validation procedures for cell analyzers dr archana vazifdar dept. of hemato-pathology, super...
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![Page 1: Validation procedures for cell analyzers Dr Archana Vazifdar Dept. of Hemato-Pathology, Super Religare Laboratories Limited, Mumbai](https://reader038.vdocuments.mx/reader038/viewer/2022103007/56649c9e5503460f9495eb84/html5/thumbnails/1.jpg)
Validation procedures for cell analyzers
Dr Archana VazifdarDept. of Hemato-Pathology,
Super Religare Laboratories Limited, Mumbai
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Principles of automation
• Impedance – count and size cells by change in resistance produced as they are suspended in an electrically conductive medium
• Optical scatter- measures scatter properties of cells by laser light– Single angle/ Multi-angle scatter
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• RBC & Platelets measured in one channel
– RBC volume > 30-36 fl
– Platelet volume 2-20 fl
• Hb & WBC measured in second channel
• DLC in third channel
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Interpretation of data
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Normocytic Normochromic
RBC count
Spurious increase:• Giant PLT• High WBC counts (>50)
Spurious decrease:• Cold /warm agglutinins• Very small RBC• Cryoglobulins
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ADVIA 120
CELL-DYN
COULTER
Platelet count
Spurious increase:• RBC/ WBC fragments• Cryoglobulins• Lipids
Spurious decrease:• Platelet clumps• Giant platelets
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neutrolympho
Baso,mono, eos, blasts
WBC (FCM)
Normal WBC scatterplot
Normal WBC histogram
Impedance- VCS
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Optical scatter: ADVIA120 DLC by Peroxidase method
Spurious increase
• PLT clumps & large platelets • Nucleated red cells• Resistant RBC’s
Spurious decrease:
• Clotted sample• Fragile cells- CLL• Lymphoid aggregates- UTI, B- cell NHL,
CMML• Storage associated degeneration
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Flags
• A signal to the operator that the analyzed sample may have a significant abnormality/ does not meet acceptance criteria/ cannot be displayed
• Cause of errors:– Analyzer– Sample– Random run error
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RBC flags
Suspect flags• N’rbc, R’rbc, Micro RBC, RBC fragments,
– interfere with WBC & platelet counts
• H & h errors• short sample, aged sample
Definitive flags• Anemia, anisocytosis, microcytosis, macrocytosis,
poikilocytosis• Erythrocytosis
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FLAG:Anemia, Microcytosis, anisocytosis
Hb 8.5RBC 3.2
Left shift of curve:
MicrocytosisIron Deficiency Anemia
β thalassemia trait Anemia of chronic diseases
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Conclusion:
s/o Iron Deficiency AnemiaAdvise Iron studies
ACTION:
RBC indicesMentzer’s index (MCV/RBC)=
18.3MI ≤ 13- BTT, ≥ 13- IDA
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Flags:• N’rbc, Micro RBC/ RBC
fragments• Giant plt• Thrombocytopenia
Lt of curve not touching baseline:NoiseSchistocytes &/ extremely small rbcGiant platelets
PLT 140MPV 7.9PCT .148PDW 15
Hb 6.4
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Conclusion:
RBC count falsely ↓Platelets falsely ↑ (mask t’penia)
Hemolytic anemia
Action:
• RBC Indices- MCV, RDW• PLT Histogram- MPV & PDW • Review PS- RBC morphology
-PLT count (100)
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Bimodal peak: Dimorphic RBC population
Transfused cellsCombined deficiencyTherapeutic response in IDA
Hb- 8.6, MCH- 26.5, MCHC- 32.2
Flags:Dimorphic RBC population, anisocytosis
Action:
Review PS to identify cause
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50/ F, Hb-8.9, MCV-73, MCH- 25.6, RDW-26.8
Blood transfusion
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Dual/Combined deficiency
45/F, Severe pallorHb-5.1, MCV-96.7, MCH- 29.6, MCHC-31.4, RDW-24.5 TLC/Plt-Normal
S. Fe- 25TIBC- 144
S. Fe saturtn- 20.8S. B12- 158
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Right portion of curve extended:RBC agglutinationN’rbcsLeukocytosis
Flags:H&H error, N’rbc, dimorphic redsAnemia, macrocytosis, anisocytosis
H&H
• Sample related problems- turbidity-↑ Hb– Lipemia/ TPN– Cryoglobulins
• Autoagglutination• Hemolysis (in-vitro/vivo)• Spurious ↓ Hct• Clotted sample
Spurious ↑MCHC:
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corrected
Conclusion:False ↓ RBC, Hct, False ↑ MCV, MCH & MCHC
Cold agglutinin disease
After warming in H2O bath @ 37ºC for 15 mins
Action:Review PS: L/F agglutination vs n’rbc’s
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Short sample (microtainer)Repeat collection
Causes of H&H mismatch:
• partial sample aspiration/ improper mixing
• Hb/ MCV measurement error/ very low• High WBC counts (interfere with Hb
measurment)• Cold agglutinins
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PlateletsSmallest guys largest culprits!!
• As platelet counts fall, reliability of analyzer decreases.
• Conventional methods are unable to provide consistently accurate results in lower range
• Clinicians using thresholds of 5-10 X 109/l must be aware of the limitations in precision and accuracy of cell counters
Linearity : 10–1,000 X 109/l
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Common platelets flags
• PLT Clumps – ↓Plt counts– Interferences with WBC Results (↑WBC
counts)• Giant platelets• Small platelets• PIC/POC delta- difference > 20,000• Thrombocytopenia- true/false
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Increased small sized particles:
Noise, debris, lipids, bacteria, fungi ? Wiskott Aldrich syndrome
Conclusion:
Falsely elevated platelet counts
Flags:Small platelets
Debris/ noise
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Action:
Review PS for platelet count
Conclusion:
Falsely ↑RBC countFalsely ↑WBC count
Falsely ↓ Plt count, ↑MPV
Giant platelets
Flags:Giant platelets, platelet clumpsCellular interference
Non fitted curve with increase in large cells:
Large platelets, clumps
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PIC/POC delta
• Excessive noise included in impedance count
• Debris, bacteria, fungi• Plt clumps• Giant plt
45/M
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IG, Band, BlastsAty ly, Variant lyMPO, non viable WBCN’RBC, rst RBCPlt clumpOutside Reportable RangeLeukocytosis, monocytosis, basophilia, eosinophiliaUnable to Find Clear Separation between WBC subpopulations
WBC Flags
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Shoulder on the left of curve:
N’rbcLyse resistant RBCPlatelet clumps/ Giant platelets FibrinImpedance noise
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Flags: IG, Blasts, eosinophilia,monocytosis, lymphopenia
CML
LeukocytosisThrombocytosisAnemia
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Flags:Aty lymphocyte, Variant lymphocyteNon-viable wbcLeukocytosisT’penia
Acute Leukemia
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38/F, k/c/o DM
Flag: leukocytosis, n’rbc, dimorphic reds
Conclusion:
21 nrbc’s/100 wbc- corr WBC= 17.35
DM in sepsis with liver abscess
Plt 100
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VCS:• Quantitative • Operator independent• Routinely available• Inexpensive
INCREASE MEAN NEUTROPHIL VOLUME (MNV)DECREASE MEAN NEUTROPHIL SCATTER (MNS) – left shift
– Lacking leukocytosis or neutrophilia
Newer Aspects: VCS-Neutrophil population data
Suggestive of acute bacterial sepsis
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Automated malaria detection
• “Gold standard” - thick & thin smear • Need for rapid, sensitive & cost-effective
screening technique
• Hemazoin pigment• Activation of neutrophils & monocytes• Increase volume heterogeneity (anisocytosis) of
monocytes & lymphocytes, detected by VCS
• ‘Positional parameters’, used as objective criteria for detecting presence of plasmodium
Clin. Lab. Haem., 26, 367–372 Automated detection of malaria
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Normal Plasmodium falciparum
Monocytes
Reactive LY
Parasitized RBC
Vol SD lymphocyte X SD Monocyte / 100 > 3.7
Am J Clin Pathol 2006;126:691-698Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY
shoulder
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• 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
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Reticulocyte Indices
• most 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 acid
• Available in very few analyzers, not standardized
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Case 1 38/M, No history available
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Result after treatment in H20 bath @ 37 C �
Cold agglutinin disease
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27/M, Hb 7, MCV 94, MCH 32, MCHC 35.7, RDW 14.6, Plt 158
Flags: Blasts, IG, n’rbc, rbc fragments, giant platelets
Case 2
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Conclusion:
Severe hemolysis following Primaquine ingestion in G6PD deficiency
50 nrbc’s/100 WBCSpherocytes +Giant platelets
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Case 3 : 33/M, Thrombocytopenia X 6 mnths, no bleeding. All other parameters WNL, ? ITP
Flags: n’rbc, micro rbc/ rbc fragments
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Action:
Change anticoagulant to Sodium Citrate
Platelet count- 243
Conclusion
EDTA dependant pseudothrombocytopenia(EDP)
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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 EDTA
• In-vitro phenomena• Associated with autoimmune/ neoplastic pathology, but
also seen in healthy individuals• Abnormal plt from CMPD, more prone to clumping by
EDTA• Alternate anticoagulants; 10% trisodium citrate/ ACD
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Case 4: 15/M, Fever
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Conclusion:
Plasmodium falciparum , PI 15%Thrombocytopenia
Malaria discriminant factor= 6.3
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THANK YOU
Archana Vazifdar, M.D.SRL RELIGARE LTD.