validation procedures for cell analyzers dr archana vazifdar dept. of hemato-pathology, super...
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Validation procedures for cell analyzers
Dr Archana VazifdarDept. of Hemato-Pathology,
Super Religare Laboratories Limited, Mumbai
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
• 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
Interpretation of data
Normocytic Normochromic
RBC count
Spurious increase:• Giant PLT• High WBC counts (>50)
Spurious decrease:• Cold /warm agglutinins• Very small RBC• Cryoglobulins
ADVIA 120
CELL-DYN
COULTER
Platelet count
Spurious increase:• RBC/ WBC fragments• Cryoglobulins• Lipids
Spurious decrease:• Platelet clumps• Giant platelets
neutrolympho
Baso,mono, eos, blasts
WBC (FCM)
Normal WBC scatterplot
Normal WBC histogram
Impedance- VCS
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
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
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
FLAG:Anemia, Microcytosis, anisocytosis
Hb 8.5RBC 3.2
Left shift of curve:
MicrocytosisIron Deficiency Anemia
β thalassemia trait Anemia of chronic diseases
Conclusion:
s/o Iron Deficiency AnemiaAdvise Iron studies
ACTION:
RBC indicesMentzer’s index (MCV/RBC)=
18.3MI ≤ 13- BTT, ≥ 13- IDA
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
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)
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
50/ F, Hb-8.9, MCV-73, MCH- 25.6, RDW-26.8
Blood transfusion
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
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:
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
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
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
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
Increased small sized particles:
Noise, debris, lipids, bacteria, fungi ? Wiskott Aldrich syndrome
Conclusion:
Falsely elevated platelet counts
Flags:Small platelets
Debris/ noise
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
PIC/POC delta
• Excessive noise included in impedance count
• Debris, bacteria, fungi• Plt clumps• Giant plt
45/M
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
Shoulder on the left of curve:
N’rbcLyse resistant RBCPlatelet clumps/ Giant platelets FibrinImpedance noise
Flags: IG, Blasts, eosinophilia,monocytosis, lymphopenia
CML
LeukocytosisThrombocytosisAnemia
Flags:Aty lymphocyte, Variant lymphocyteNon-viable wbcLeukocytosisT’penia
Acute Leukemia
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
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
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
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
• 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
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
Case 1 38/M, No history available
Result after treatment in H20 bath @ 37 C �
Cold agglutinin disease
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
Conclusion:
Severe hemolysis following Primaquine ingestion in G6PD deficiency
50 nrbc’s/100 WBCSpherocytes +Giant platelets
Case 3 : 33/M, Thrombocytopenia X 6 mnths, no bleeding. All other parameters WNL, ? ITP
Flags: n’rbc, micro rbc/ rbc fragments
Action:
Change anticoagulant to Sodium Citrate
Platelet count- 243
Conclusion
EDTA dependant pseudothrombocytopenia(EDP)
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
Case 4: 15/M, Fever
Conclusion:
Plasmodium falciparum , PI 15%Thrombocytopenia
Malaria discriminant factor= 6.3
THANK YOU
Archana Vazifdar, M.D.SRL RELIGARE LTD.