Fragmented Red Cell Research Articles

Quantification of Schistocytes from the Peripheral Blood Smear in Thrombotic Thrombocytopenic Purpura (TTP) Compared to Non-TTP Thrombocytopenic Hospitalized Patients

IntroductionThe diagnosis of TTP requires thrombocytopenia without an underlying cause and microangiopathic hemolytic anemia (MAHA)1. TTP is usually confirmed with a low ADAMTS level. However, the turnaround time for an ADAMTS level is several days and treatment for this rare disease cannot be delayed, since it is uniformly fatal if left untreated but highly responsive to plasma exchange therapy. A rapid diagnosis is required and still must be made on clinical findings and peripheral smear morphology. The degree of MAHA that is seen with TTP, though, has not been well defined, or correlated with ADAMTS results. Automated fragmented red cell analyzers are not yet widely available or accepted, so the determination of MAHA still relies on manual smear reviews. Presenting smears from pts with TTP over a 1 ½ year period were reviewed, with manual counting of schistocytes, and were compared to smears from in-patient non-TTP cases of thrombocytopenia where hematology was consulted to rule out TTP.Methods29 cases were identified and peripheral smears from date of presentation were reviewed, initially by a hematologist and experienced special hematology technician, then by 2 medicine residents after they were given a 1 hour tutorial on smear interpretation. All smear reviewers were instructed on schistocyte identification as defined by the International Council for Standardization in Hematology (ICSH)2. The number of schistocytes per 1000 red cells was counted at 1000 power magnification. The blood smears were coded/de-identified and then read “blindly” by the 4 observers. Degree of agreement between the 1st 2 observers was measured (using Pearson correlation coefficient), as was the their average as compared to the average #schistocytes counted between the 2 med residents. Time taken to review smears was also measured.ResultsAve age 54 (19-83); 59% female, 41% male; 62% Caucasian; 35% AA; 3% Hispanic. 9 had TTP and 20 did not. All cases of TTP had a low ADAMTS level (med <3%, range <3-13%) and all but one had a +inhibitor (one had congenital TTP, confirmed by ADAMTS mutation analysis). The non-TTP cases included sepsis (8/20), DIC (3/20), metastatic cancer (2/20), scleroderma crisis (2/20) and single cases of HIT, EtOH, gestational, MPN/MDS and unknown causes for thrombocytopenia. For TTP cases: med plt count was 22k (4-52k); LDH 760 (309-1175); hgb 9.4 (7.1-10.9); creat 1.59mg/dL (0.6-4.85). For non-TTP cases: med plt count was 44k (3-126k); LDH 300 (155-1111); hgb 8.1 (5.9-14.7); creat 1.36mg/dL (0.9-9.1). 3 non-TTP pts had ADAMTS levels sent (med= 69%, 57-100%).The med #schistocytes in TTP cases was 6% (ave 6.3, 1.85-11.95%) compared to 0.625% (ave 1.52%, 0.25-7.5%) in the non-TTP group (p= 0.00002). The R-coefficient for the 1st 2 observers was 0.93 (95% CI 0.85-0.96) and for their average compared to the 2 medicine residents it was 0.82. The average time to read smears overall was 10.5 minutes.Conclusions:This retrospective study showed that manual quantification of schistocytes is feasible, may be done in a timely fashion and with excellent interobserver reproducibility. All pts had some degree of MAHA but the TTP cases, not surprisingly, had a significantly higher percentage of schistocytes/1000 RBCs than non-TTP thrombocytopenic in-pt consult cases (6% vs 0.625%, respectively, p= 0.00002). All cases of low ADAMTS, positive inhibitor confirmed TTP had >2.5% schistocytes, compared to 4/20 non-TTP cases. A larger cohort of acquired TTP should be studied to determine if there is a cut-off % schistocytes which, in the appropriate clinical setting, should warrant urgent plasma exchange while waiting for ADAMTS levels to return.1George JN and Al-Noun ZL. Diagnostic and therapeutic challenges in the TTP and HUS syndromes. ASH Education Book. 2012, p. 604-609.2Zini G et al. ICSH recommendations for identification, diagnostic value, and quantitation of schistocytes. International J of Lab Hematology. 2011; 34: 107-116. DisclosuresNo relevant conflicts of interest to declare.

THE AUTOMATED SCHISTOCYTE COUNT REQUIRES MICROSCOPIC CONFIRMATION

Background: The automated fragmented red cell (FRC) is a promising diagnostic tool for the rapid diagnosis of patients with a suspected thrombotic micro-angiopathy (TMA). Aim: To evaluate the FRC in comparison to the microscopic schistocyte percentage in order to determine its diagnostic utility. Methods: Schistocytes were evaluated in 136 samples by microscopy by two competent morphologists according to International Council for Standardisation in Haematology (ICSH) recommendations. This was compared with the FRC on the Sysmex XN-9000 (Sysmex Corporation, Kobe, Japan). The degree of agreement was measured using the Bland and Altman method and Deming-regression statistical methods. Results: Schistocytes were observed in patients with TMA (8.82%), malignancies (11.03%), haemodialysis (34.56%), haemoglobinopathies (8.82%), nutritional anaemias (13.24%) and in neonates (1.47%). The correlation co-efficient between the two morphologists was 0.97 (CI, 0.92 to 1.01) and the mean of the differences was -0.02 (CI, -0.53 to 0.50). The Sysmex overestimated the schistocyte count (2.61, CI, 2.15 to 3.07) and revealed a poor correlation with microscopy (0.21 CI, 0.05 to 0.38). There was no correlation between the percentage of microcytic (%Micro-R) and hypochromic red cells (%Hypo-He) and the FRC. Conclusion: This study confirms that observer bias between morphologists can be reduced by implementing the ICSH guidelines. Measurement of the automated FRC by the Sysmex XN 9000 analyser overestimated the schistocyte percentage. The FRC requires confirmation by microscopy.

The Clinical Significance of Schistocytes: A Prospective Evaluation of the International Council for Standardization in Hematology Schistocyte Guidelines.

Objective:The presence of ≥1% schistocytes on a peripheral blood smear (PBS) is an important criterion for the diagnosis of thrombotic microangiopathy (TMA). The reporting of schistocytes has been standardized by the International Council for Standardization in Hematology (ICSH). Despite the availability of guidelines, however, the assessment of schistocytes remains subjective. More recently, the automated fragmented red cell (FRC) parameter has been evaluated. However, local studies are not available.Materials and Methods:A prospective study was performed at the Charlotte Maxeke Johannesburg Academic Hospital in order to evaluate the ICSH recommendations for schistocyte measurement in 146 PBSs with schistocytes. Schistocytes were evaluated by microscopy and ADVIA 2120 automated hematology analyzers.Results:Schistocytes were frequently observed in patients with TMA (n=76), infection (n=20), hematologic malignancy (n=10), renal failure (n=5), and hemoglobinopathy (n=15), and in neonates (n=11). Schistocytes were ≥1% in all PBSs with TMA (n=76) with a mean of 3.44±1.84. Schistocytes of ≥1% were also observed in cases of renal failure and hemoglobinopathy, and in neonates. In these conditions, schistocytes were mainly observed in conjunction with moderate red blood cell changes. The agreement between two morphologists gave a correlation coefficient of 0.63 [confidence interval (CI): 0.52-0.75], while the correlation coefficient between the average of the morphologists and the FRC percentage was -1.97 (CI: -1.60 to -2.34). The ADVIA 2120 underestimated the schistocyte count in patients with TMA.Conclusion:Observer bias can be decreased by implementing the standardized procedures recommended by the ICSH. However, estimation of schistocytes by the ADVIA 2120 analyzer requires further evaluation as a screening tool. A higher threshold for schistocytes in thrombotic thrombocytopenic purpura is recommended to distinguish this hematological emergency from other conditions associated with ≥1% schistocytes.

Adult patent Ductus Arteriosus complicated by endocarditis and hemolytic anemia

An adult with a large patent ductus arteriosus may present with fatigue, dyspnea or palpitations or in rare presentation with endocarditis. The case illustrated unique role of vegetation of endocarditis in hemolytic anemia in adult with patent ductus arteriosus (PDA). Despite treatment of endocarditis with complete course of appropriate antibiotic therapy and normality of C- reactive protein, erythrocyte sedimentation rate and leukocytosis and wellness of general condition, transthoracic echocardiography revealed large vegetation in PDA lumen, surgical closure of PDA completely relieved hemolysis, and fragmented red cell disappeared from peripheral blood smear. The 3-month follow-up revealed complete occlusion of PDA and abolishment of hemolytic anemia confirmed by clinical and laboratory examination.

Fragmented Red Cell as a Possible Favorable Prognostic Marker of Hematopoietic Stem Cell Transplantation Associated Thrombotic Microangiopathy

Fragmented red cell (FRC) by automated hematologic analyzer is known to detect schistocyte. In this study, it is noted that FRC might be a favorable prognostic marker of hematopoietic stem cell transplantation associated thrombotic microangiopathy (TA-TMA). The peripheral blood samples and clinical data of 89 patients were collected. The diagnosis of TA-TMA was defined by the Blood and Marrow Transplant Clinical Trials Network's criteria and schistocyte or both schistocyte- and FRC-positive cases and other parameters fulfilled are regarded as TA-TMA. Schistocyte and FRC displayed a correlation coefficient of 0.461 (P < 0.001) by Spearman's method. The diagnostic concordance of TA-TMA using schistocyte and FRC was 92.1% with kappa index of 0.531 (P < 0.001). The number of diagnosed patients and mean survival month were as follows: TA-TMA by schistocyte, 8 (8.9%), 13.5 month; TA-TMA by schistocyte and FRC, 7 (7.8%), 40.4 month; No TMA, 74 (83.1%), 38.3 month, respectively. Kaplan-Meier survival analysis by log-rank method of the patient with TA-TMA by schistocyte and rest of the group showed statistical significance (P < 0.01). As evidenced by the data, FRC might be a favorable prognostic marker for TA-TMA, but additional studies with larger patients groups are required for validation of clinical applications.

Behavior of ADAMTS13 and Von Willebrand factor levels in patients after living donor liver transplantation

IntroductionThrombotic microangiopathy (TMA) is one of the important complications occurring after liver transplantation (LT), and it is suggested that a Von Willebrand factor (VWF) and ADAMTS13 may play an important role in the onset of TMA and poor outcome after LT. Materials and MethodsIn 81 patients after living donor LT (LDLT), 17 patients who had both severe thrombocytopenia and hemolytic anemia with fragmented red cell were diagnosed as TMA- like syndrome (TMALS) and 10 patients died. ResultsADAMTS13 activities were slightly low, and plasma levels of VWF and VWF propeptide (VWFpp) antigens and the ratio of VWFpp/VWF were significantly high before LDLT. ADAMTS13 activities were significantly reduced from day 1 to day 28 after surgery, and plasma levels of VWF antigen slightly decreased on day 1 and plasma levels of VWFpp continued to be high. The ratio of VWFpp/VWF was significantly high on day 1 after surgery.The mortality was high in the patients with TMALS and the frequency of TMALS was high in non-survivors. VWF levels were significantly low and the ratio of VWFpp/VWF was significantly high in those with TMALS on day 1 after surgery. The ADAMTS13 activity was significantly low, and the VWFpp and the VWFpp/ADAMTS13 ratio were significantly high in non-survivor on day 28 after surgery. ConclusionThese findings suggest that VWF and ADAMTS13 might therefore play an important role in the onset of TMA and poor outcome after LT. The VWFpp may therefore be a more useful marker for the diagnosis of TMALS than VWF.

Evaluation of fragmented red cell (FRC) counting using Sysmex XE‐5000 – Does hypochromia play a role?

Schistocytes are major signs of micro- and macroangiopathic haemolytic anaemia. The aim was to evaluate automated fragmented red cell (FRC) count compared to visual microscopy, and to assess FRCs in the presence of microcytosis and hypochromia using Sysmex automated counters. Schistocytes were determined with visual microscopy after the observation of 1000 erythrocytes, and the automated counting with Sysmex XE-5000. Indices of microcytosis (%MicroR) and hypochromia (%Hypo-He) were also analysed in the XE-5000 analyser. Linear regression analysis showed a good correlation between automated and manual FRC% count (r = 0.824, P < 0.0001), but Bland-Altman's plot revealed an overestimation of FRC of 0.82%. There is a global correlation between %MicroR and FRCs. In subgroup analysis of %MicroR (reference value: 0.3-3%, mild microcytosis: 3.1-7.2% and severe microcytosis: 7.3-56.7%), no correlations with automated %FRC were noticed (P > 0.05). Based on %Hypo-He subgroups (mild hypochromia: 1.2-5.2%, and severe hypochromia: 5.3-35.4%), a significant correlation of automated %FRC with mild hypochromia was found (r = 0.621, P < 0.01). Despite the agreement between FRC count and the manual method, the overvaluation of FRC was high, leading to false-positive results. Microcytosis appeared to have no impact on FRC count, whereas mild hypochromia seemed to be related with FRC count. Particular attention is required to assess automated FRCs in samples with mild hypochromia.

Evaluation of schistocytes measurement guidelines

The schistocytes are fragmented red blood cells mainly observed in the setting of hemolytic anemias and particularly among the thrombotic microangiopathies. The presence of schistocytes is an important criterion for the diagnosis of mechanical anemias, though the identification of these cells remains problematic. As a high variability of the morphologic identification criteria of the schistocytes among morphologists has been observed, some guidelines have been proposed after workshops (French and Italian groups). The International council for standardization in hematology published a consensus in November, 2011. The French group of cellular hematology (GFHC) aimed to recover the opinion of French biologists directly confronted to schistocytes measurements. 169 out 500 (34%) answered 10 questions dealing with the identification and measurements of schistocytes as proposed by the ICSH guidelines. A consensus was reached for the urgent need of guidelines documents, moreover in the current background of the European accreditation NF EN ISO 15189 rules. A traduction in native (French) language as warmly wished in order to facilitate the diffusion of the information. New fragmented red cell parameter recently provided by 2 manufacturers of automated blood cell counters remained doubtfull for routine use for half of the biologists.

Quantification of red blood cell fragmentation by the automated hematology analyzer XE-2100 in patients with living donor liver transplantation

The fragmented red cell (FRC) is a useful index for diagnosing and determining the severity of thrombotic thrombocytopenic purpura (TTP), thrombotic microangiopathy (TMA) and other similar conditions, as it is found in peripheral blood in patients with these diseases. The FRC expression rate has conventionally been determined by manual methods using smear samples. However, it is difficult to attain accurate quantification by such methods as they are time consuming and prone to a great margin of error. With cases of living donor liver transplantation, the current study examined the possibility of using a multi-parameter automated hematology analyzer, the XE-2100 (Sysmex Corporation) for FRC quantification. While there was a notable correlation between the manual and automated measurements, the manual measurement resulted in higher values. This suggested remarkable variations in judgment by individuals. The FRC values had a significant correlation with the reticulocyte count, red blood cell distribution width (RDW), fibrin/fibrinogen degradation products (P-FDP) and lactate dehydrogenase (LDH) among the test parameters, and this finding was consistent with the clinical progression in patients. The automated method can offer precise measurements in a short time without inter-observer differences, meeting the requirement for standardization. The determination of FRC count (%) by the XE-2100 that enables early diagnoses and monitoring of TTP or TMA will be useful in the clinical field.

Sensitive measurement of fragmented red cell population using flow cytometry, and its application for estimating thrombotic microangiopathy after stem cell transplantation.

Thrombotic microangiopathy (TMA) is one of the lethal complications after hematopoietic stem cell transplantation (SCT). The levels of fragmented red cells (FRCs), thrombomodulin (TM), and factor VIII-related antigen in the blood are the most important markers for estimating TMA. However, the FRC level has been measured by using microscopy and the naked eye; therefore, an improvement in technology to objectively count FRC is necessary. We established a novel technique to sensitively measure FRC as glycophorin A dull-positive small particles using a flow cytometer and estimated its reliability in patients treated with SCT. The blood level of FRC was compared with other clinical data in 257 blood samples in 16 clinical courses after SCT of 15 patients. Sorted glycophorin A dull-positive small particles morphologically showed FRC. Measured FRC percentage had a weak correlation with serum levels of lactate dehydrogenase (LDH) and total bilirubin but not with TM level, whereas TM showed a weak correlation with the levels of aspartate aminotransferase and LDH. In a patient with fulminant TMA, decrement of the FRC level led to improvement in liver parameters after treatment, presumably due to the rapid clearance of FRC, and increased simultaneously with the levels of LDH and bilirubin by the TMA recurrence. Levels of FRC percentage and TM were independent parameters of TMA. This novel technique may be used as a standard methodology in diagnosing TMA.

Quantification of red blood cell fragmentation by automated haematology analyser XE-2100.

The extent of red blood cell fragmentation in peripheral blood is useful for diagnosis and follow-up in many diseases, e.g. haemolytic uremic syndrome, transplantation-associated thrombotic microangiopathy (BMT-TMA). However, this quantification still relies on manual counting of fragmented red cells on blood smears. We have developed a quantification system by gating a fixed area of fragmented red blood cells (Gate 1) on an automated haematology analyser (XE-2100, Sysmex Co., Kobe, Japan). The fragmented red cell percentage (FRC%) calculated with this system, from 100 samples, was highly correlated with the manual count (r=0.902, P < 0.0001). Because microcytic anaemia specimens usually occupy a lower position on the XE-2100 scattergram, with microcytic cells overlapping Gate 1 and causing a spuriously high FRC% calculation, a supplementary gate (Gate 2) was added. Using the particle number in this gate as well as in Gate 1, a revised method for such samples was developed and its validity confirmed (revised FRC% correlated with a manual count for 10 subjects (P < 0.001). Because this gating system can be programmed on any XE-2100, it is likely to prove useful for accurate quantification of red blood cell fragmentation and for the monitoring of the development of BMT-TMA.

Equilibrium binding of calcium to fragmented human red cell membranes and its relation to calcium-mediated effects on cation permeability

We have measured Ca binding to fragmented human red cell membranes under equilibrium conditions in the presence of low concentrations of EGTA-buffered, ionized Ca. The ionic strength of the assay medium was maintained at 0.16. Two high affinity Ca binding sites were identified: Site I was pH-sensitive. Its apparent dissociation constant (K′) increased from 2×10−7 to 6×10−7m as the pH was shifted from 6.8 to 6.0. Between pH 8.5 and 6.8K′ remained constant. The capacity of the same site decreased between pH 8.5 and 6.8 from 0.16 to 0.04 nmoles/mg protein. Site II was insensitive to pH changes between 8.5 and 6.0. It had aK′ value of ∼3×10−6m and a capacity of ∼0.2 nmoles/mg protein. Mg and the local anesthetic propranolol (but not tetracaine) inhibited Ca binding to site I competitively and to site II noncompetitively. The properties of the high affinity Ca membrane binding sites are consistent with the assumption that site I corresponds to the site at which Ca initiates an increase in K permeability in resealed red cell ghosts. Site II is possibly involved in the Ca-mediated resealing of red cell ghosts after osmotic hemolysis. In the presence of MgATP, only a single saturable high affinity Ca binding site was observed (K′∼6×10−7 at pH 6.8). The capacity of this site (∼1.8 nmoles/mg protein) was almost 10 times higher than the combined capacities of sites I and II under control conditions. The results are discussed in the light of inevitable but severe shortcomings due to the evaluation of binding constants from nonlinear Scatchard plots by a curve-fitting procedure.

Interaction of thallous ions with the cation transport mechanism of erythrocytes

1. 1. Tl + can replace K + in the activation of the ATPase of fragmented red cell membranes. In the presence of Na +, equal activation is obtained with Tl + concentrations only 1/10 of those of K +. In all other respects the ( Na + + K + )- and (Na + + Tl +)-ATPases were found to be identical. 2. 2. The transport of Tl + into erythrocytes was found to be independent of ouabain and glucose. The stationary cell/medium distribution of 204Tl + was only 1.8–2.0, i.e. close to reciprocal values of the passive distribution of small anions. The inward rate constants were 9.36 h −1 for 204Tl + and 0.153 h − for K +. The corresponding outward rate constants were 4.68 h −1 and 0.0066 h −1, respectively. 3. 3. Tl + could substitute for K + in supporting the ouabain-sensitive outflux of 22Na + from red cells. A similar outflux of 22Na + was observed at 1 mM Tl + and 10 mM K +. At higher concentrations Tl +, inhibited the ouabain-sensitive 22Na + outflux. The inhibition was greater in fresh erythrocytes than in cells stored in the cold for about 1 week or more. The difference in the degree of the Tl + inhibition may be attributed to the different Na + concentrations in fresh and old erythrocytes. 4. 4. On the basis of the results obtained it is suggested that the mechanism of active ion transport involves a destruction of the ion hydration shells.