Presentation on theme: "Validation procedures for cell analyzers"— Presentation transcript:
1 Validation procedures for cell analyzers Dr Archana VazifdarDept. of Hemato-Pathology,Super Religare Laboratories Limited, Mumbai1
2 Principles of automation Impedance – count and size cells by change in resistance produced as they are suspended in an electrically conductive mediumOptical scatter- measures scatter properties of cells by laser lightSingle angle/ Multi-angle scatter
3 RBC & Platelets measured in one channel RBC volume > flPlatelet volume 2-20 flHb & WBC measured in second channelDLC in third channelRBC-Plt- separated by Volume & refractive index based counting/sortingMCV does not exceed 150–160 fl and, as there is noparticle above that size in the blood stream in healthor in disease.
4 Interpretation of data 3 steps for performing a CBC- interpretation of numeric, graphic & flag data, Delta checks, Review of peripheral smear
5 Normocytic Normochromic RBC countSpurious increase:Giant PLTHigh WBC counts (>50)Spurious decrease:Cold /warm agglutininsVery small RBCCryoglobulinsAs cell passes through an orifice, the change in resistance produced is measured as a single pulsNo. of pulses= no. of cellsSize of pulse= volume of cellAll info is then depicted on a histogramRBC histo- X axis, volume, Y axis, No. of RBC, MCV- mean of distribution curve
7 WBC (FCM) Impedance- VCS Baso,mono, eos, blasts neutro lympho The traditional microscopic method based on the count of 100 cells has 3 types of error: statistical error, distributional error owing to unequal distribution of cells in the smear, and error in identifying cells related to the subjective interpretation of the examiner. Newer analyzers analyze thousands of cells per sample and can produce morphologic and quantitative flags, which have significantly reduced error and allow reliable absolute counts at low and high concentrations.WBC analyzed by fcm, using impedance (VCS), Optical scatter (cytochem)VCS- Simultaneous measurement of volume, conductivity and scattered laser light, CELLS R ANALYZED IN NEAR- NATIVE STATE. x-axis represents laser light scatter, the y-axis volume and the z-axis conductivityNormal WBC histogramNormal WBC scatterplot
8 Optical scatter: ADVIA120 DLC by Peroxidase method Spurious increasePLT clumps & large plateletsNucleated red cellsResistant RBC’sSpurious decrease:Clotted sampleFragile cells- CLLLymphoid aggregates- UTI, B- cell NHL, CMMLStorage associated degenerationEDC include immature or atypical cells such as blasts, IGs, atypical lymphocytes, hematopoietic progenitor cells (HPCs), and NRBCs.70 The principal aims of the EDC are to further reduce the need for microscopic revision, to obtain more precise and accurate countswhen elevated basophil/ monocyte counts are produced, they must be examined with caution because they can be artifacts due to the presence of abnormal cells such as blasts, plasma cells, and lymphoma cells.
9 FlagsA signal to the operator that the analyzed sample may have a significant abnormality/ does not meet acceptance criteria/ cannot be displayedCause of errors:AnalyzerSampleRandom run error
11 Anemia, Microcytosis, anisocytosis Hb 8.5RBC 3.2FLAG:Anemia, Microcytosis, anisocytosisLeft shift of curve:MicrocytosisIron Deficiency Anemiaβ thalassemia traitAnemia of chronic diseasesIDA & ACD- NORMAL RDW
12 s/o Iron Deficiency Anemia Advise Iron studies ACTION:RBC indicesMentzer’s index (MCV/RBC)=MI ≤ 13- BTT, ≥ 13- IDAConclusion:s/o Iron Deficiency AnemiaAdvise Iron studies
13 N’rbc, Micro RBC/ RBC fragments Giant plt Thrombocytopenia Hb 6.4PLT 140MPV 7.9PCT .148PDW 15Flags:N’rbc, Micro RBC/ RBC fragmentsGiant pltThrombocytopeniaFRBCs are identified only on the basis of size and hemoglobin content, independent of their shapeLt of curve not touching baseline:NoiseSchistocytes &/ extremely small rbcGiant platelets
15 Dimorphic RBC population, anisocytosis Hb- 8.6, MCH- 26.5, MCHC- 32.2Flags:Dimorphic RBC population, anisocytosisBimodal peak:Dimorphic RBC populationTransfused cellsCombined deficiencyTherapeutic response in IDANN anemias: Hypoplastic, malignancy, Sideroblastic (low retic)Acute bld loss, Sev hemolysis, AIHA, G6PD, Sickle,Action:Review PS to identify cause
16 50/ F, Hb-8.9, MCV-73, MCH- 25.6, RDW-26.8 Blood transfusion Volume/Hemoglobin Concentration (V/HC) cytogram hemoglobin concentration (chromia) is plotted along the x axis and cell volume/size is plotted along the y axis, Nine zone grid, central zone NNBlood transfusion
19 Review PS: L/F agglutination vs n’rbc’s Action:Review PS: L/F agglutination vs n’rbc’sAfter warming in H2O 37ºC for 15 minscorrectedspuriously low RBC counts and to abnormally high MCV (each small RBC clump is considered as one single particle Haematocrit (RBC · MCV) is erroneous and spuriously low, contrasting with Hb that is measured after RBC lysis and is unaffected by agglutinins.As a rule, the MCHC is spurious, usually >36 g/dl.Conclusion:False ↓ RBC, Hct,False ↑ MCV, MCH & MCHCCold agglutinin disease
20 Causes of H&H mismatch: partial sample aspiration/ improper mixingHb/ MCV measurement error/ very lowHigh WBC counts (interfere with Hb measurment)Cold agglutininsShort sample (microtainer)Repeat collection
21 Platelets Smallest guys largest culprits!! As platelet counts fall, reliability of analyzer decreases.Conventional methods are unable to provide consistently accurate results in lower rangeClinicians using thresholds of 5-10 X 109/l must be aware of the limitations in precision and accuracy of cell countersLinearity : 10–1,000 X 109/lThresholds for use of prophylactic plt tx has reduced frm 20 ×103/µL to 10 × 103/µL (20 × 109/L to 10 × 109/L). OtherAuthors have suggested that in patients without feveror bleeding, there may be even lower values. However, theutilization with confidence of these new thresholds requiresknowledge of the limitations in precision and accuracy of theanalyzers at these count levels.
22 Common platelets flags PLT Clumps↓Plt countsInterferences with WBC Results (↑WBC counts)Giant plateletsSmall plateletsPIC/POC delta- difference > 20,000Thrombocytopenia- true/false
24 Falsely ↓ Plt count, ↑MPV Flags:Giant platelets, platelet clumpsCellular interferenceAction:Review PS for platelet countNon fitted curve with increase in large cells:Large platelets, clumpsConclusion:Falsely ↑RBC countFalsely ↑WBC countFalsely ↓ Plt count, ↑MPVGiant platelets
25 45/M PIC/POC delta Excessive noise included in impedance count Debris, bacteria, fungiPlt clumpsGiant pltCAUSE- Plt clumps, giant plt, ….?
26 WBC Flags IG, Band, Blasts Aty ly, Variant ly MPO, non viable WBC N’RBC, rst RBCPlt clumpOutside Reportable RangeLeukocytosis, monocytosis, basophilia, eosinophiliaUnable to Find Clear Separation between WBC subpopulations
27 Shoulder on the left of curve: N’rbc Lyse resistant RBC Platelet clumps/ Giant plateletsFibrinImpedance noiseN’RBC: neonatal hemolytic disease, in premature neonates and neonates affected by hypoxia in the perinatal period.adults: thalassemic syndromes, myeloproliferative diseases (myelofibrosis), bone marrow metastases of solid tumors, extramedullary hematopoiesis, and all of the conditions of hematopoietic stress (eg, septicemia, massive hemorrhage, and severe hypoxia)Resistant RBC’s: physiological (neonates) pathological (abnormal hb, liver disease, uraemia, chemotherapy)extended lysis mode
30 38/F, k/c/o DM Plt 100 Flag: leukocytosis, n’rbc, dimorphic reds it is useful not only to identify the presence of NRBCs, but also to estimate the NRBC count: 1.to evaluate the efficacy of transfusion therapy, as with thalassemic syndromes in which it is advisable to maintain an NRBC concentration of less than 5/100 WBCs2. persistence of NRBCs in the peripheral blood of subjects undergoing stem cell transplantation has been shown to be a poor prognostic factor3. hospitalized patients after general or cardiothoracic surgery or with other nonhematologic disease, the mortality rate was 21.1% for patients with NRBCs, whereas it was 1.2% for patients withoutFlag: leukocytosis, n’rbc, dimorphic redsConclusion:21 nrbc’s/100 wbc- corr WBC= 17.35DM in sepsis with liver abscess
31 VCS-Neutrophil population data Newer Aspects:VCS-Neutrophil population dataVCS:QuantitativeOperator independentRoutinely availableInexpensiveINCREASE MEAN NEUTROPHIL VOLUME (MNV)DECREASE MEAN NEUTROPHIL SCATTER (MNS) – left shiftLacking leukocytosis or neutrophiliaSuggestive of acute bacterial sepsis
32 Automated malaria detection “Gold standard” - thick & thin smearNeed for rapid, sensitive & cost-effective screening techniqueHemazoin pigmentActivation of neutrophils & monocytesIncrease volume heterogeneity (anisocytosis) of monocytes & lymphocytes, detected by VCS‘Positional parameters’, used as objective criteria for detecting presence of plasmodiumClin. Lab. Haem., 26, 367–372 Automated detection of malaria
33 Vol SD lymphocyte X SD Monocyte / 100 > 3.7 Plasmodium falciparumNormalMonocytesReactive LYParasitized RBCshoulderVol SD lymphocyte X SD Monocyte / > 3.7Am J Clin Pathol 2006;126:Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY
34 Specificity is 94% and sensitivity 98% PPV is 70% and NPV 99.7%.A flag indicating potential presence of malaria is a valuable diagnostic method for detection of malaria and may become a routine parameter in it’s diagnosis
35 Reticulocyte Indicesmost promising from a clinical viewpoint are the CHr and the MCVr.CHr:directly reflects hemoglobin synthesis in marrow, & measures iron availability.↓ IDA & BTT (independent of iron stores)MCVr: ↑rapidly following iron therapy↓ with the development of iron-deficiency↓ in macrocytosis after therapy with B12 &/or folic acidAvailable in very few analyzers, not standardizedCHr- reduction indicates iron-deficient erythropoiesis, even in conditions in which traditional biochemical markers such as ferritin and transferrin are inadequate (eg, in cases of inflammation oranemia from chronic disease), and, on the other hand, it is useful for monitoring early response to intravenous iron therapy because it increases significantly after only 48 hours. exceptions are heterozygotes for β-thalassemia whose CHr is always reduced independent of iron stores.
39 50 nrbc’s/100 WBCSpherocytes +Giant plateletsConclusion:Severe hemolysis following Primaquine ingestion in G6PD deficiency
40 Case 3 : 33/M, Thrombocytopenia X 6 mnths, no bleeding Case 3 : 33/M, Thrombocytopenia X 6 mnths, no bleeding. All other parameters WNL, ? ITPFlags: n’rbc, micro rbc/ rbc fragments
41 Action:Change anticoagulant to Sodium CitratePlatelet count- 243ConclusionEDTA dependant pseudothrombocytopenia(EDP)
42 EDP EDTA dependant pseudothrombocytopenia (EDP): Hypothesis- antigen-binding site in the GPIIb/IIIa complex , normally hidden/cryptic, is modified by or exposed only in presence of EDTAIn-vitro phenomenaAssociated with autoimmune/ neoplastic pathology, but also seen in healthy individualsAbnormal plt from CMPD, more prone to clumping by EDTAAlternate anticoagulants; 10% trisodium citrate/ ACD