises the use of laser Doppler perfusion measurements of neonates has been carried out and is discussed in Example 2.

(16) The present inventor was the first to appreciate the prognostic utility of analysis of the microcirculation of ICU patients. Thus, in a further aspect, the present invention provides a method of making a prognosis for a patient with circulation failure being considered for, or undergoing, intensive care therapy, comprising assessing the state of the patient's microcirculation.

(17) This aspect of the invention is particularly applicable to patients with systemic circulation failure, for example following acute cardiac pump failure, hypovolemia or sepsis, and the supportive treatment may comprise extra-corporeal life support treatment (ECLS), e.g. extra-corporeal membrane oxygenation (ECMO). The invention extends to monitoring the effect of the supportive treatment for said patient by the same means.

(18) Examinations according to the invention can be used to make a prognosis and hence improve selection of patients for life supporting treatments such as ECMO/ECLS and/or to guide such therapy as well as providing an indication of the effect of additional supportive therapy and the benefit in continuing with treatment i.e. to provide stop criteria for life supporting treatment.

(19) An embodiment of this aspect invention will now be described, by way of example only, with reference to the accompanying further drawings:—

(20) FIG. 5 is an overview of eight ECMO patients;

(21) FIGS. 6 (a) and (b) are microscope images showing pen-capillary bleedings and dark haloes respectively;

(22) FIGS. 7 (a) and (b) are graphs showing functional capillary density for three groups of ECMO patients and healthy controls; and

(23) FIG. 8 is a histogram showing the proportions of capillaries in various blood flow categories for three groups of ECMO patients.

(24) With reference to FIG. 1, there is shown a part of a patient's body 1, a hand-held video microscope head 2, video microscope controller 3, computer 4, keyboard 5 and display unit 6.

(25) The microscope head 2 is shown in contact with the patient's body 1 as it would be when acquiring images therefrom under the control of microscope controller 3. Once images have been obtained, the microscope head 2 is removed from the patient's body 1.

(26) Images are passed via the microscope controller 3 to the computer 4 for processing. This involves analysis of the images to identify and measure/quantify the following:— (a) pericapillary bleedings and/or dark haloes (number per unit area); (b) functional capillary density (FCD) (number per unit area); (c) heterogeneity of the FCD (coefficient of variation); (d) capillary flow-categorical velocity profiles (e) mean flow-categorical velocity (i.e. speed of flow in capillaries).

(27) In one variant of the embodiment, these characteristics are displayed on a screen for identification/analysis by a human operator who then makes appropriate entries of representative values via the keyboard 5. In another variant, image recognition software identifies capillaries (and associated bleedings and haloes) and speed of blood flow therein and assigns values automatically.

(28) The computer then calculates a weighted sum of these values and outputs this to the display 6, along with the values on which it is based. This score is indicative of the degree of pathology of the microcirculation.

(29) The invention is further illustrated in the following Examples in which:

(30) FIGS. 9(a) and (b) are graphs showing (a) oxygen saturation as a mean % value over time as measured by DRS and described in Example 5; and (b) the CoV of the values for oxygen saturation presented in (a).

(31) FIG. 10 is flow chart showing the timeline for the patient protocol being applied.

EXAMPLE 1

(32) 1 Patients and Methods

(33) 1.1 Patients

(34) Over a period of two years, patients treated with ECMO for cardiogenic shock in a medium sized cardiac surgical unit (approximately 550 open heart operations per year) were candidates for inclusion. Eight consecutive patients (E1-E8), two females and six males; median age 59 years (range 27-78), were included. No patients were excluded from the study. Four patients (E4-E7) were in a state of cardiogenic shock before reaching the operating theatre: one secondary to massive pulmonary embolism with ongoing mechanical cardiac compression (E4), one with massive myocardial infarction (MI) (E5), one with acute MI and complication to percutaneous coronary intervention (PCI) (E6) and finally one with endocarditis (E7). The remaining four patients suffered from post cardiotomy cardiogenic shock. Eight healthy, non-smoking male students (21-29 years old) served as controls.

(35) 1.2 Extra-Corporeal Membrane Oxygenation (ECMO)

(36) Arterial cannulation was performed in the groin in seven patients, in two of them via an end to side Dacron graft. One patient had arterial cannulation via the right subclavian artery. Seven patients had vein drainage via the femoral vein and one via the right atrium. The ECMO-circuit consisted of a centrifugal pump (Medtronics Incorporated, Minneapolis, Minn., USA), a heparin coated membrane oxygenator and tubes (Maquet Cardiovascular 72145 Hirrlingen, Germany). After establishing ECMO, all patients were initially treated with a flow of at least 4.0 I/min. Maintenance therapy on ECMO was guided by a standard protocol for the unit [38]. Weaning from ECMO was guided by trans-oesophageal ECCO Doppler in addition to clinical parameters.

(37) 1.3 Microvascular Techniques

(38) Skin microcirculation was evaluated with video microscopic measurements in eight patients and Laser Doppler measurements in six.

(39) 1.3.1. Computer Assisted Video Microscopy (CAVM)

(40) This technique involves a hand-held video-microscope applied gently on the surface of the region of interest (ROI). Immersion oil is used. Pictures or film sequences are projected and stored on a computer. For the first patient, E1, a less advanced microscope, with a 1.3 megapixel CCD, (ProScan, Bodelin technologies, OR, USA), magnifying 200 times was used. With this microscope pericapillary pathology, functional capillary density (FCD) and heterogeneity could be evaluated, but not capillary flow patterns. The remaining patients (E2-E8) were examined with another microscope (Microvision 2100, Finlay Microvision Co. Ltd., Warwickshire, UK) with higher resolution and a 500 times magnifying lens. An analogue to digital converter (Canopus, Kobe, Japan) was used to project and store the film sequences on a Mac Book pro, using the software iMovie (both Apple, Cupertino, USA).

(41) To study pericapillary pathology, calculate functional capillary density (FCD), heterogeneity of FCD and microvascular flow patterns in a ROI, the first five captured film sequences with adequate quality were used. A grid with four equally sized rectangles was made to facilitate visual analysis. The software Xscope (the Iconfactory, Greensboro, N.C., USA) was used for creating the grids.

(42) Adequate film quality was defined as focused capillaries in all four quadrants of the frame and sequence duration of at least ten seconds. FCD was defined as the mean number of visible capillaries per square millimetre. Heterogeneity of FCD was expressed as the coefficient of variation (CoV=SD/mean) of the density of capillaries in each of the four rectangles in the five film sequences (n=20). For analysis of flow velocity, each capillary of the five film sequences were visually scored into one of five groups from “no flow” (Category 0) to “brisk flow” (Category 4), and expressed in fractions (Fr=number of capillaries in each category/total number of capillaries). Based on the fractions of capillaries in each flow category, mean flow-categorial velocity was calculated in the following way:
Mean flow-categorical velocity={Fr(1)×1}+{Fr(2)×2}+{Fr(3)×3}+{Fr(4)×4}.

(43) 1.3.2 Laser Doppler Perfusion Measurements (LDPM)

(44) LDPM is a technique for quantification of microvascular perfusion. The output of the technique is given in a semi-quantitative scale of flux, defined as the product of number of moving blood cells and their mean velocity in the measured volume (<1 mm.sup.3).

(45) A Moor Blood Flow Monitor (MBF 3D) for perfusion measurements with Moorsoft (both Moor instruments, Axminster, Devon, England) was used for recording and analysis. Flux in a ROI was given as the mean value of seven consecutive measurements of a ten-second duration.

(46) 1.2.4. Measuring Procedure

(47) FIG. 6 shows skin microcirculation in fossa tabatiere in two ECMO patients. To the left (a) several pericapillary bleedings are seen, and hardly no dot-liked capillaries are present (Patient E1, ProScan (×200)). To the right (b) circular dark haloes are surrounding capillaries with no flow or extremely slow sluggish flow are seen (Patient E2 Microvision 2100 (×500)).

(48) The first microvascular examinations were performed as soon as possible, usually within 24 hours after establishing ECMO. The second measurement was performed on day 3 when possible. E4 was examined twice with an interval of only two hours before the ECMO was turned off due to ceased cerebral blood flow. Four survivors (Group 2) were controlled 18-65 days after weaning off ECMO. E3 was examined seven times. The skin on the dorsum of the hand, between the first and second metacarpus (fossa tabatiere) was examined by CAVM in all patients. From patient 3 and onwards we also assessed the skin perfusion of the medial side of the right foot, with the ROI being located one third of the distance on an imaginary line from the medial malleolus to the caput of the first metatarsus. In the patient with pericapillary bleedings (E1), additional skin areas, such as over arm, thigh, chest, and face were also examined. Six patients were measured by LDPM. Patient E1 was only measured in fossa tabatiere. From patient 4 and onwards LDPM was measured on the foot as well. CAVM and Laser Doppler measurements were performed in both locations in all controls. At the time of microvascular measurements, the corresponding central hemodynamic parameters, results from blood tests and clinical parameters were noted.

(49) 1.5 Ethical Considerations

(50) The decision to establish ECMO was taken by the responsible surgeon solely on clinical grounds. All microvascular measurements were non-invasive, and performed only after approval from the responsible surgeon and anesthesiologist. No results of microvascular findings influenced maintenance therapy on ECMO. Since patients on ECMO could not give an informed consent, next of kin, when available, consented to the microvascular assessments. Long-term survivors (n=3) gave their consent to follow-up measurements and to publication of their data. The Regional ethics committee has approved publication of data from all eight patients.

(51) 1.6 Statistics Data are presented as means with range. The coefficient of variation (CoV) was used as a parameter for heterogeneity of functional capillary density. For comparing results between outcome groups, independent t-test was used.

(52) 2 Results

(53) 2.1 Clinical Information and Outcome

(54) An overview of clinical information of the individual patients, laboratory data, and performed examinations is given in tables 1, 2 (see Annex at end of this Example) and FIG. 5. FIG. 5 is an overview over the eight ECMO patients, from start of the ECMO treatment till death or last control measurement. Measurement periods are marked according to measuring technique.

(55) The patients were grouped according to clinical outcome into two groups: patients dying on ECMO (Group 1) and patients surviving ECMO (Group 2). Mean age for group 1 was 44 years, for group 2 it was 58 years. Both sexes were present in each group. In group 2, one patient had a recovery of heart function and a maintained cerebral function but died from bleeding complications in the intensive care unit. Of the remaining four patients in group 2, one patient (E3) was transferred to another hospital where he died from multi organ failure on day 51 after establishing ECMO. The remaining three were still alive and out of institutions two years after the ECMO treatment.

(56) 2.2 Clinical Measurements

(57) White blood cell count, during the first microvascular measurements, was lower among survivors as compared with non-survivors, table 2.

(58) 2.3 Microvascular Measurements

(59) 2.3.1. Computer Assisted Video Microscopy (CAVM)

(60) 2.3.1.1 Pericapillary Pathology:

(61) E1 had numerous pericapillary bleedings in several skin locations (fossa tabatiere, volar side of forearm, leg and face) on both examinations, FIG. 3 (a). No other patients had any visible pericapillary bleedings. Patients E2 and E4 had circular dark haloes at a distance of 12±1 microns around some skin capillaries (65% in E2 and in 14% in E4), see FIG. 3 (b).

(62) 2.3.1.2 Functional Capillary Density (FCD):

(63) FIG. 4 shows functional capillary density (in capillaries/mm.sup.2) for three groups of ECMO patients and eight healthy controls (b) and coefficient of variation of functional capillary density for the same patients (a). For patients measured more than once, the first, second and follow-up measurements are given. The follow-up measurements were performed 18-65 days after the patient was successfully weaned off ECMO (n=4).

(64) The three patients dying on ECMO had significantly lower FCD in fossa Tabatiere compared with patients surviving ECMO (p=0.002), FIG. 4 (b). No difference in FCD was seen between patients in group 2 and the controls (p=0.24).

(65) From patient E3 onwards, measurements were also performed on the medial side of foot (n=6). In this location the patients had an FCD of 66.2 (range 55.6-75.8) capillaries/mm.sup.2, similar to mean FCD of the hand in patients surviving ECMO (70.8 capillaries/mm2) and controls (65.5 capillaries/mm2), as well as in the foot of controls (66.2 capillaries/mm2). Since only one patient in group 1 (E4) was measured on the foot, no further analysis was done on data from this location. Still, E4 had the lowest FCD and the highest CoV of FCD of the measured patients in this location.

(66) The second measurements of FCD in fossa tabatiere gave values similar to the first measurements.

(67) Final follow-up measurements of four (patient E5 had already died) patients in group 2 were performed 18-65 days after weaning off ECMO, FIGS. 5 and 7. One patient showed a reduced FCD on final assessment (E3, see later), while the rest of the survivors had a mean FCD of 65.5 (range 56.9-76.5) on final assessment, values in the same range as their first measurement and in controls in this location.

(68) 2.3.1.3 Heterogeneity of FCD:

(69) Patients dying on ECMO (group 1) had significantly higher CoV of skin capillaries compared with patients in group 2 (p<0.005), FIG. 7 (a). No difference was seen between the latter group and controls (p=0.74).

(70) 2.3.1.4 Capillary Flow Patterns:

(71) The mean flow-categorial velocity during the first measurements in fossa tabatiere showed no difference between patients surviving ECMO, 2.67 (range 2.53-2.87)), and controls with 2.76 (range 2.65-2.88) (p=0.17). The two patients that died on Zo ECMO had mean flow velocities of 0.5 and 1.76, significantly lower than the patients surviving ECMO (p=0.007). In one of these patients with pericapillary dark haloes (E2), erythrocyte movement in the capillaries was hardly detected in any of the film sequences, although the ECMO circuit gave an output of 4.5 litre/min. All capillaries in healthy controls had flow patterns 2 or 3, FIG. 8. Patients in group 2 had a similar flow category distribution as the controls. In the two patients in group 1 where capillary flow assessments were performed (E2, E4); the capillaries had a broadened and a left-shifted spectrum. Flow category 0 (no flow) and 1 (sluggish flow) was hardly seen in patients surviving ECMO and never seen in controls. E3 had a prolonged and complicated stay in the ICU with lung infections, sepsis episodes, and progressive renal failure after ECMO treatment. The patient died 51 days after ECMO was established due to multi organ failure. The patients final microscopy assessments on day 44 showed that FCD was reduced with 30%, CoV was increased with 64% and the mean flow-categorial velocity was reduced to 1.75, a reduction of 34%, compared with the initial measurements. On one occasion the patient was examined during an episode with gram negative sepsis (day 24); at that time the FCD was markedly reduced to 49 capillaries/mm2 (−29%), the CoV was 0.17 (−23%), and 22% of the skin capillaries had brisk flow (category 4). Capillaries in this flow category were not observed for any other patients at any time.

(72) 2.3.2 Laser Doppler Perfusion Measurements (LDPM)

(73) The controls had a mean flux value of 48 Au (range 22-96) in fossa Tabatiere, while the corresponding values at the medial side of the foot were 31.5 (range 17.4-60.1). The mean coefficient of variation was 0.20 (range 0.13-0.35) in fossa Tabatiere, and 0.25 (range 0.12-0.34) at the medial side of the foot.

(74) The laser Doppler data sets for patients are incomplete. The patients that died on ECMO (E1 and E4) had lower flux values in both locations and on both measuring occasions than any of the surviving patients or controls (tab. 3), but the differences did not reach significance, probably due to small numbers and large variability. The coefficient of variation for flux in fossa Tabatiere was significantly higher in the survivors as compare with the controls at the first measurement (p=0.03), but not for the second and third measurement at the same locations (p=0.17 and 0.74 respectively). On the medial side of the foot, coefficient of variation did not show significant changes between survivors and controls (p=0.22, 0.44 and 0.82 respectively).

(75) 3 Discussion

(76) The study was undertaken in a medium sized unit for heart surgery, with approximately 550 open heart operations a year. Both the incidence of patients in need of ECMO (0.6%) and survival of patients treated with ECMO (28%) are comparable with data from the literature. Except for white blood cell count no clinical parameters showed significant differences between the survivors and non-survivors. Significant changes were seen for all parameters of CAVM. Skin microvascular anatomy is complex with subpapillary capillaries mainly serving nutrition for epithelial proliferation and deeper vascular plexus mainly serve body temperature regulation. In adult skin, only the sub-papillary capillaries are seen in most locations with the microscopy equipment used in this study. The Laser Doppler techniques measure both superficial and the deeper plexus perfusion.

(77) 3.1 Observations Made with Computer Assisted Video Microscopy (CAVM).

(78) 3.1.1 Functional Capillary Density and Heterogeneity of Capillary Distribution:

(79) In 1920 the Danish physiologist August Krogh was awarded the Nobel Prize in Physiology or Medicine. One of his main achievements was the identification of the “Krogh cylinder”, postulating that all cells need to be located within a critical radius of a perfused capillary to survive. Cells outside this radius would experience insufficient oxygenation independent of the flow rate and erythrocyte oxygen saturation in the nearest capillary. An uneven distribution of perfused capillaries may give low oxygen tension to some cells in spite of normal Sa0.sub.2.

(80) The patients that died on ECMO had reduced FCD and increased CoV of FCD compared with patients surviving ECMO and healthy controls. The patients surviving ECMO had stable FCD and CoV within the reference levels for the controls (FIG. 7). The exception was patient E3, who had a complicated post ECMO course with several septic episodes. The last assessments, seven days before he died from multi organ failure, showed values comparable to the patients dying on ECMO see FIG. 7.

(81) In 1922 Freedlander used a microscope to show a decreased capillary density in skin in septic patients. Later studies confirmed Freedlanders findings and demonstrated increased heterogeneity of FCD in different tissues and mammalian species with systemic diseases. In patients with septic and cardiogenic shock a persistent severely reduced FCD for 24 hours in the sublingual area, is associated with increased mortality. Reduced FCD in the rectal mucosa is associated with poor prognosis in patients with severe malaria.

(82) 3.1.2 Capillary Flow-Patterns:

(83) FIG. 7 shows distribution of capillaries in each of five flow categories for patients dying on—(group 1), and patients successfully weaned off ECMO (group 2) as compared to eight healthy controls. Flow categories were defined as: 0=no flow; 1=sluggish flow (very slow cell movement, sometimes backward flow); 2=continuous low flow (cells moving continuously forward, mostly slowly); 3=continuous high flow (cells moving continuously forward, mostly rapid); 4=brisk flow (rapidly moving cells throughout the entire film sequence).

(84) In the two patients in group 1, in whom capillary flow had been analysed, a significant number of capillaries with “no flow” or “sluggish flow” were seen. These categories were hardly seen in controls or survivors. Patients in group 2 had capillary flow pattern similar to the controls. An increased number of no-flow capillaries have been described in different diseases and a positive correlation between capillaries with no-flow and high mortality has been shown. All capillaries in patient E2 had flow category 0 or 1, fifty percent in each category. Interestingly the patient at that point had biochemical markers indicating disseminated intravascular coagulation (DIC).

(85) 3.1.3 Circular Dark Haloes

(86) Dark haloes were found in two of the patients who died on ECMO (E2 and E4), with the halo edges 12±1 microns from the capillaries. In E2 numerous haloes were present, prominently around capillaries with “no flow”. E4 had fewer haloes, and they were also seen surrounding perfused capillaries. The cause of these haloes is uncertain, but one possibility is that they represent precipitated proteins or erythrocyte degradation products leaking from injured capillaries. Another possibility is that they are caused by pericapillary oedema.

(87) 3.1.4 Peripapillary Bleedings

(88) Bleeding capillaries have been described in patients with von Willebrand disease, in patients with critical lower limb ischemia, in patients with connective tissue disorders and in patients on anticoagulants. We have reported pericapillary bleedings in the tongue of septic pigs four hours after injection of N. meningitides antigen. Pericapillary bleedings was found in several skin locations of patient E1 (died on ECMO). This patient suffered from systemic lupus erythematosus. He had not been treated with anticoagulants. No macroscopic bleedings could be detected. Capillary erythrocyte leakage is the result of severe damage to the capillary wall and increased fluid leak and oedema would be expected. Patient E1 gained 30 kilos body weight during the first 24 hours on ECMO, and gained another 12 kilos between the first and second measurement.

(89) 3.2 Laser Doppler Perfusion Measurements

(90) The major part of skin perfusion takes place in the deeper thermoregulatory plexuses where perfusion is mainly regulated by sympathetic activity. Since perfusion in these plexuses mainly serve a thermoregulatory function, skin nutrition can not be assessed by the LDPM technique. Even though the two patients that died on ECMO (E1 and E4) had the lowest perfusion values of all in both locations and measuring periods, no significant differences were demonstrated du to small numbers and large data variation.

(91) 3.3 Limitations

(92) The number of included patients is small and represents a heterogeneous group with acute heart failure, while the reference data were collected from healthy young male students. Since capillary erythrocyte velocities in healthy subjects are not age dependent, we assume that our control group of young students can be used.

(93) 3.4 Possible Implications of the Findings

(94) In USA, 40% of the Medicare expenditures occurred in the last month of life and inpatient expenses accounted for over 70% of the decedents' total costs. This indicates increased use of high-tech intensive care facilities when approaching the end of the patients' lives, without gaining much improvement of life expectancy. ECMO and other circulation assist devices are costly in use. Generally accepted criteria for selection of patients for such treatment are missing. Assist devices improve central hemodynamics, but often without improving life expectancy. It is therefore a strong need for diagnostic techniques that can be used to select patients for expensive extracorporeal life support techniques, for estimation of prognosis early after establishing such treatment and to assess the effects of supportive treatment during the use of an assist device.

(95) The idea that the microscopic examination of sublingual microcirculation may serve as a prognostic indicator of critically ill patients with sepsis or cardiogenic shock seems to be accepted. In a study on 68 patients with cardiogenic shock, reduced sublingual functional capillary density was associated with development of organ failure.

(96) A case report on one patient on ECMO examined sublingually by a microscopic method (OPS) showed that capillary flow velocity varied with varying ECMO flow. The changes were most prominent in the smallest capillaries. The small sublingual capillaries correspond to the size of the nutritive skin papillary capillaries examined in our study.

(97) Our study indicates that techniques for bedside assessments of skin microcirculation can be developed to valuable clinical tools for improved handling of patients on assist devises.

(98) 4 Conclusion

(99) Microvascular examinations of skin nutritive capillaries in patients on ECMO show major structural and functional pathology in patients dying on ECMO, while patients surviving ECMO have results similar to healthy controls. The finding of intact skin microcirculatory morphology and function in survivors early after establishment of ECMO appears to be a robust and clinically useful finding implying a good prognosis. Pericapillary bleedings or dark haloes, micro-thrombi/capillaries with “no flow”, low capillary flow velocity and low functional capillary density are associated with poor prognosis.

(100) Annex—Tables

(101) TABLE-US-00001 TABLE 1 Clinical information and outcome for all study patients. Indication and Indication for ECMO Patient Gender Risk factors type of surgery ECMO duration Outcome E1 Male SLE chronic kidney Aortic and mitral Post-cardiotomy 11 days Death on graft rejection stenosis. AVR shock ECMO and MVR E2 Male NYHA III Type A aortic Post- cardiotomy 2.5 days Death on EF: 15-20% dissection. Aortic shock ECMO graft E3 Male Previous AMI and Type A aortic Post- cardiotomy 4 days Death 51 lung embolism. dissection. Aortic shock days post Redo graft operative E4 Female Leyden mutation Shock due to Cardiogenic shock 5 hours Death on Oral contraceptives massive lung ECMO embolism E5 Male Pre operative Mitral Post- cardiotomy 3 days Death 8 cardiogeneic shock insufficiency and shock hours with recent AMI CAD post MVR + CABG ECMO E6 Male Reduced EF Cardiogenic Post- cardiotomy 7 days Long- Heavy smoker shock following shock term failed PCI survivor CABG E7 Female Previous cerebral Mitral Post- cardiotomy 4 days Long- stroke. Re-do after endocarditis. shock term AVR 3 years Cardiotomy and survivor previously deposit removal E8 Male DM Type II Type A aortic Post- cardiotomy 4 days Long- Cerebral stroke dissection. Aortic shock. term Pulmonary graft survivor hypertension

(102) TABLE-US-00002 TABLE 2 Clinical and laboratory data in the ECMO patients at the time of the first microvascular measurement. Group 1 Group 2 (N = 3) (N = 5) Hemoglobin (g/dl) 9.3 (7.8-10)  10.1 (9.5-11.2) NS Erythrocytes .sup. 21 (10.0-37) 16.4 (10.0-20) NS transfusions (no. of units) Heart rate 74 (68-80)  82 (55-107) NS (beats/minute) MAP (mm Hg) 53 (45-63) 53 (45-60) NS CVP (mm Hg) .sup. 19 (13.0-28) 10 (8-18)  NS ECMO (in 3.5 (3-4)    4.1 (3.5-4.5) NS liters/minute) Intra Aortic 2 of 3 4 of 5 NS Balloon Pump? Vasoactive 3 of 3 3 of 5 NS medication? FiO.sub.2 .sup. 0.68 (0.45-1.00) 0.60 (0.5-0.7)  NS SaO.sub.2 (in percent) 86 (61-99) 98 (98-99) NS SvO.sub.2(in percent)   .sup. 70 (70 and 70) 65.4 (55-72).sup.  NS Lactate (mmol/l) .sup. 5.5 (1.2-12.5)  3 (1.3-3.6) NS pH .sup. 7.34 (7.23-7.41) .sup. 7.42 (7.36-7.45) NS pCO.sub.2(in kPa)  5.0 (4.7-5.4)  4.8 (4.4-5.5) NS Base excess   −4.3 (−12.2-(+0.3)) .sup. −0.6 (−3-(3.5)) NS Temperature 36.8 (36.5-37) .sup. 37.1 (36.9-37.5) NS (in ° C.) CRP (mg/l)   77.3 (22.0-140.0)  86.5 (22.0-173) NS WBC (10.sup.9    14 (12.0-16.0) .sup. 7.2 (5.0-12.5) P = cells/liter) 0.02 Urinary output 3 (0-10) 48 (0-100) NS (ml/hour) Dialysis (numbers 2 of 3 1 of 5 NS of patients) Cumulative positive 21 (8-30)  16.6 (10-26).sup.  NS fluid balance (in liters)

EXAMPLE 2

(103) 1. Material and Methods

(104) 1.1. Study Population

(105) During a six month period twenty-five healthy term newborns of Caucasian race with healthy mothers were enrolled within the first twenty-four hours after delivery (Table 3).

(106) TABLE-US-00003 TABLE 3 Demographic data of the study population (n = 25) Values Mean (range) Gestational age (weeks) 40.3 (38.3-42.6) Birth length (cm) 50.0 (48.0-56.0) Head circumference (cm) 35.0 (32.0-38.0) Birth weight (gram)  3425 (2946-4536) Apgar scores at 1 min 9 (7-10).sup.  Apgar score at 5 min 9 (8-10).sup.  Male gender (%) 48 Age at first measurement (hours) 15 (4-23)   Mothers age (years) 31.8 (20.0-40.0)
1.2. Microvascular Techniques
1.2.1. Computer Assisted Video Microscopy (CAVM)

(107) In vivo studies of microvascular morphology and physiology were performed by use of a hand-held digital video-microscope (Optilia, D1, Sundbyberg, Sweden) with enlargement 250×, resolution 640×480 pixels and frame rate 15 frames per second. Film sequences were projected and stored on a computer (Mac OS X, QuickTime Player). Five to seven film sequences were taken from each skin area the first three days of life. A high quality frame from each film sequence was used for analysis of capillary density and heterogeneity of distribution of capillaries within the region of interest (ROI). The frames were analyzed off-line in a quantitative way where three equidistant horizontal and three equidistant vertical lines were drawn. The software Xscope (the Iconfactory, Greensboro, N.C., USA) was used for creating the grids. Functional capillary density (FCD) was calculated as the number of microvessels crossing a grid of lines/mm line (c/mm) (De Backer et al., Am. J Respir Crit Care Med, 2002, 166(1) pp. 98-104).

(108) For analyses of inter-observer reliability of the FCD assessments, results were compared from two independent researchers (SF,TW) who blindly assessed visually judged good quality films of eight infants. For examination of the intra-observer reliability, one researcher (SF) performed the same analyses twice several months apart.

(109) Individual capillary flow patterns were analyzed in eight infants with film sequences validated as particularly good. The five best ten-second sequences with no or limited movement artefacts were selected. One experienced researcher performed the analyses (TW). The flow velocity in individual capillaries was scored in a semi quantitative five categories scale (Table 4).

(110) TABLE-US-00004 TABLE 4 Flow categories with description of flow in each category. Flow Category Description of flow 0-No flow Erythrocytes visible, no movement 1-Sluggish flow Slow cell movement, sometimes backward flow 2-Continuous low flow Continuous forward movement, mostly slowly 3-Continuous high flow Continuous forward movement, mostly rapid 4-Brisk flow Rapidly moving cells throughout the entire film sequence

(111) Data was expressed as the fraction of capillaries (Fr) in a particular flow category (number of capillaries in this category/total number of counted capillaries). Mean capillary flow-categorical velocity (MFCV) was calculated according to the formula: Fr (1)×1+Fr (2)×2+Fr (3)×3+Fr (4)×4 (Wester et al. Clin Physiol Funct Imaging 31, 2011, pp 151-8).

(112) 1.2.2. Laser Doppler Perfusion Measurements (LDPM)

(113) Microvascular perfusion was assessed with a Moor Blood Flow Monitor (MBF 3D) with Moorsoft (Both Moor instruments, Axminster, Devon, England) for recordings and analyses. The output was given in a semi quantitative scale of flux (arbitrary unit, AU) defined as the product of the number of moving blood cells and their mean velocity in the measured volume (approximately 1 mm.sup.3). Seven ten-second sequences with no or limited movement artifacts were taken from each ROI.

(114) 1.2.3. Diffuse Reflectance Spectroscopy (DRS)

(115) For measurement of microvascular oxygen saturation, a setup consisting of a spectrometer operating in the visible wavelength region (S2000, Avantes, The Netherlands) and a tungsten halogen light source (AvaLight-HAL, The Netherlands) having an effective spectral range of 450 to 800 nm was used. A polytetrafluoretylene tile (WS-2, Avantes, Netherland) enclosed in a black plastic housing was used as reference. A custom-built fiber optic probe was used for measurements with a fiber composition of three adjacent illuminating fibers (fiber diameter 400μηη) and one receiving fiber (fiber diameter 400μηη) resulting in an emitting-receiving distance for the probe of approximately 800μηη (Meglinsky et al. Med Biol Eng Comput, 2001. 39(1): pp. 44-50). Twelve spectra were collected from each ROI in 20 neonates.

(116) Analyses of the spectra were done by adapting a tissue model based on a diffusion approximation (Farrell et al., Med Phys, 1992. 19(4): pp. 879-88; Jacques, IEEE Transactions on Bio-Medical Engineering, 1989. 36(12): pp. 1 155-1 161). The model included the chromophores melanin, hemoglobin derivatives, water and a Mie and Rayleigh scattering factor. Decomposition of the spectral signature was done by a least square fit of the model to the measured spectra. The decomposition of reflected light spectra was then used to estimate the apparent content of oxy- and deoxy-hemoglobin. Microvascular oxygen saturation was compared with arterial oxygen saturation to estimate oxygen extraction.

(117) 1.2.4. Pulse Oxymetry

(118) The arterial oxygen saturation was measured using a pulse oxymeter (Masimo Set, Rad-5v, Irvine, USA) with the probe located on the right hand.

(119) 1.2.5. Skin Temperature Measurements

(120) Skin temperature was measured using a surface temperature scanner (Omega Medical, Model no. STS-101-C, USA) attached to the ROI just before measurements with the microvascular techniques. Axillary temperature was taken with an ordinary thermometer (Digitemp, Microlife Asia, Mt1671, Taiwan).

(121) 1.2.6. Bilirubin Measurements

(122) A transcutaneous bilimeter (Drager, J M 103, Drager medical, Lubeck, Made in Japan) was used to estimate bilirubin values, a factor in the DRS analysing algorithm.

(123) 1.3. Measuring Procedure

(124) To make recordings possible, it was important to have a quiet and satisfied baby. Recordings were made in a room with stable temperature around 21° C. and dimmed light. The baby was lying in its bed. CAVM, LDF and DRS were all recorded at postnatal day one, two and three. The two same researchers conducted all measurements (SF, EH). Two skin regions were defined (ROI): The skin in the centre of the dorsal side of the left hand (H) and the skin in the chest in the midline between the jugulum and left mammilla (C). During examinations the sequence of recordings were always CAVM, followed by LDF and finally DRS measurements. The chest was examined first. Oxygen saturation and skin temperature were recorded before each new technique was applied. Finally axillary temperature and transcutaneous bilirubin were measured. Baby oil (Natusan) was used as immersion oil for the video-microscope. All equipment was gently applied on the skin surface.

(125) 1.4. Ethics

(126) Written parental consent was obtained. The study was approved by the Regional Committee for Medical and Health Research Ethics, South-Eastern Norway and by the Scientific Committee of the Hospital.

(127) 1.5. Statistics

(128) Demographic data are presented as mean with range. All other variables are reported as mean with standard deviation (SD). For continuous variables, paired t-test was conducted to compare means. P-value<0.05 was considered significant.

(129) Intra-class correlation coefficient (ICC) and Bland-Altman plot were used to analyze test-retest reliability of continuous variables. The heterogeneity was expressed as the coefficient of variation (CoV=SD/mean). Statistical analyses were performed using SPSS for Windows (Statistical Package for the Social Sciences, version 18.0 SPSS Inc., Chicago, Ill., USA).

(130) 2. Results

(131) It was possible to obtain data with the three noninvasive techniques in a non-traumatic way during a time period of 30 to 45 minutes with parents present in a standard patient room. Seventeen complete data sets with all three methods were obtained. Spectroscopic examinations were not performed in the first five infants due to technical problems at the start of the study. Three infants were lost for follow up on day two and/or three due to early discharge from the maternity ward.

(132) 2.1. Computer Assisted Video Microscopy

(133) Functional capillary density (FCD) was significantly higher in the hand compared to the chest all three days (Table 5).

(134) TABLE-US-00005 TABLE 5 Functional capillary density (capillary crossings per mm line) (n = 25). Day p-value (day 1 1 2 3 versus day 3) Chest (C) 11.3 (1.5) 11.0 (1.7) 10.7 (1.6) p = 0.14 Hand (H) 13.2 (2.0) 13.2 (1.9) 12.4 (1.6) p = 0.05 p-value p < 0.001 p < 0.001 p < 0.001 Data given as mean (SD)

(135) There was a slight tendency towards a reduction in the number of FCD both in chest and hand from day one to three. The heterogeneity of FCD expressed as CoV of five repeated measurements was 11-13%.

(136) 2.1.1. Test-Retest Reliability of FCD

(137) Intra-observer reliability of the prime investigator (SF) was high (ICC 0.72 (0.54-0.83)), although there was a difference in mean scores (11.5 versus 10.7 c/mm, p<0.001). When the second set of FCD values from SF was compared with data from the more experienced researcher (TW) the mean FCD scores were similar (10.7 versus 10.8 c/mm, p=0.70). Inter-observer reliability was also good (ICC 0.54 (0.31-0.72)). The Bland-Altman plots, both for inter- and intra-observer tests, showed increased difference between the two data sets with increasingly capillary density (FIGS. 3a and b).

(138) 2.1.2. Capillary Flow Patterns

(139) The dominant capillary flow category was category three (continuous high flow), but flow category two (continuous low flow) was also represented (Table 2, FIGS. 4a and b). Flow categories zero (no flow) and four (brisk flow) were not found. The mean flow-categorical velocity (MFCV) was similar both in the chest and hand at all the three days, varying between 2.57 (0.10) and 2.71 (0.11).

(140) 2.2. Laser Doppler Perfusion Measurements

(141) The skin laser Doppler perfusion was significantly higher in the chest compared with the hand (Table 6).

(142) TABLE-US-00006 TABLE 6 Laser Doppler perfusion (AU) (n = 25). Day p-value (day 1 1 2 3 versus day 3) Chest (C) 109.1 (26.0) 101.4 (24.6) 100.8 (25.3) p = 0.62 Hand (H)  58.9 (17.5)  54.3 (15.8)  46.9 (14.8) p = 0.09 p-value p < 0.001 p < 0.001 p < 0.001 Data given as mean (SD)

(143) There was a non-significant trend towards a reduction in skin laser Doppler perfusion from day one to three both in chest and hand. The heterogeneity of perfusion expressed as CoV in seven repeated measurements at three consecutive days was 24-32%.

(144) 2.3. Diffuse Reflectance Spectroscopy

(145) The oxygen saturation of microvascular erythrocytes (Smv0.sub.2) was significantly higher in the chest compared to the hand all three days (Table 7) with no changes over time.

(146) TABLE-US-00007 TABLE 7 Microvascular oxygen saturation (SmvO.sub.2) (%) (n = 20) Day p-value (day 1 1 2 3 versus day 3) Chest (C) 88.1 (5.2)  87.8 (10.0) 86.7 (9.0)  P = 0.07 Hand (H) 79.9 (15.2) 82.7 (11.8) 82.2 (12.1) P = 0.77 p-value p < 0.05 p < 0.05 p < 0.05 Data given as mean (SD)
The heterogeneity of twelve repeated DRS measurements, expressed as CoV, was 9-18%.

(147) Oxygen extraction defined as Sa0.sub.2 minus Smv0.sub.2, showed significant difference between chest and hand with higher oxygen extraction in the hand on all three days (mean (SD)); Chest day 1-3: 14.5 (1.6), 14.1 (3.1), 11.7 (2.7); Hand day 1-3: 23.5 (3.4), 20.2 (2.8), 24.3 (3.5), There were no changes with time.

(148) 2.4. Other Results

(149) As expected, bilirubin levels increased from day one to three. Temperature, pulse and arterial oxygen saturation were stable all three days, without differences between the sexes (data not shown).

(150) 3. Discussion

(151) It was possible to obtain reproducible non-invasive skin microvascular data in a non-traumatic way in healthy term newborns using Computer Assisted Video Microscopy, Laser Doppler Perfusion Measurements and Diffuse Reflectance Spectroscopy.

(152) 3.1. The Model

(153) The cardio-pulmonary adaptation in neonates with closure of the fetal shunts mainly occurs during the first hours of life, but is not completed until days to months after birth. Other adaptive responses such as reduction of total body water accompanied by 4-7% weight loss, hemolysis of fetal erythrocytes and production of erythrocytes with adult hemoglobin, occurs during the first days to weeks. The hematocrit peaks at two hours of age and then decreases steadily over the next weeks. Our neonates were examined the first, second and third day of life when many of these adaptive processes affecting the central hemodynamics and hemorheology, and thereby microvascular perfusion, occur.

(154) 3.2. CAVM

(155) 3.2.1. Microvascular Anatomy

(156) Our newborns were not sedated and spontaneous movements sometimes reduced quality of recordings. Training was required to record high quality film sequences. In adult skin microvessels are arranged into superficial papillary nutritive capillaries ensuring the metabolic need for epithelial proliferation, and a deep and a superficial horizontal plexus mainly serving body thermoregulation. In adult hands and feet, the regions with the most differentiated structure, the superficial nutritive capillaries are seen as “dots” or “comma shapes” in the microscope. The newborn epithelium is thinner than in adults and the vascular architecture is not fully differentiated. With the microscope a disorderly network with horizontal microvessels were seen. Functional capillary density assessments were therefore assessed as number of microvessels crossing a grid of lines per mm line (c/mm).

(157) 3.2.2. Functional Capillary Density (FCD)

(158) Oxygen has a limited capacity for diffusion in biological tissues, in contrast to the diffusion capacity for C0.sub.2. August Krogh, the Nobel Prize winner in Physiology or Medicine in 1920, postulated that all cells need to be located within a critical cylinder of a perfused capillary to get sufficient oxygen supply. Within this cylinder the oxygen availability falls exponentially with increasing distance from the centre of the capillary. Cells outside such cylinders will experience lack of oxygen delivery independent of the erythrocyte oxygen saturation in the nearest capillary. This means that FCD and heterogeneity of microvessels have to be within defined limits to ensure nutrition and oxygen availability to all cells in a tissue.

(159) 3.2.3. FCD Values

(160) In the chest FCD varied between 10.7 and 11.3 c/mm, and in the hand between 12.7 and 13.2 c/mm, significantly higher in the hand compared to the chest on all three days (Table 3). There was also a clear tendency towards reduction in FCD from day one to day three for both locations.

(161) 3.2.4. Heterogeneity of Microvessels

(162) An uneven distribution of perfused capillaries may give low oxygen tension to some cells in spite of a normal Sa0.sub.2. The heterogeneity of FCD expressed as CoV in five repeated measurements was 11-13%. Healthy adults measured in fossa Tabatiere (CoV: 15-30%) had values in the same range.

(163) 3.2.5. Capillary Flow Patterns

(164) In our healthy newborns the dominant microvessels flow categories were flow category three (60-70%) and flow category two (25-35%) (table 2, FIG. 4). Flow categories zero and four were not seen. The velocities were similar in the chest and the hand all three days. We have previously found similar flow categories in the skin (Fossa Tabatiere) of healthy young adult controls (75% of skin microvessels were in flow category three, 25% in category two) using the same equipment and the same examiners.

(165) The mean capillary flow-categorical velocity (MFCV) was similar in the chest and the hand with little variation on the consecutive days (varying between 2.57 and 2.71). MFCV in the newborns were also similar to what we previously found in the healthy young adults in the skin of Fossa Tabatiere (2.56-2.88),

(166) 3.2.6. Reproducibility

(167) Reproducibility analyzes were performed by one experienced investigator (TW) and one less experienced at the start of the study (SF). The inter-observer variation in FCD was small when the second set of results from SF was compared with the results of TW (FIG. 2a). The intra-observer reliability test from SF showed a statistically significant decrease in mean FCD values from the first to the second analysis done several months apart, probably due to the effect of the learning curve (FIG. 2b), indicating the need for a training program for frame and film analysis to obtain reproducible results.

(168) 3.2.7. OPS/SDF Versus CAVM

(169) Human intra-vital microvascular microscopy has been performed for many years. For more than 10 years OPS (Orthogonal Polarization Spectral Imaging) and SDF (Side-stream dark field imaging) have been used. These systems consist of polarized green light (wavelength 550 nm) and a filtration system to visualize the microcirculation. The light is absorbed by hemoglobin and red blood cells therefore appear dark. The systems can, however, only be used on mucous membranes (the tongue), and on some areas with thin skin in human newborns. In contrast CAVM uses white light, which gives pictures in colors and the possibility to examine different skin types.

(170) 3.3. LDPM

(171) The laser Doppler principle for quantifying microvascular perfusion in tissue volumes in the range of 1 mm.sup.3, has been commercially available for nearly 40 years, but the technique has hardly any routine applications in clinical medicine. This is partly explained by the fact that the method assesses a mix of both superficial papillary capillaries, mainly serving the nutritive perfusion, and deeper plexuses, mainly serving the thermoregulatory function.

(172) In this study perfusion in the chest skin was significantly higher as compared to the hand, corresponding to a higher temperature in the chest (+2.8° C.) (Table 4). The heterogeneity of repeated measurements expressed as CoV was 24-32%, considerably higher than for the CAVM and DRS data.

(173) 3.4. DRS

(174) 3.4.1. The DRS Method

(175) Diffuse Reflectance Spectroscopy was used to assess the oxygen saturation of erythrocytes in the microcirculation (Smv0.sub.2). The measuring volume of DRS is dependent on the emitting light spectrum, the optical properties of the tissue and the design of the measuring probe. The equipment used in this study is estimated to have a measuring volume of <1 mm.sup.3 corresponding to a measuring depth of approximately 0.8 mm in skin (Meglinsky et al., supra).

(176) 3.4.2. DRS Results

(177) Microvascular oxygen saturation (Smv0.sub.2) represents the balance between oxygen supply and consumption in the measuring volume. The supply is again dependent on the product of perfusion and arterial oxygen saturation, while the consumption is dependent on the metabolic rate of the tissue. Since Sa0.sub.2 in our newborns was near to 100%, our Svm0.sub.2 data showed extraction between 12 and 20%. In the newborns both thermoregulatory and nutritive perfusion takes place in the DRS measuring volume in contrast to in adult skin where only the papillary nutritive perfusion is assessed. Parts of the perfusion in the measuring volume in the newborn may also have a transport function (the horizontal structure in newborns as compared with the vertical papillary loops in the DRS measuring volumes of adult skin).

(178) Higher Smv0.sub.2 in the chest (86-88%) compared to the hand (76-80%) may reflect the differences in the microvascular architecture, but also the lower perfusion in the hand as demonstrated by the LDPM. The lower perfusion in the hand with a compensatory higher oxygen extraction may also be a way of preventing unnecessary loss of heat.

(179) 4. Conclusion

(180) It is believed that microscopy and spectroscopy, together, are the most useful techniques for assessing skin microcirculation in neonates. They both have measuring volumes of fractions of 1 mm.sup.3 and a resolution corresponding to individual microvessels. Small measuring volumes give high resolution, but at the price of a larger variation in measured values, i.e. a larger spread in repeated measurements. This problem can be handled by using the mean of repeated measurements to express an average value from a tissue, and the spread of repeated measurements can be used to describe the heterogeneity of the microcirculation.

(181) All cells are dependent on delivery of nutrients and oxygen from the microcirculation, but assessments of microvascular function are not done in routine clinical practise. In this study we have shown that it is feasible to obtain reproducible information from the skin microvasculature in newborns. The techniques used in this study gives information on the quality of delivery of oxygen for the metabolic process necessary for growth and development.

EXAMPLE 3

Erythromelalgia and Microcirculation

(182) Erythromelalgia (EM) is a clinical syndrome characterized by erythema, increased skin temperature and burning pain in the extremities. The pain is relieved by cooling and aggravated by warming. EM is commonly divided into primary and secondary cases, depending on whether or not there is an underlying disease. Symptoms vary from mild discomfort to limbs threatening hypoxia and amputation.

(183) The pathogenesis of EM is debated. The inventor and others have proposed a hypothesis of a common final pathway of the pathogenesis: maldistribution of skin microvascular perfusion through anatomical or functional microvascular arteriovenous shunts, with increased thermoregulatory perfusion and a relative lack of nutritive capillary perfusion in affected skin. The tissue consequently becomes hypoxic, causing supplying arterioles to dilate, leading to a paradoxical situation with coexistence of hyperaemia and hypoxia. This hypothesis gives an explanation for why cooling universally reduces pain. The cooling reduces metabolism, and thereby the hypoxia; the improvement of tissue oxygenation reduces the arteriolar dilatation, and hyperaemia is less pronounced: the vicious cycle is reversed.

(184) The study described below utilises the 6 parameters of the invention as described herein to investigate erythromelalgia, an example of localised circulatory failure.

(185) Material and Methods

(186) Material

(187) Our group has gathered a group of 207 patients, the largest seen by a single group or institution in the Western world.

(188) Methods

(189) Computer Assisted Video Microscopy (CAVM).

(190) This technique involves a hand held video-microscope applied gently on the surface of the region of interest. Pictures or film sequences are projected and stored on a computer. A digital video microscope (Optilia, D1, Sundbyberg, Sweden) with enlargement 250×, resolution 640×480 and frame rate 15 fps (frames per second) is used. An analogue to digital converter (Canopus, Kobe, Japan) is used to project and store the film sequences on a Mac Book pro, using the software iMovie (all Apple, Cupertino, USA).

(191) Five to seven recordings are taken from each site. We select the five best 10 seconds sequences with no or limited movement artefacts. These sequences are being analyzed in a semi quantitative way; three equidistant horizontal and three equidistant vertical lines were drawn. The software Xscope (the Iconfactory, Greensboro, N.C., USA) is used for creating the grids. Vessel density (FCD) is calculated as the number of vessels crossing these lines divided by the total length of the lines. In each patient, the data from the five best records are averaged. Heterogeneity of FCD is expressed as the coefficient of variation (CoV=SD/mean) of the density of capillaries in the five film sequences.

(192) The red blood cells' flow pattern in the capillaries varies over time. For analysis, the flow velocity of each capillary of the five film sequences are visually scored into one of five groups from “no flow” to “brisk flow” and expressed as mean flow velocity and fraction of capillaries in each flow category (n=10). The heterogeneity of the mean flow values is expressed as CoV (Wester et al., supra).

(193) Diffuse Reflectance Spectroscopy (DRS).

(194) A spectrometric set-up is used with a spectrometer operating in the visible wavelength region (S2000, Avantes, Netherland) and a tungsten halogen light source (AvaLight-HAL, Netherland) having an effective spectral range of 450 to 800 nm. A polytetrafluoretylene tile (WS-2, Avantes, Netherland) enclosed in a black plastic housing was used as reference. A custom-built fiber optic probe is used for measurements with a fiber composition of three adjacent illuminating fibers (E 400μηη) and a receiving fiber (E 400μηη) resulting in an emitting-receiving distance for the probe of approximately 800μηη (Meglinsky I et al. supra). Twelve records are taken from each region. Spectral analysis are done by adapting a tissue model based on a diffusion approximation (Farrell J, et al. supra). The model included the chromophores melanin, hemoglobin derivatives (Zijlistra W G, et al. Visible and Near Infrared Absorption Spectra of Human and Animal Haemoglobin: Determination and Application: Brill Academic Publishers; 2000), water (Hale G M, et al. Appl Opt. 1973; 12:555-63) and a scattering factor accounting for both Mie and Rayleigh components. Decomposition of the spectral signature is made by a least square fit of the model to the measured spectra. The decomposition of reflected light spectra is then used to estimate the apparent content of oxy- and deoxyhemoglobin.

(195) Patient Protocol

(196) Shown in FIG. 10.

(197) Normal medications are allowed. Acetylic salicylic acid acid is discontinued one week before the examinations and misoprostol is optimally discontinued for 4 weeks.

(198) A maximum of 225 minutes will be allowed to complete the examinations. 1. The patient is welcomed, and anamnestic data are rechecked. Severity score during the last week and the last month will be evaluated. Menstrual cycle may influence the vascular function and is therefore recorded. It will be noted if fertile patients use oral contraceptive pills. Outside temperature is recorded. 2. The subject rests in a supine position for 15 min in a room with an ambient temperature of 23±1° C. protected from physical and psychological stress. The test extremity is stabilized with soft pillows to avoid gross movement artefacts. 3. Clinical assessments are recorded: VAS (visual analogue scale) score Skin colour/photo documentation 4. Baseline parameters are recorded: Skin temperature is measured on the pulp of the first toe on the left foot. Baseline DRS will be recorded at the pulpa and between the 1.sup.st and 2.sup.nd toe/finger. Eight 10-second sequences will be recorded. Baseline CAVM will be recorded on the terminal phalanx of the first toe, just proximal to the nail bed where the capillary loops are perpendicular to the skin surface. An alternative site is the arch of the foot containing fewer AV anastomoses. Approximately 20-second sequences of film and 8 frames (to determine capillary density and heterogeneity) are recorded. 5. The subject is heated according to the core body heating procedure (Mork C et al. J Invest Dermatol. 2004 March; 122(3):587-93) 6. Step 4 is repeated. 7. Skin needle biopsies are taken from the foot arch, and stored in deep freezer. 8. Blood samples for haematological examination and for genetic testing will be collected.

(199) We believe that microvascular arterio-venous shunting in affected skin leads to tissue hypoxia and secondary compensatory hyperperfusion.

EXAMPLE 4

Skin Microvascular Assessments to Provide a Prognosis in a Patient with Traumatic Limb Ischemia

(200) Acute traumatic limb ischemia is a challenge for trauma surgeons. Is the life and function of the limb salvageable or has the period of ischemia given irreversible tissue damage? The surgeons may have to make quick decisions being aware that wrong decisions may cause either amputation of a savable limb or development of necrosis and sepsis in a non-savable limb where a salvage procedure was tried. In the worst case the latter may contribute to a fatal outcome in a multi-traumatized patient. Currently surgeons lack predictors for limb prognosis. A way of monitoring the circulation of a re-perfused limb is therefore of great importance.

(201) To monitor these patients, clinical tests/signs like capillary refill time, skin temperature and skin color together with arterial blood pressure measurements are mainly used to evaluate the circulation of affected limbs. Blood tests like lactate, creatine kinase (CK) white blood cell count (WBC) and C-reactive protein may also be helpful, but none of these tests/signs are particularly reliable in predicting outcome in reperfused limbs.

(202) Materials and Methods:

(203) The Patient

(204) A 33 year old female patient was involved in a high-energy car crash. At the site of injury she could move the fingers of the injured arm and has maintained intact sensory function. She had a moderate severe brain concussion corresponding to a Glasgow Coma scale of 12, sixty minutes after the initial injury. She arrived at our hospital (a level I trauma center), nearly 120 minutes after the accident and was examined according to guidelines for advanced trauma and life support (ATLS).

(205) At hospital admittance, the left upper extremity was cold, pale and had no palpable pulse. X-ray showed a humerus shaft fracture, an elbow fracture with luxation, a proximal ulnar fracture and a distal antebrachi fracture. In addition CT showed an occluded left subclavian artery. Injury to the descending thoracic aorta Aortic was also shown, but this injury was not in need of repair.

(206) An axillo-brachial bypass with autologous vein and an embolectomia was done to reperfuse the arm. Arterial flow to the arm was re-established 250 minutes after the injury. The fractures were initially fixed with an external fixator. Prophylactic fasciotomy was done in the limb's entire length. At the operating theatre, the ulnar and median nerves were visualized without signs of injury After the embolectomia and by-pass procedure, the flow in the brachial artery was 180 millilitres per minute, the left hand was warm and had capillary re-fill time of two seconds.

(207) Microvascular Measurements

(208) Computer assisted video microscope (CAVM) and diffuse reflectance spectroscopy (DRS) were used. The first microvascular examinations were performed only a few hours after reperfusion surgery on the day of admittance (Day 0), and repeated on days 1, 2, 5 and 9. The skin on the dorsum of the hand, between the first and second metacarpus (fossa Tabatie're) was examined by CAVM and DRS. And each time repeated measurements were performed. Measurements of the uninjured hand served as a control. The time delay between examinations of the same monitoring modality on the two hands was only a few minutes.

(209) Computer Assisted Video Microscopy (CAVM)

(210) A hand-held digital microscope (Mediscope, OP-120 01 1), (Optilia Instruments AB Sollentuna, Sweden) with a 200× magnifying lens, a resolution 640×480 and frame rate of 15 frames per second was used. The use of the microscope and analysis of parameters is described elsewhere.

(211) CAVM parameters of interest are functional capillary density (FCD), heterogeneity of FCD, microvascular flow-patterns and pericapillary pathology. CAVM films were analyzed blindly by an experienced investigator and also by the software Java Cap (Eye catcher technologies, Linkoping, Sweden). Baby oil (Natusan) was used as immersion oil for the video-microscope. All equipment was gently applied on the skin surface.

(212) Diffuse Reflectance Spectroscopy (DRS)

(213) For measurement of microvascular oxygen saturation, a spectrometric setup consisting of a spectrometer operating in the visible wavelength region (S2000, Avantes, The Netherlands) and a tungsten halogen light source (AvaLight-HAL, The Netherlands) was used. The setup has an effective spectral range of 450 to 800 nm. A polytetrafluoretylene tile (WS-2, Avantes, Netherland) enclosed in a black plastic housing was used as reference. A custom-built fiber optic probe was used for measurements with a fiber composition of three adjacent illuminating fibers (fiber diameter 400μηη) and one receiving fiber (fiber diameter 400μηη) resulting in an emitting-receiving distance for the probe of approximately 800μηη. The estimated measuring volume of the equipment has been estimated to be in the range of 0.1 mm.sup.3. Seven spectra were collected from each hand at each measurement.

(214) Analyses of the spectra were done as described in Example 3. Microvascular oxygen saturation was compared with arterial oxygen saturation to estimate oxygen extraction.

(215) Data Presentation

(216) The results from one hand were compared to the results from the other hand taken the same day. Results from one hand were also compared to results from the same hand taken another day. A group of eight healthy male students serve as a control group.

(217) Results

(218) Clinical Course from Admittance to Day 13 (Amputation Day).

(219) The patient was treated in the intensive care unit. Except for wounds in her forehead and knee, the only injuries needing surgery were in her left arm. She was hemodynamically stable through the entire course, but received norepinephrine initially to maintain a mean arterial pressure above 70 mm Hg. She did not suffer from lung ventilation problems. She did not develop kidney failure or other organ failures. Attempts were made to wake her up at day 3, but due to confusion and exaggeration she was slept down again. On day 5 she had a fever episode (maximum temp 39.2° C.), but no other signs of infection.

(220) On day 9 she got a fever (maximum temp 39.5° C.), tachycardia (125 beats/minute) and her white cell blood count rose to 16.6×10.sup.9 cells/l. However, the concentration of C-reactive protein fell and there was not found other signs of infection. Bacteriologic tests from blood and wounds taken this day were all negative. She stabilized to day 13 when a planned inspection of the split skin graft was done. This operative procedure showed necrotic muscles in the forehand and no pulse distal of elbow. At this time there were no verified information of voluntary movements of the hand or fingers. Sensory function had not been tested The patient was brought back to the theater for an above elbow amputation. From here to discharge 12 days later the patient developed no further complications.

(221) At follow-up three months after hospital discharge all her wounds has healed. She has a left arm with amputation level 20 centimeters below the acromio-clavicular joint.

(222) Computer Assisted Video Microscopy (CAVM) Results

(223) Functional capillary density: On the first measurement, only a few hours after the reperfusion procedure, the functional capillary density was 33 percent lower in the left hand as compared to the opposite side. The next day, this difference had increased to 42 percent. At day five, no difference was seen in FCD between the two hands, neither was there a difference at day 9. The FCD values of both hands after day five were in the same range as for the healthy volunteers in Example 1.

(224) The heterogeneity of the FCD: The coefficient of variation varied between 0.20 and 0.36 on the right arm and between 0.15 and 0.35 on the left side, for both hands the highest value was the day after the accident and reperfusion.

(225) Capillary flow pattern: For all measurements except on the left arm on day 9, an overwhelming majority of capillaries were in category 2 and 3 (continuous flow). The flow pattern and mean-categorial flow index were in the same range as our healthy controls.

(226) No pericapillary pathology or dark haloes were seen at any measurements.

(227) All CAVM parameters are summarized in the table below.

(228) TABLE-US-00008 TABLE CAVM parameters during the observation period. For FCD the measured value from the right hand is 1, while the left hand is the quotient of FCD(Left)/FCD(Right). Right hand Left hand CoV of CoV of Day FCD FCD MCFV FCD FCD MCFV 0 1 0.26 2.50 0.67 0.29 2.62* 1 1 0.35 2.86 0.58 0.36 2.58 5 1 0.21 2.90 1.05 0.20 2.71 9 1 0.22 2.73 0.96 0.15 3.64
Discussion

(229) In an arm there are five main kinds of tissues: bone, fat, skin, muscle and nervous tissue. The tolerance for hypoxia varies between cells from different tissues, and is related to varying metabolic rate. For a limb to survive and function after an ischemic trauma, a minimum of cells from all kinds of tissues has to survive. In cases with traumatic ischemia the decision whether to amputate or to try limb-salvage surgery may be hard, and is related to the time from ischemia to reperfusion. After reconstructive surgery it is difficult to predict the final functional outcome related to survival of cells in the different tissues, based on clinical examination, or by assessments by transcutaneous oxygen tension measurements, or Laser Doppler perfusion assessments.

(230) In this patient skin, bone and fat tissue survived, muscle tissue partly became necrotic, while nerve tissue lost all function. An early amputation would have been safer for the patient, and resources, including 12 hours of operating time following the initial operation (vascular reconstruction and external fixation), would have been saved.

(231) Our skin microvascular examinations were able to quantify a circulatory insufficiency in the injured arm after the vascular reconstruction, as compared with reference data and data from the uninjured arm. The diagnostic sensitivity of our system is therefore sufficient to discover a nutritional problem. This case indicates that the diagnosed circulatory failure was too severe for nervous tissue to survive (nerve tissue has the highest metabolic demands and the lowest tolerance to hypoxia), the circulatory failure was at a critical level for muscle tissue survival, but was below a critical level for skin survival. Our innovation may be used to guide clinical decision-making on the difficult question of whether to perform an early amputation, or to use time and resources and add a risk of life threatening complications (like sepsis), associated with complicated reconstructions.

EXAMPLE 5

Case Study and Prognosis of a Patient Receiving Ecmo

(232) A 54 year old woman received irradiation treatment against the chest at the age of 16 because of a malignancy. She was successfully treated, but in recent years has experienced progressive chronic heart failure due to complications from the previous irradiation: Aortic valve stenosis, mitral valve stenosis and insufficiency, moderate to severe left ventricular diastolic dysfunction, in addition to moderately reduced pulmonary function. She was operated on with prosthetic replacement of the two valves. During the primary operation she came off cardio-pulmonary bypass and was transferred to the postoperative unit. In the postoperative period she was treated for insufficient heart function with inotropes and Intra-aortic balloon pumping, and with hemodialysis secondary to renal failure. On postoperative day 2 the heart failure became critical and she was connected to an ECMO system. After establishment of ECMO, clinical assessments as well as standard monitoring parameters, (central hemodynamic parameters (pressures and cardiac output), arterial oxygen saturation, blood lactate, acid base balance and cerebral oxygenation assessed with NIRS (Near infrared spectroscopy) were within reference levels, and she was regarded a candidate for heart transplantation (HTx).

(233) After establishment of ECMO, and daily thereafter she was examined according to the present invention by assessment of the 6 microvascular parameters discussed herein, namely:

(234) (a) functional capillary density (FCD);

(235) (b) heterogeneity of the FCD;

(236) (c) capillary flow velocity;

(237) (d) heterogeneity of capillary flow velocity;

(238) (e) oxygen saturation of microvascular erythrocytes (Smv0.sub.2); and

(239) (f) heterogeneity of Smv0.sub.2.

(240) Clinical course: The patient was treated with veno-arterial ECMO for 10 days. The ECMO was then converted to veno-venous ECMO and the patient died during this circulatory support on day 11. During this course she had multiple surgical revisions due to a bleeding tendency, partly related to the anticoagulation needed for the two mechanical heart valves, partly to a multi-transfusion syndrome with accompanying coagulopathy. The standard monitoring techniques showed acceptable values, and only on day 10 she was taken off the transplant list due lack of clinical progress and development of extensive ulcerations, skin necrosis, on the buttocks and the back. On day 10 the conversion from veno-arterial to veno-venous ECMO was decided because of lack of clinical progress, and ECMO was turned off on day 11, while the heart was still beating due to progression of the skin necrosis on the back and deterioration of central hemodynamic readings, and critical NIRS values indicating irreversible brain damage. The patient died shortly thereafter.

(241) The microvascular data were continually reviewed shortly after collection. The CAVM collected frames and films were scored by two independent and experienced examiners according to the impression of FCD (parameter (a)), heterogeneity of FCD (parameter (b)), CFV (parameter (c)) and heterogeneity of FCV (parameter (d)), and a written report was made. For all the films and frames at all examinations there was total agreement between the two examiners that all parameters showed values outside reference values, and it was concluded that existed a severe circulatory failure in the nutritive skin perfusion.

(242) The DRS data, parameter (e) and (f), are shown in FIGS. 9(a) and (b).