Validation procedures for cell

analyzers
Dr Archana Vazifdar
Dept. of Hemato-Pathology,
SRL Religare Ltd.
Validation
A documented act of demonstrating that a
procedure, process, & activity will
consistently lead to expected results
FDA 1987
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
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
Validation of automated CBC analyzers:
interpretation of numeric, graphic & flag data
Delta checks
Review of peripheral smear
Interpretation of data
Normocytic
Normochromic
RBC count
Spurious increase:
Giant platelets
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
neutro
lympho
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 RBCs
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
Nrbc, Rrbc, 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 8.5
RBC 3.2
Left shift of curve:
Microcytosis
Iron Deficiency Anemia
thalassemiatrait
Anemia of chronic diseases
Conclusion:
s/o Iron Deficiency Anemia
Advise Iron studies
ACTION:
RBC indices
Mentzersindex (MCV/RBC)=
18.3
MI 13- BTT, 13- IDA
Flags:
Nrbc, Micro RBC/ RBC
fragments
Giant platelets
Thrombocytopenia
Lt of curve not touching
baseline:
Noise
Schistocytes &/ extremely
small rbc
Giant platelets
PLT 140
MPV 7.9
PCT .148
PDW 15
Hb 6.4
Conclusion:
RBC count falsely
Platelets falsely (mask
tpenia)
Hemolytic anemia
Action:
RBC Indices- MCV, RDW
PLT Histogram- MPV & PDW
Review PS- RBC morphology
-PLT count (100)
Bimodal peak:
Dimorphic RBC population
Transfused cells
Combined deficiency
Therapeutic 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 pallor
Hb-5.1, MCV-96.7, MCH- 29.6, MCHC-31.4, RDW-24.5
TLC/Plt-Normal
S. Fe- 25
TIBC- 144
S. Fe saturtn- 20.8
S. B12- 158
Right portion of curve extended:
RBC agglutination
Nrbcs
Leukocytosis
Flags:
H&H error, Nrbc, dimorphic reds
Anemia, 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
@ 37C for 15 mins
Action:
Review PS: L/F agglutination vs nrbcs
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
Platelets
Smallest 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 10
9
/l
must be aware of the limitations in
precision and accuracy of cell counters
Linearity : 101,000 X 10
9
/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 count
Falsely WBC count
Falsely Plt count, MPV
Giant platelets
Flags:
Giant platelets, platelet
clumps
Cellular 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, Blasts
Aty ly, Variant ly
MPO, non viable WBC
NRBC, rst RBC
Plt clump
Outside Reportable Range
Leukocytosis, monocytosis, basophilia, eosinophilia
Unable to Find Clear Separation between WBC
subpopulations
WBC Flags
Shoulder on the left of curve:
Nrbc
Lyse resistant RBC
Platelet clumps/ Giant platelets
Fibrin
Impedance noise
Flags: IG, Blasts, eosinophilia,monocytosis, lymphopenia
CML
Leukocytosis
Thrombocytosis
Anemia
Flags:
Aty lymphocyte,
Variant lymphocyte
Non-viable wbc
Leukocytosis
Tpenia
Acute Leukemia
38/F, k/c/o DM
Flag: leukocytosis, nrbc, dimorphic reds
Conclusion:
21 nrbcs/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
Hemazoinpigment
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, 367372 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;126:691-698
Briggs 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 its diagnosis
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, nrbc, rbc fragments, giant
platelets
Case 2
Conclusion:
Severe hemolysis following Primaquine
ingestion in G6PD deficiency
50 nrbcs/100 WBC
Spherocytes +
Giant platelets
Case 3 : 33/M, Thrombocytopenia X 6 mnths, no
bleeding. All other parameters WNL, ? ITP
Flags: nrbc, micro rbc/ rbc
fragments
Action:
Change
anticoagulant to
Sodium Citrate
Platelet count- 243
Conclusion
EDTA dependant pseudothrombocytopenia
(EDP)
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.