Dynamic observation of bone marrow suppression and chromosomal aberrations in patients with acute colchicine poisoning

Yan-qing Liu, Xiao-xia Lu,2, Kai-xun Hu, Xiao-bo Peng, Yun Jiang, Li-mei Han, Zhi-qiang Ma, Ming-fei Peng,Kun Wan, Xi-gang Zhang, Ze-wu Qiu

1 Poisoning Treatment Department, Department of Hematology, the Fifth Medical Center of Chinese PLA General Hospital,Beijing 100039, China

2 Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China

Colchicine is a neutral lipophilic tricyclic alkaloid toxic to human cells containing tubulin,and primarily stops the proliferation of active cells in the mitotic metaphase, such as intestinal mucosal cells and hematopoietic cells. It is widely used in treating gout and familial Mediterranean fever, while it is also used in establishing animal and plant experimental model.In the recent study, treatment of patients affected by coronavirus disease 2019 (COVID-19) with colchicine may prove a viable path.The course of colchicine poisoning can be divided into three stages,and most of the patients died in the second stage (2-7 d, the multiple organ injury stage). The cause of death was heart failureand bone marrow hematopoietic failure in the terminal phase.Hematopoiesis is a classic sign of colchicine poisoning.Many cases of colchicine poisoning have been comprehensively reported, and the target organs included heart, digestive tract, muscle, lung, kidney, hematopoietic system, etc.This study aimed to investigate the injury of bone marrow hematopoietic system after colchicine poisoning and evaluate the therapeutic effect of bone marrow hematopoietic stimulation drugs.

METHODS

More than 50 cases of colchicine poisoning have been diagnosed and treated by the Fifth Medical Center of Chinese PLA General Hospital in the last decade. Patients with poison doses less than 0.5 mg/kg survived, and their degree of bone marrow hematopoietic injury was minor.Seven patients with toxic doses ≥0.5 mg/kg were included between December 2017 and May 2018, and 14 liquid samples were collected by puncture of the posterior superior iliac spine at different time points. These patients were previously healthy and had not taken or injected any drug within two weeks before colchicine overdose. A volume of 0.2 mL bone marrow sample was smeared for Swiss staining. Under low magnification, the degree of bone marrow proliferation, the number of cells in each line and the presence or absence of abnormal cells were observed.Under high magnification, 200 nucleated cells were counted and classified in the well-stained and well-distributed sites to ensure quality and comparability of smears and stains in all cases. Moreover, 3-5 mL bone marrow sample was collected for toxicological examination, smear,classification, and culture. All these cases provided informed consent, and this study was approved by the Ethics Review Committee of the Fifth Medical Center of Chinese PLA General Hospital.

Clinical records

Clinical characteristics including age, gender, poison dosage, visceral damage, peripheral blood and bone marrow colchicine concentration were recorded (poison concentration was detected with liquid chromatographymass spectrometer [API-3200, ABI, USA]). Bone marrow smear, chromosome karyotype + short-term lymphocyte culture during 2.5-10 d of poisoning were analyzed. Flow cytometry analysis was performed for immunophenotyping of bone marrow samples, and the expression of Wilms’ tumor gene 1 (WT1) was measured by fluorescence quantitative polymerase chain reaction(FQ-PCR). The positive rate of neutrophil alkaline phosphatase (NAP, %), score, manual classification of white blood cells and blood routine examination were examined by neutrophil alkaline phosphatase staining.

Treatment

Two patients with ingestion dose 0.5-0.8 mg/kg and four patients with ingestion dose ≥0.8 mg/kg were treated by granulocyte-colony stimulating factor (G-CSF)when their peripheral white blood cell counts (WBC)were ≤3.5×10/L and ≤9.5×10/L, respectively. Routine treatment included gastric lavage and catharsis, active carbon absorption, protection of liver and gastrointestinal tract, anti-infection, steroid therapy, component transfusion plasma, platelet (PLT), suspended red blood cells,low molecular weight heparin to improve coagulation function, aminomethylbenzoic acid, human fibrinogen(FIB), analgesia, etc. The prognoses (survival/death) were recorded. Outpatient follow-up was conducted.

RESULTS

General data of the case series

The general data is listed in Table 1. Of these seven patients, four are males. Their age was 22.29±5.88 years.The median colchicine dosage was 0.84 (interquartile range 0.5-1.0) mg/kg. The blood concentration of colchicine was 41.86±32.25 ng/mL for 1.00±0.64 d after poisoning, and their treatment duration was 13.5±7.1 d. All the patients presented with varying degrees of nausea, vomiting, abdominal pain, and diarrhea. Two of them died of developed acute respiratory failure and acute renal failure when receiving mechanical ventilation and renal replacement therapy.

Bone marrow aspiration

The results of bone marrow aspiration are shown in Table 2. The bone marrow cell hyperplasia level was examined under a low magnification microscope and evaluated by a five-grade estimation standard.The results of chromosomal abnormalities are shown in Figure 1.

Prognosis and follow-up

Of these seven patients, five survived. Six cases were treated with G-CSF. One case was given 0.5 mg/kg G-CSF, and the WBC was not recorded. Two cases were given 0.5-0.8 mg/kg G-CSF, the peripheral WBC was 1.39×10/L in 4.0-4.5 d after poisoning, and returned to normal in 4.5-5.0 d. In four patients whose G-CSF dose≥0.8 mg/kg, the peripheral blood leukocyte count was 0.05×10/L in 3.3-5.5 d, and the leukocyte count in two patients returned to normal in 5.0-5.5 d, from lowest to normal in 0.5-1.8 d. The WBC did not recover in these two patients. The leading cause of death was bone marrow hematopoietic failure. The changes of peripheral blood cells are shown in Figure 2. During the follow-up,the WBC of bone marrow and peripheral blood of two patients returned to normal 3-4 months after poisoning.

DISCUSSION

The injury characteristics of bone marrow hematopoietic system were usually analyzed with bone marrow smear, peripheral blood smear, blood coagulation function and blood routine. In this case-series study,the bone marrow smears revealed: (1) At 3-5 d after poisoning, the proliferation of granulocytes, erythroid and megakaryocytic cells in bone marrow decreased, the ratio of granulocytes to erythroid cells was abnormally high, the inhibition on erythroid cells was stronger than that on granulocytes. After 5 d, the counts of granulocytes and PLT in bone marrow and peripheral blood gradually increased, the hematopoietic function began to recover,the proliferation of bone marrow was extremely active after 10 d, the count of granulocytes in peripheral blood reached the peak, and then gradually returned to normal. (2)Granulocytes were binucleate, annular nucleus and annular rod nucleus, with rod-shaped, globular and teardrop-shaped processes, multiple globular lobulated cells, poor nuclear lobulation, mature cytoplasm and vacuoles. In one of our patient, 1 ng/mL of colchicine was detected in the bone marrow sample at 10 d after poisoning (poison dose 1 mg/kg), no marrow granules were found in the bone marrowsmear. Fifty nucleated cells were counted, 0.14×10/L of peripheral blood leukocytes, 0.04×10/L of neutrophils (NE)and 10×10/L of PLT were counted, and the sample dilution was excluded. This indicated that colchicine accumulated in the bone marrow after entering the human body, directly inhibited the mitosis of hematopoietic cells, and had obvious toxic effect on the most active primitive cells, resulting in bone marrow hematogenesis failure.

Table 1. General information, treatment and prognosis of the seven cases with colchicine poisoning

Figure 1. Results of chromosomal abnormalities. A and B: dosage 2.00 mg/kg; A: 65 h after taking the poison, there were two single gaps, three monomer fractures and one acentric centromere (red arrow); B:117 h after taking the poison; C and D: dosage 0.59 mg/kg; C: 114 h after taking the poison. D: 229 h after taking the poison, one monomer fracture (red arrow).

Table 2. Results of bone marrow sample analysis of the seven cases with colchicine poisoning

Analyses of blood coagulation function and blood routine showed that all seven patients had disseminated intravascular coagulation (DIC) in 1.15±0.72 d after poisoning. Among them, six patients were infused with fresh frozen plasma to supply coagulation factor, 2,500-5,000 U/12 h of high coagulation period without gastrointestinal bleeding and low molecular weight heparin sodium usage. FIB was injected if it was ＜ 1.5 g/L. Five patients had normal coagulation function after 4.67±1.21 d, two patients entered fibrinolytic stage 5 d after intravenous poisoning, and the administration of aminomethylbenzoic acid was ineffective. PLT consumption decreased during DIC, and reached the lowest in 4.43±1.13 d, intravascular PLT life period was 4.2 d, and bone marrow hematopoiesis injury led to the reduced PLT formation,intermittent infusion machine was used to collect PLT. The WBC in peripheral blood was mainly neutrophils, which reached the lowest in 4.84±1.06 d, the life span was 6-8 h,and six patients were treated with G-CSF. The WBC returned to normal in four survivors in 0.5-1.8 d, while it continued to decrease in two patients until death. The decreased counts of NE and PLT could lead to the aggravation of infection and bleeding. The red blood cell life span was 120 d, and after 5 d the bone marrow hematopoiesis began to recover.Patients without subcutaneous or gastrointestinal bleeding had normal red blood cell counts. Peripheral blood smear showed that some of the granulocytes had poor lobulation,and the morphology of red blood cells was normal. The results indicated that the positive rate of NAP (%) and the score of NAP were higher than normal within 10 d. NAP is an intracellular hydrolase of mature neutrophils (lobulated nucleus and rod nucleus). In peripheral blood cells, NAP is not tissue-specific for the following reasons: (1) Colchicine has direct cytotoxicity effect on cells. (2) On day 1.50±0.66,the levels of WBC 18.69 (10.03-24.59)×10/L, C-reactive protein (CRP) 36 (34-40) mg/L, and procalcitonin (PCT)2.05(1.34-2.47) ng/mL in peripheral blood were higher than normal. The expression of NAP was up-regulated to enhance the immune function and play an anti-inflammatory role. (3)In six cases with G-CSF therapy,the cell proliferation was not controlled by the body, which promoted NE maturation,stimulated the release and activation of mature granulocytes from bone marrow, enhanced NE chemotactic and phagocytic functions, and prolonged the survival time of mature NE. (4)Seven patients were intravenous adrenocortical hormones. All these factors could significantly increase the positive rate and integral of neutrophils. After 10 d, the positive rate and integral score of NAP returned to normal with the control of infection,discontinuation of G-CSF and hormone, and the clearance of colchicine.

Figure 2. The changes of peripheral blood cells. A: case 1 at 10 d; B: case 1 at 10 d; C: case 2 at 4.75 d; D: case 3 at 2.5 d; E: case 3 at 5 d; F: case 4 at 3.25 d; G: case 5 at 2.75 d; H: case 5 at 5.25 d; I: case 6 at 8 d; J: case 7 at 4 d; K: case 7 at 15 d (time after taking poison). A/E: vacuoles; C:with rod-shaped, globular and teardrop-shaped processes; D: metamyelocyte; F: annular nucleus; G/H/I/J/K: archeocyte (red arrow showed). Case 1/3/7: archeocyte; Case 2/5: poor nuclear lobulation, mature cytoplasm and vacuoles; Case 4/6: granulocytes were binucleate, annular nucleus and annular rod nucleus, with rod-shaped, globular and teardrop-shaped processes.

In addition to the injury of bone marrow hematopoietic system, our seven patients suffered from different degrees of gastrointestinal tract, liver, muscle and myocardial injuries.After supportive treatment, their condition improved, and two cases with lung and kidney failure were administered tracheal intubation mechanical ventilation and hemodiafiltration. There is no specific treatment for colchicine poisoning currently,and it is essential to recover the bone marrow hematopoietic function. G-CSF can shorten the duration of neutrophils in patients with bone marrow hematopoietic suppression,but cannot in those with bone marrow failure. In our study, two cases died. The cause of death was bone marrow hematopoietic failure followed by septic and hemorrhagic shock, and finally multiple organ dysfunctions. Since colchicine can accumulate in the bone marrow, and these patients have other organ injury,bone marrow hematopoietic failure cannot be treated with bone marrow transplantation. The feasibility of hematopoietic stem cell infusion is subject to clinical confirmation.

There is no report of secondary leukemia and genetic toxicity after colchicine poisoning. Immunophenotyping and WT1 are effective indexes for diagnosis and prognosis prediction.WT1 can inhibit or activate the gene transcription regulating the proliferation and differentiation of hematopoietic cells.A chromosome is the carrier of genetic material in the metaphase of cell mitosis. Chromosomal aberrations may predict the risk of leukemia.In this study, the immunophenotyping of bone marrow samples of the patients with acute colchicine poisoning was normal. At the same time, the expression of WT1 was increased in one case, but it had no clinical significance.

Colchicine was not considered clastogenic. Colchicine was negative for mutagenicity in the bacterial reverse mutation assay. In a chromosomal aberration assay in cultured human white blood cells, colchicine intervention resulted in micronuclei formation; these micronuclei were formed by mitotic nondisjunction without structural DNA changes. In this study, chromosome karyotype was 46XY in four cases and 46XX in the other three cases. Structural variations were found in two cases, among which one case was poisoned with 2 mg/kg colchicine, and 26 metaphase mitotic cells were observed under oil microscope 2.5 d after poisoning. Four of them were aberrant cells, including monomeric break, single space, acentric body, pyknosis,karyolysis, and chromatin agglutination in bone marrow smear. Thus, the possibility that colchicine can induce chromosomal structural abnormalities is not ruled out.The bone marrow and peripheral blood of two patients recovered to normal 3-4 months after poisoning. However,the follow-up time was short in our study, and whether there was genetic toxicity needed further clinical observation. The limitation of this study was that the bone marrow biopsy was performed at different time points (phase 2 and even phase 3) and after G-CSF treatment was given. Thus, the degree of bone marrow recovery could vary.

CONCLUSIONS

According to the dosage and body constitution,colchicine poisoning can lead to different changes from transient inhibition of bone marrow hematopoiesis to bone marrow hematopoiesis failure. In addition,some patients can develop chromosomal structural aberrations. G-CSF may be effective in patients with myelosuppression, but not in those with hematopoietic failure.

This research was supported by grants from the National Key R&D Program of China (No. 2019YFC16063000).

All these cases provided informed consent, and the study was approved by the Ethics Review Committee of the Fifth Medical Center of Chinese PLA General Hospital.

All authors declared no conflicts of interests.

ZWQ had full access to all of the data reported in this study and had final responsibility for the decision to submit this report for publication. YQL, XXL and XGZ designed research study; KXH, XBP, YJ, LMH, ZQM, MFP and KW performed research; YQL and XXL analyzed data and wrote the article. YQL and XXL contributed equally to this work.