Ethical aspects of this study were reviewed and approved by the Human Research Ethics Committee at Ashikaga Red Cross Hospital. This study was performed after obtaining informed consent from all participants upon admission. For patients below the age of 18 years, informed parental consent was also obtained. Diagnosis was based on criteria in the ICD-10, and each patient was diagnosed by two of the three psychiatrists, each of whom is a board-certified specialist for psychiatry and had >10 years of experience in psychiatry at the time of the study. Participants were recruited from the neuropsychiatric unit in Ashikaga Red Cross Hospital during the period from October 1999 to March 2018, during which there were 101 admissions with anorexia nervosa (F50.0) from 55 individual patients that were managed in our unit, all of whom had no hematological malignancies. These were categorized into the restrictive type (F50.01, anorexia nervosa, restricting type) and the binge-purge type (F50.02, anorexia nervosa, binge eating/purging type) by two of the three psychiatrists. Among a total of 55 individual patients, all of whom were Japanese, 19 had two or more consecutive admissions, which added up to a total of 101 admissions. These 101 admissions were used for hemoglobin values at admission (Table 1). Regarding the lowest levels and the rate of decrease in these three cell measures, we gathered data from admissions for which the lowest level of each blood cell measure was confirmed, that is, individual patients who showed a V-shaped recovery during the refeeding period. Conversely, we excluded the admissions in which each blood cell measure of the individual patients did not show a V-shaped recovery, who were discharged from hospital before reaching their lowest hematological values. This is because our aim in this study is to precisely investigate backgrounds behind the nadir hematological values. Thus, of 101 admissions from 55 patients, 78 admissions from 45 patients were used for the nadir hemoglobin value, 76 admissions from 43 patients for the nadir white blood cell count, and 75 admissions from 43 patients for the nadir platelet count (Table 1).
Collection of patient information
Electronic medical records of eligible participants were retrospectively reviewed. As outcome indicators, the following three measures were used: the hemoglobin value, white blood cell count, and platelet count. We determined these values at admission, determined their nadir hematological values during the refeeding period, and determined the rate of decrease (the extent of decrease divided by the value at admission). Explanatory variables included demographics (duration of illness, sex, body mass index, presence of chronic kidney diseases, anorexia nervosa subtype, i.e., restrictive or binge-purge), laboratory data at admission (blood urea nitrogen/creatinine [BUN/Cr] ratio and aspartate aminotransferase [ALT]), presence of infectious diseases, and an indicator involving treatment, i.e., the amount of caloric intake. Body mass index was calculated as the weight of the individual (in kilograms) divided by the square of the height of the individual (in meters). The BUN/Cr ratio was used to indicate the degree of volume depletion or hemoconcentration, although other conditions, e.g., protein-energy malnutrition and the catabolic state, might contribute to a higher BUN/Cr ratio in patients with anorexia nervosa . The presence of chronic kidney disease (glomerular filtration rate(GFR)＜60mL/min/1.73m2for three months or longer) was counted as an explanatory variable only for hemoglobin analysis because anemia is a common complication of chronic kidney disease . Values of ALT, an indicator of liver function, were also evaluated as an explanatory variable only for platelet count analysis, low levels of which are often resulted from liver dysfunction [22,23]. Because elevation of transaminases in patients with anorexia nervosa is common not only at admission but also during the refeeding period even with a worsening [24-26], ALT values at admission were used for platelet values at admission while the maximum ALT values during the hospital stay were used for the nadir platelet cell counts and the rate of the decrease in the values. As the ALT values were not normally distributed, they were used for this analysis only after logarithmic transformation . Regarding the presence of infectious diseases, when a patient already contracted an infectious disease at the time of admission, the presence of infectious complications at admission was used as an explanatory variable for hematological values at admission. For hematological values during the refeeding period (the nadir hematological values and the rate of decrease in the values), the presence of infectious diseases during the hospital stay (both at admission and during the refeeding period) was counted. As a treatment-related indicator, the caloric intake was measured because it frequently affects laboratory data during the refeeding period [20,28,29]. Caloric intake (in kilocalories) refers to the average total caloric intake from day 1 through day 7 [20,28,29], including both oral intake and intravenous infusion therapy. If the patient ate only half the provided 1200-kcal meal, the actual amount of total caloric intake was recorded as 600 kcal. To accurately investigate the effect of energy intake on an individual patient depending on his or her weight, we used the total caloric intake per body weight at admission for this analysis [20,29]; this measure is widely used for diet therapy for diabetes mellitus .
A laboratory panel was carried out upon admission. Regarding testsused for the measurement of the nadir level and the rate of decrease in the three blood cell measures as well as the measurement of ALT values during the refeeding period, each blood test from the second examination onward was conducted at 7:30 in the morning before breakfast. To precisely identify the nadir levels, the patients frequently underwent serial laboratory tests: 52 admissions (64.2%) were tested on the second hospital day, 43 (53.1%) on the third and fourth hospital days, 35 (43.2%) on the fifth hospital day, 32 (39.5%) on the sixth hospital day, 27 (33.3%) on the seventh hospital day, 31 (38.2%) on the eighth hospital day, 17 (21.0%) on the ninth hospital day, and 14 (17.3%) on the tenth hospital day. The second laboratory panel was carried out for 66 admissions (81.5%) by the fourth hospital day and in 75 admissions (92.6%) by the seventh hospital day. These patients basically continued to have blood tests until their blood cell counts went up or showed a V-shaped recovery. The measurement of vitamin B12, folate, and reticulocytes was not routinely conducted, but were performed as needed.
Protocol for refeeding
The initial caloric prescription for each patient was determined by the degree of malnutrition, caloric intake preceding admission, and the weight of each patient. Although caloric intake was administered mainly through oral food, intravenous infusion therapy was sometimes used and, less frequently, nasogastric feeding was also carried out. Normally, the total initial caloric prescription consisted of ~600–1400 kcal/day and was usually increased by ~200 kcal every day.
Associations between the three hematological cell measures and patient factors were investigated using values at admission, nadir values during the refeeding period, and the ratio of the decrease within each patient for these values. Regarding data from patients who received red blood cell transfusion, the hemoglobin levels immediately before transfusion were used for their nadir hemoglobin levels. The same applies to data from patients who received platelet transfusion. The general mixed model was used to deal with repetitive admissions, in which individuals were used as random intercepts and other variables as explanatory variables, i.e., demographics (duration of illness, sex, body mass index, and anorexia nervosa subtype), laboratory data (BUN/Cr ratio and ALT), presence of infectious diseases, presence of chronic kidney disease for hemoglobin analysis, and the treatment-related indicator (caloric intake). No single numerical variable had a correlation of >0.35 with other variables, indicating that all numeric variables were relatively independent, such that all variables were included in the general mixed model. For analysis of the hematological value at admission, the treatment-related indicator was excluded from the explanatory variables. The Bonferroni adjustment was used for multiple comparisons correction by multiplying the p-values by the number of comparisons, which was 9 in these analyses. We also investigated associations between the need for red blood cell transfusion and those explanatory variables using mixed effects logistic regression model.
Given the frequent occurrence of pancytopenia, correlations among the three cell measures were also investigated by using multilevel correlation analyses, which were conducted for the values at admission (N = 101), the nadir values during the refeeding period (n = 73), and the rate of the decrease in the values (n = 73). The latter two cohorts included 73 admissions with confirmed nadir levels for all three hematological cell measures. The Bonferroni adjustment was again used for multiple comparisons correction by multiplying the p-values by the number of comparisons, which was also 9. For all statistical tests, two-tailed p-values of <0.05 were considered significant. All statistical analyses were conducted with R (4.1.1).