1. High Resolution Protein Electrophoresis
A Clinical Overview with Case Studies By
Lawrence M. Killingsworth, Ph.D.

2. Human Proteins
Only ~200 of the vast array of human proteins have been characterized.
Clinical knowledge is limited to 25 – 30 relatively high concentration components of plasma, CSF, urine and other fluids.
Of these, 15 or so can be visualized by high resolution agarose electrophoresis.

3. Normal Control Pre-albumin Albumin 1-Acid Glycoprotein 1-Antitrypsin
-Lipoprotein
1-Antichymotrypsin
Haptoglobin
C-3
IgA
IgG
Albumin
1-Antitrypsin
2-Macroglobulin
Transferrin
-Lipoprotein
Fibrinogen
IgM

4. Monoclonal Gammopathies
Uncontrolled proliferation of a single clone of plasma cells at the expense of other clones.
Protein analysis is valuable in diagnosing and monitoring lymphoproliferative diseases.

5. Evaluation of Monoclonal Gammopathies
Serum and Urine High Resolution Protein Electrophoresis (24-hour urine preferred)
Quantitative Serum Immunoglobulins
Serum and Urine IFE

6. SPIFE ImmunoFix
IFE is the method of choice to identify suspicious serum or urine electro-phoretic bands.
Periodic evaluation by serum and urine electrophoresis and by quantitative Ig assay can help monitor therapy.

7. Monoclonal Gammopathies
Variable mobility and band appearance.

8. Case 1: IgG Kappa Monoclonal
Control
Patient
Patient: 78 year-old male
History & Physical: Severe pain right leg and right lumbar region.
SPE: Monoclonal band in gamma region.
IFE: IgG Kappa.
Hospital Course: Bulging disk surgically decompressed; referred to hematologist/ oncologist for follow-up & treatment.

9. Case 2: IgG Kappa Monoclonal
Patient: 78 year-old male
History & Physical: Recent chemotherapy for lymphadenopathy.
SPE: Hypoalbuminemia and monoclonal band in 2 region.
IEP: IgG Kappa.
Hospital Course: Treated with transfusions and plasmapheresis. Symptomatic improvement. Discharged for outpatient re-evaluation.
Control
Patient

10. Case 3: IgA Lambda Monoclonal
Patient: 74 year-old female
History & Physical: Myeloma. Pain in lower thoracic and upper lumbar spine, right shoulder and left anterior ribs.
SPE: Large monoclonal band in 2 region.
IEP: IgA Lambda.
Hospital Course: Radiation therapy. Transferred to hospital closer to home for continued radiation and chemotherapy.
Control
Patient

11. Case 4: IgM Kappa Monoclonal
Patient: 68 year-old male
History & Physical: Anemia, elevated IgM, edema in ankles, petechiae.
SPE: Marked M-component in 2 region; also in urine.
IEP: IgM Kappa in serum; free Kappa light chains in urine.
Hospital Course: Bone marrow biopsy non-diagnostic. Discharged for out-patient treatment and followup for possible macroglobulinemia, chronic lymphatic leukemia or lymphoma.
Control
Patient

12. Case 5: Lambda Light Chain
Patient: 78 year-old male
History & Physical: Anemia, azotemia; admitted for dialysis.
SPE:  1AT and Hp,  pre-albumin, albumin & transferrin (consistent with acute inflammation); 2 small M-proteins in  region. Urine:  albumin,1AT, transferrin; large M-protein in  region and smaller cathodal band.
IEP: Monoclonal lambda light chain in urine and serum. No heavy chain.
Hospital Course: Bone marrow biopsy confirmed multiple myeloma. Hemodialysis, plasmapheresis and chemotherapy improved symptoms.
Control
Patient

13. Case 6: Lambda Light Chain
Patient: 82 year-old male
History & Physical: Fractured left hip, suspected frontal lobe infarction.
SPE:  albumin, acute inflammation,  Hp (consistent with in-vivo hemolysis or  RBC turnover), mild hypogamma-globulinemia. Urine: Several small monoclonal bands in  region.
IEP: Monoclonal free lambda light chain in urine; no monoclonal proteins in serum.
Hospital Course: Acute left cerebral infarction confirmed. B12 anemia treated. Died due to post-operative pneumonia following hip surgery.
Control
Patient
Serum
Urine

14. Multiple Myeloma Clinical Presentation
Bone pain, especially in spine, pelvis or ribs
Renal failure of unknown etiology
Recurrent bacterial infections
Physical exam usually unremarkable – no lymphadenopathy or hepatosplenomegaly

15. Macroglobulinemia Clinical Presentation Fatigue Generalized weakness
Skin and mucosal bleeding
Visual disturbances
Headache
Other neurological signs and symptoms
Cardiopulmonary abnormalities due to increased plasma volume and viscosity
Recurrent bacterial infections
Physical exam may reveal purpura, lymphadenopathy and hepatosplenomegaly

16. 1-Antitrypsin Deficiency
Genetic Deficiencies
1-Antitrypsin Deficiency
Linked to hepatitis and cirrhosis in neonates; chronic obstructive pulmonary disease and hepatic cirrhosis in adults
Electrophoresis useful in initial evaluation
Quantitative immunochemical assays and phenotyping required

17. Immunoglobulin Deficiencies
Genetic Deficiencies
Immunoglobulin Deficiencies
Isolated IgA deficiency
Isolated IgM deficiency
X-linked immunodeficiency with  IgM
Wiskott-Aldrich Syndrome
Transient hypogammaglobulinemia of infancy
Ataxia Telangiectasia
Severe combined immunodeficiency (SKID)
Common variable immunodeficiency
Pan hypogammaglobulinemia
IgG and IgA deficiency
Isolated IgG deficiency

18. Inflammatory Response
ACUTE
SUBACUTE
CHRONIC
Pre-albumin  
Albumin
ALPHA-1
-lipoprotein
1-acid glycoprotein 
 
1-antitrypsin
ALPHA-2
Cerulopasmin
 or N
Haptoglobin
BETA-1
Transferrin
 
BETA-2
C-3
Fibrinogen
IgA
GAMMA
IgM
IgG
CRP
  

19. Chronic Hepatocellular Disease & Cirrhosis
Liver Diseases
Chronic Hepatocellular Disease & Cirrhosis
Most common pattern includes diffuse increase in IgG with proportionally greater increases in IgA and sometimes IgM
1-antitrypsin is the most sensitive indicator for hepatocellular disease
Pre-albumin is the most sensitive monitor in cirrhosis; 2-macroglobulin and ceruloplasmin also very elevated
All other proteins usually normal or decreased

20. Case 7: Chronic Hepatocellular Disease
Patient: 39 year-old male
History & Physical: Long-term alcohol abuse; ascites, leg swelling, shortness of breath, right side pain, enlarged liver and spleen.
SPE: Hypoalbuminemia with normal migration.  pre-albumin, -lipoprotein and transferrin, consistent with chronic disease. Diffuse IgA, IgG .
Hospital Course: Ascites , right lung abcess treated. Liver tests normal at 4 weeks. Discharged in good condition.
Pre-albumin
Albumin
Haptoglobin
Transferrin
Immunoglobulins
C-3
2-Macroglobulin
1-Antitrypsin
-Lipoprotein
Normal
Patient

21. Liver Diseases Hepatitis
Often associated with acute phase inflammatory response in the early stages
Diffuse elevations in one or more of the immunoglobulins with chronic disease

22. Case 8: Cirrhosis
Pre-albumin
Albumin
Haptoglobin
Transferrin
Immunoglobulins
C-3
-Lipoprotein
1-Antitrypsin
2-Macroglobulin
-Lipoprotein
Normal
Patient
Patient: 54 year-old female History & Physical: Chronic alcoholism; deeply jaundiced, rapid pulse, hepatomegaly, splenomegaly. SPE: Hypoalbuminemia ( anodic mobility due to bilirubin binding).  pre-albumin,  -lipoprotein and  transferrin, consistent with chronic disease. Diffuse IgA, IgG . Hospital Course: Rehydrated and stabilized. Discharged in good condition.

23. Protein Losing Disorders
Selective Protein Loss
Nephrosis can result in elevations in serum concentrations of large proteins with decreases in smaller components.
Serum pattern shows:
Increased 2-macroglobulin, -lipoprotein and polymeric forms of haptoglobin.
Decreased pre-albumin, albumin, 1-acid glycoprotein, 1-antitrypsin, transferrin.
IgM usually elevated, IgG usually decreased.

24. Case 9: Acute Renal Failure
Patient: 57 year-old male History & Physical: Rapid onset abdominal pain and enlargement, edema, decreased urine output. SPE:  albumin,  2-macroglobulin and  -lipoprotein, consistent with selective protein loss due to glomerular-type proteinuria. Hospital Course: Received albumin and hemodialysis. Discharged in improved condition with limited outpatient dialysis. Final diagnosis: Acute renal failure due to tubular necrosis, possibly of a toxic nature.
Albumin
Haptoglobin
Transferrin
Immunoglobulins
C-3
-Lipoprotein
1-Antitrypsin
2-Macroglobulin
-Lipoprotein
Normal
Patient

25. Protein Losing Disorders
Nonselective Protein Loss
Whole blood loss
Congestive heart failure
Liver failure
Hemodilution
Malnutrition
Protein-losing enteropathies – greater decrease in immunoglobulins than other plasma proteins

26. Pregnancy & Hyperestrogenism*
Moderate decreases in prealbumin, albumin, 1-acid glycoprotein and IgG
Large relative increases in 1-antitrypsin, ceruloplasmin, transferrin and fibrinogen
Moderate increase in -lipoprotein
Slight increase in 2-macroglobulin and hemopexin
Haptoglobin and C-3 essentially normal
*Hyperestrogenism (i.e. contraceptive pills, estrogen medications) can mask pathological changes

27. Case 10: Infant w/ Hepatic Involvement
Patient: 5 month-old female History & Physical: Previously healthy; 104º fever/24 hrs, jaundice, hepatomegaly. SPE: Hypoalbuminemia ( anodic mobility due to bilirubin binding),  1-AT,  Hp,  -lipoprotein,  transferrin; consistent with acute inflammation. Age-appropriate hypogammaglobulinemia. Hospital Course: Blood culture positive for gram negative rods. Treated with antibiotics and discharged.
Pre-albumin
Albumin
Haptoglobin
Transferrin
Immunoglobulins
-Lipoprotein
1-Antitrypsin
2-Macroglobulin
C-3
Normal
Patient

28. Case 11: Acute Renal Failure
Patient: 52 year-old male History & Physical: Weakness, progressive shortness of breath, tachycardia, anemia, azotemia, mild hepatomegaly, edema. SPE: Hypoalbuminemia,  1-AT,  Hp,  -lipoprotein,  transferrin; consistent with acute inflammation. Low normal gammaglobulins. CRP? Hospital Course: Blood culture positive for Staph aureus. Vigorous antibiotic therapy. Died 5 days post-admission.
Immunoglobulins
C-reactive protein
Pre-albumin
2-Macroglobulin
Albumin
Haptoglobin
Transferrin
C-3
-Lipoprotein
1-Antitrypsin
Normal
Patient

29. Case 12: Chronic Renal Failure
Patient: 68 year-old male History & Physical: Chronic bladder outlet obstruction; abdominal pain, hematuria, urinary or bladder infection. SPE: Hypoalbuminemia with  anodal mobility;  1-AT,  Hp,  transferrin; consistent with acute inflammation. Random urine pattern consistent with mixed glomerular-tubular proteinuria. Hospital Course: Continued antibiotics, hemodialysis.
Pre-albumin
Haptoglobin
Transferrin
C-3
-Lipoprotein
1Antitrypsin
Albumin
2-Macroglobulin
-Lipoprotein
Immunoglobulins
Normal
Patient

30. Case 13: Acute & Subacute Inflammation
Pre-albumin
Haptoglobin
Transferrin
C-3
-Lipoprotein
1-Antitrypsin
Albumin
2-Macroglobulin
Immunoglobulins
-Lipoprotein
Normal
Patient
Patient: 74 year-old male
History & Physical: Pneumonia, 2 weeks duration.
SPE: Hypoalbuminemia with  anodal mobility;  1-AT,  Hp,  transferrin,  a-lipoprotein, and  C-3; consistent with acute and subacute inflammation.
Hospital Course: Antibiotics, discharged in good condition.

31. Case 14: Diffuse Hypergamma-globulinemia with Lymphoma
Patient: 63 year-old female History & Physical: Pruritis, sweats and fatigue, multiple dermal nodules. SPE: Non-specific findings – diffuse increase in immuno-globulins, suggesting chronic inflammation. Hospital Course: Biopsy reports consistent with lymphoma; patient discharged for outpatient treatment and followup.
2-Macroglobulin
Immunoglobulins
Pre-albumin
Haptoglobin
Transferrin
C-3
-Lipoprotein
1-Antitrypsin
Albumin
-Lipoprotein
Normal
Patient

32. Urinary Proteins of Plasma Origin
Normal Urinary Protein < 150 mg/day
Primarily Filtered Plasma Proteins – albumin, low MW species, immunoglobulin components
Remainder – derived from urinary tract
Electrophoretic pattern of normal urine – trace albumin, sometimes transferrin
Urine
Serum
Albumin
Sometimes Transferrin

33. Urinary Proteins of Plasma Origin
Proteinuria
Glomerular – results from increased passage of proteins through the glomerulus; characterized by loss of plasma proteins the size of albumin or larger
Tubular – results from decreased capacity of tubules to reabsorb proteins; characterized by inceased excretion of very small proteins such as 2-microglobulin
Systemic – exercise, postural, pregnancy, overflow

34. Urinary Proteins of Plasma Origin
High Resolution protein electrophoresis developed with a sensitive protein stain
Excellent analytical technique
Easily distinguishes & characterizes the various types of proteinuria
Provides useful insight on specific functions within the nephron
“Biochemical biopsy”
Differential diagnosis & monitoring of patients with renal dysfunction

35. Urinary Proteins of Plasma Origin
Glomerular Proteinuria
Renal glomeruli are ultrafilters for macromolecules
Damage to renal glomeruli leads to increased urinary excretion of proteins (30,000 to 100, daltons) which are normally retained
Some selectivity remains – very large proteins (>500,000 daltons) still retained by glomerulus
In early disease, very LMW proteins (<15, daltons) are still reabsorbed by tubules and absent from urine

36. Urinary Proteins of Plasma Origin
Glomerular Proteinuria
Urine Protein Pattern strong band of albumin strong, broad 1 zone due to 1-acid glycoprotein and 1-antitrypsin strong band of transferrin (1)
Serum Protein Pattern marked decrease inalbumin marked decrease in 1-acid glycoprotein and 1-antitrypsin increases in large proteins: 2-macroglobulin, -lipoprotein

37. Urinary Proteins of Plasma Origin
Severe Proteinuria / Nephrotic Syndrome
Total Protein > 3.5 g/day
Hypoalbuminemia and hyperlipidemia
Massive edema

38. Urinary Proteins of Plasma Origin
Disorders Associated with Nephrotic Syndrome
Glomerular diseases
Proliferative glomerulonephritis
Other diseases Infections - Drugs Neoplasia - Multisystem diseases Miscellaneous - Hereditary disorders

39. Case 1: Nephrotic Syndrome with Glomerular Proteinuria
Patient: 45 year-old white male
History & Physical: Diabetes mellitus / nephrotic syndrome.
SPE:  pre-albumin, albumin and transferrin with  2-macro-globulin and -lipoprotein; consistent with selective renal protein loss.
Urine Elp:  albumin, 1-antitripsin and transferrin with trace pre-albumin and 2-components; consistent with sieving glomerular-type protein loss.
Serum Urine
Transferrin
Albumin
1 acid glycoprotein
1 AT
1AT
2-Macro HP
-Lippo
C-3
IgA
IgM
IgG
Normal

40. Urinary Proteins of Plasma Origin
Tubular Proteinuria
Normal tubules reabsorb and catabolize 95 to 99% of proteins from glomerular filtrate
Tubular disease reduces capacity to reabsorb and catabolize, resulting in increased urinary excretion
Causes of tubular proteinemia: Chronic metal exposure (cadmium, gold, lead, mercury) Acute and chronic pyelonephritis Renal transplant rejection Toxicity due to aminoglycoside therapy Balkan nephropathy Uremic medullary cystic disease

41. Urinary Proteins of Plasma Origin
Tubular Proteinuria
Serum Protein Pattern little or no change since LMW proteins are present in very low levels
Urine Protein Pattern faint albumin band double band in 2 area due to 2-microglobulin strong band in mid- region due to 2-microglobulin diffuse background in  region due to free light chains

42. Urinary Proteins of Plasma Origin
Mixed Glomerular/Tubular Proteinuria
Chronic renal disease or renal failure
Combined pattern with both “glomerular-type” and “tubular-type” proteins in the urine

43. Case 2: Heavy Metal Toxicity with Tubular Proteinuria
Serum Urine
Normal
Albumin
2Micro- globulin
2Micro globulin
1-AT
2-Macro HP
Transferrin
-Lipo
C-3
IgA
IgM
IgG
Patient: 52 year-old black male
History & Physical: Metal worker.
SPE: Essentially normal.
Urine Elp: Trace albumin,  2-microglobulin and 2-microglobulin; consistent with tubular-type proteinuria.

44. Urinary Proteins of Plasma Origin
Other Conditions with Increased Urinary Protein Excretion
Exercise Proteinuria – strenuous muscular exercise increases excretion of HMW and LMW proteins
Postural or Orthostatic Proteinuria – present/upright, absent/recumbent – benign or underlying cause?
Pregnancy – usually transitory, may be associated with toxemia, delivery, UTI, or asymptomatic
Overflow Proteinuria – increased plasma concen- tration of LMW proteins, e.g. BJP, myoglobin, hemoglobin, acute phase reactants

45. Case 3: Septicemia with Overflow Proteinuria
Patient: 65 year-old white female
History & Physical: High fever, chills, sweats, joint and muscle aches.
SPE:  pre-albumin, albumin, -lipoprotein and transferrin,   1-antitrypsin, haptoglobin, C3 & C-reactive protein; consistent with acute inflammation.
Urine Elp: Trace albumin and transferrin,  1-acid glycoprotein, faint acute phase reactants; consistent with overflow proteinuria
Acute phase
reactants
Albumin
1-acid
glycoprotein
1-AT
1AT
2-Macro HP
Transferrin
C-3
IgA
IgM
IgG
-Lipo
Serum Urine
Normal

46. Cerebrospinal Fluid Proteins CSF Proteins versus Plasma Proteins
CSF Total Protein: Much less than in plasma – 450 mg/L, ages 10 – 40 years, lumbar Slightly higher, ages Slightly higher for verticular & cisternal specimens
CSF Production: Primarily ultrafiltration and active transport of proteins, ions, water and other components through the choroid plexus. Small amount produced within CNS

47. Cerebrospinal Fluid Proteins CSF Protein Composition
Albumin – major protein present, 55 – 75% of the total
1 – primarily 1-antitrypsin, -lipoprotein absent
2 – essentially absent
1 – transferrin detectable
2 – carbohydrate-deficient “CSF-specific” transferrin
 – almost exclusively IgG, faint “-trace”
Normal CSF
Abnormal
Patient CSF
Pre-albumin
Albumin
1-Antitrypsin
Haptoglobin
Transferrin
CSF TF
Oligoclonal Bands

48. Cerebrospinal Fluid Proteins Permeability of Blood-CSF Barrier
Increased permeability caused by bacterial or viral menigitis neoplastic infiltration of meninges polyneuropathies disk herniations cerebral infarctions
Integrity of blood-CSF barrier Total CSF protein 2-macroglobulin Protein ratios

49. Cerebrospinal Fluid Proteins Abnormal CNS Protein Production
Demyelinating Diseases Increased IgG synthesis in Multiple Sclerosis and Subacute Sclerosing Panencephalitis
IgG as Percentage of Total Protein Considers increased permeability vs. synthesis IgG >10% suspicious; >13% abnormal production likely.
CSF/Serum Ratios Considers increased plasma concentration % of MS patients show values above reference range.
Oligoclonal Banding Indicative of MS; new more sensitive procedure is IEF.

50. Cerebrospinal Fluid Proteins
Oligoclonal Banding
Multiple, restricted bands in the gamma fraction
Detectable by high resolution electrophoresis and IEF methods
90% of MS patients exhibit oligoclonal banding
New CSF IEF methods have become the gold standard for diagnosing MS in Europe.

51. Cerebrospinal Fluid Proteins
Isoelectric Focusing
IEF followed by immunoblotting
Oligoclonal banding in unconcentrated CSF but not serum is diagnostic of MS
Gold Standard in Europe
S = Serum C = CSF

52. Cerebrospinal Fluid Proteins Laboratory Protocol to Rule Out MS
Draw both CSF and serum samples.
Note CSF color and appearance. Determine total protein, glucose, white cell count, differential, red cell count.
Perform high resolution protein electrophoresis on concentrated CSF.
Perform high resolution protein electrophoresis on serum for presence or absence of banding.
In borderline cases, determine CSF and serum albumin and IgG ratios.

53. Cerebrospinal Fluid Proteins Laboratory Findings in Multiple Sclerosis
NSC
Patient Serum
Patient CSF
Normal CSF
CSF Appearance: Clear
Leukocytes: Usually normal
Total Protein: Usually normal
Glucose: Normal
Electrophoresis: Oligoclonal banding in 90% of cases
IgG Ratio: Elevated in 90% of cases

54. Cerebrospinal Fluid Proteins Clinical Manifestations of MS
40% of patients present with optic neuritis
60 to 70% present with evidence of spinal cord or brainstem lesion

55. Case 1: Multiple Sclerosis
Patient: 33 year-old female
History & Physical: left-side numbness - 9 months; fuzzy vision - 2 weeks.
SPE: Normal.
CSF: Clear, colorless. Cell count, differential, glucose and total protein all normal.
CSF Elp: Increased gamma globulins, strong oligoclonal banding. Consistent with demyelinating disease.
Normal CSF
Patient CSF
Patient Serum
NSC

56. Case 2: Multiple Sclerosis
Patient: 31 year-old male
History & Physical: Unsteady walk, leg numbness, inability to concentrate, irritability, slurred speech, disturbed vision, urinary urgency.
SPE: Normal.
CSF: Clear, colorless. Normal glucose. 29 WBC/mm3 (9% lymphocytes, 1% moncytes). Total Protein 55 mg/dL.
CSF Elp: Gamma globulin increase, strong oligoclonal banding; consistent with MS.
Normal CSF
Patient CSF
Patient Serum
NSC

57. Case 3: Multiple Sclerosis
Patient: 36 year-old female
History & Physical: numbness in legs – 1 month; urinary urgency and blurred vision – 4 months; tires easily.
SPE: Normal.
CSF: Clear, colorless. Normal total protein, WBC count, and glucose.
CSF Elp: Profound gamma globulin increase, very strong oligoclonal banding. (IgG & ratios markedly elevated.) Consistent with MS.
Normal CSF
Patient CSF
Patient Serum
NSC

58. Case 4: Suspected Viral Meningitis
Patient: 25 year-old white female
History & Physical: Progressive headache, lethargy, altered mental status; “flu” – 4 weeks prior.
SPE: Borderline hypoalbuminemia.
CSF: Clear, colorless. Normal glucose. 158 WBC/mm3 (1oo% moncytes). Total Protein 83 mg/dL.
CSF Elp: Increased gamma globulins, prominent oligoclonal banding. Considering  total protein; consistent with viral encephalitis. Discharged for outpatient followup.
Normal CSF
Patient CSF
Patient Serum
NSC

59. Case 4: Suspected Viral Meningitis (cont’d)
Patient: Readmitted 8 days later with redeveloped headache.
Normal CSF
Patient CSF
Patient Serum
NSC
CSF: Clear, colorless. Normal glucose and protein. 103 WBC/mm3 (90% lymphocytes, 10% neutrophils).
CSF Elp: Gamma globulins and oligoclonal banding decreased from previously. Transient increase consistent with CNS infection / inflammation.

60. SPIFE 3000 Analyzer
Automated sample application, separation and staining of visible analytes
Automated reagent application for enzymatic assays
On-board cooling for excellent separation and resolution
On-board availability of two stains

61. SPIFE Assays Cholesterol Profile Serum Proteins Split-Beta SPE
Urine Proteins
Immunofixation
Urine Immunofixation
Hi Resolution Proteins
Alkaline Hemoglobins
Acid Hemoglobins
CK* & LD Isoenzymes
Alk Phos Isoenzymes*
CSF IgG IEF*
Lipoproteins
*Pre-market approval pending 4/02

62. SPIFE Hi Res Proteins Excellent separation
Active cooling preserves pattern integrity
Acid violet stain for added sensitivity
Urine

63. SPIFE IFE 15, 9, 6 & 3 Up to 210 profiles in 8 hours
Highest sensitivity antisera
Acid violet for best sensitivity
Built in QC wells

64.
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