Automated measurement of reticulated platelets in estimating thrombopoiesis

Abstract: We described a fully automated measurement of reticulated platelets using a fluorescent dye, auramine O, and a reticulocyte counter, the R-3000, equipped with special software. Reproducibility and linearity were shown to be good. In the normal subjects studied (n = 60), the mean value for reticulated platelets was 0.98% ± 0.41% and the mean absolute count was 2.12 ± 0.69 × 10⁹/l. The absolute count for reticulated platelets was significantly lower (p < 0.05) in patients with reduced thrombopoiesis as seen in acute myeloblastic leukemia, aplastic anemia or chemotherapy-induced thrombocytopenia and it was elevated (p < 0.05) in essential thrombocythemia and in chronic myelocytic leukemia with thrombocytosis. All 20 patients with chronic idiopathic thrombocytopenic purpura had a high percentage of reticulated platelets. The percentage of reticulated platelets was significantly increased (p < 0.05) in patients with impaired thrombopoiesis despite the reduction in the absolute count. In 2 leukemic patients, an apparent rise was noticed in the percentage of reticulated platelets which preceded by several days a progressive increase in the platelet count at the recovery phase of thrombocytopenia. The results suggest that an automated measurement of reticulated platelets can be applied to routine laboratories for clinical use.     

High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis

To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20-fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0.05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.     

Characterization of autotransplant-related thrombocytopenia by evaluation of glycocalicin and reticulated platelets

Thrombocytopoiesis of 21 multiple myeloma patients undergoing single or double transplant regimen was characterized by measuring the level of reticulated platelets and plasma glycocalicin. Since reticulated platelets are an index of thrombopoietic activity and glycocalicin plasma values are related to platelet damage and turnover, it may be possible to perform a novel type of analysis of the thrombopoietic compartment during the mobilizing regimen and during transplant-related chemotherapy. Patients underwent mobilizing therapy and first transplant. Some randomized patients also underwent a second transplant with mobilized peripheral blood stem cells. The results show that the percentage of reticulated platelets decreased after therapy and then gradually increased in the recovery phase either during first or second transplant. By contrast, the percentage of reticulated platelets increased until day +8 and then gradually decreased during the mobilizing regimen. The glycocalicin index (glycocalicin plasma value normalized for the individual platelet count) increased significantly both during the course of mobilization and after transplant-related chemotherapy when the platelet number was at its nadir. However, the glycocalicin index was more elevated after transplant-related chemotherapy than after the mobilizing regimen. Our findings suggest that chemotherapy-related thrombocytopenia may be due to a dual mechanism: thrombocytopenia results from decreased platelet production in addition to increased platelet damage and possible destruction.     

Plasma thrombopoietin (TPO) levels and expression of TPO receptor on platelets in patients with myelodysplastic syndromes

Data on endogenous thrombopoietin (TPO) levels and their regulation in myelodysplastic syndromes (MDS) are sparse. We examined the plasma TPO level of 85 MDS patients by a sensitive enzyme immunoassay and the platelet expression of TPO receptor (TPO-R) protein, which metabolizes endogenous TPO, in 19 MDS patients with an equilibrium binding assay using ¹²⁵I-TPO. The MDS patients had higher plasma TPO levels (7.0 ± 9.3 fmol/ml) than 52 normal subjects ( P < 0.0001). Refractory anaemia (RA) patients ( n  = 39) had higher plasma TPO levels than patients ( n  = 28) with RA with excess blasts (RAEB) or RAEB in transformation (RAEB-t) ( P  = 0.0002), irrespective of similar platelet counts in these groups. The plasma TPO level correlated inversely with the platelet count in RA patients ( P  = 0.0027) but not in RAEB and RAEB-t patients ( P  = 0.7865). These data suggest that the physiological pathway for TPO production and metabolism is conserved, at least partially, in RA, but deranged in RAEB/RAEB-t. The number of TPO-R per platelet was significantly smaller in 19 MDS patients (17.5 ± 13.3) than in normals ( P  = 0.0014), but similar between RA patients and patients with RAEB and RAEB-t. Further, the bone marrow megakaryocyte count, determined in 31 MDS patients, was quite similar between RA patients and patients with RAEB or RAEB-t. Thus, in addition to thrombocytopenia, a reduced platelet TPO-R number may contribute to elevated plasma TPO levels in MDS, and a regulatory pathway for circulating TPO other than platelet TPO-R and marrow megakaryocytes, such as blasts expressing TPO-R, may operate in RAEB/RAEB-t.     

Elevated numbers of reticulated platelets in hyperthyroidism: direct evidence for an increase of thrombopoiesis

We studied thrombopoietic activity in hyperthyroidism by determination of reticulated platelet counts. At the time of hyperthyroidism 14/15 patients had higher reticulated platelets than after achievement of euthyroidism ( P  < 0.001). There was no difference in peripheral platelet counts and mean platelet volumes at the time of hyperthyroidism when compared to euthyroidism. Three patients had pan- and auto-reactive platelet antibodies during hyperthyroidism. These antibodies were directed against GPIIb/IIIa in two patients and against GPIb/IX in one patient. Our findings provide direct evidence that hyperthyroidism is associated with increased platelet production, as reflected by an increase in reticulated platelets.     

Perioperative management of MYH9 hereditary macrothrombocytopenia (Fechtner syndrome)

OBJECTIVE: Hereditary thrombocytopenias characterized by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA) are known as MYH9-related hereditary macrothrombocytopenia, and include the May-Hegglin anomaly, Sebastian platelet syndrome, Fechtner syndrome, and Epstein syndrome. Despite the presence of thrombocytopenia, these patients often have only mild or non-bleeding phenotypes. A major risk for these patients can be inappropriate treatment with long-term corticosteroids or splenectomy for misdiagnosed chronic autoimmune thrombocytopenia, as well as inadequate peri- and postoperative management. METHODS: Using the case of a 44-yr-old male with Fechtner syndrome (macrothrombocytopenia, leukocyte inclusions, sensorineural deafness, glomerulonephritis) who underwent neurosurgery for an intracerebral arteriovenous malformation, we describe current methods to diagnose hereditary MYH9-related macrothombocytopenia by analysis of the blood smear, immunofluorescence staining of the NMMHC-IIA in leucocytes, and by MYH9-gene sequencing. RESULTS: Clusters of NMMHC-IIA in granulocytes and a R1165C mutation in the MYH9-gene in two macrothrombocytopenic family members confirmed the diagnosis of a MYH9-related disease. The patient had no bleeding diathesis by history or physical examination. Thus no perioperative prohemostatic pharmacologic therapies or transfusions were given, with only minimal bleeding observed. Postoperative antithrombotic thromboprophylaxis was not given because of anticipated enhanced risk for bleeding. However, the patient developed symptomatic pulmonary embolism on postoperative day 6, which was successfully managed with 8 months of anticoagulation. CONCLUSION: MYH9-related hereditary macrothrombocytopenia does not necessarily protect against postoperative venous thromboembolism, and affected patients who do not evince bleeding diathesis should be considered for routine postoperative pharmacologic thromboprophylaxis.     

Thrombopoietin (TPO) levels in hepatic patients with thrombocytopenia

Thrombocytopenia is a common problem complicating the course of liver disease. One of the postulated mechanisms in chronic liver disease is impaired production of the hormone, thrombopoietin (TPO). The aim of present study was to evaluate the role of TPO on the occurrence of thrombocytopenia. Serum TPO levels was determined by ELISA in 40 patients with liver disease (11 seropositive with hepatitis C; 10 with mixed liver cirrhosis; 19 with bilharzial hepatic fibrosis), plus 14 normal healthy subjects as a control group. The sTPO levels were unevenly distributed among the liver disease subgroups being the highest in the group with HCV (median 1232.0, range 154.7-2042.0 pg/ml) followed by the mixed cirrhosis group (556.5; 342.0-1497.0 pg/ml) and lowest among the bilharzial hepatic fibrosis group (130.0; 22.0-204.0 pg/ml) (P<0.01). While sTPO levels in HCV and cirrhotic group were significantly higher when compared to the control group (97.0; 19.0-377.0 pg/ml), those in the Bilharzial hepatic fibrosis group were not significantly elevated (P>0.05). There is significant negative correlation between sTPO levels and spleen size (R=-0.3, P=0.043); but there was no correlation with platelet count (R=0.09, P>0.05). In addition, sTPO levels were significantly higher in patients with platelet counts >or=60x10(9)/l as compared to those with platelet counts <60x10(9)/l (P=0.04). Using the receiver operating curve (ROC) at sTPO cut off value >or= vs. <368 ng/ml, most of HCV and cirrhotic patients had higher sTPO levels (81.8 and 80.0%, respectively), while all Bilharzial hepatic fibrosis group (100%) had lower sTPO levels. In conclusion, sTPO levels had no role in the occurrence of thrombocytopenia in liver disease patients and other factors appear to be more important. It also appears that the mechanism controlling sTPO levels might be different in cirrhotic patients compared to Bilharzial hepatic fibrosis patients.     

Comparison of reticulated platelet count with plasma glycocalicin concentration as a marker of platelet turnover in patients with thrombocytopenic disorders

Measurements of plasma glycocalicin (GC) and reticulated platelets (RP) have been reported to be useful for classifying thrombocytopenic disorders. However, there have been no reports comparing the clinical usefulness of the two methods. We measured GC and RP levels simultaneously in 39 patients with idiopathic thrombocytopenic purpura (ITP), 15 patients with aplastic anemia (AA), and 17 patients with hypoplastic thrombocytopenia (HypoT) due to chemotherapy. The GC index (GC level normalized for the individual platelet count) and the percentage of RP (%RP), a parameter of platelet life span, were very high (7.5 +/- 11.4 and 20.8 +/- 13.0%, respectively) in patients with ITP as compared with those of healthy subjects (1.3 +/- 0.5 and 7.9 +/- 2.5%, respectively). However, 6 AA patients and 14 HypoT patients, in whom platelet life span is thought to be normal, also had an elevated GC index, suggestive of a false positive result. The RP, a parameter of platelet production, was low in all AA and HypoT patients except for one in each case. However, the GC level, an additional parameter of platelet production, was normal in 4 AA and 8 HypoT patients, indicating that it is not a sensitive indicator. We conclude that the RP and %RP are more feasible markers of thrombopoiesis and platelet life span, respectively, than the GC level and GC index.     

网织血小板检测在血小板减少症中的临床意义

目的 :探讨网织血小板 (RPs)检测在血小板减少症中的临床意义。方法 :采用流式细胞仪技术测定36例血小板减少性疾病患者 [其中特发性血小板减少性紫癜 (ITP) 2 2例 ,慢性再生障碍性贫血 (CAA) 9例 ,脾功能亢进 5例 ]全血中 RPs的比例和绝对值 ,观察 RPs在血小板减少性疾病中的变化 ,并与 2 3例健康对照组进行比较。结果 :健康对照组 RP s百分率和 RPs绝对值为 (2 .5 2± 1.12 ) %和 (3.32± 1.6 0 )× 10 9/ L;ITP患者的 RPs百分率为 (10 .77± 8.42 ) % ,明显高于健康对照组 ,但 RPs绝对值 [(1.74± 3.19)× 10 9/ L]却明显低于对照组 (P<0 .0 1;P <0 .0 5 ) ;CAA及脾功能亢进者的 RPs百分率接近健康对照组水平 ,分别为 (2 .92± 2 .19) %和 (1.82±0 .97) % ,但 RPs绝对值明显低于对照组 [(0 .5 8± 0 .86 )× 10 9/ L ;(0 .5 9± 0 .11)× 10 9/ L ]。结论 :采用本方法测定RPs可靠易行 ,在血小板破坏增多或血小板生成不足所致的血小板减少性疾病中 ,RPs的比例和绝对值均有相应的显著变化。在临床上可作为 ITP诊断的辅助条件 ,并可与其他血小板生成不良性疾病相鉴别     

Comparison of glycocalicin,thrombopoietin and reticulated platelet measurement as markers of platelet turnover in HIV+ samples

It is important to distinguish between decreased platelet/megakaryocyte production or increased peripheral platelet destruction as causes of thrombocytopenia. The measurement of reticulated platelets, plasma glycocalicin and thrombopoietin (TPO) levels are potentially of use as discriminators. Thrombocytopenia occurs in many HIV+ patients, and plasma glycocalicin has previously been shown to be elevated in this patient group. Reticulated platelets, glycocalicin and TPO were measured in samples from 56 HIV+ subjects and 20 healthy normal controls. The glycocalicin index (GCI - the glycocalicin levels adjusted for the platelet count) measured in HIV+ subjects was found to be significantly elevated when compared to normal controls (mean GCI 1.5 and 1.27, p = 0.04), while the percentage of reticulated platelets and TPO levels were not. Thrombocytopenic HIV+ subjects had significantly elevated mean GCI (2.8 and 1.4, p < 0.0001), TPO (85.2 and 27.2 pg/ml, p = 0.002), percentage of reticulated platelets (15.3 and 10.8%, p = 0.01), and significantly reduced absolute numbers of reticulated platelets (16.2 and 24.5 2 10 9 /l, p = 0.0004) when compared to non-thrombocytopenic HIV+ subjects. GCI and percentage of reticulated platelets exhibited a significant positive correlation ( r = 0.4, p = 0.002) in HIV+ subjects. The reticulated platelet, TPO and GCI data suggests that thrombocytopenic HIV+ subjects have normal platelet production, and increased peripheral platelet destruction.     

Evaluation of thrombopoiesis kinetics by measurement of reticulated platelets and CD34(+) cell subsets in patients with solid tumors following high dose chemotherapy and autologous peripheral blood progenitor cell support

BACKGROUND AND OBJECTIVES: The transplantation of mobilised peripheral progenitor cells has resulted in shortening of neutrophil and platelet engrafment times following high-dose chemotherapy. Since reticulated platelet percentage (PR%) has been established as a measure of bone marrow platelet production, we performed this type of analysis on the thrombopoietic compartment during transplant-related chemotherapy. DESIGN AND METHODS: Kinetics of thrombopoiesis of 19 patients with solid tumors undergoing a single or double autologous peripheral blood progenitor cell transplant was characterized by evaluating the level of RP. The correlation between CD34(+) cell subsets and the time of highest percentage of RP was also evaluated. RESULTS: The percentage of RP increases since day +8 after single transplant reaching the peak (3.4%) at day +10. In the group of patients receiving double transplant, the RP value of peak observed after second transplant is not significantly different from that one observed after the first transplant (3 vs 3.7%). In a subgroup of patients both the number of CD34(+) cells/Kg infused and the percentage of CD34(+) CD61(+) cell subsets correlate with the day of RP peak. INTERPRETATION AND CONCLUSIONS: These results suggest that RP measurement is an early indicator of engraftment. Additionally, the observation that RP percentage is high at the time of platelet transfusion in 13 out of 20 cases of transfusions (the 7 cases with low RP value being transfused during the period of obligate thrombocytopenia) suggests that the evaluation of this parameter, together with the platelet count, can be used to monitor the need for platelet transfusion.     

Comparison of glycocalicin, thrombopoietin and reticulated platelet measurement as markers of platelet turnover in HIV+ samples

It is important to distinguish between decreased platelet/megakaryocyte production or increased peripheral platelet destruction as causes of thrombocytopenia. The measurement of reticulated platelets, plasma glycocalicin and thrombopoietin (TPO) levels are potentially of use as discriminators. Thrombocytopenia occurs in many HIV+ patients, and plasma glycocalicin has previously been shown to be elevated in this patient group. Reticulated platelets, glycocalicin and TPO were measured in samples from 56 HIV+ subjects and 20 healthy normal controls. The glycocalicin index (GCI--the glycocalicin levels adjusted for the platelet count) measured in HIV+ subjects was found to be significantly elevated when compared to normal controls (mean GCI 1.5 and 1.27, p = 0.04), while the percentage of reticulated platelets and TPO levels were not. Thrombocytopenic HIV+ subjects had significantly elevated mean GCI (2.8 and 1.4, p < 0.0001), TPO (85.2 and 27.2 pg/ml, p = 0.002), percentage of reticulated platelets (15.3 and 10.8%, p = 0.01), and significantly reduced absolute numbers of reticulated platelets (16.2 and 24.5 x 10(9)/l, p = 0.0004) when compared to non-thrombocytopenic HIV+ subjects. GCI and percentage of reticulated platelets exhibited a significant positive correlation (r = 0.4, p = 0.002) in HIV+ subjects. The reticulated platelet, TPO and GCI data suggests that thrombocytopenic HIV+ subjects have normal platelet production, and increased peripheral platelet destruction.     

特发性血小板减少性紫癜患者网织血小板的测定

目的 :观察慢性血小板减少性紫癜 (c ITP)患者网织血小板百分率 (RP% )和绝对值水平及其在治疗过程中的变化。方法 :采用噻唑橙染色 ,流式细胞仪检测 6 4例正常人和 47例 c ITP患者的 RP%和 RPs绝对值。结果 :c ITP组 RP%〔(2 9.31± 16 .30 ) %〕明显高于对照组〔(5 .83± 1.81) %〕,RPs绝对值明显低于对照组 ,分别为 (6 .33± 4.5 0 )× 10 9/ L 和 (8.70± 2 .6 0 )× 10 9/ L,分别与对照组比较 ,差异有显著性意义 (P <0 .0 5 ) ;患者在接受治疗后 ,RP%和成熟巨核细胞明显降低 ,产板巨核细胞明显增高 ,分别与治疗前比较 ,差异有显著性意义 (P <0 .0 5 ) ;还观察到 ,在血小板升高前 ,RP%升高达峰值 ,此后随血小板升高 ,RP%逐渐下降。结论 :RP%和 RPs绝对值能反映 c ITP患者骨髓血小板生成活性 ,且 RP%能作为血小板恢复的预测指标     

Genetic linkage studies of thyroid peroxidase (TPO) gene in families with TPO deficiency.

We have conducted biochemical and genetic studies in five unrelated families (denoted A, C, R, P, and G), which included nine goitrous subjects (five borderline euthyroid and four hypothyroid) with complete (n = 6) or partial (n = 3) thyroid peroxidase (TPO) deficiency. Thyroid tissue was obtained from four subjects, respectively, in families A, C, and R. No iodide organification or iodide incorporation into protein was present in families A and C. The two affected siblings in family R had a low normal tissue thyroperoxidase activity. Using a 0.8-kilobase (kb) cDNA clone (pM5) encoding 30% of the cDNA of human TPO gene down-stream from basepair 730 and four restriction enzymes (TaqI, PstI, BglI, and BglII), we were unable to find any polymorphisms in family A. In another family (C) blood samples were obtained from only two family members, and consequently, it was not possible to determine linkage. DNA from families G, P, and C showed biallelic polymorphisms when digested with BglII, at 8.7 and 8.5 kb. In family R we detected two biallelic polymorphisms at 9.0 and 8.5 kb, and the 9.0-kb bands were clearly larger than 8.7-kb bands found in the other subjects. Also, there was an absence of a 4.0- to 3.9-kb band that may represent a partial gene deletion. With PstI-restricted DNA a possible deletion of 5.5-kb band was also present in affected siblings of family R. The logarithm of odds (Lod) score analyzed from the family with inbreeding (R) was compatible with linkage of disease and the TPO gene (Lod = 2.08). When this method was used with families G and P, the Lod score was inconsistent with linkage between disease and the TPO gene. These data suggest that the cause of TPO deficiency in these families is heterogeneous. However, the restriction fragment length polymorphism pattern of BglII-restricted DNA in the R family strongly suggests that a partial TPO gene deletion has occurred in this family.     

Sebastian platelet syndrome: A new variant of hereditary macrothrombocytopenia with leukocyte inclusions

Summary This report describes a new variant of hereditary macrothrombocytopenia combined with the presence of neutrophil inclusions that differ from those found in patients with May-Hegglin anomaly, the Chediak-Higashi syndrome or individuals with septicaemia and toxic Döhle bodies in polymorphonuclear leukocytes (PMN). The PMN inclusions in the family described in this report are similar to those found in patients with the Fechtner syndrome, a variant of Alport's syndrome. However, other features of Alport's syndrome, including high frequency deafness, congenital cataracts, and chronic interstitial nephritis are absent in the members of the family described here. We have named this anomaly the Sebastian platelet syndrome. The macrothrombocytopenia and neutrophil inclusions observed in this family can occur in the absence of other congenital anomalies and therefore represent a unique syndrome.     

Detection of reticulated platelets: estimating the degree of fluorescence of platelets stained with thiazole orange

The primary problem in the measurement of reticulated platelets (RP) stained with thiazole orange (TO) by flow cytometry is the definition of a threshold limit for fluorescence positivity. We evaluated settings for the threshold gate for TO positivity based on two principles: a fluorescence histogram (median FL1, Relative FL1) or a plot of forward light scatter (FSC; reflecting the distribution of the platelet size) versus fluorescence intensity (% RP). These methods were applied prospectively in examination of 54 healthy blood donors (16 females) and a total of 50 blinded patient samples: pregnant women with thrombocytopenia (Group 1A, n = 11), thrombocytopenic women after delivery (Group 1B, n = 9) and healthy women with a thrombocytopenic newborn (Group 2, n = 30). Group 1A displayed higher median FL1 (mean 306, CI 279-332) as compared to that of Group 2 (mean 266, CI 255-277; p = 0.0038) or to that of the female controls (mean 249, CI 231-268; p < 0.001). Relative FL1 was also higher in the patients of Group 1A than those of Group 2 (p = 0.037). When analysing the % RP, the difference between these groups was not significant. In the patients (n = 50), the median FSC (mean 407, SD 40, CI 395-418) was also higher than that of the controls (n = 54; mean 383, SD 25, CI 376-390; Mann-Whitney U-test, p = 0.0015). In Group 1A, a significant correlation was observed between the Patient median FL1 and Patient median FSC (r = 0.62, p = 0.043). When developing methods for the measurement of RP, it seems to be useful to analyse the data with more than one principle to define the threshold limit for TO positivity.